Ana M G6mez

Heping Cheng

W Jonathan Lederer

Donald M Bers

Cheng

& Lederer

Cannell

; Cannell

Cheng & Lederer

; L6pez-Lopez

Balke Weir

; Lopez-Lopez

Ca2O diffusion and sarcoplasmic reticulum transport both contribute to [Ca2+]i decline during Ca2O sparks in rat ventricular myocytes

We sought to evaluate the contribution of the sarcoplasmic reticulum (SR) Ca2P pump (vs. diffusion) to the kinetics of [Ca2+], decline during Ca2+ sparks, which are due to spontaneous local SR Ca2+ release, in isolated rat ventricular myocytes measured using fluo-3 and laser scanning confocal microscopy. 2. Resting Ca2+ sparks were compared before (control) and after the SR Ca2+-ATPase was either completely blocked by 5/M thapsigargin (TG) or stimulated by isoprenaline. Nae-Ca2+ exchange was blocked using Na+-free, Ca2+-free solution (0 Na+, 0 Ca2+) and conditions were arranged so that the SR Ca2+ content was the same under all conditions when Ca2+ sparks were measured.

3. The control Ca2P spark amplitude (281 + 13 nM) was not changed by TG (270 + 21 nM) or isoprenaline (302 + 10 nM). However, the time constant of [Ca2+]i decline was significantly slower in the presence of TG (29-3 + 4-3 ms) compared with control (21P6 + 1P5 ms) and faster with isoprenaline (14X5 + 0 9 ms), but in all cases was much faster than the global

[Ca2+], decline during a control twitch (177 + 10 ms).

4. The spatial spread of Ca2+ during the Ca2P spark was also influenced by the SR Ca2P pump.

The apparent 'space constant' of the Ca2+ sparks was longest when the SR Ca pump was blocked, intermediate in control and shortest with isoprenaline.

5. We conclude that while Ca2P diffusion from the source of Ca2+ release is the dominant process in local [Ca2+]i decline during the Ca2P spark, Ca2P transport by the SR contributes significantly to both the kinetics and spatial distribution of [Ca2+], during the Ca2P spark.

[Ca2P], (Cheng, Cannell & Lederer, 1995). Six factors could contribute to the decline of the Ca2P spark: (1) closure of the SR Ca2P release channel (i.e. RyR); (2) Ca2+ binding to buffers; (3) Ca2+ diffusion away from the release site in free solution or on buffers; (4) Ca2+ pumping back into the SR via the SR Ca2+-ATPase; (5) Ca2P accumulation by the mitochondria; and (6) Ca2+ extrusion from the cell by the Na+-Ca2+ exchanger and sarcolemmal Ca2+-ATPase.

Global elevation of [Ca2+],, produced by a normal twitch [Ca2P], transient, arises as Ca2+ sparks are recruited by the activation of L-type Ca2P channels (Cannell et at. 1994, 1995; L6pez-Lopez et al. 1995; Santana, Cheng, G6mez, Cannell & Lederer, 1996). Such global increases in [Ca2+]i remove diffusion as a factor contributing to the fall of [Ca2+]i because the spatial distribution of [Ca2+], is uniform (Cheng et al. 1995). In this situation, the dominant processes contributing to global [Ca2+], decline are SR Ca2+-ATPase activity and the Na+-Ca2+ exchange (Bassani, Bassani & Bers, 1994). In rat ventricular myocytes, the proportion of Ca2+ transported by the SR Ca2+ pump vs. that by Nae-Ca2+ exchange during relaxation is about 92 % vs.7 %, whereas in rabbit ventricular myocytes, owing to slower SR Ca2+ pumping and faster NaW-Ca2+ exchange this is 70 % vs.28% (Bassani, Bassani & Bers, 1992, 1994; Negretti, O'Neill & Eisner, 1993). In contrast, the Ca2+ spark represents a local t To whom correspondence should be addressed.

This manuscript was accepted as a Short Paper for rapid publication.

elevation of [Ca2+], which is free to diffuse from its site of origin to other regions of the cardiac myocyte where [Ca2+] is lower. Thus, the rapid decay of the Ca2+ spark (time constant, r, -1%,-25 ms) when compared with the global [Ca2+]i transient (T, -200 ms) may arise largely from a diffusional component that only the Ca2+ spark has. However, as Ca2+ diffuses away from the release site it is subject to transport by the same Ca2P transport systems mentioned above. In this paper we have investigated the role of SR Ca2+-ATPase in the decline of the Ca2+ spark. We have made use of the specific inhibitor of SR Ca2P-ATPase thapsigargin (Sagara, Fernadez-Belda, de Meis & Inesi, 1992). We examined Ca2+ sparks when SR Ca2+-ATPase was completely inhibited, but without depleting the SR of Ca2P (Bassani, Bassani & Bers, 1993; Bassani, Yuan & Bers, 1995). We also used isoprenaline to stimulate SR Ca2+-ATPase via cyclic AMP and the phosphorylation of phospholamban [START_REF] Tada | The stimulation of calcium transport in cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase[END_REF]. The results indicate that either blocking or stimulating SR Ca2+-ATPase significantly alters the kinetics of local [Ca2P]i decline during a Ca2+ spark, as well as its spatial spread.

METHODS

Cells

Single ventricular cardiac myocytes were prepared from male, Sprague-Dawley rats (-2 months old) by standard methods (Cheng et al. 1993; Cannell et al. 1995). Briefly, animals were killed by an overdose of sodium pentobarbitone (100 mg kg-' i.P.) after which hearts were quickly removed and perfused using the Langendorff method with a series of physiological solutions, including one that contained collagenase (Worthington, Type II; 1 mg ml-'). Following isolation of single cells, extracellular [Ca2+] ([Ca2'].) was increased to 1I0 mm and the cells were stored at room temperature (20-23 0C) until needed.

Measurement of [Ca2]i

Cells were loaded with the Ca2P indicator fluo-3 either by diffusion of the fluo-3 (100 /sM) salt from the voltage-clamp pipette into the cell or by using the acetyoxymethyl (AM) ester derivative of fluo-3. This second method involves dissolving fluo-3 AM (50 ,sg) in 100 #1 of a DMSO-Pluronic F-127 mixture (4: 1 by weight) and incubating cells in a physiological saline containing 5 ,M fluo-3 for 20 min and then allowing 15 min in physiological saline alone for de-esterification. Fluorescence was measured using a confocal microscope (see below) with 488 nm excitation from an argon ion laser [START_REF] Cheng | Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle[END_REF].

Images from the confocal microscope

A laser scanning confocal microscope (MRC 600; Biorad, Cambridge, MA, USA) with a x 63 magnification Zeiss Neofluar lens (numerical aperture, 1 25) was used to acquire fluorescence images. Two kinds of image were obtained, both with a resolution of 0 4 ,sm (in the horizontal plane) and 0'7 ,um (in the vertical plane). Images of the whole cell in the x-y plane at full spatial resolution were obtained at 1 s-'. Line-scan images were obtained at 500 s-' but with resolution only along a single line (Cheng et al. 1993); these images were used for all of the kinetic data obtained here. Images were analysed using SOM and COMOS software (Biorad) and IDL (Research Systems Inc., Boulder, CO, USA).

The fluo-3 fluorescence was transformed to [Ca2+]i by a pseudoratio method (Cheng et al. 1993; Cannell et al. 1994) using the following equation:

[Ca2+],

Kd(F/FO)

(Kd/[Ca]i,rest) + 1 -(F/FO)
where Kd is the dissociation constant for fluo-3 (400 nM), F is the fluorescence intensity and Fo is the intensity at rest at each position determined as the mean fluorescence intensity of the lowest fifty pixels along each time line. The [Ca2+], at rest ([Ca2]rest) for this Fo was assumed to be 100 nm.

Solutions

Changes in superfusion solutions were carried out in less than 3 s using a bath equipped for rapid solution change. Three basic solutions were used in our experiments. The normal Tyrode solution (NT) contained (mM): 140 NaCl, 5-4 KCl, 2 CaCl2, 1 MgCl2, 10 Hepes and 10 glucose, pH 7*4. The Na+-free, Ca2+-free (0 Na+, 0 Ca2+) solution was the same, except Na+ was replaced by Li+ and CaCl2 was replaced by 1 mm EGTA. A Nae-free solution (O Na, 2 Ca2+) had the same composition as NT, but Nae was replaced with equimolar Li+. Thapsigargin (TG) was added from a 10 mm stock in DMSO to the appropriate solution immediately before use to give a final concentration of 5 juM. Caffeine and isoprenaline were added directly to the appropriate solution to produce final concentrations of 10 mm and 1 uSM, respectively. In experiments that used patch clamping to control membrane potential (in whole-cell mode), the pipette-filling solution contained (mM): 60 CsCl, 60 KCl, 5 K2ATP, 5 MgATP, 10 Hepes and 0-10 fluo-3, pH 7-2. Patch-clamp electrodes were pulled from 1-5 mm filamented thin-walled borosilicate glass capillaries (World Precision Instruments, New Haven, CT, USA). All experiments were carried out at room temperature. Statistical comparisons were carried out using Student's t test or one-way analysis of variance. P < 0 05 was considered significant and n refers to the number of analysed Ca2' sparks.

RESULTS

Protocol to allow assessment of Ca2+ sparks with SR Ca2+-ATPase blockade by thapsigargin

To assess the contribution of SR Ca2P-ATPase to the [Ca2P],

decline during Ca2+ sparks we compared control Ca2+ sparks with those observed when SR Ca2+-ATPase was completely blocked by TG. Comparison of Ca2+ sparks in the absence and presence of SR Ca2+-ATPase requires two fundamental conditions. First, the SR Ca2+-ATPase must be completely inhibited and second, Ca2P sparks of comparable amplitude must be observable in the presence of Ca2+-ATPase blockade. To achieve this we used the experimental strategy developed by Bassani et al. (1993, 1994, 1995), where SR Ca2+-ATPase could be completely blocked without significantly changing the SR Ca2P content. The results were obtained as follows.

After steady-state stimulation at 1 Hz, the amplitude of a caffeine (10 mM)-induced [Ca2P], transient in 0 Nae, 0 Ca2P solution was used to assess the SR Ca2+ content. Caffeine was washed away in NT and the cell returned to steadystate stimulation. Then stimulation was stopped and the cell was superfused in 0 Na+, 0 CaW+ solution to abolish CW+ flux via Na+_Ca2+ exchange and maintain constant SR Ca2+ load during rest. Control Ca2+ sparks were acquired and then the cell was exposed to 5 ,UM TG for 2 min (in 0 Nae, 0 Ca2P solution), which was sufficient to block completely the SR Ca2P pump [START_REF] Bassani | Twitchdependent SR Ca accumulation and release in rabbit ventricular myocytes[END_REF], and see below). During this time resting [Ca2P]i did not change. After Ca2P pump blockade, Ca2+ sparks were again acquired in 0 Na+, 0 Ca2+ solution for 1-2 min until the SR Ca2+ content was assessed by a second caffeine exposure in 0 Na+, 0 Ca2+ solution. After 2 min of TG treatment there was no significant decrease in the peak [Ca2+]i during the caffeine-induced contracture (control, 1077 + 187 nM; TG, 1087 + 198 nM; see Fig. 1A). While the amplitude of caffeine-induced [Ca2+]i transients in rat ventricular myocytes are generally not affected by the presence or absence of [Na+]o (Bassani et al. 1994), 0 Na+, 0 Ca2+ solution with caffeine was used to assess SR Ca2+ content in the present study Isoprenaline (Iso) values were obtained after a series of 5-10 voltage-clamp pulses to load the SR in the presence of 1 /SM Iso (n = 15). Control values were under similar conditions but without TG or Iso present (n = 17). B, steady-state twitch [Ca2+]i transients under control conditions and after exposure first to TG (to inhibit the SR Ca2+ pump) and then caffeine (to deplete the SR). Time constants (T) of [Ca2+]1decline are indicated. C, the peak of the control caffeine exposure (in 0 Na+, 0 Ca2+ solution) provides an estimate of SR Ca2+ content [START_REF] Bassani | Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms[END_REF]). The lower trace shows a caffeine exposure after TG application, caffeine- induced depletion and pacing at 1 Hz in an attempt to reload the SR. transients by -90% (Fig. 1B). Figure 1C shows that TG completely prevented reloading of the SR as assessed by a third caffeine exposure (after TG exposure, caffeine-induced SR Ca2+ depletion and extensive twitches in an effort to reload). This confirms that the TG concentration used here was sufficient to completely prevent SR CW+ uptake. In addition, the r of [Ca2+]1 decline during the twitch was increased by TG from 177 + 10 to 1285 + 215 ms, similar to previous results where SR CW+ uptake was inhibited by either TG or caffeine (Bassani et al. 1994). Thus, this protocol has allowed acquisition of Ca2+ sparks at comparable SR Ca+ content in both the presence and absence of SR Ca2+ pump function.

Protocol to measure Ca2+ sparks in the presence of SR Ca2+ pump stimulation by isoprenaline An additional group of cells was studied when the SR Ca2+ pump was stimulated by isoprenaline (1 FM). In this series, cells were voltage clamped and loaded with fluo-3 via the patch pipette. After a series of five to ten conditioning pulses (from -80 to 0 mV for 200 ms at 0 03 Hz in 0 Na+, 2 Ca2+ solution) either Ca2+ sparks were measured or caffeine was applied (in 0 Na+, 0 Ca2+ solution) to assess the SR Car+ load. This sequence was repeated after exposure to 1 FM isoprenaline for 10 min. In the presence of isoprenaline, the pulse protocol was modified so that the steady-state SR Cat+ load was comparable to that for control (steps were to only -10 mV). The amplitude of caffeineinduced [Ca2+]i transients in isoprenaline was not significantly different from control levels (control, 1077 + 188 nM; isoprenaline, 1040 + 161 nm; The amplitude and kinetics of control Ca2+ sparks were statistically indistinguishable between the control series for the TG and isoprenaline experiments above. Thus, the control sparks were pooled for further analysis. Figure 2A shows signal-averaged Ca2+ sparks for control, TG and isoprenaline experiments. Figure 2B shows that the amplitude of Ca2P sparks was not significantly altered by either TG or isoprenaline in these experiments (control, 281 + 13 nm; TG, 270 ± 21 nM; isoprenaline, 302 + 10 nM; n= 27, 10 and 86 Ca2+ sparks from 8, 5 and 6 cells, respectively). This agrees with the similar SR Ca2+ load above and observations of a stereotypical Ca2P spark amplitude (Cheng et al. 1993).

Isoprenaline increased the frequency of Ca2+ sparks (from 0-63 to 1P9 s-'), despite an unchanged SR Ca2P load.

Figure 2C shows that the r of [Ca2P], decline during Ca2P sparks was different among the three groups. The T was longest after TG (29f3 + 4*3 ms), shortest in isoprenaline (14-5 + 09 ms) and intermediate in control conditions (21-6 + 1.5 ms). This indicates that the SR Ca2P-ATPase contributes significantly to the [Ca2P], decline of the normal Ca spark.

Spatial spread of Cae' sparks with thapsigargin and isoprenaline The slower local [Ca2+]i decline observed with SR Ca2+ pump blockade might also result in a greater spatial spread, since the SR Ca2+ pumps are expected to be broadly distributed throughout the cell. Figure 3A shows the time and width A. M. Gomez, H. Cheng, W J Lederer and D. M. Bers of the cell. The CaZ+ spark spreads in both directions along the line being scanned without obvious asymmetry. While the precise spatial resolution along the length of the scan line is somewhat limiting, the full-width half-height of the Ca2+ sparks in the three conditions was significantly different (see Fig. 3B andC). The spatial spread of the Ca2P sparks was largest with TG (2-4 ± 019 ,um), shortest in isoprenaline (1P56 ± 0 07 #sm) and intermediate in control conditions (1P9 ± 0 09 /sm). Thus the SR Ca2+-ATPase function modulates both the spatial as well as temporal spread of Ca2+ sparks.

DISCUSSION

Ca2+ sparks appear to represent relatively stereotypical elementary events of SR Ca2P release both during excitation-contraction coupling and at rest (Cheng et al. 1993; Cannell et at. 1994, 1995; Lopez-Lopez et al. 1995).

Furthermore, the apparently homogeneous global [Ca2+]i transient during a twitch probably reflects the temporal and spatial summation of many individual Ca2+ sparks triggered and synchronized by depolarization and consequent Ca2P influx. Indeed, most of the L-type Ca2P channels must be blocked so that spatially distinct Ca2+ sparks can be observed upon activation of the Ca2P current. It is still unclear how many individual SR Ca2P release channels contribute to the functional unit during an individual Ca2+ spark (e.g. see Cannell et al. 1994; Tsugorka, Rio & Blatter, 1995; Klein, Cheng, Santana, Jiang, Lederer & Schneider, 1996; Lipp & Niggli, 1996). Results in the present study also illustrate that characteristic Ca2+ sparks occur in the complete absence of [Ca2+], (with EGTA), indicating that Ca2+ influx is not essential for triggering the characteristic Ca spark.

Since the SR Ca2P content and the Ca2P spark amplitude

were not different, this helps to rule out potential complications due to (1) intrinsic amplitude effects on T (Bers & Berlin, 1995), (2) slowed kinetics from Ca2P sparks which originate from out of the plane of focus, or (3) fluorescence obtained from outside the nominal voxel space. Nevertheless, these sort of technical constraints place some limitations on the precise quantitative interpretation. Thus we restricted our analysis to semiquantitative terms.

As a first approximation, the rate constants (k) of [Ca2+]i decline due to SR Ca2P-ATPase activity and diffusion during a Ca2P spark could be considered to be additive allowing some initial comparisons to be made (where k = 1/i from data in Fig. 2C). Thus, the rate constant for

[Ca2+], decline due to diffusion only (kD, with TG in 0 Nae, 0 Ca2P solution) is 34-1 s-' and that for both diffusion and the SR Ca2+-ATPase activity (kD + kSR) is 46-3 s-.

From these numbers it can be inferred that diffusion would account for 74% of the rate of [Ca2P], decline and SR Ca2+- ATPase activity for 26%. When SR Ca2+-ATPase is stimulated by isoprenaline (lD + ksR ISO = 69-0 s-') this simplified analysis would indicate that the balance is changed to about 50 % due to diffusion and 50 % due to the stimulated SR Ca2P-ATPase. This is obviously an oversimplification since the above would imply that kR iS -12 s-, whereas the value based on global [Ca2P], decline would give a rate constant of only about 6 s-5 (i.e.

T= 177 ms for [Ca2P]1 decline during a twitch).

Another simplified quantitative consideration uses the number of Ca2+ ions released during a Ca2+ spark based on an SR Ca2+ release flux of 2 pA for 5 ms (31 191 Ca2+ ions;

Tinker, Lindsey & Williams, 1993). In a 2 ,sm diameter sphere this would be sufficient to raise local total [Ca2+] by 12-3 /uM or local mean free [Ca2+] by 123 nm (assuming 100: 1 Ca2+ buffering; Hove-Madsen & Bers, 1993). This is comparable to the Ca2+ spark. The number of Ca2+ ions transported by SR Ca2+-ATPase within this same sphere can also be calculated using the SR Ca2+-pump function estimated by Bassani et al. (1994) in intact rat myocytes (208 UM s-'/(1 + (184 nM/[Ca2+]1)3 9)). The [Ca2+], which serves as the driving function (in time and space) was taken as an exponentially declining function of time (T, 20 ms) with a Gaussian spatial profile (full width at half-maximum, 2 ,um) analogous to Fig. 3A. Numerical integration of this Ca2+ pump flux yields a transport of 6616 ions or 21 % of the total Ca2P ions released as mentioned above. More precise mechanistic calculations would require a detailed spatiotemporal model of Ca2+ movements (including diffusion, buffers, pumps and leaks) beyond the scope of the present work.

We have used thapsigargin to block completely SR Ca!+-ATPase during Ca2+ sparks. The Na+-Ca2+ exchange did not contribute to [Ca2P], decline during the Ca2+ sparks in this study since they were measured in 0 Na+, 0 Ca2+ solution.

However, Nae-Ca2+ exchange participation during control Ca2+ sparks seems unlikely because [Ca2+]i decline during control Ca2+ sparks in 0 Na+, 0 Ca2+ solution were similar to those in normal Na+-and Ca2+-containing solution (i.e. T, 19-6 + 1-6 s, n = 20, not significantly different from control 0 Na+, 0 Ca2+ solution). This is not surprising since the Na+-Ca2+ exchange is about ten times slower than SR Ca2+-ATPase in rat ventricular myocytes (Bassani et at. 1992, 1994). Thus, the effect of Na+-Ca2+ exchange on Ca2+ sparks ought to be an order of magnitude smaller than attributed here to SR Ca2+-ATPase. If Ca2+ released during a Ca2+ spark has preferential access to the Na+-Ca2+ exchanger rather than the SR Ca2+ pump, the effect could be greater.

Since the SR Ca2+-ATPase and mitochondrial Ca2P transport are both more than ten times slower than the Nae-Ca2+ exchange (Bassani et al. 1992, 1994), these

systems are not likely to contribute quantitatively to the decline of [Ca2+]i during Ca2+ sparks. Slow cytosolic Ca2+ buffering could also contribute to the decline of [Ca2+],

during Ca2+ sparks (e.g. at sites from which Mg2+ or protons might first have to dissociate before Ca2+ can bind), but assessment of this contribution would again require more detailed data and modelling. At higher temperature it might be expected that the SR Ca2+ pump rate would increase to a greater extent than diffusion, so the SR Ca2+ pump would probably contribute at least as much to [Ca2+]i decline during Ca2+ sparks at 37 °C as described here.

Isoprenaline decreases the sphere of influence of a given Ca2+ spark, such that SR Ca2+ release from one area will be less likely to induce Ca2+ release from a neighbouring region.

In principle this aspect of SR Ca2P pump stimulation would tend to decrease the propagation of Ca2P waves. From a practical standpoint, however, this effect will be counter- balanced by the fact that isoprenaline stimulates SR Ca2+-ATPase and load which would increase the likelihood of Ca2+ sparks or CaP+ waves. Conversely, decreased SR Ca2+- ATPase activity would increase the spread of a given Ca2+ spark.

  Figure 1. SR Ca2+ content and twitch [Ca2+]i transients before and after thapsigargin A, SR Ca2P content assessed as the amplitude of the [Ca2+]i transient induced by rapid exposure to 10 mM caffeine under the 3 conditions used to measure Ca2+ sparks. Thapsigargin (TG) values were obtained after 1 Hz steady-state stimulation and 2 min of rest in 0 Na+, 0 Cad+ solution with 5/M TG (n = 6).

  I ................... A. M. C6mez, H. Cheng, W J Lederer and D. M. Bers (1'7 + 0 4 s in NT vs. 8-9 + 3-2 s in 0 Na+, 0 Ca2+ solution), in agreement with the results of Bassani et al. (1994). The second caffeine exposure after acquisition of Ca2+ sparks in TG caused depletion of SR Ct content and reduced the amplitude of subsequent twitch [Ca2+],

  Fig. IA).

Figure 2 .

 2 Figure 2. Ca2O spark characteristics after SR CaO' pump blockade or stimulation A, signal-averaged Ca2+ sparks in the 3 experimental conditions. Sparks were temporally aligned by matching their initial rising phase and the vertical lines indicate + 1 S.E.M. (n = 10, 27 and 86 CaP+ sparks, respectively). B, mean amplitude of Ca2+ sparks and C, mean time constant of Ca2+ spark decline under the 3 conditions. * P < 0 05 and ** P < 0 001, significantly different from control conditions.

Figure 3 .

 3 Figure 3. Spatial spread of Ca2e sparks after SR Ca2+ pump blockade or stimulation A, 3-D plot of local [Ca2+]i during a control line scan as a function of time and distance along the transverse scan line. B, mean values of the Ca2+ spark width at half-maximal amplitude under the 3 experimental conditions. * P < 0'05 and ** P < 0-01, significantly different from control conditions. C, signal-averaged Ca2P spark widths in the 3 experimental conditions. Sparks were aligned by placing their maximal value at x = 0 and the vertical lines indicate + 1 S.E.M. (n = 10, 27 and 86, respectively). The dashed horizontal lines indicate the full width at half-maximal amplitude.

J Phy8iol.496.2

J Physiol.496.2

Acknowledgements

This work was supported by grants from the United States Public Health Service (HL-30077, HL-25675 and HL-36974) to D.M.B. and W.J.L. A.M.G. is supported by the Ministerio de Educaci6n y Ciencia (EX94-03838550). Author's present address H. Cheng: Laboratory of Cardiovascular Sciences, National Institute on Aging, Baltimore, MD 21224, USA. Author's email address D. M. Bers: dbers@luc.edu Received 30 July 1996; accepted 14 August 1996.


Garcia Julien

Psa

Dominique Millet

Pierre Tonnelier

STATE OF THE ART IN THE ECO-DESIGN FIELD: TOWARD SPECIFICATIONS FOR THE INTEGRATION OF ECO-DESIGN IN THE AUTOMOTIVE SECTOR

This paper lies within the integration of an eco-design method adapted to the Innovation structure at a car manufacturer. The environmental constraints in the automotive industry are more and more important (European emission standards for exhaust emissions, European directive on end-of life vehicles …). Eco-design is a new manner to design products related to the concept of sustainable development, which combines economy and ecology and put the environmental criterion alongside the classical criterions of design. The goal of this study is to identify the specifications of a strategy for integrating the dimension "Environment". This strategy is applied in the innovation process thanks to ecodesign tools which are the learning vectors for an organization, and therefore support a learning process. This process is structured with the interactions between the management of firm, the environment department, and the design team. Therefore we first make a synthesis of the different classifications of eco-design tools and use two categories: diagnosis and improvement. Second, as our goal is the integration in the Innovation structure and within a design process, we analyze some design process models and highlight the RID (Research, Innovation structure, Development) concept. Third, the main practices of several car makers are synthetized and a focus on three of them (Volvo, Ford, and Volkswagen) is made; we link their strategies with the concept of RID. Finally in the fourth part, we propose a model of a strategy for integrating eco-design practices based on the three examples and supported by a learning process.

INTRODUCTION

The concept of sustainable development has been defined for the first time in 1987 in the report "Our Common Future" [START_REF]Our Common Future[END_REF] of the United Nations World Commission on Environment and Development chaired by Gro Harlem Bruntland. It is a "development that meets the needs of the present without compromising the ability of future generations to meet their own needs." In the business world, this theoretical concept occurs notably with a new manner to design products which combines economy and ecology [START_REF] Karlsson | EcoDesign: what's happening? An overview of the subject area of EcoDesign and of the papers in this special issue[END_REF] and put the environmental criterion alongside the classical criterions of design such as costs, time, or quality, namely eco-design. The goal is to design more environmentally friendly products thanks to a multi-stage and multi-criteria approach, i.e. an approach which takes into account the entire lifecycle of the product, "from cradle to grave", and a set of environmental impacts.

To reach this goal, there are some methods for integrating the dimension "Environment" (or dimension "E") in the scientific literature. Two methodological trends exist: the majority methods are supported by the technical resources such as tools and software, the others are supported by the training of the design team and learning [START_REF] Jacqueson | Integration of the Environment in the Product Design Process: Proposal for a Learning Driving Tool[END_REF]. In the first category, Bauman et al. highlight that there are a lot of eco-design tools, more than 150 in 2002 [START_REF] Baumann | Mapping the green product development field: engineering, policy and business perspectives[END_REF]. However, most of them are not used in companies because they are not appropriate for the design process [START_REF] Fargnoli | The Optimization of the Design Process for an Effective Use in Eco-Design[END_REF]. In the second category, Bras [START_REF] Bras | Incorporating Environmental Issues in Product Design and Realization[END_REF] considers that learning is "a necessary prerequisite" to use the eco-design tools.

FIGURE 1 -COMPLETE INTEGRATION PROCESS [9]

Millet et al. consider that the tools are the learning vectors for an organization and remark that the dimension "E" can have different status according to the apprehension of the actors of design: it can be perceived as a constraint, a criterion, or a value [START_REF] Millet | The firm faced to sustainable development: change of paradigm and learning process[END_REF], and it generates respectively a partial, classical, or innovative eco-design [START_REF] Millet | Intégration de l'environnement en conception -L'entreprise et le développement durable[END_REF]. In this context, Jacqueson et al. proposed a "Learning Driving Tool" which assures an enhanced value monitoring of the dimension "E", i.e. to pass from constraint to criterion and value according to formalization and diffusion actions [START_REF] Jacqueson | Integration of the Environment in the Product Design Process: Proposal for a Learning Driving Tool[END_REF]. It is based on the interactions between the management of firm, the environment department, and the design team.

Therefore, Millet et al. bring together both methodological trends into a complete integration process of the dimension "E" in the design process (Figure 1) which is supported by three interdependent sub-processes [START_REF] Millet | Intégration de l'environnement en conception -L'entreprise et le développement durable[END_REF][START_REF] Millet | Integration of new dimensions in design process. Application to the environmental dimension[END_REF]:

• "The creation of an environmental knowledge through the elaboration an environmental evaluation tool, • The creation of new eco-design practices based on a system of tools, • The creation of new governing values and new learning schemes modifying the principles, the mental models, the faiths to which every actor in his activity within the company refers."

The integration of other dimensions has been subject to studies: for example, the integration of ergonomics at the car manufacturer Renault has been analyzed [START_REF] Millet | Intégration de l'ergonomie dans la conception de produits mécaniques[END_REF]. Thanks to an analogy with the integration of eco-design, the goal of this study was to deduce several principles from the observation so as to propose an integration process which supports the perception of designer (constraint criterion value), and which is supported by the interactions between the management of firm, the ergonomics department, and the design team. The authors highlight a downstream integration approach, i.e. from the downstream parts of a project to the upstream parts, for every field of research in ergonomics. The analogy between ergonomics and environment is used in this paper, and the downstream approach is exploited.

Our project research is to integrate an eco-design method for the Innovation structure at a car manufacturer. One of the particularities of the automotive industry is that there is a "clear distinction between the vehicle development project and the innovation project" [START_REF] Buet | Integrating innovations into vehicle projects -towards a robust "touch down" process[END_REF]. Although there is less information on the final product in the Innovation structure than in the vehicle development projects, the levers for action and improvement are higher.

In the first part of this paper, a state of the art of eco-design tools is done so as to categorize them. Then, the analysis of design and innovation process is detailed in order to determine the different possibilities to integrate the eco-design. In the third part, we make a synthesis of the main actions of car or parts manufacturers, and we make three focuses on Volvo, Volkswagen and Ford in order to highlight a strategy. Finally, the fourth part concludes the paper with the requirements and specifications for an eco-design method for the Innovation structure.

CATEGORISATION OF ECO-DESIGN TOOLS

Generally, the eco-design tools are an implemented result of the application of a specific eco-design method/approach which forms a way to manage to improve the environmental efficiency of products for equivalent criterions e.g. cost, time, quality. A lot of authors have classified and categorized the numerous eco-design tools. The goal of this part is to synthetize the various classifications of tools available in the literature.

According to Millet et al. [START_REF] Millet | Intégration de l'environnement en conception -L'entreprise et le développement durable[END_REF], one "toolbox environment" of an eco-design method should contain at list tools of type of:

• Illustration for having a good picture of the environmental issue, • Diagnosis to identify the environmental hotspots of the reference product, • Definition of the objectives from the diagnosis, • Recommendation to find areas for improvement, • Evaluation/Classification to make the best choices thanks to a multi-criteria analysis (cost, time, quality, environment …). Le Pochat et al. [START_REF] Pochat | Integrating ecodesign by conducting changes in SMEs[END_REF] analysed the classifications of Janin [START_REF] Janin | Démarche d'éco-conception en entreprise -Un enjeu : Construire la cohérence entre outils et processus[END_REF], Tischner and Nickel [START_REF] Tischner | Eco-design in the printing industry -Life cycle thinking: Implementation of Eco-design concepts and tools into the routine procedures of companies[END_REF], Van Berkel et al. [START_REF] Van Berkel | Development of an industrial ecology toolbox for the introduction of industrial ecology in enterprises-I[END_REF] and Dewulf [START_REF] Dewulf | A proactive approach to ecodesign: framework and tools[END_REF]. The function e.g. evaluation, improvement, or decision help, the efficiency, the usability, etc. are parameters for classifying. However, Le Pochat et al. enhanced that "the ease of these tools being used by non-experts was not considered during their development." Therefore, they developed the software EDIMS (EcoDesign Integration Method for SMEs) based on "the environmental analysis of the product" and "assistance in conducting changes in the business." This method resulted in the French standard NF E01-005 [START_REF] Afnor | Mechanical products -Eco-design methodology[END_REF].

Bellini and Janin [START_REF] Bellini | Ecoconception : état de l'art des outils disponibles[END_REF] realized a mapping of the main classes of tools according to their evaluation level and their recommendation level on two scales from 1 to 5. The marks of the evaluation tools are attributed according to their evaluation mode i.e. qualitative or quantitative, and their approach i.e. number of lifecycle stages and environmental criterions. The same is true for the recommendation tools which are marked according to the type of recommendation.

The study of Bovea and Pérez-Belis [START_REF] Bovea | A taxonomy of ecodesign tools for integrating environmental requirements into the product design process[END_REF] takes three studies [START_REF] Byggeth | Handling trade-offs in Ecodesign tools for sustainable product development and procurement[END_REF][START_REF] Knight | Adopting and applying eco-design techniques: a practitioners perspective[END_REF][START_REF] Ilgin | Environmentally conscious manufacturing and product recovery (ECMPRO): A review of the state of the art[END_REF] completed by focusing on the early steps of the design process and the authors propose a more general classification. One of the contributions of this study is that it mentions methods for integrating classical requirements in the product design but adapted for integrating the environment. Those eco-design methods link most of the time one classical tool of design e.g. Quality Function Deployment matrix for quality, with a diagnostic or evaluation tool e.g. Life Cycle Assessment (LCA).

TABLE 1 -CLASSIFICATION OF ECO-DESIGN METHODS AND TOOLS

Diagnosis

Improvement • Analysis Life Cycle Assessment [START_REF]Environmental management -Life cycle assessment -Principles and framework[END_REF][START_REF]Environmental management -Life cycle assessment -Requirements and guidelines[END_REF][START_REF] Finnveden | Recent developments in Life Cycle Assessment[END_REF] Simplified Life Cycle Assessment [START_REF] Graedel | Streamlined life-cycle assessment[END_REF][START_REF] Wenzel | Application dependency of LCA methodology: Key variables and their mode of influencing the method[END_REF] • Evaluation/Classification Matrix LCA : MECO [START_REF] Wenzel | Environmental Assessment of Products, Vol. 1: Methodology, Tools and Case Studies in Product Development[END_REF] ERPA [START_REF] Graedel | Weighted matrices as product life cycle assessment tools[END_REF] MET [START_REF] Brezet | Ecodesign, A promising approach to sustainable production and consumption[END_REF] Indicator MIPS [START_REF] Ritthoff | Calculating MIPS : Resource Productivity of Products and Services[END_REF] Energetic content [START_REF] Lenau | Design of environmentally friendly products using indicators[END_REF] Environmental indicators [START_REF] Jasch | Environmental performance evaluation and indicators[END_REF] Eco-indicator 99 [START_REF] Goedkoop | The Eco-indicator 99 -A damage oriented method for Life Cycle Impact Assessment -Methodology Report[END_REF] Classical methods Green-QFD [START_REF] Zhang | Green QFD-II: A life cycle approach for environmentally conscious manufacturing by integrating LCA and LCC into QFD matrices[END_REF], QFDE [START_REF] Masui | Applying Quality Function Deployment to environmentally conscious design[END_REF], ECQFD [START_REF] Vinodh | Integration of ECQFD and LCA for sustainable product design[END_REF] Eco-VA [START_REF] Sakao | Evaluating Customer Requirements in Eco-VA[END_REF] EEA [START_REF] Lindhal | Environmental Effect Analysis (EEA) -An approach to Design for Environment[END_REF] Others Environmental material selection charts [START_REF] Holloway | Materials selection for optimal environmental impact in mechanical design[END_REF] Eco-functional matrix [START_REF] Lagerstedt | Functional and environmental factors in early phases of product development -Eco functional matrix[END_REF] Lifecycle brick [START_REF] Gehin | Integrated design of product lifecycles -The fridge case study[END_REF] • Recommendation DFE, DFX [START_REF] Holt | Towards an integrated approach to ''Design for X'': an agenda for decision-based DFX research[END_REF]

Guidelines

The Ten Golden Rules [START_REF] Luttrop | EcoDesign and The Ten Golden Rules: generic advice for merging environmental aspects into product development[END_REF] Checklist The EcoDesign Checklist [START_REF] Brezet | Ecodesign, A promising approach to sustainable production and consumption[END_REF] PILOT [START_REF] Wimmer | ECODESIGN Pilot -Product-Investigation-, Learning-and Optimization-Tool for Sustainable Product Development[END_REF] Lists Volvo's black, grey, and white material lists [START_REF] Volvo | Chemical substances which must not be present in processes or products within the Volvo Group -Volvo's black list[END_REF][START_REF] Volvo | Chemical substances which should not be present in processes or products within the Volvo Group -Volvo's grey list[END_REF][START_REF] Volvo | Substitutes for hazardous chemical substances -Volvo's white list[END_REF] •

Objectives and Ideas generation TRIZ [START_REF] Chen | An eco-innovative design approach incorporating the TRIZ method without contradiction analysis[END_REF] ecoASIT [START_REF] Tyl | Stimulate Creative Ideas Generation for Eco-Innovation: an Experimentation to Compare Eco-Design and Creativity Tools[END_REF] EcoMal'in [START_REF] Samet | Ecoinnovation Tool for Mal'in Software, Application on a Waffle Iron[END_REF] Lifecycle Design Strategy wheel [START_REF] Brezet | Ecodesign, A promising approach to sustainable production and consumption[END_REF] PIT diagram [START_REF] Jones | Applying structured methods to Eco-innovation. An evaluation of the Product Ideas Tree diagram[END_REF] Eco-ideation and assessing the implementation difficulty and emissions benefits of innovations [START_REF] Bocken | Development of an eco-ideation tool to identify stepwise greenhouse gas emissions reduction options for consumer goods[END_REF][START_REF] Bocken | Development of a tool for rapidly assessing the implementation difficulty and emissions benefits of innovations[END_REF] Some authors [START_REF] Hochschorner | Evaluation of Two Simplified Life Cycle Assessment Methods[END_REF][START_REF] Hur | LCA and matrix methods in identifying the environmental aspects of a product system[END_REF] compared evaluation tools with diagnosis tools. While the firsts are generally analysis tools i.e. they allow a detailed study of the entire product system, the seconds are approximate and their evaluation modes differ: quantitative, semi-quantitative, and qualitative. Nevertheless, these evaluation modes are complementary. It is therefore relevant to check the reliability of the results of evaluation tools.

The list of eco-design tools and methods in Table 1 is not exhaustive. This classification is inspired from the previous classifications. We consider two classes: the tools for diagnosis which can be analysis or evaluation tools, and the tools for improvement which can be recommendation or ideation and objectives generation. The next part of this paper deals with the models of design process, particularly the RID model which seems relevant for our goal to integrate the dimension "E" in the Innovation structure.

TOWARD THE CONCEPT OF RID

Eco-design methods and tools must lie within the design and innovation processes. The complexity of these processes asks to use models in order to have a better illustration. In her PhD thesis, Lahonde [START_REF] Lahonde | Optimisation du processus de conception : Proposition d'un modèle de sélection des méthodes pour l'aide à la décision[END_REF] carried out a synthesis of five models of design process: Axiomatic Design [START_REF] Suh | The Principles of Design[END_REF], Design for Six Sigma [START_REF] Mader | Design For Six Sigma[END_REF], New Product Design [START_REF] Aoussat | La pertinence en Innovation: Nécessité d'une Approche Plurielle[END_REF], Systematic Design [START_REF] Pahl | Engineering Design: A Systematic Approach[END_REF], and TRIZ [START_REF] Cavallucci | L' approche altshullerienne de la créativité[END_REF]. Although each model has its own characteristics and objectives, Lahonde highlighted similarities: per phase breakdown, or iterations. Her comparison table identifies five phases: Preparation, Identification, Design 1, Design 2, Manufacturing. The international standard ISO 14062 [START_REF]Environmental management -Integrating environmental aspects into product design and development[END_REF] about the integration of environmental aspects into the product design and development introduces one generic model in six stages: Planning, Conceptual design, Detailed design, Testing/prototype, Production/Market launch, Product review. These models are not sufficient to establish the specifications for integrating an eco-design method for a designer of complex systems; that leads us to explore the innovative nature of design.

From the study of innovative companies, Hatchuel et al. [START_REF] Hatchuel | From R&D to RID : Design Strategies and the Management of Innovation Fields[END_REF] searched for understanding how the organization of a company must change in order to generate innovations. They recommend the use of the concept of RID (Research, Innovation structure, and Development) instead of the usual R&D concept, where I is "a structured process, with its specific management principles which are different from respectively R and D activities". They defined the three functions from a managerial point of view: "if research is a controlled process of knowledge production that is

not in charge of values definition, and if development requires competencies and value definition, we can define the Innovation structure as being in charge of a twofold design work:

• A Value definition process • New competencies identification process".

Figure 2 shows all interactions between the three functions. According to the authors, the companies in the automotive industry are good examples of the application of the concept of RID; Buet et al. [START_REF] Buet | Integrating innovations into vehicle projects -towards a robust "touch down" process[END_REF] remind that innovative design projects are distinguished from the vehicle development projects. Therefore, the specifications of an eco-design method for the development are different from those for the Innovation structure. This method should meet the twofold activity defined by Hatchuel et al.: the dimension "E" must be perceived as a new value for the designers and generate new concepts and knowledge. The ecodesign method for the Innovation structure must generate:

• ideas and products ready for development, and emerging product concepts at various formalization stages, thanks to one "toolbox environment" discussed in the first part of this paper, • new emerging competencies allowing the valorization of the dimension "E". Moreover, the method must be adapted to the different technology readiness levels of the innovation which are measurable thanks to the scale defined by the NASA [START_REF] Mankins | Technology readiness assessments: A retrospective[END_REF].

In the following part, we make a synthesis of the main actions of different car makers and link the practices of three of them with the concept of RID.

INTEGRATION STRATEGY FROM THE AUTOMOTIVE INDUSTRY

Car makers and part suppliers must increasingly take into account the dimension "E" within the design process. Nevertheless, their publications are still rare. The nonexhaustive list in the Table 2 gives some actions of companies in the worldwide automotive industry. We make a distinction between LCA studies and eco-design methods: the bibliographical references for LCA studies concern case studies in the automotive sector or improvement of the methodology itself, while the aim of those of the eco-design method list is to improve the product by using a system of tools which may include the LCA and the relations of these tools within the design process.

Conducting LCA may lead car manufacturers to publish Environmental Product Declarations (EPD). For example, since 2007 Volkswagen has published on its website 1 eleven Environmental Commendations for vehicles (Passat, Golf, Polo …), one engine (TSI Engine) and one gearbox (Dual-Clutch 1 http://www.volkswagen.com/vwcms/master_public/virtualmaster/en2/unt ernehmen/mobility_and_sustainability0/service/download/Download_en.html (accessed 12/07/2011) gearbox). Volvo2 has made a simulator of EPD available to the website visitors.

Chanaron made a benchmark of best practices in LCA from European car makers: DaimlerChrysler, Ford Motor and Volkswagen [START_REF] Chanaron | Life Cycle Assessment practices: benchmarking selected European automobile manufacturers[END_REF]. One of his conclusions is that car manufacturers use the LCA methodology for "assessing innovative technologies and vehicles of the future." Nunes and Bennett analyzed the "green" initiatives of three manufacturers namely Toyota, General Motors and Volkswagen, particularly concerning the eco-design [START_REF] Nunes | Green operations initiatives in the automotive industry: An environmental reports analysis and benchmarking study[END_REF]. They observed that they mainly focus on the improvement of internal combustion engines, e.g. fuel reduction, alternative driving chain, or alternative fuels. However, eco-design approaches and methods do not appear.

Furthermore, some common projects bring together several car makers such as the LIRECAR project about the LCA of lightweight and end-of life scenarios [START_REF] Schmidt | Life cycle assessment of lightweight and end-of-life scenarios for generic compact class passenger vehicles[END_REF], or the project on an "Environmentally Friendly Vehicle concept" [START_REF] Schmidt | Feasibility of a globally harmonised Environmentally Friendly Vehicle concept[END_REF] where they recommend to avoid a single score for the environmental assessment of a vehicle.

In order to build a strategy for integrating the dimension "E" in the innovative structure, we propose a focus on the strategies adopted by three car makers: Volvo, Ford and Volkswagen. The strategies are connected with the concept of RID. Volvo The Swedish car maker began researches on LCA in 1989 [START_REF] Louis | Life Cycle Assessment and Design -Experience from Volvo Car Corporation[END_REF]. This action aimed to product knowledge through a controlled process, which is the definition of the function R. Then, these researches resulted in the EPS method (Environmental Priority Strategies in design) in co-operation with the Federation of Swedish Industries. This method enables the agglomeration of "all the data coming from the inventory phase of a LCA to one single value, expressed in Environmental Load Unit (or ELU)" [START_REF] Karlsson | EPS as a life cycle oriented system assessment tool to facilitate industrial learning about relations to the environment[END_REF]. The EPS method was computerized for being used by LCA non-experts such as designers. In 1996 [START_REF] Curran | Summary of Global Life Cycle Inventory Data Resources[END_REF], the SPINE database (Sustainable Product Information Network for the Environment) is developed in order to "facilitate the exchange of LCA data and improve the operational implementation of LCA methods". The EPS method, its computerization, and the SPINE database fulfill the requirements of the function D i.e. the activation of environmental knowledge so as to specify the product for evaluating its environmental profile. In 1997, the company began to work with the Environmental Effect Analysis (EEA) which comes from an adaptation of the "Failure Mode Effect Analysis" to the environment. It clearly transforms the environment into a new value and brings to designers new concepts for improving their products, which are the requirements of the function I. Tingström and Karlsson [START_REF] Tingström | The relationship between environmental analyses and the dialogue process in product development[END_REF] analyzed the relations between LCA and EEA by focusing on the integration in the design process. From the interviews they made with Volvo's teams, they concluded that "the problem was to make the environmental information effective, by making it readily available early enough to make it useful to influence the environmentally relevant design decisions"; Volvo's teams therefore prefer using EEA early in the design process. Moreover, Tingström [START_REF] Tingström | The Challenge of Environmental Improvement in Different Types of Innovation Projects[END_REF] remarked that "a radical innovation should use EEA as a supporting tool, and incremental innovation should use LCA". Volvo's strategy is shown in Figure 3.

FIGURE 3 -VOLVO'S STRATEGY TABLE 2 -ENVIRONMENTAL ACTIONS OF CAR MANUFACTURERS AND PART SUPPLIERS

Car maker / Part supplier

Country

Studies in LCA Eco-design methods

Daimler AG Germany Supplying databases on manufacturing processes (gear wheel of the front-shift transmission for Daimler AG; valve injector for Bosh GmbH) [START_REF] Schlosser | Assessment of Energy and Resource Consumption of Processes and Process Chains within the Automotive Sector[END_REF] Application of Life Cycle Assessment for the Environmental Certificate of the Mercedes-Benz S-Class [67] Faurecia France Learning Driving Tool for the design for the valorization of the dimension "E" [START_REF] Jacqueson | Integration of the Environment in the Product Design Process: Proposal for a Learning Driving Tool[END_REF][START_REF] Jacqueson | Integration of the environment in product design by a learning process: proposal for a piloting tool[END_REF] Evaluation method of environmental impacts for the design team [69] Ford-Werke GmbH / Ford Motor Germany / USA Streamlined LCA of the recovery of battery-housing [START_REF] Schmidt | Environmental considerations on battery-housing recovery[END_REF] Weighting in Life Cycle Assessments in a Global Context [START_REF] Schmidt | Weighting in Life Cycle Assessments in a Global Context[END_REF] Product Sustainability Index explanations, Demonstration of the validity of a spreadsheet tool for non-experts in LCA [START_REF] Schmidt | Life Cycle Tools within Ford of Europe's Product Sustainability Index -Case Study Ford S-MAX & Ford Galaxy[END_REF] Life Cycle Costing as part of design for environment [START_REF] Schmidt | Life Cycle costing as part of design for environment -Environmental business cases[END_REF] Electrical and electronic components in the automotive sector: Economic and environmental assessment [START_REF] Alonso | Electrical and electronic components in the automotive sector: Economic and environmental assessment[END_REF] Enhancement of the application efficiency of LCA [START_REF] Rebitzer | Enhancing the application of life cycle assessment for industrial uses[END_REF] General Motors USA Comparison of two fuel tank system designs [75] PSA Peugeot Citroën

France

Comparative LCA of several end-of life scenarios for a polypropylene bumper skin [START_REF] Borgne | End-of-life of a polypropylene bumper skin -Some key elements for a pragmatic environmental management[END_REF] Preliminary study for a comparative LCA of a classical vehicle and a hybrid electric vehicle [START_REF] Garcia | Preliminary study for a Life Cycle Assessment of Hybrid Electric Vehicle[END_REF] Methodology for improving the recovery rate of end-of life vehicles [START_REF] Coppens | Evaluating and improving the recovery aptitude of an automobile function: The PSA approach[END_REF] Evaluation tool of the recovery potential earlier in the design process [START_REF] Tonnelier | Is it possible to evaluate the recovery potential earlier in the design process? Proposal of a qualitative evaluation tool[END_REF] Siemens AG Germany Quantitative streamlined LCA: Parameterised inventories and interactions with design parameters [80] Toyota Japan Eco-VAS system including a LCA database and an evaluation tool for designers during the design process [START_REF] Nakano | Collaborative activity with business partners for improvement of product environmental performance using LCA[END_REF] Volkswagen AG Germany On the calculation of fuel savings through lightweight design in automotive life cycle assessments [START_REF] Koffler | On the calculation of fuel savings through lightweight design in automotive life cycle assessments[END_REF] An integrated approach for environmental assessments using fuzzy system [START_REF] Thiel | An integrated approach for environmental assessments -Linking and integrating LCI, environmental fate models and ecological impact assessment using fuzzy expert systems[END_REF] Comparative Life Cycle Assessment of Remanufacturing and New Manufacturing of a Manual Transmission [START_REF] Warsen | Comparative Life Cycle Assessment of Remanufacturing and New Manufacturing of a Manual Transmission[END_REF] Comparative LCA of two methods for recycling end-of life vehicles [START_REF] Krinke | The Volkswagen-SiCon Process: Eco-efficient solution for future end-of life vehicle treatment[END_REF] Life Cycle Inventory for the Golf A4 [START_REF] Schweimer | Life Cycle Inventory for the Golf A4[END_REF] A procedure for streamlined inventory modeling within life cycle assessment of vehicles [START_REF] Koffler | Volkswagen slimLCI: a procedure for streamlined inventory modelling within life cycle assessment of vehicles[END_REF] Incorporating sustainability into supply management in the automotive industry [START_REF] Finkbeiner | Application of Life Cycle Assessment for the Environmental Certificate of the Mercedes-Benz S-Class[END_REF] AB Volvo Sweden Fitting LCA for the design process [START_REF] Louis | Life Cycle Assessment and Design -Experience from Volvo Car Corporation[END_REF] EPS as a Life Cycle oriented System Assessment Tool to Facilitate Industrial Learning about Relations to the Environment [START_REF] Karlsson | EPS as a life cycle oriented system assessment tool to facilitate industrial learning about relations to the environment[END_REF] Material lists [START_REF] Volvo | Chemical substances which must not be present in processes or products within the Volvo Group -Volvo's black list[END_REF][START_REF] Volvo | Chemical substances which should not be present in processes or products within the Volvo Group -Volvo's grey list[END_REF][START_REF] Volvo | Substitutes for hazardous chemical substances -Volvo's white list[END_REF] Using the Environmental Effect Analysis method [START_REF] Tingström | The relationship between environmental analyses and the dialogue process in product development[END_REF] Ford Ford developed in 2006 the Product Sustainability Index (PSI) by conducting researches in the LCA and Life Cycle Costing (LCC) fields. The oldest research in LCA that we found was made in 1999 and aimed to compare end-of life scenarios for a battery housing; the authors conclude that "from a strategic point of view, the simplified LCA is suitable for answering the questions" [START_REF] Schmidt | Environmental considerations on battery-housing recovery[END_REF]. In 2002, Schmidt and Sullivan [START_REF] Schmidt | Weighting in Life Cycle Assessments in a Global Context[END_REF] had a part in the LCA methodology by recommending to not using weighting in Life Cycle Impact Assessment. In 2003, Schmidt [START_REF] Schmidt | Life Cycle costing as part of design for environment -Environmental business cases[END_REF] conducted a research on LCC but concluded that LCC cannot be used by non-experts in life cycle. The thesis of Rebitzer [START_REF] Rebitzer | Enhancing the application of life cycle assessment for industrial uses[END_REF] aimed "to simplify the methods of LCA application and to use LCA as a basis for life cycle costing." That leaded in 2006 to the first publication of the PSI applied to Ford's car [START_REF] Schmidt | Life Cycle Tools within Ford of Europe's Product Sustainability Index -Case Study Ford S-MAX & Ford Galaxy[END_REF]; three PSI reports are available on the website of the company3 for S-Max and Galaxy, Kuga, and Mondeo. The PSI tool "allows non-experts to manage key environmental, social and economic aspects in the product development, also on a vehicle level." Finally, in 2007, LCA and LCC were applied to "define optimum design and end-of-life scenarios" for electrical and electronic components. Ford's strategy is represented in Figure 4. The need to pass from diagnosis to improvement is obvious through the opening of the LCC research field and placing a tool at non LCA experts' disposal.

FIGURE 4 -FORD'S STRATEGY

Volkswagen

Volkswagen has begun making Life Cycle Inventory (LCI) since 1992, which leaded to some Environmental Commendations. The company had a part in the LCA methodology in 2008 by developing the slimLCI method to automate LCI [START_REF] Koffler | Volkswagen slimLCI: a procedure for streamlined inventory modelling within life cycle assessment of vehicles[END_REF], and in 2010 by improving the use stage modeling with the Fuel Reduction Value parameter [START_REF] Koffler | On the calculation of fuel savings through lightweight design in automotive life cycle assessments[END_REF]. Moreover, Volkswagen opened a research field on end-of life and compared in 2006 two recycling methods [START_REF] Krinke | The Volkswagen-SiCon Process: Eco-efficient solution for future end-of life vehicle treatment[END_REF]. Finally, Warsen et al. [START_REF] Warsen | Comparative Life Cycle Assessment of Remanufacturing and New Manufacturing of a Manual Transmission[END_REF] assessed in 2011 the benefits of an innovative end-of life strategy namely the remanufacturing thanks to the LCA methodology; this highlights the complementary aspects of the tools. Volkswagen's strategy is shown in Figure 5. By using the LCA results, Volkswagen tried to strive toward the improvement of the end-of life step with the process SiCon [START_REF] Krinke | The Volkswagen-SiCon Process: Eco-efficient solution for future end-of life vehicle treatment[END_REF] and the remanufacturing strategy [START_REF] Warsen | Comparative Life Cycle Assessment of Remanufacturing and New Manufacturing of a Manual Transmission[END_REF].

FIGURE 6 -MODEL OF A STRATEGY FOR INTEGRATING THE DIMENSION "E"

By synthetizing the three examples, it seems that the main evolution for integrating the dimension "E" passes from the diagnosis research field to the improvement research field. So it seems relevant first to open a research field which may lead to create, adapt or use methodological elements for analyzing and/or evaluating. The diagnosis leads the company to "know what they do". This is the first sub-process of the complete integration process of Millet et al. [START_REF] Millet | Integration of new dimensions in design process. Application to the environmental dimension[END_REF]. After detecting the forces and weaknesses of their product, the three previous examples seem to act in order to improve their product and so "do what they know". The variety of action kinds is wide and seems to be specific to the company's strategy. This is the second subprocess of Millet et al.. Therefore, a second specification would be the building of both types of tools: diagnosis and improvement.

Finally, the chosen strategy for integrating the dimension "E" must support learning and the creation of knowledge. Each research field generates methodological elements for the Development and the Innovation structure. Therefore, we can define a third specification: according to Millet et al. [START_REF] Millet | Intégration de l'ergonomie dans la conception de produits mécaniques[END_REF], the transfer in the same research of methodological elements from D to I, e.g. D 1 to I 1 , must be coherent, and the methodological elements from one research field to another, e.g. D 1 to D 2 or I 1 to I 2 , must be complementary. As the learning process is driven by the organization and the interactions between the management of firm, the environment department and the design team, collaborations between the environment department and the design team would promote the development of the methodological elements.

CONCLUSION AND PERSPECTIVES

In this paper we first proposed one synthesis of categorization of eco-design tools; we chose to classify the tools according to their function: diagnosis with analysis and evaluation tools, and improvement with recommendation tools and objective and idea generations. Second, we introduced the concept of RID which highlights the specificities of the Innovation structure. This led us third to look for the practices of car makers and part suppliers and their strategies for integrating the dimension "E". We made a focus on three car manufacturers and connected their strategies with the concept of RID. Then in the fourth part, we proposed one model based on the analysis of the three examples for an integration strategy of the dimension "E" supported by a learning process.

We did not propose any methodological elements and we highlighted that they are specific to the company. They may be extracted from the scientific literature, adapted, or created. Nevertheless, they should be developed by collaboration between the environment department and the design team. Moreover, the trajectory of the use of the tools has not been studied, only mentioned in the Volvo's example. In perspectives, these observations ask for some researches and a collaborative work for building the methodological elements, so as to be tested in a company.

FIGURE 2 -

 2 FIGURE 2 -RELATIONSHIPS BETWEEN R, I AND D [64]

http://www.volvotrucks.com/dealers-vtc/en-gb/VTBC-EastAnglia/aboutus/environment/environmental_product_declaration/Pages/Intr oduction.aspx (accessed 12/07/2011)

http://www.ford.co.uk/AboutFord/Sustainability/ProductSustainabilityIn dex (accessed 12/13/2011)

FIGURE 5 -VOLKSWAGEN'S STRATEGY

Looking at the three examples above, as the environment was seen as a constraint, they conducted researches (function R) in the LCA field and developed some tools e.g. EPS, PSI, or slimLCI, which leaded the designers to comprehend the dimension "E" as a criterion (function D). Lastly, thanks to tools for the Innovation structure e.g. EEA, the dimension "E" became a value (function I). Therefore, the specifications for our integration method of eco-design in the Innovation structure may be based on these observations.

SPECIFICATIONS FOR INTEGRATING ECO-DESIGN

The valorization of a dimension has already been studied in the ergonomics field. Millet et al. [START_REF] Millet | Intégration de l'ergonomie dans la conception de produits mécaniques[END_REF] proposed one model of integration of ergonomics in the design process. This model is based on a downstream approach. Ergonomics must "pass from the newly knowledge coming from the research process to credibility." It is therefore an "intervention based on the downstream parts of design process."

Here we make an analogy between the downstream integration approach of ergonomics and the integration approach of the dimension "E" in the Innovation structure and try to make a model (Figure 6), based on the concept of RID and the previous examples. The level of maturity of a technology increases during its design process, and generally, the phase Development follows the phase Innovation, which follows the phase Research. Therefore, one of the specifications for our downstream integration approach should first integrate methodological elements in the Development phase to go back to the Innovation structure. These methodological elements are built by the Research function.


D Hurther

E Terray

Dynamics of shear layers induced by hairpin packets in shallow free surface flows: implication for gas transfer

.

INTRODUCTION

Over the past forty years, research on shear turbulence has focused on the dynamics of intermittent quasiorganized flow events, such as streaks, omega and hairpin structures, bursts, vortex heads, ejections, sweeps, boils, upwellings, downwellings, spiral eddies, occurring intermittently at scales that vary with distance from the wall. As pointed out by [START_REF] Robinson | Coherent motions in the turbulent boundary layer[END_REF] in the review of this topic, most of the studies have been devoted to the flow organization in the inner region z + <200, where z + is the normal coordinate normalized by the viscous length. Until the end of the eighties, flow observation capabilities of the measuring tools were limited. The experimental studies resulted in a large number of conceptual models on the bursting phenomenon [START_REF] Theodorsen | Mechanism of turbulence[END_REF][START_REF] Hinze | Turbulence. McGrawHill[END_REF][START_REF] Blackwelder | Streamwise vortices associated with the bursting phenomenon[END_REF][START_REF] Bogard | Burst detection with singlepoint velocity measurements[END_REF]. The concept diversity led to some confusion which has been partially overcome by DNS results. The success of this technique in the study of coherent structures relies on its capability to provide a Lagrangian view of the flow field over arange pf scales extending from the viscous to the integral lengthscales. In turn, important limitations of DNS methods persist in terms of bedroughness, moderate to high Reynolds and Froude numbers and stratification effects. These effects appear to be crucial in the study of geophysical shear currents.

Recent studies by [START_REF] Tardu | Characteristics of single and clusters of bursting events in the inner layer[END_REF], Zhou et al. (1999) [START_REF] Zhou | Mechanisms for generating coherent packets of hairpin vortices in channel flow[END_REF] (hereafter referred to as ATM00) significantly contributed to the progress in understanding organized motion in the outer region of flows with high Reynolds number. Their results suggest the existence of hairpin packets in the outer region. A subdivision of the entire boundary layer into three zones is proposed depending on the mean advection velocity spatiallyaveraged over zones of uniform, retarded streamwise momentum. The major advantage of the hairpin packet model lies in its ability to explain most of the conceptual models of the bursting phenomenon in the inner region as well as the presence of coherent structures in the outer region, the socalled "packets" which scale with outer parameters of the boundary layer. The change of scales with distance from the wall is thereby taken into account. Furthermore, good agreement between numerical (Zhou et al. 1999) and experimental results (ATM00) on the hairpin packets organization is found which reinforce the validity of this concept.

In the present work, we check if the hairpin packet concept occurs in the outer region of highly turbulent openchannel flows (Reynolds number with O(10 4 -10 5 )) under fully rough bed situations. Therefore, we aim to determine the sensitivity of this concept to the wall roughness in order to verify whether this concept can or cannot be applied to shear flows under geophysical conditions. Furthermore, we are interested to determine the influence of the freesurface on the hairpin packets organization. Unconventional quadrant threshold techniques are used to investigate these aspects. We will analyze the structure of shear stress and its dependence on the hairpin packets organization. Finally, we will discuss the parameterization of the TKE budget in the intermediate and free Surface Influenced Layer (SIL).

From the previous results, the implications for such processes as the gas exchange at the airwater interface are addressed. A better understanding of turbulence near the free surface is of primary importance for the improvement of gas transfer velocity predictions. We will analyze the effects of the hairpin packets organization on the Turbulent Kinetic Energy (TKE) dissipation rate in the SIL.

We propose a mechanism underlying the gas transfer velocity models considering horizontal inhomogeneities at the free surface. Several studies [START_REF] Komori | The relationship between surfacerenewal and bursting motions in an openchannel flow[END_REF][START_REF] Rashidi | Mechanisms of heat and mass transport at gasliquid interfaces[END_REF][START_REF] Nimmo Smith | Surface effects of bottomgenerated turbulence in a shallow tidal sea[END_REF] have shown that horizontal inhomogeneities are due to the impinging of large coherent flow structures emanating from the bulk region. As was demonstrated by [START_REF] Moog | Airwater gas transfer in uniform channel flow[END_REF] these models lead to inconsistencies for shear flows with Reynolds above 500 (Reynolds number based on the turbulent macroscale and bedfriction velocity). We will discuss these aspects by revisiting the conceptual model called the "Chain Saw Model" (CSM), derived in [START_REF] Moog | Airwater gas transfer in uniform channel flow[END_REF].

EXPERIMENTAL SETUP

Experiments were carried out in a laboratory openchannel under uniform flow conditions (28m long, 2.4m wide, 1m deep) over a rough bed. A sand bed with a mean grain size of d 50 =9mm was used. The measurement section is placed 13m downstream from the entrance where the turbulent flow is well developed. All velocity data presented here were taken in the center of the channel. The hydraulic parameters are given in Table 1. These indicate subcritical highly turbulent flow. The standard roughness is normalized with the corresponding viscous lengths and is higher than 70, corresponding to a fully rough bed. The aspect ratio of the flow is larger than 14 ensuring that secondary currents are negligible at the centerline of the flume. The turbulent microscale is equal to 1.47cm in the present flow conditions. The spatial resolution of the instrument used herein is less than 4mm which allows to cover the inertial subrange of the turbulent flow.

The 3D Acoustic Doppler Velocity Profiler (3D ADVP) offers the unique possibility of profiling the 3D quasiinstantaneous velocity field simultaneously over the entire boundary layer thickness, corresponding to 99% of the flow depth in uniform openchannel conditions. This instrument is well adapted to the study of turbulent quasicoherent flow motion in clearwater freesurface flows (Hurther andLemmin 2000, Blanckeart and[START_REF] Blanckeart | Turbulence structure in an openchannel bend[END_REF]. Compared to conventional Acoustic Doppler Velocity measurement, we use a correction method to increase the bandwidth of the resolved velocity field [START_REF] Hurther | A correction method of mean turbulence measurements with a 3D acoustic Doppler velocity profile[END_REF]. TKE budget terms are estimated with an uncertainty below 15%.

HAIRPIN PACKETS ORGANIZATION

The occurrence of multiple hairpin structures or clusters of hairpins that form a coherent group or packet, has been anticipated by several pioneering studies on the inner region of zero pressure gradient boundary layers with low Reynolds number (Acarlar and Smith 1987a and b; Zhou et al. 1999). The demonstration of the existence of hairpin packets in the outer region of shear flows at moderate Reynolds number (which is of more interest herein) relies on the unique study by ATM00. This work clearly exhibits the presence of packets composed by several units of hairpin heads arranged along streamwise lines of uniform streamwise momentum. A subdivision of the boundary layer into four zones has been proposed. The first zone is siutated in the inner region, nearly corresponding to the buffer layer where the bursting phenomenon occurs and associated Omega shaped structures emanate. Between this zone and the second one (called Zone I after ATM00's terminology which will be used hereinafter), groups of Omega shaped structures are deformed (vortex stretching occurs under the effect of shear) into packets of hairpins that propagate together with a constant streamwise velocity. Zone I is located at the beginning of the logarithmic layer and therefore is part of the outer region. At this stage, the size of the packets is relatively small compared to the size of the packets in Zones II and III. Zone II which is still located in the logarithmic layer is characterized by packets of hairpins that are well delimited by domains of uniformly retarded fluid. The typical size of the packets in this zone is 1.2δ (where δ corresponds to the boundary layer thickness). The characteristic growth angle of the packet is found to be smaller than the growth angle of the individual hairpin structures in a packet. It has been shown that the growth angle of the individual hairpins in the packets appears to increase with distance from the wall. Close to the head the individual hairpin inclination varies between 45° and 90°. The inclination of the packet, however, is found to remain linear quite frequently over the three zones. The mean angle varies between 3° and 35° with a mean value of around 10°. In Zone III, the packets are less retarded compared to the packets of Zone II. They represent older packets that have been generated upstream by packets of Zone II. The characteristic features found by ATM00 in Zones I to III are observed in Figure 1. It shows the data taken at the centerline of the openchannel flow. The grey zones represent regions of uniform streamwise velocity defined as u-U d /u * . u is the instantaneous streamwise velocity component. U d is the depth averaged velocity. Three ranges of levels are represented. The dark gray range corresponds to values lower than -2.5, the lighter grey region ranges from -2.5 to -1.8 and the white levels corresponds to values higher than -1.8. The three zones show the presence of regions of uniform streamwise momentum that are very similar to the zones observed in ATM00. The ranges of the values corresponding to the different zones also agree to the values found by ATM00 for their high Reynolds number experiment. As shown in Table 1, the Reynolds numbers of both experiments are very similar. Furthermore, the circles in Figure 1 denote regions of high transverse vorticity supposed to correspond to the heads of hairpin structure in the outer region. Three packets called P1, P2 and P3 are observed over a streamwise distance of 3δ. Here δ, the boundary layer thickness, corresponds to 99% of the flow depth. A very robust criteria of similarity between ATM00 observations and our data is observed in the location of the hairpin heads of P1 and P2 on the separation line between Zone II and Zone III. The size of the packets in Zone II again matches well with the size indicated in ATM00 (roughly equal to δ). The approximation of the growth angle of P2 is given by the inclination of the dashed dotted line. We find a value of 18° which lies in the range found in ATM00. The inclination of P1 is seen to be negative. However, this occurs rarely in our data.

Overall we deduce that we find good agreement between the zone subdivision in the openchannel flow and in the freestream boundary layer of ATM00. Nevertheless, it seems that the lines of separation between the different zones are smoother in AMT00's results. Since these discrepancies can not be attributed to Reynolds numbers differences between the two experiments (considering the high Reynolds experiment in AMT00), we suspect bed roughness effects.

The previous discussion was based on qualitative aspects. A more quantitative proof that the hairpin organized motions in the outer region are induced in three zones of uniform streamwise momentum can be attempted by evaluating the probability density function (PDF) of the instantaneous longitudinal velocity. As discussed in ATM00, the size of the data sample should be small, roughly equal to the mean length of hairpin packets, i.e. around δ. Such a PDF is shown in Figure 2. The averaging can be interpreted as a spatial average over the entire boundary layer thickness and over a time period roughly corresponding to t=δ/U d . As can be seen, three local maxima (U z1 /U ∞ =0.45, U z2 /U ∞ =0.79, U z3 /U ∞ =0.97, U ∞ is the time averaged velocity at 99% of the flow depth) can be distinguished. They correspond to the first order moment of three approximated probability density subfunctions. ATM00 have found very similar PDF From the good agreement between ATM00's data and the present openchannel flow data we conclude that hairpin packet organized motion is also found in open channel flows over completely rough beds. At this stage we cannot distinguish any effects of the freesurface on the turbulent motion of the outer region.

STRUCTURE OF SHEAR STRESS IN THE OUTER REGION

The quadrant threshold technique proposed by [START_REF] Lu | Measurements of the structure of the Reynolds stess in a turbulent boundary layer[END_REF] has been used originally to study the bursting phenomenon in the buffer region of shear flows. Since then, many studies (Bogard andTiederman 1987, Sechet and[START_REF] Séchet | Near wall turbulent structures and bedload transport[END_REF] have applied this technique to point measurements throughout the entire boundary layer. The ability of the 3D ADVP to profile quasiinstantaneously over the entire boundary layer, offers a greatly extended possibility to study organized flow motion.

The quadrant threshold technique, which provides quantitative information on the momentum flux structure, can be used in visualizing instantaneous flow field features (Figure 3). Figure 3a shows the effect of the conditional sampling on the relative amplitude of u′w′(z, t) in the (u′, w′) plane at a given depth z. The thin and thick hyperbolic curves correspond to the functions are called the holeevents. Figure 3b shows the effect of the threshold selection on the instantaneous momentum flux field. The flow patterns delimited by the thin and thick lines again correspond to the regions for H equal to 1 and 5 respectively. In Figure 3b, the thick and thin lines represent isolines of high and low relative momentum flux, respectively. As suggested by the results in Figure 3b, only ejections and sweeps contribute significantly to the total shear stress for H=5. Outward and inward interactions events can not be distinguished in this figure. Furthermore, several high shear stress patterns in Zone III are not attached to the wall layer which suggests that they might not be a direct consequence of the bursting phenomenon occurring in the wall layer. Instead they may depend on a large scale hairpin organization of the outer region. It is also seen that for larger H values the smallscale shear stress structures (ejections and sweeps) are not selected and are generally detached from the high shear structures with much larger spatial scales. This aspect is clearly indicated by the important number of smallscale structures delimited by the thin lines and the very few smallscale structures delimited by the thick lines in Figure 3b (except at coordinates (0.6, 0.95) and (0, 0.22)). The patterns in Figure 3b show elongated shear layers which are composed of an extended core region surrounded by smaller scale shear stress structures corresponding to structures with lower H value. One may speculate from these observations that the frontiers of the large coherent shear layers interact with the surrounding less coherent fluid because of their relatively low momentum flux. The latter are probably more isotropic structures since they are not detected by the quadrant threshold technique. The interaction seems to occur at much lower scales than the mean scale of the core region corresponding to high H values.

In order to quantitatively verify the existence of high shear stress events with a uniform core region, Figure 4 represents the vertical gradient of the momentum flux of ejections and sweeps as a function of the threshold H. In this figure, the momentum flux is normalized by the squared bed friction velocity rather than by the mean local shear stress value. The brackets < > denote that the quantity has been spatially averaged over the region 0.2≤z/δ≤0.75. It can be seen that ejections and sweeps reach a negligible vertical gradient for 8≤H≤9. The average value of the vertical gradient is equal to 5.9 and is taken at 10% of its maximum. This result strongly supports the existence of elongated shear layers with a uniform vertical core region in Zones II and III.

The combination of an unconventional quadrant technique with instantaneous flow field observations has provided insight into the dynamical details of the shear layers that exhibit a core region of uniform shear in the vertical direction. The vertical size of the shear layers is observed to scale with the vertical dimensions of Zones II and III corresponding to outer flow length scales. The uniform core regions of the shear structures are typically delimited by isocontours of values equal to 5 or above. The shear layers are found to be surrounded by smaller scale shear stress structures selected by threshold values typically lower than 2. This observation suggests that these events are related to the largescale organization of the outer region (hairpin packets) and depend weakly on the internal smallscale organization of the wall layer. Their uniformity also implies that they weakly depend on the internal structures of the hairpin packets.

TKE BUDGET IN THE OUTER REGION: THE ROLE OF ELONGATED SHEAR LAYERS

Figure 5 shows the profile of the normalized difference between the shear production and the TKE dissipation rate (dashed line). The profile of the normalized TKE transport term (solid line) is also presented in Figure 5. The TKE dissipation is compensating the TKE transport in the near surface region. The shear production is negligible in this region because the vertical gradient of mean longitudinal velocity is negligible. From this observation we also infer that the SIL can be defined as the region corresponding to z/δ≥0.7. The value of the normalized dissipation very close to the free surface tends towards unity. This agrees well with the measurements by [START_REF] Nezu | Turbulence in openchannel flows[END_REF]. In order to investigate the dynamical details of this balance in the outer region, we present the profile of the normalized vertical TKE flux (called F k ) in Figure 6. A region of constant F k is observed over an extended region corresponding to 0.15≤z/δ≤0.7. As discussed by [START_REF] Lopez | Wall similarity in turbulent open channel flow[END_REF] and [START_REF] Hurther | Shear stress statistics and wall similarity analysis in turbulent boundary layers using a high resolution 3D ADVP[END_REF], this region reveals the presence of a dynamical equilibrium between production and dissipation in the outer region. In the SIL, the equilibrium is found to be affected by the free surface for z/δ≥0.7. The constant slope of the F k curve in the layer explains the nearly constant TKE dissipation profile shown in Figure 5.

We intent to determine to what extend the shear layers detected by the quadrant threshold technique for high H values contribute to F k in the outer region. As can be deduced from the literature results on the quadrant contribution [START_REF] Nakagawa | Prediction of the contribution to the Reynolds stress from bursting events in openchannel flows[END_REF][START_REF] Raupach | Conditional statistics of Reynolds stress in rough wall and smooth wall turbulent boundary layers[END_REF][START_REF] Hurther | Shear stress statistics and wall similarity analysis in turbulent boundary layers using a high resolution 3D ADVP[END_REF], it is not likely that the sum of ejections and sweeps contributes to F k since they are mainly responsible for the shear production. [START_REF] Raupach | Conditional statistics of Reynolds stress in rough wall and smooth wall turbulent boundary layers[END_REF] has pointed out, however, that the difference between the contribution of ejections and sweeps can be strongly correlated to F k in the outer region. The following expression has been proposed:

(1) ΔS is equal to the difference between the relative shear stress (relative to the local mean value) due to ejection and sweep events for H=0. Eq.( 1) is a semitheoretical derivation of F k because it relies on the experimental evidence that the third order moments (skewness, kurtosis, crossedthird order moments) are found to be linearly related over the outer region of the turbulent boundary layer (Raupach 1981, Hurther and[START_REF] Hurther | Momentum flux and particle flux statistics in suspension flows[END_REF].

Our motivation to analyze the dynamical details of the relation between F k and ΔS relates the following -ΔS is found to be positive in the outer region [START_REF] Nakagawa | Prediction of the contribution to the Reynolds stress from bursting events in openchannel flows[END_REF][START_REF] Raupach | Conditional statistics of Reynolds stress in rough wall and smooth wall turbulent boundary layers[END_REF][START_REF] Hurther | Shear stress statistics and wall similarity analysis in turbulent boundary layers using a high resolution 3D ADVP[END_REF] which results in an ascendant momentum flux.

The TKE flux F k is also ascendant in the outer region (see Figure 6).

-The depth where F k changes its sign coincides very well with the depth where the sign of ΔS changes (see [START_REF] Hurther | Shear stress statistics and wall similarity analysis in turbulent boundary layers using a high resolution 3D ADVP[END_REF].

-For H>6, corresponding to the value for which the vertical gradients of ejections and sweeps are negligible, the difference between ejections and sweeps, i.e. the ΔS for values of H>6, is uniform over a region roughly corresponding to 0.2<z/δ<0.75 (Figure 4). As can be seen in Figure 6, this region coincides with the region where F k is constant. Based on these arguments, we suggest that the residual ascendant momentum flux resulting from the elongated shear layers detected with the quadrant technique for H values roughly above 5, (discussed in Section 4, Figure 3) could be responsible for the TKE flux shown in Figure 6.

To verify this hypothesis on a more quantitative basis, we compare the curve of ΔS as a function of H to the curve of F k conditionally sampled as a function of H.

Note that as H increases, ΔS is equal to the difference between mean ejection and sweep contributions for do not contribute to F k . Furthermore; it is seen that F k varies from 0 to 1 as H increases. This shows that the shear stress is the leading turbulent stress in the generation of F k in the outer region. Normal stresses seem to provide a weak contribution.

Finally, we also observe in Figure 7 that the H value for which the elongated shear stress events have a uniform vertical region agrees very well to H max . The dashed dotted curve in Figure 7 has been redrawn from Figure 4.

We find very similar trends for the curves in the region 0.2≤z/δ≤0.7 (not shown here). This region corresponds well to the region of constant F k (see Figure 6).

Above and below this domain, the ΔS curve is very weakly correlated to the F k curve (not shown here).

IMPLICATIONS FOR GAS TRANSFER AND CONCLUSIONS

As discussed in section 5, we suggest that the elongated shear stress layers associated to the hairpin organization of the outer flow region are responsible for the TKE dissipation rate in the SIL. The TKE implied in this transport results from a residual ascendant momentum flux induced by the asymmetrical contribution to shear stress from ejections and sweeps. As a consequence, small scale turbulence in the outer region makes a very negligible contribution to F k in the region 0.2≤z/δ≤0.7. In the SIL, however, the TKE diffused through the equilibrium region is dissipated into heat, evidently dominated by small scale isotropic eddies. This concept is very different from the commonly accepted concept which explains the values of TKE dissipation close to the free surface. Generally it is assumed that the residue of TKE resulting from the difference between shear production and dissipation in the inner region is transported by turbulent diffusion towards the free surface where it interacts. It appears from our results that the vertical transport of TKE mostly depends on the organization of the large turbulent flow structures in the outer region. In this region hairpin packets and associated elongated shear layers dominate the TKE transport between the bottom and the top of the boundary layer.

This result has important implications for the prediction of gas transfer velocities in shear flows. [START_REF] Moog | Airwater gas transfer in uniform channel flow[END_REF] have discussed the validity of the different approaches. They distinguished between two categories of models for the gas transfer velocity estimation. The first one considers the renewal of the water in the CBL (Concentration Boundary Layer of size O(10µm)) through the action of turbulent micro structures that scale with the Kolmogorov scale. The second category considers the characteristics of intermittent coherent flow structures impinging on the free surface [START_REF] Komori | The relationship between surfacerenewal and bursting motions in an openchannel flow[END_REF][START_REF] Rashidi | Mechanisms of heat and mass transport at gasliquid interfaces[END_REF]. Their detailed analysis revealed that models with burst characteristics are unreliable for shear flows with Re * ≥400. The smallscale models on the other hand, give good results for highly turbulent flows. As pointed out in their study, these models "do not provide a comprehensive depiction of turbulence interactions with the freesurface".

To advance the understanding, [START_REF] Moog | Airwater gas transfer in uniform channel flow[END_REF] proposed a conceptual mechanism that includes the nonhomogeneity of turbulence at the freesurface due to coherent flow structures: the socalled "Chain Saw Model" (CSM). In this model, the TKE dissipation ε is written as follows: must be independent of Re * for Re * ≥400, for the model results to match the small scale model results. From the previous results on the shear layer dynamics we propose to evaluate this quantity indirectly as: where in a flow where Re * =O(10 6 ). In another openchannel flow experiment, [START_REF] Kumar | An experimental investigation of the characteristics of the free surface turbulence in open channel flows[END_REF] have estimated the area of the active gas tranfer zone (patterns of large surface divergence at the freesurface). From their surface fraction measurement, the mean surface fraction is close to 0.8. This result agrees as well with our estimation. In their experiments, Re * is (2)

(3) equal to 411 whereas Re * is equal to 6800 in our flow. The good agreement found between the three is independent of Re * as proposed by [START_REF] Moog | Airwater gas transfer in uniform channel flow[END_REF].

Further investigations are conducted to validate this result by direct measurement of the surface fraction of shear layers impinging at the freesurface and its relation to the hairpin packets organization in the outer region. This mechanism is of primary importance for freesurface processes in geophysical shear currents.

Figure 1 .

 1 Figure 1. Hairpin packets and subdivision into zones of uniform streamwise momentum of the outer region of openchannel flow Histogram.

Figure 2 .

 2 Figure 2. Histogram of the relative instantaneous velocity component u. and corresponding modal velocities, U z1 /U ∞ =0.59, U z2 /U ∞ =0.76, U z3 /U ∞ =0.94, for the high Reynolds number flow.

Figure 3 .

 3 Figure 3. The quadrant threshold technique and its effects on the instantaneous flow velocity field. (a) Representation of constant isolines of relative momentum flux u′w′ in the (u′, w′) plane. (b) Representation of conditionally sampled momentum flux for low and high threshold values, respectively for H=1 (thin lines) and H=5 (thick lines).

Figure 4 .

 4 Figure 4. Vertical gradient of momentum flux u′w′ corresponding to ejection and sweeps as a function of H. The brackets denote a spatial average over the domain 0.2<z/δ<0.7.

Figure 5 .

 5 Figure 5. Profile of the difference between normalized shear production and TKE dissipation rate and profile of the TKE transport term.

Figure 6 .

 6 Figure 6. Profile of the normalized TKE flux F k and the vertical TKE transport term T k .

Figure 7 .

 7 Figure 7. Conditionally sampled AS (dotted line), F k (solid line) and curve in Figure 4 (dashed dotted line, see Figure 4 for details). The dashed line represents the relative derivative in H of the conditionally sampled Fk curve.

Figure 8 .

 8 Figure 8. Determination of from the conditionally sampled ΔS (dotted line) and F k (solid line) at z/δ=0.55 and z/δ=0.9.

Table 1 .

 1 Flow conditions.

	Run	δ (cm)	U ∞ (cm/s)	u * (cm/s)	Re δ (×10 3 )	Re θ (×10 3 )		λ (cm)
	A	17.2	39.8	2.8	68.45	7.9	280	1.47

ACKNOWLEDGEMENTS

The first author expresses his gratitude to the Postdoctoral Scholar Program at the Woods Hole Oceanographic Institution, with funding provided by the J. Seward Johnson Fund/Allyn Vine Senior Technical Staff Award. This is a Woods Hole Oceanographic Institution contribution No. 10911.