Hakim Abdulhamid
email: hakim.abdulhamid@isae.fr

Christophe Bouvet

Laurent Michel

Jacky Aboissière

Clément Minot

Influence of internally dropped-off plies on the impact damage of asymmetrically tapered laminated CFRP

Keywords: A, Carbon fibre B, Impact behaviour C, Damage mechanics Ply drop-off

This paper presents an experimental study of low velocity impact response of carbon/epoxy asymmetrically tapered laminates. The tests are realised at energy between 10 and 30 J on two types of layup with multiple terminated plies. The type and localisation of damage are analysed using C-scan and micrographs. Then, the data is compared with the response of corresponding respective plain laminate. The effects of some tapering parameters (taper angle, drop-off disposition and configuration) on the impact damage mechanisms are also investigated. Very similar impact damage phenomena are found between tapered and plain laminates. The presence of material discontinuity due to the resin pocket affects less the damage mechanism than the structural difference between the thick and the thin sections.

Introduction

In order to achieve optimum weight reduction of a structure like thin walled or beam structural elements, part thickness needs to be tailored according to local loads distribution. In composite structures, thickness tapering cannot be achieved in a continuous way. Plies must be terminated or dropped off at discrete location over the part. Additionally, as an effect of the manufacturing process, a triangular-shape resin-rich pocket is generally present at the end of a dropped ply. Effects of those geometry and material discontinuities on the damage mechanism under static and fatigue loadings have already been significantly studied [START_REF] Cui | New model to predict static strength of tapered laminates[END_REF][START_REF] He | The study of tapered laminated composite structures: a review[END_REF][START_REF] Hitchen | The effect of stacking sequence on impact damage in a carbon fibre/epoxy composite[END_REF][START_REF] Curry | Effect of dropped plies on the strength of graphiteepoxy laminates[END_REF][START_REF] Wisnom | Delamination in asymmetrically tapered composites loaded in tension[END_REF][START_REF] Murri | Fatigue life methodology for tapered composite flexbeam laminates[END_REF][START_REF] Xing | An experimental study of failure mechanisms in laminates with dropped plies[END_REF]. In the tapered region, ply drop-off generates high inter-laminar stress concentration [START_REF] Hitchen | The effect of stacking sequence on impact damage in a carbon fibre/epoxy composite[END_REF][START_REF] Vizzini | Damage analysis of composite tapered beams[END_REF] since the load carried by the dropped plies must be transferred to continuous plies through the inter-plies resin layers and the resin-rich pocket. This stress concentration triggers premature failure initiated by matrix cracking and delamination [START_REF] Curry | Effect of dropped plies on the strength of graphiteepoxy laminates[END_REF][START_REF] Wisnom | Delamination in asymmetrically tapered composites loaded in tension[END_REF][START_REF] Murri | Fatigue life methodology for tapered composite flexbeam laminates[END_REF][START_REF] Xing | An experimental study of failure mechanisms in laminates with dropped plies[END_REF][START_REF] Wisnom | Failure of tapered composites under static and fatigue tension loading[END_REF]. Both tensile and compressive strength are reduced when compared with the corresponding strengths of the thin section [START_REF] Steeves | Compressive strength of composite laminates with terminated internal plies[END_REF]. For instance, Curry and Johnson [START_REF] Curry | Effect of dropped plies on the strength of graphiteepoxy laminates[END_REF] found that the compressive strength ratio between the thin section and the tapered specimen is equal to the ratio of compliance between the thin and the thick section. Under fatigue loading, Weiss et al. [START_REF] Weiss A | Influence of ply-drop location on the fatigue behaviour of tapered composites laminates[END_REF] reported that the propagation of delamination initiated at the resin pocket nearest to the thin section leads to the final rupture of the specimen.

Another important element in composite structures design is the ''impact damage tolerance'' (i.e. the capacity to sustain load in presence of damage). Here, it is worth noting the difference with ''impact damage resistance'' which is more the concern for high energy impact. In fact, low velocity/low energy impact loading is likely to happen during the lifecycle of a given structure. Minor impact events like maintenance tool drop or in-service debris impact on the runway are likely to happen. Many studies have shown how dangerous out-of-plane loading can be to composite laminates [START_REF] Abrate | Impact on composite structures[END_REF][START_REF] Freitas | Composite failure: mechanisms on composite specimens compression after impact[END_REF][START_REF] Hongkarnjanakul | Validation of low velocity impact modelling on different stacking sequences of CFRP laminates and influence of fibre failure[END_REF][START_REF] González | Simulation of dropweight impact and compression after impact tests on composite laminates[END_REF][START_REF] Reis | Damage growth analysis of low velocity impacted composite panels[END_REF][START_REF] Rivallant | Failure analysis of CFRP subjected to compression after impact: FE simulation using discrete interface elements[END_REF]. In fact, impact loading creates complex internal damage, generally considered as matrix cracking, delamination and fibre rupture. Even in the absence of visible external damage, the internal damage can propagate far beyond the impact point and significantly reduces the resistance of the structure below design loads. For thin composite laminates, the energy threshold for BVID (Barely Visible Impact Damage) at which external damage is detectable, generally corresponds already to a substantial loss in the plate compression strength [START_REF] Richardson | Review of low velocity impact properties of composite materials[END_REF].

Many authors have highlighted the importance of interactions between matrix cracking and delamination onset and propagation on the impact damage mechanism [START_REF] Abrate | Impact on composite structures[END_REF][START_REF] Bouvet | Low velocity impact modelling in laminate composite panels with discrete interface elements[END_REF][START_REF] Allix | Mesomodeling of delamination: towards industrial applications[END_REF][START_REF] Renault | Compression après impact d'une plaque stratifiée carbone époxyde -etude expérimentale et modélisation éléments finis associée[END_REF]. Since, the ply drop-off discontinuity is found to be prone to damage, it is therefore justified to investigate its response to impact loading. In literature, there are not many reports concerning the study of impact damage of ply drop-off. To the authors' knowledge, only one study done by Kairouz and Ball [START_REF] Kairouz | Impact of ply drops[END_REF] is available in the open literature. They have tested T300/914C asymmetrically tapered specimens with 6.88 mm and 6.02 mm thickness respectively in the thick and thin sections. One sub-laminate of three plies, situated near the nonimpacted side is terminated at the mid-length of the specimen. They presented the evolution of delamination area for energy between 14 J and 42 J and concluded on the vulnerability regarding the initiation of delamination of the 3 plies drop-off using the stress field from a 2D bending numerical simulation. However, that unique study remains limited and there is a need for a more in-depth understanding of the subject. In this work, specimens with multiple dropped plies are considered to match with industrial type structures. Firstly, the location of delaminated interfaces and their propagation are discussed with regards to the tapered region. Secondly, results of plain and tapered laminates are compared. And finally, the effects on the damage mechanism of drop-off disposition, configuration and the taper angle are analysed.

Experimental setup

Material and specimens

Two tapered laminate layups (A and B) are considered in this study. Both specimens have the same range of thickness: 6 mm (A) and 6.25 mm (B) in the thick and 4 mm in the thin sections. This range of thickness is a classical characteristic of aircraft fuselage. For such application, one side of the structure needs to be flat and smooth to provide good aerodynamic properties. This requirement has led to choose a specimen geometry with only one tapered side as shown in Fig. 1. Test specimens are 250 mm long and the tapering starts at 125 mm from the edge of the thin section (Fig. 1). This size is 100 mm longer compared with the standardized 100 Â 150 mm 2 specimen commonly used in low velocity impact because some space needs to be allocated for the attachment of tabs at each end of the specimen in anticipation of future CAI tests. During the CAI tests, tabs will compensate for the thickness difference between thick and thin sections.

In this work, the dropped-plies of the specimens are interspersed with continuous plies. Such configuration of ply drop-off presents a good resistance under static loadings according to the work of Vizzini and Lee [START_REF] Vizzini | Damage analysis of composite tapered beams[END_REF] and is advocated by the aeronautical regulation [START_REF] Industrie | Reference structure design principles for A350 XWB: composite design principles[END_REF]. The specimens are made from 0.25 mm thick T700GC/M21 carbon-epoxy unidirectional prepreg tapes manufactured by HEXCEL Ò . The mechanical properties of the material obtained from experimental testing [START_REF] Guillon | Etude des mécanismes d'absorption d'énergie lors de l'écrasement progressif de structures composites à base de fibre de carbone[END_REF][START_REF] Prombut | Caractérisation de la propagation de delaminage des stratifies composites multidirectionnels[END_REF] are provided in Table 1.

In the case of type A specimens, 8 in 24 plies are dropped-off. All the terminated plies are oriented in the 0°longitudinal direction of the laminate. In this layup, the number of potential interface of delamination is reduced by grouping plies by pairs. The layup in the thin section is chosen to be the same as a previously studied plain plate [START_REF] Bouvet | Discrete modelling of inter-and intra-laminar failure for impact in composites[END_REF] to enable the comparison of damage. For type B specimens, the layup contains 25 plies, 9 of which are droppedoff. It was designed following AIRBUS composite design rules [START_REF] Industrie | Reference structure design principles for A350 XWB: composite design principles[END_REF]. Plies with all orientations (0, 45, À45, 90) are terminated. Compared with type A specimens, the number of ply interfaces is increased by separating some plies of the same orientation. Also, there are some partially dropped groups of plies (Fig. 2(a)) which are obtained by terminating only one ply in a group of two or three plies of the same orientation. To assess the damage vulnerability of tapered laminates, for both layups, plain laminate with the corresponding thin section layup is also considered.

Regarding the parametric study, two taper angles are tested: a steep slope of 17% and a shallow slope of 10%. Since ply thickness is 2.5 mm, the step spacing is then respectively equal to 1.5 and 2.5 mm. To modify the drop-off configuration in layup A, the groups of plies are dropped-off in two different manners: in a staggered way (Fig. 2(b)) for A st specimens and simultaneously (Fig. 2(c)) for A si . For type B layup, two configurations B 1 and B 2 are also manufactured by changing the order of dropping the plies. The nomenclature of the tapered specimens is presented in Table 2. It is defined as the type (A st , A st , B 1 , B 2 ) followed by the drop-off spacing (1.5, 2.5) in subscript. The plain laminates are respectively designated as A P and B P . Detailed descriptions of the layups are provided in Table 3.

The consistency of the taper angle is ensured by controlling the step spacing between terminated plies with a microscopic screw driven ruler during layup; no mould was placed against the tapered face during the curing process. After the manufacturing process, the transition zone was observed under a microscope to assess the state of the drop-off. A triangular shape resin pocket is observed at the end of dropped plies, except for those close to a 90°ply. For such plies, the 90°ply material migrates inside the pocket. Curry and Johnson [START_REF] Curry | Effect of dropped plies on the strength of graphiteepoxy laminates[END_REF] have also reported similar observation for their specimens. In-plane shear strength 110 MPa

Test procedure

Impact tests were performed using a drop weight testing rig. A guided 4 kg-mass with a hemispherical indenter of 16 mm diameter is dropped from a specific height onto the specimen placed below. Specimens were impacted on the flat face; the opposite side being simply supported by a tapered base with a rectangular opening of 75 Â 125 mm 2 . This boundary condition is very similar to the Airbus standard AITM 1-0010 [START_REF]Fiber reinforced plastics determination of compression strength after impact[END_REF] defined for plain specimens. The impact target is the centre of the specimen which is located at the end of the thin section. The schematic diagram of the test setup is provided in Fig. 1.

Regarding the test measurement, a piezoelectric sensor is attached to the impactor to measure the impact force history and an optical sensor is placed at the level of the specimen to measure the initial impact velocity. Also, the deflection of the non-impacted face is measured using another optical sensor positioned below the specimen. All the data is acquired and recorded at a sampling rate of 200 kHz. The initial velocity and impact force history were then used to compute the evolution of velocity and displacement of the indenter throughout the test. A detailed description of the integration and data processing procedure is available in [START_REF] Petit | Impact and compression after impact experimental study of a composite laminate with a cork thermal shield[END_REF]. Post-impact damage analysis was conducted using C-scan techniques and microscopic observations.

Results

Force curves

Force versus displacement curves of four different specimens impacted at 30 J are plotted in Fig. 3 for comparison. Tapered specimens (A st 1.5 and B 1 1.5 ) curves are very similar to the respective corresponding thin section plain laminate (A P or B P ). The impact phases, from the damage mechanism point of view, are reflected in the changes of the force-displacement curve. Similarly to composite plain laminates, three major phases can be distinguished. The first phase corresponds mainly to the linear dynamic response of the system: impactor and plate. It lasts until the onset of first severe damage -in this case, delamination -when the force reaches around 4 kN. The second phase of the impact is characterized by the propagation of ply interfaces delamination. This propagation is as stable as for plain specimen given the strong similarity between the curves. The final phase is characterized by a drastic drop in the force which is commonly associated with fibre rupture. It happens shortly before the return of the indenter.

Global results

A summary of the impact results for tapered and plain specimens is presented in Table 4. For each test, data is presented in terms of: the impact and absorbed energies, the maximum filtered impact force and the maximum deflection of the plate during the test. Also provided are the projected delaminated area obtained from the C-scan image and the indentation measured with a stereo-correlation system at least 48 h after the test. In all, 20 tests have been realised and the results are fairly repeatable from one test to another. Some tests are performed twice in order to provide a specimen for microscopic observation.

For the plain laminates impacted at 30 J, absorbed energy is comparable between A P and B P , 65% and 62% of the initial energy respectively. However, the projected delaminated area is 60% lower for B P compared with A P as a result of the higher number of plies interfaces in B P layup. As the number of interface increases, smaller projected area is needed to dissipate the impact initial energy. For both cases, the maximum impact force of the specimen is reached because there is a sudden drop of the peak force for B P and a plateau for A P (Fig. 3).

In the cases of tapered laminates, the tests are performed for a range of increasing nominal energy of 10, 20 and 30 J. For low impact energies (10 and 20 J), the damage consists mainly of diffused matrix cracking and ply interface delamination. For those cases, the absorbed energy is around 40% of the impact energy. At 30 J, a severe drop in the plate stiffness due to fibre rupture is observed and the absorbed energy increases up to 65-70%. Furthermore, the dissipated energy of steeper specimens (A 1.5 , B 1.5 ) is slightly higher compared with the shallower ones (A 2.5 , B 2.5 ) but they still remain in the same range as for plain specimens. Regarding the delamination of tapered specimens, the damage area of A specimens remains higher (around 50%) compared with B specimens. This observation is consistent with the data of plain laminates. However when comparing specimens A and B, impacted at 30 J, with their respective plain laminate, the damage area is quite similar for type A specimens. This is contrary to type B results where the damage area is increased up to 60% for tapered specimens (Fig. 4(a)).

Permanent indentation is an important parameter to compare with the BVID (0.25-0.5 mm) and to assess the impact damage tolerance of composite plates. For tapered specimens it ranges from 0.07 to 0.4 mm. At 30 J, an important scattering of the indentation is observed for the B tapered specimens (Fig. 4(b)) and the values are smaller compared with the corresponding plain laminates.

Internal damage

NDT such as Ultrasonic system is widely used to identify the impact damage in CFRP laminates. It can provide information regarding both the damage area and through thickness localisation of material discontinuities like interfaces debonding. However, in the presence of ply drop-off, the ultrasonic wave signal can be disturbed by material discontinuities and plies inclination which makes it problematic to localise deep delamination. In the case of pristine specimen for example, it was difficult for the transceiver to record the reflected signal in the tapered region from both sides of the specimen. Another difficulty inherent to tapered specimen is the identification of the delaminated interface. Depending on the interface location in the thickness, its position may change on either side of the tapered region. Therefore, a particular interface can be represented by two different colours in the C-scan image, or a given colour may represent two different interfaces which makes the interpretation not straightforward.

In Fig. 5, a detailed C-scan image of specimen A st 1.5 is presented. In this case for example, two different colours represent the interfaces between the group of plies 4/5 [0, 90] and 8/9 [90, 0]. To facilitate the interpretation, delamination profile in the transition zone is represented next to the C-scan. The scan is carried out from the impacted face, in a rectangle area of 150 Â 100 mm 2 , centred at the impact point. Plies drop-off are represented by the horizontal dotted lines situated in the middle of the image. The dashed red line rectangle represents the impact boundary condition. The thick section is situated below the dotted lines and the thin one is in the upper part of the image.

Globally, the orientation of delaminated interfaces is governed by the same rule as for plain laminates: i.e. delamination propagates in the direction of the underlying ply. The conic evolution of delamination throughout the thickness, generally observed in plain laminates, is still verified apart from the propagation in the transverse direction of the specimen. The identified delamination profile shows that interfaces between continuous plies are more critical. Although a 1 mm thick 0°terminated sub-laminate (plies drop-off [START_REF] Cui | New model to predict static strength of tapered laminates[END_REF]) is present at the mid-thickness, it does not trigger any delamination. Otherwise, there would have been another The micrograph of specimen A st 1.5 , in Fig. 6a, provides a comprehensive view of internal defects. The cutting plane is parallel to the longitudinal direction of the plate and passes through the impact point. Dense matrix cracking characterized by both small and big cracks are found below the indenter and farther in the thin section. Nevertheless, in the transition zone, the cracks are much less dense and consist of large trans-laminar failure that pilots the interface jump of delamination. The presence of multiple plies fibre rupture below the indenter correlates with the drop in the force curve in Fig. 3. Apart from the interface between the groups of plies 11/ 12 [0, À45] situated near the non-impacted face, all the delaminated interfaces involve continuous plies (Fig. 6(a)). The micrograph confirms the absence of any damage around the 0°terminated mid-thickness sub-laminate observed in the C-scan. Even though the ply drop-off is quite far from the impact point, the absence of any cracks around it induces that it is less critical to impact load.

For B 1 1.5 specimens, the interpretation of the C-scan (Fig. 7) is much more complicated due to the existence of a higher number of ply interfaces and their proximity. Identification of interfaces using only the C-scan data is less accurate and it was conducted with information from micrographs. In order to lighten the view, only large delaminations are identified on the C-scan. These delaminations are found in the interfaces of both continuous and partially dropped plies. Despite the presence of dropped-off plies of different orientation, it is not possible to distinguish the most critical one in terms of delamination. It is only observed that, in the thin section, the largest delaminations propagate toward the 90°transverse direction and that in the transition region they propagate toward the 45°a nd À45°directions of the specimen.

The micrograph in Fig. 8 presents internal damage of B 1 1.5 specimen impacted at 30 J. Three cross sections (A-A, B-B and 8(a-c) and (e) are schematic diagrams of local damage at different sections to show the evolution of damage in the transverse direction.

For this specimen, multiple matrix cracks and fibre ruptures are observed below the indenter (Fig. 8(d)). In the transition zone (Fig. 8(d)), there are some trans-laminar matrix cracks located far from the impact point. Those matrix cracks which pilot the interface jump of delamination are found in the proximity of ply drop-off. However, there is no crack observed in the resin pocket. Therefore, it is the inclination of ply due to the presence of a ply drop-off that generates additional out-of-plane shear responsible for crack initiation. Regarding the delamination, in addition to the ones identified in the C-scan, there are some smaller delaminations involving dropped plies. Actually, the presence of partially dropped layers has led to small delamination between two plies of the same orientation like the 0/0 (Fig. 8(a)-ii) and À45/À45 (Fig. 8

(e)-ii). This type of [h/h] interface delamination

has not yet been reported in the case of impact damage. The micrograph shows that it does not propagate much farther and switches to a neighbour [h/b] interface.

The analysis of delamination around ply drop-off at different cross-sections reveals that the delamination does not initiate at the ply drop-off (Fig. 8(e)). Dropped ply interfaces delaminate only when delamination of continuous plies interface reaches the resin pocket (Fig. 8(a), (b) and(e)) and that the orientation of the underlying ply is toward the resin pocket. To illustrate this idea, let's consider the zone represented in Fig. 8(a). In Fig. 8(a)-ii, obtained from cross-section B-B, the delamination of [90, 0] interface reaches the resin pocket [START_REF] Cui | New model to predict static strength of tapered laminates[END_REF]. Since its propagation is in the longitudinal direction of the specimen, this delamination can progress and trigger a delamination between the continuous and terminated 0°p lies. However, at 45°from the impact point, Fig. 8(a)-i obtained from cross section C-C, there is no delamination near the resin pocket (1) and this layer is not damaged.

Discussion

Comparison with plain laminate

Based on the force-displacement curves and the C-scan images, damage from tapered and plain laminates is compared to the effect of the tapering in the damage mechanism. Note that plain laminate specimens are impacted at 30 J only. Regarding the force-displacement curves (Fig. 9), the response of tapered laminates are compared to the corresponding thin section plain laminate. For both specimens, the presence of the tapering does not modify the critical force for the onset of first damage. Nonetheless, the appearance of major fibre rupture seems to be affected by the tapering, but it is difficult to draw a conclusion given the scattering of composite materials. The existence of major fibre rupture, recorded on the force-displacement curve, affects significantly the value of permanent indentation as it was already reported by He et al. [START_REF] He | Prediction of permanent indentation due to impact on laminated composites based on an elasto-plastic model incorporating fiber failure[END_REF]. Besides that, permanent indentation increases with the value of additional deflection recorded after the fibre rupture during the impact. This additional deflection corresponds to the difference between the maximum displacement of the impactor and its displacement at major fibre rupture as presented in Fig. 11(a). Fig. 11(b) shows the correlation between permanent indentation and additional deflection of specimens impacted at 30 J. Consequently, the variation of onset of fibre rupture and additional deflection after rupture explain the variation of permanent indentation between plain and tapered laminates. Although permanent indentation of tapered laminates is found to be lower compared to plain laminates, it is not possible to highlight a tendency of the effect of the tapering angle on fibre rupture or permanent indentation.

As regards to delamination in Fig. 10, the same interfaces are affected between plain laminates and the thin section of tapered laminates. For type A specimens particularly, delamination shape is comparable with a slightly larger propagation in the 90°transverse direction of tapered specimens. Nonetheless, for type B specimens, differences in delamination shape are clearly visible. They are mainly observed in the first interfaces, especially for the ones that propagate in the transverse direction. As a result of the ply drop-offs, delamination is no more confined in a small area around the impact point as it should be for type B plain laminate layup because it has many ply interfaces. Additionally, some new interfaces [h/h] involving partially dropped plies are also delaminated for tapered B specimens.

For A P specimen, a micrograph from Bouvet et al. [START_REF] Bouvet | Discrete modelling of inter-and intra-laminar failure for impact in composites[END_REF] is also available in Fig. 6(b). This specimen is named A P⁄ . The damage was created by quasi-static indentation test under the impact boundary conditions. The maximum deflection was 6.10 mm which is equivalent to the displacement of a 30 J impact on tapered specimen. For T700GC/M21, Bouvet et al. [START_REF] Bouvet | Discrete modelling of inter-and intra-laminar failure for impact in composites[END_REF] have demonstrated that quasi-static indentation and low velocity impact give very similar damage. Note the strong similarity existing between the two specimens in terms of matrix cracks, delamination and fibre rupture.

Effects of the tapering parameters

The effects of three drop-off parameters: step spacing, configuration and disposition are investigated in this section. The comparison of the force-displacement curves of various specimens (Fig. 9(a) and (b)) shows that those three parameters have no significant effect either on the onset of damage or on the curve evolution. The only observed variation is related to the instant and the severity of tensile fibre rupture at the third phase of the impact loading between steep and shallow tapered specimens. For example, for specimens B 1 1.5 and B 2 1.5 impacted at 30 J, there is a sudden 4 kN loss when the impact force reaches 10.5 kN due to simultaneous fibre rupture in multiple plies (Fig. 9). Nonetheless, specimens with shallow angle reveal less severe loss, i.e. B 1 2.5 presents a 1.5 kN drop at 9.5 kN and B 2 2.5 has no force drop until 11.5 kN. Yet, it is difficult to draw a clear conclusion with the small number of tests and the scattering of composite material.

Regarding the delamination, its evolution with respect to the impact energy is presented for all specimens in Fig. 10. The comparison of A 1.5 with A 2.5 specimens shows that the propagation of delaminations toward the transverse direction is more pronounced for steep specimens. This comparison is only valid when impact energy is not enough to lead to major fibre rupture (10 and 20 J). In fact, a steeper angle gives a smaller length of the zone and therefore the speed of delamination propagation toward the thick section is likely reduced earlier. As a result, more energy is available to be dissipated by delamination propagation through the transverse direction.

Concerning the disposition of ply drop-offs, Weiss et al. [START_REF] Weiss A | Influence of ply-drop location on the fatigue behaviour of tapered composites laminates[END_REF] showed that the order of dropping plies is an important parameter in the fatigue strength of tapered laminates. However, in this study, there is not any significant influence observed on the impact damage mechanism (Fig. 10). Even though there seems to be some differences in the delaminated interfaces of the transition zone between B 1 and B 2 , those differences concern mainly the first interfaces close to the impact side and can be explained by the order in adding new plies in the layup. For the case of type A specimens, where the group of plies were dropped in two different manners: simultaneous (A si 1.5 ) and stagger (A st 1.5 ), a noticeable difference is observed on the delamination area. At 20 J impact energy, despite both configurations have the same tapering slope, the delamination area is 25% higher when the group of plies is dropped simultaneously (A st 1.5 ). The propagation in the specimen transverse direction is the most affected by this modification. When two plies are dropped simultaneously, the neighbouring continuous plies are highly curved. As a result, the load passing through the inter-plies resin in the transition region is higher which explains the increase in delamination area.

Tapered specimens damage mechanism

The synthesis of information gathered in this study allows establishing the impact damage mechanism of tapered laminates. At low impact energy, as the micrographs have confirmed the absence of damage at ply drop-off, first damage must take place within the continuous plies in terms of matrix cracking. This leads to the delamination of the interfaces of continuous plies as found in the C-scan of low energy impacted specimens (Fig. 10). Delamination propagates in both the thin and thick sections at more or less the same rate in all directions. The shape and the propagation of delamination are governed by the same rules as for plain specimens. Resin pocket is only damaged if a delamination reaches its location and that the conditions of delamination propagation are fulfilled. Then, when delamination reaches the end of the transition region near the thick section, its propagation toward that direction is slowed down. The deceleration is due to the increase in local bending stiffness as more new plies are loaded. Simultaneously, the propagation in the transverse direction accelerates and the damage can reach the boundary conditions in presence of enough impact energy. Finally, major fibre rupture appears at the end of the impact load below the indenter.

The observation of micrographs reveals that ply drop-off resin pocket is less critical to damage than normal ply interface during impact. Even though, the resin pocket creates stress concentration, as it is located far enough from the impact point, the stress increase is not high enough to trigger any crack. The resin pocket is only damaged when delamination reaches its position and even then the crack does not propagate much far away as the stress concentration is confined near to the indenter. To understand this phenomenon, consider the stress field in a bending of a simply supported plate; the highest shear and normal stresses responsible for matrix cracking is localised below the indenter. This notion of confinement of the critical area explains the difference of resilience of tapered laminates when subjected to traction/compression loading and impact loading. In a traction/compression test, excluding the boundary effects, the stress is constant across the width of the specimen and therefore the first damage will be triggered by the stress concentration at the resin pocket. In the case of impact, as the location of ply drop-offs is quite far from the indenter, the stress concentration is not high enough to create cracks. Consequently, two main effects of the ply drop-off regarding delamination are observed: first, the creation of trans-laminar cracks in the neighbour plies that pilots the jump of delamination interface and second, the increase of delamination parallel to the ply dropoff axis which is essentially attributed to the structural effects of the thickness variation.

Conclusion

Impacts tests have been conducted on two typical tapered layups with a thickness varying from 4 mm to 6.25 mm. Some taper parameters like drop-offs disposition, configuration and taper-angle were varied to investigate any effects on the damage. The impact energy level ranges from 10 to 30 J. Impact damage assessment was realised using C-scan and micrographs. The results show that the global response of a tapered specimen is quite similar to the response of a plain specimen in terms of the force-displacement curve. The effects of ply drop-offs on the delamination shape is limited when the layup has few number of ply interfaces (A specimens). However in presence of laminate with many interfaces (B specimens); ply drop-off generates larger delamination area compared to plain laminates. In both cases, the overall delamination shape is elliptical with the major axis parallel to the ply drop-off. At high impact energy, the propagation of delamination is biased toward the thin section. Microscopic observations have shown that cracks do not necessarily initiate at the resin pocket. This behaviour is attributed to the confinement and complexity of the stress state in the impact loading. However, ply drop-off can trigger trans-laminar matrix cracking in the neighbour plies that can modify the propagation of delamination. The presence of partially dropped group of plies leads to small delamination between plies of the same orientation. The variation of the tapering angle and the order of adding new ply do not affect much the final damage of the specimen. However, delamination area increases when groups of plies are terminated simultaneously.

Compared to plain laminates, permanent indentation of tapered laminates is reduced due to the delay in fibre rupture. From a damage tolerance point of view, this difference can make tapered laminates more critical to impact. In the future, this research will be pursued to investigate the compression after impact of tapered laminates. Additionally, numerical analysis will be carried out to investigate the impact behaviour of tapered laminates, more specifically predicting the onset and growth of damage in matrix cracking, delamination and fibre rupture.

Fig. 1 .

 1 Fig. 1. Impact boundary conditions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2 .

 2 Fig. 2. Drop-off configurations (a -partially dropped-off, b -staggered drop-off, csimultaneously dropped). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3 .

 3 Fig. 3. Force-displacement curves for specimens impacted at 30 J. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

between the interfaces 4

 4 /5 [0, 90] shown in green and the ones shown in yellow 8/9 [90, 0].

Fig. 4 .Fig. 5 .

 45 Fig. 4. (a) -Evolution of delamination area. (b) -Evolution of permanent indentation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6 .

 6 Fig. 6. Micrograph of (a) -A st 1.5 specimen, (b) -A P⁄ specimen [26]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7 .Fig. 8 .Fig. 9 .

 789 Fig. 7. C-scan from the impacted side of specimen B 1 1.5 impacted at 30 J. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 10 .Fig. 11 .

 1011 Fig. 10. Evolution of interface delamination for specimens A and B (C-scan from the impacted side). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1

 1 Mechanical properties of T700GC/M21[START_REF] Guillon | Etude des mécanismes d'absorption d'énergie lors de l'écrasement progressif de structures composites à base de fibre de carbone[END_REF][START_REF] Prombut | Caractérisation de la propagation de delaminage des stratifies composites multidirectionnels[END_REF].

	q	Density	1600 kg/m 3
	E T 1	Tensile Young's modulus in fibre direction	130 GPa
	E C 1	Compressive Young's modulus in fibre direction	100 GPa
	E 2	Transverse Young's modulus	7.7 GPa
	G 12	Shear modulus	4.8 GPa
	m 12	Poisson's ratio	0.33
	e T0 1	Tensile failure strain in fibre direction	0.016
	e C0 1	Compressive failure strain in fibre direction	À0.0125
	r T0 2	Transverse tensile strength	60 MPa
	r 0 12

Table 3

 3 Specimens

	Layup A	[45 2 0 2	-45 2 0 2	90 2	0 2 ] s
	termination order -	2 nd -	-	-	1 st
	Layup B	[45 -45 90 0 0 0 45 45 90 -45 -45 0 0 1/2 ] s
	termination order for B 1	-	-	3 rd 1 st --4 th -	-	2 nd	-	-5 th
	termination order for B 2	-	-	1 st 5 th --4 th -	-	2 nd	-	-3 rd

layup. (For interpretation of the references to colour in this table legend, the reader is referred to the web version of this article.)

Table 4

 4 Specimen results summary. (For interpretation of the references to colour in this table legend, the reader is referred to the web version of this article.)

	Specimens	Impact	Delaminated	Dissipated	Maximum	Maximum	Permanent
	(1)	Energy	Area (2)	Energy	Force	Deflection	Indentation
	A si 2.5 A si 1.5	10.3 J 10.5 J	594 mm² 684 mm²	3.9 J (37 %) 4.5 J (43 %)	6.6 kN 6.6 kN	3.0 mm 3.1 mm	0.09 mm 0.09 mm
	A si 2.5 A st 1.5 A si 1.5	20.4 J 19.9 J 20.6 J	1828 mm² 1800 mm² 2288 mm²	11 J (53 %) 8.9 J (44 %) 11.8 J (57 %)	7.5 kN 8.0 kN 7.5 kN	4.7 mm 4.6 mm 4.6 mm	0.16 mm 0.23 mm 0.20 mm
	A P	30.1 J	2827 mm²	19.8 J (65%)	7.8 kN	6.0 mm	0.40 mm
	A si 2.5 A st 1.5	30.6 J 29.1 J	2655 mm² 2730 mm²	19.7 J (64 %) 18.7 J (65 %)	8.2 kN 8.4 kN	6.1 mm 5.9 mm	0.29 mm 0.40 mm
	B 1 B 2 B 1 B 2	2.5 2.5 1.5 1.5	10.3 J 10.1 J 10.3 J 9.7 J	421 mm² 540 mm² 387 mm² 343 mm²	4.6 J (45 %) 4.0 J (40 %) 5.9 J (58 %) 4.6 J (48 %)	6.4 kN 6.4 kN 6.3 kN 6.4 kN	2.9 mm 3.0 mm 2.9 mm 2.8 mm	0.07 mm 0.07 mm 0.08 mm 0.07 mm
	B 1 B 2	2.5 2.5	20.4 J 20.3 J	1106 mm² 999 mm²	10.4 J (51 %) 9.0 J (44 %)	8.9 kN 9.0 kN	4.3 mm 4.3 mm	0.13 mm 0.12 mm
	B P	30.2 J	1178 mm²	18.8 J (62%)	9.4 kN	5.4 mm	0.47 mm
	B 1 B 2 B 1 B 2	2.5 2.5 1.5 1.5	30.6 J 30.5 J 31.1 J 30.9 J	1357 mm² 1911 mm² 1596 mm² 1734 mm²	20.3 J (66 %) 14.3 J (47 %) 21.5 J (69 %) 23.6 J (76 %)	9.5 kN 11.5 kN 10.6 kN 10.8 kN	5.5 mm 5.2 mm 5.4 mm 5.5 mm	0.31 mm 0.19 mm 0.24 mm 0.24 mm

Note: (1) the subscripts 1.5 and 2.5 correspond to the spacing between the ends of two successive dropped-off plies. (2) Area of projected delamination is computed from C-scan image.

Acknowledgement

The authors gratefully acknowledge SOGETI HIGH TECH for the financial support of this Ph.D. program.


G Parésys

C Rigart

B Rousseau

A W M Wong

F Fan

J.-P Barbier

J Lavaud

Quantitative and qualitative evaluation of phytoplankton communities by trichromatic chlorophyll fluorescence excitation with special focus on cyanobacteria

Keywords: Bloom monitoring, Chlorophyll fluorescence, Fluorescence induction kinetics, Phytoplankton, Southeast Asia, Water supply Chl, Chlorophyll, FIK, Chl a fluorescence induction kinetics, F o and F m , , respectively, HPLC, Highpressure liquid chromatography, LED, Light-emitting diode, PP, photosynthetic potential, PS II, Photosystem II

We present results that were obtained with a newly developed fluorometer, the 'PhytoSensor'. They are based on multi-wavelength excitation of chlorophyll fluorescence to detect the phytoplankton biomass and to identify main taxons (among cyanobacteria, green and brown microalgae). A method to evaluate the photosynthetic potential of the phytoplankton was established. Attention was focused on the measurement of the cyanobacterial biomass. A modelling to distinguish between the two spectral groups (blue and red) of cyanobacteria as a function of their pigments and physiological status is proposed. The main innovation of the device results in the recording of the fluorescence induction kinetics of the phytoplankton to confirm and refine the evaluation of the taxonomic composition. The PhytoSensor abilities were compared with pigment analysis, commercial fluorometers, particle and microscopic counting and identification. The PhytoSensor has been used with success to monitor the dynamics of phytoplankton in drinking-water supply reservoirs in Southeast Asia.

Introduction

Chlorophyll a (Chl a) is an ubiquitous photosynthetic pigment present in all eukaryotic (algae) and prokaryotic (cyanobacteria) phytoplankton organisms. In vivo Chl a fluorescence has became an increasingly important tool for the assessment of both biomass and photosynthetic activity of phytoplankton in situ [START_REF] Falkowski | Aquatic photosynthesis[END_REF]. Very sensitive techniques have been developed [START_REF] Wilhelm | The application of chlorophyll fluorescence in the aquatic environment[END_REF] among which multi-wavelength fluorometers that allow in vivo and/or in situ taxonomic identification. The current systems are based on the discrimination of 'spectral groups' of phytoplankton (see [START_REF] Beutler | A fluorometric method for the differentiation of algal populations in vivo and in situ[END_REF]). These groups have specific accessory pigments which absorb light efficiently in different ranges of the visible light spectrum [START_REF] Millie | Using absorbance and fluorescence spectra to discriminate microalgae[END_REF]. When they absorb a photon, these pigments become excited and they transfer their excitation energy from pigment to pigment to the Chl a molecules, the terminal acceptor of the excitation transfer channel, which emit fluorescence (reviewed in [START_REF] Krause | Chlorophyll fluorescence and photosynthesis: the basics[END_REF]). By use of light sources of different excitation wavelengths, an evaluation of the systematic composition of a mixed phytoplanktonic community can be obtained. With the progress in light-emitting diode optic, the feasibility of such an approach has been demonstrated [START_REF] Schreiber | PAM fluorometer based on medium-frequency pulsed Xe-flash measuring light: a highly sensitive new tool in basic and applied photosynthesis research[END_REF]. Two devices, the Phyto-PAM (Heinz Walz, Germany) and the Fluoroprobe (BBE Moldaenke, Germany) [START_REF] Beutler | A fluorometric method for the differentiation of algal populations in vivo and in situ[END_REF], are currently available. They have from four to five excitation wavelengths and can detect from three to four spectral groups (green algae, cyanobacteria, 'brown microalgae' (mainly diatoms and dinoflagellates) and a 'mixed group' (cryptophytes)). Recent approaches have been developed to refine the discrimination between the two spectral groups of cyanobacteria (blue and red) and the cryptophytes [START_REF] Beutler | A reduced model of the fluorescence from the cyanobacterial photosynthetic apparatus designed for the in situ detection of cyanobacteria[END_REF][START_REF] Beutler | Algorithms and practical fluorescence models of the photosynthetic apparatus of red cyanobacteria and Cryptophyta designed for the fluorescence detection of red cyanobacteria and cryptophytes[END_REF].

One of the applications of these instruments is the monitoring of the algal and cyanobacterial proliferations (so-called blooms) in water resources used as drinking-water supplies and/or recreational areas [START_REF] Leboulanger | Application of a submersible spectrofluorometer for rapid monitoring of freshwater cyanobacterial blooms: a case study[END_REF]. Blooms are due to eutrophication and more than 40 % of worldwide lakes are eutrophic [START_REF] Briand | Health hazards for terrestrial vertebrates from toxic cyanobacteria in surface water ecosystems[END_REF]. Because they can impair the water quality, blooms have been of major concern for the water resource managers [START_REF] Mouchet | Solving algae problems: French expertise and word-wide applications[END_REF][START_REF] Pitois | Problems associated with the presence of cyanobacteria in recreational and drinking waters[END_REF]. One of the sources of awareness is the synthesis of toxins by cyanobacteria which are responsible for human health hazards [START_REF] Haider | Cyanobacterial toxins: a growing environmental concern[END_REF].

The present work is based on the measurement and interpretation of in vivo Chl a fluorescence emission by freshwater phytoplankton. The fluorometer, the 'PhytoSensor', uses the trichromatic excitation of Chl a fluorescence to differentiate three spectral groups: green and brown microalgae and cyanobacteria. A mathematical modelling based on pigment content and physiological status differences to discriminate between the blue and red groups of cyanobacteria is tentatively presented. A rough evaluation of the photosynthetic potential of the phytoplankton was also reached. The main novel aspect proposed by this device is the simultaneous combination of two independent measuring principles, the minimum level of fluorescence and the fluorescence induction kinetics, for a better identification of taxonomic groups. The PhytoSensor has been tested in natural and man-made (sub-) tropical reservoirs in Southeast Asia. Tropical water resources are more sensitive to eutrophication, they show a higher proportion of cyanobacteria and blooms occurring year round are often unpredictable [START_REF] Jr | Basis for the protection and management of tropical lakes[END_REF][START_REF] Oliver | Freshwater blooms[END_REF]. The detection of the phytoplanktonic biomass, its proliferation, and the identification of the responsible taxa have been possible.

Materials and Methods

Culture of algae and cyanobacteria reference species

The strains of diatoms came from CCMP (Provasoli-Guillard Center for Culture of Marine Phytoplankton, USA), UTEX (University of Texas, USA) and Laboratoire Arago (France) collections, green algae from the University Technology of Malaysia (UTM) collection and cyanobacteria from the UTM and PCC (Pasteur Culture Collection, France) collections. All strains were grown photoautotrophically in continuously sterile air flushed recipients. The following conditions were used. For marine isolates of diatoms: natural seawater F/2 medium, 18°C, light intensity of 40 µE.m -2 .s -1 with a 16 h light / 8 h dark cycle.

The freshwater isolate of Phaeodactylum tricornutum was grown in a Bristol medium. For green algae and the cyanobacteria Anabaena sp. and Pseudoanabaena sp.: Bold Basal medium, 27°C, 25 µE.m -2 .s -1 , 12 h light/12 h dark cycle. For the cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803, GN medium, 30°C, 60 µE.m -2 .s -1 in continuous light.

Cells were harvested during the exponential phase, or when indicated during the stationary phase of growth.

Cell enumeration, pigment analyses and spectroscopy

Algae and cyanobacteria were identified and counted as units with a Malassez hemocytometer counting chamber as described [START_REF] Apha | Biological examination[END_REF]. Particle counting was performed with a Particle Counter (PCX Hach) (resolution 1 µm, maximum size 500 µm) on 500 mL at 100 mL. min -1 . Chl a concentration was measured with a spectrophotometer (Varian DMS 90) after acetone 90 % extraction [START_REF] Jeffrey | New spectrophotometric equation for determining chlorophyll a, b, c1 and c2[END_REF] and with a Datalink fluorometer (FL 100, Datalink, France). Pigment analyses were performed by HPLC [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF], and the relative proportions of algae and cyanobacteria were determined as previously described [START_REF] Wilhelm | The HPLC-aided pigment analysis of phytoplankton cells as a powerful tool in water quality control[END_REF]. Room temperature 680 nm fluorescence excitation spectra were measured with a F-4500 Hitachi spectrofluorometer [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF].

Water sampling

Water was sampled in 1 L bottles at 0.5 m depth. In order to avoid the high lightdependent quenching of fluorescence [START_REF] Leboulanger | Application of a submersible spectrofluorometer for rapid monitoring of freshwater cyanobacterial blooms: a case study[END_REF][START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF], the water was not sampled around noon but instead in the morning (9 AM) and the evening (5 PM). Additionally, a preliminary dark incubation of 30 min before the measurement reduced most of the fluorescence quenching that could have developed [START_REF] Lavaud | In diatoms, a transthylakoid proton gradient alone is not sufficient for non-photochemical fluorescence quenching[END_REF] in spite of sampling precautions.

PhytoSensor device and data processing

1) PhytoSensor prototype: designed to be compact and portable in a suitcase (40 x 30 x 16 cm) for easy transportation. It consists of three parts (Fig. 1a). i) a data acquisition card (PCL 818, Advantech) inserted in a PC computer together with the 'Fluo' software (see § 2) );

ii) the electronic devices; iii) a detector unit (Fig. 1b). It includes a sample compartment containing the water sample illuminated from three sides by blue, red and green lights. The The light intensity was measured inside each light source by a photodiode (BPW 34, Siemens) and was kept constant during the illumination by a feed-back electronic loop (Fig. 2a). This was necessary since for a constant current a decrease of up to 20 % of the LED light intensity was recorded during the first second of illumination (Fig. 2b). This decrease is due to an increase in the temperature of the LED. The electric signal generated by the photodiode was injected at the entry of the circuit by the feed-back electronic loop which compensates the variations in light intensity by variations in the current.

The water samples were contained in cuvettes (Greiner, ref. 613101) placed as close as possible (1 cm) to the PM to improve the detection which can be disturbed by attenuation or diffusion of excitation and/or fluorescence lights. It was tested that the sensitivity of the measurement was not decreased up to a turbidity of 150 NTU artificially created with a bentonite powder (Sigma) (data not shown, the maximal turbidity during field assay period was 20 NTU). Since the bio-optical characteristics of phytoplanktonic organisms can modify the fluorescence emission signal [START_REF] Fujiki | Variability in chlorophyll alpha specific absorption coefficient in marine phytoplankton as a function of cell size and irradiance[END_REF], cyanobacteria and algae with different shapes (unicellular, colonies and filaments) and sizes (from 2 µm to hundreds of µm) were tested to not alter the sensitivity of the measurement (data not shown).

2) Software for the experimental procedure, data acquisition and data treatment: 'Fluo' software was developed in the Delphi 5 language (Borland, USA) and consists of three parts. i) the 'protocol' of the experiment; it was controlled through a set of specific macroinstructions. For each illumination sequence, the excitation wavelength, light intensity, time of illumination and PM voltage were pre-adjusted. ii) the 'evaluation table' of the Chl a concentration and of the relative proportions of algae and cyanobacteria; they were calculated by a mathematical modelling from the fluorescence signals obtained under blue, green and red low intensity excitations. The modelling was based on pre-determined proportionality factors between fluorescence and Chl a concentration measured on reference species. iii) the 'calculation table' of the photosynthetic potential (PP) index of the phytoplankton; it was calculated from the variable part of the fluorescence between the minimum fluorescence signal (F o ) and the maximum fluorescence signal (F m ).

3) Experimental protocol: measurements were done in three replicates for each sample.

Samples were dark-adapted for 30 min before the start of the experiment; cuvettes were then shaken to resuspend cells that had settled. A typical measurement lasted around 4-5 min.

Three successive flash sequences of blue, green and red light of low intensity followed by a flash of high light were fired. Flash duration and irradiances were: i) blue: 0.2 s / 1 µE.m -2 .s -1 , ii) green: 0.5 s / 7 µE.m -2 .s -1 , iii) red: 0.2 s / 13 µE.m -2 .s -1 , and iv) red + blue: 0.2 s / 200 µE.m -2 .s -1 . The illumination sequence was first done on a blank of distilled water and thereafter on the water sample. At the end of the sequences, the signal from the blank was subtracted and the values were averaged. For low light intensity excitations, the best compromise was fixed between the light intensity/duration of illumination and signal-to-noise ratio in order to keep the flash as non-actinic as possible to obtain the closer and better approximation of the 'true' F o value. The same remark is true for the F m level; the high light intensity used here is not sufficient in some conditions to ensure a measurement of the 'true' F m value but instead an approximation of it. Short illumination times were used to prevent variations in the fluorescence emission induced by the photochemistry processes [START_REF] Krause | Chlorophyll fluorescence and photosynthesis: the basics[END_REF]. The influence of one flash on the next was minimised by inserting between flashes dark periods from 5 s between low light flashes to 20 s after high light flash: they were tested to be long enough to allow the reoxidation of the plastoquinone pool between two successive flashes (data not shown).

4) Acquisition and software treatment of the fluorescence induction kinetics (FIK): a FIK represents the variation of Chl a fluorescence emission as a function of time during exposure to a continuous light intensity (see [START_REF] Krause | Chlorophyll fluorescence and photosynthesis: the basics[END_REF]). After 60 s dark adaptation following the last flash sequence, the FIK was recorded under a red light illumination of 130 s / 125 µE.m -2 .s -1 .

Experimental FIK obtained from natural sample was compared with a software accessible library containing the kinetics of algae and cyanobacteria reference species (Fig. 3). The reference FIK were decomposed in elementary components susceptible to enter the experimental curve. The sum of the reference inductions was then fitted to the experimental curve and the best combination chosen with a statistical test (the reduced quadratic error). By this approach, relative proportions of algae and cyanobacteria were obtained. Results were embedded in the 'evaluation table'.

Results and Discussion

The distinction between the spectral groups of phytoplankton [START_REF] Beutler | A fluorometric method for the differentiation of algal populations in vivo and in situ[END_REF] is based on one or two specific dominant accessory pigments. While green algae are rich in Chl a and b (which absorb in the blue and red ranges of the visible spectrum), brown microalgae (in this work diatoms were chosen as representative of the group) and cyanobacteria are rich in xanthophylls (which absorbs in the green range) and in phycobilins, respectively. There are two main types of phycobilins: phycocyanin present in all strains absorbs in the orange-red range, phycoerythrin absorbs in the green range. Two spectral groups of cyanobacteria can thus be defined: blue cyanobacteria (or BC) which have only phycocycanin and red cyanobacteria red (RC) which have both. Hence, the fluorescence excitation spectra of the four main spectral or taxonomic groups (Fig. 4) reflect the wavelength-dependence of fluorescence emission due to differences in pigment composition.

Modelling for the evaluation of the phytoplankton biomass and the taxonomic identification

Fluorescence emission intensity is a function of Chl a concentration. As it is complementary to the photochemical activity, the fluorescence emission is also depending upon the incident light intensity [START_REF] Krause | Chlorophyll fluorescence and photosynthesis: the basics[END_REF]. Under weak light and for dark-adapted cells, fluorescence emission is minimal (F o ); under saturating light (the photochemical activity being maximal), fluorescence increases to a maximum (F m ) when photosynthesis is saturated.

The F o level is proportional to the number of photosystems (PS) II, which is not always the case for F m because of fluorescence quenching mechanisms that develop under high light [START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF][START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF]. Therefore, the only, most reliable and simple base for the evaluation of Chl a concentration is the F o level. F o was measured under three low intensity wavelengths of blue (430  30 nm), green (540  15 nm) and red (625  20 nm) (see Fig. 4) for the four reference organisms chosen (Synechococcus sp. and Pseudoanabaena sp. for cyanobacteria blue and red respectively, Chlorella sp. for green algae, Phaeodactylum sp. for diatoms, representative of the brown microalgae) and as a function of a range of Chl a concentration (0-50 µg Chl a.L -1 ) likely to occur when a bloom starts. An example of results is shown in Figure 5. The sensitivity of the device was 2 µg Chl a.L -1 in laboratory conditions (see Fig. 5). The slope of the linear relationship was the proportionality factor (Pf) between the F o fluorescence emission and the Chl a concentration. It was determined for each organism at each excitation wavelength (Table 1).

The mathematical model used to evaluate the total Chl a concentration and the relative proportions of each group was a matrix of three equations:

Fb = (Pfb G x G) + (Pfb D x D) + (Pfb C x C) Fg = (Pfg G x G) + (Pfg D x D) + (Pfg C x C) Fr = (Pfr G x G) + (Pfr D x D) + (Pfr C x C)
Pfb G, etc are the proportionality factors of Table 1 determined under blue (b), green (g) and red (r) lights. G, D and C are the concentrations (in µg Chl a.L -1 ) of green algae, diatoms (representative of the brown microalgae) and cyanobacteria (blue, B, or red, R) deduced from the measurement of the fluorescence levels Fb, Fg and Fr with blue, green and red excitations, respectively. The sum G + D + C is the total phytoplankton biomass (in µg Chl a.L -1 ).

The validity of the chosen reference organisms Chlorella sp. and Phaeodactylum sp.

for the determination of the Pf was tested. The values obtained for Chlorella sp. were compared to that of three other genera frequently encountered in freshwater reservoirs (Table 1). The Pf of all four genera were very similar and the reference species was determined as representative of the whole group. The same conclusion was reached for diatoms (Table 1). It confirms that under the same culture conditions, the fluorescence emission properties of different species of green algae and diatoms are very similar [START_REF] Millie | Using absorbance and fluorescence spectra to discriminate microalgae[END_REF].

Unlike for algae, one set of Pf cannot be considered as representative for all cyanobacteria. Although the blue cyanobacteria (BC) group is the most frequent, the red group (RC) can be abundant in some conditions. As the fluorescence emission properties differ between the two groups, the identification procedure was then optimised as follows (Fig. 6):

Step 1-As the presence of cyanobacteria was always correlated with a high fluorescence under red light (Table 1), they were unequivocally detected even at low concentrations. If cyanobacteria were not the dominant taxon, the Pf of BC were used as the default set. It was verified that below a threshold of 20 % BC or RC in a mixed population, the Pf of BC and the Pf of RC sets gave equivalent results (data not shown). Beyond this threshold, the modelling was settled to discriminate between the two spectral groups.

Step 2-The distinction between the two types of cyanobacteria was based on the red light excited fluorescence over green light excited ratio (Fr/Fg) (Table 1). The evaluation was refined with the Pf of RC set when Fr/Fg was below 5 by referencing to Anabaena sp. (Fr/Fg = 5.6).

It has to be point out that a non negligible biomass of the brown microalgae (especially diatoms and cryptophytes) may disturb the discrimination between BC and RC because they absorb green light [START_REF] Beutler | A reduced model of the fluorescence from the cyanobacterial photosynthetic apparatus designed for the in situ detection of cyanobacteria[END_REF][START_REF] Beutler | Algorithms and practical fluorescence models of the photosynthetic apparatus of red cyanobacteria and Cryptophyta designed for the fluorescence detection of red cyanobacteria and cryptophytes[END_REF]. For this reason, this modelling would be reliable only if cyanobacteria are dominant which is often the case under tropical latitudes [START_REF] Jr | Basis for the protection and management of tropical lakes[END_REF][START_REF] Oliver | Freshwater blooms[END_REF]. On the other hand, it has been observed that the presence of green light absorbing RC in non negligible amounts disturbs the identification of the brown microalgae (data not shown), that is why the discrimination between BC and RC is essential over a certain threshold of cyanobacterial biomass.

When phytoplankton shifts from growth to stationary phase and cell death occurs (socalled 'ageing of the cells'), and to a larger extent when they are submitted to an environmental stress, the light absorption properties and Chl a fluorescence yield of their pigment antenna are modified [START_REF] Falkowski | Aquatic photosynthesis[END_REF]. Then, the proportionality factors between Chl a concentration and fluorescence determined for cells in the exponential phase of growth (Table 1) can be changed. The process is different among taxa. For green algae, fluorescence yields were not changed (see also [START_REF] Beutler | A fluorometric method for the differentiation of algal populations in vivo and in situ[END_REF]). In contrast, aged diatom cells showed a decreased Pf under green light (data not shown) possibly due to an increase in xanthophyll amount [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF] and related fluorescence quenching [START_REF] Lavaud | Influence in the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF], and/or to a decrease in antenna energetic coupling [START_REF] Shimura | Changes in the activity of fucoxanthin-excited photosynthesis in the marine diatom Phaeodactylum tricornutum grown under different culture conditions[END_REF]. This uncertainty was not fully solved: in field experiments, diatoms were always in very low amounts as in most tropical resources [START_REF] Jr | Basis for the protection and management of tropical lakes[END_REF]. The consequences of the cyanobacteria cell ageing on the variability in fluorescence emission are more dramatic because of the structure and location of their antenna, the phycobilisomes (PBS). Nutrient deficiency generates a degradation but also a partial disconnection of PBS from PS II producing fluorescence from the PBS itself [START_REF] Campbell | Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation[END_REF]. As a consequence, in aged cyanobacteria, the fluorescence emitted under red and/or green lights was strongly increased (also observed by [START_REF] Beutler | A reduced model of the fluorescence from the cyanobacterial photosynthetic apparatus designed for the in situ detection of cyanobacteria[END_REF]) as well as the corresponding Pf (Table 1). In order to refine the identification and quantification of the cyanobacteria, the evaluation procedure was completed after Step 2 as follows (Fig. 6):

Step 3-The aged cells were distinguished with the Fr/Fg ratio (Table 1). For BC, Fr/Fg > 10 (by referencing to Synechocystis sp., Fr/Fg = 9.0) was indicative of their ageing and new Pf were used. For RC, the aged status was for Fr/Fg > 2. The choice for such thresholds was justified and illustrated by field experiments (see § 3.3.).

Laboratory and field tests

In order to test the sensitivity of the PhytoSensor and the modelling, the identification of the taxonomic groups was determined on laboratory culture mixtures of known composition and Chl a concentration (as measured spectrophotometrically) and compared to HPLC analyses. The Chl a concentration was set to 20 µg Chl a.L -1 and three relative concentrations of each group were used: 12.5 %, 33 % and 75 % (Table 2). The Chl a concentration evaluated by the PhytoSensor was not significantly different from the spectrophotometry and HPLC measurements whatever the proportions of groups (Student test, p < 0.05). When groups were in equal proportions (33 % each) or one of them two times higher (50 %), the PhytoSensor evaluation as the HPLC analysis were very similar to the expected values (no more than 8 % difference); which made the PhytoSensor and HPLC evaluations as close as 3.9  3.5 % in average (max 11 %). When a group was largely dominant (75 %), the difference between PhytoSensor evaluation and expected values was increased up to 10 % while HPLC analysis remained as close to the expected values as before.

Under these conditions, the difference between the PhytoSensor and HPLC evaluations was nearly doubled (6.5  3.5 % in average, max 12 %). Hence, evaluation of biomass and identification of the groups by the PhytoSensor was similar to that of HPLC measurements even in conditions where group proportion was low (12.5 %).

The reliability of the evaluation of Chl a concentration based on F o measurement was further tested on several reservoirs used from drinking-water supply in China and Malaysia.

PhytoSensor measurements were compared to particle counting (with a PCX, Hach) and Chl a evaluation by Turner (10-AU Digital) and Datalink (FL 100) fluorometers which are widely used to determine Chl a concentration as indicative of phytoplankton biomass in freshwaters [START_REF] Pinto | Chlorophyll-a determination via continuous measurement of plankton fluorescence: methodology development[END_REF]. An example of the results obtained is shown if Figure 7. It corresponds to the monitoring of a Malaysian reservoir with variable Chl a concentrations (by a factor of up to 6) and over a three months period. The relationships between the Chl a concentration as measured with the PhytoSensor and both the Datalink Chl a evaluation and the number of particles showed a R factor of 0.98 and 0.93, respectively, with a slope close to 1 (0.96 and 0.88 respectively). The limit of sensitivity of 2 µg Chl a.L -1 (corresponding to 2-3 10 2 particles.mL -1 ) was confirmed by field experiments (data not shown).

Bloom monitoring and the use of the chlorophyll fluorescence induction kinetics

A bloom with changes in the amounts of algae and cyanobacteria was monitored in the 'Cahaya Bahru' reservoir (Malaysia) over a one month period (Fig. 8a). The PhytoSensor Chl a concentration evaluation based on the F o measurements was in agreement with the Datalink measurement (data not shown, slope = 1.01, R = 0.99). The ability of the PhytoSensor to identify the phytoplankton groups was compared to microscopic identification and counting (Fig. 8b). Despite a low sampling frequency before and after the bloom period, these data show that the procedure for the identification of phytoplankton groups, and especially cyanobacteria, was reliable. Cyanobacteria were determined by the PhytoSensor as aged red cyanobacteria. It is reliable with the observed continuously decreasing cyanobacterial population (Fig. 8a) and with the microscopic identification which revealed a predominance of the colony forming toxic genus Microsystis sp. for which a number of species have phycoerythrin [START_REF] Otuska | 16s rDNA sequences and phylogenetic analyses of Microcystis strains with and without phycoerythrin[END_REF].

In order to confirm and refine the discrimination of phytoplankton groups from the Phytosensor F o measurement, the fluorescence induction kinetics (FIK) were recorded (Fig. 8c). A FIK (also called the 'Kautsky effect') represents the variation in the Chl a fluorescence emission as a function of time under exposure to continuous light (see [START_REF] Krause | Chlorophyll fluorescence and photosynthesis: the basics[END_REF]). The shape of the FIK is depending upon the redox state of the electron transport chain between the PS II and PS I. The amplitude of the FIK is a function of the Chl a concentration and, as shown in Figure 3, its shape is characteristic for respective taxonomic group. FIK showed a maximum of emission already after 500 ms illumination in green algae and after 5 s for diatoms. For the two spectral groups of cyanobacteria, the shape of the induction was similar: flat with a maximum emission after approximately 20 s. Comparing the shape of the FIK from natural water samples (Fig. 8c) to the ones of model organisms (Fig. 3), it is clear that there is a majority of green algae and cyanobacteria. The results of the software FIK analysis are shown in Fig. 8b in grey. The relative proportions of the taxonomic groups evaluated with the two independent approaches (F o and FIK) were similar. It was possible to monitor a change in biomass of the brown microalgae which represented less than 10 % of the phytoplankton. It is noteworthy that for the sampling of March 8, the two approaches greatly differed from the microscopic counting. It thus can be concluded that the counting might be false, which is not surprising since colony forming cyanobacteria can sometimes be over-estimated by the counting [START_REF] Rashash | An evaluation of algae enumeration[END_REF].

Evaluation of the photosynthetic potential of phytoplankton

During ageing of cells, F m is usually lowered due to a decrease in photochemical activity. As a consequence, the variable part of the fluorescence emission (F v = F m -F o ) decreases (see [START_REF] Parkhill | Fluorescence-based maximal quantum yield for PSII as a diagnostic of nutrient stress[END_REF]). The ratio F v /F m has been taken as an indicator of the photosynthetic capacity reflecting the global physiological status [START_REF] Falkowski | Aquatic photosynthesis[END_REF][START_REF] Wilhelm | The application of chlorophyll fluorescence in the aquatic environment[END_REF][START_REF] Krause | Chlorophyll fluorescence and photosynthesis: the basics[END_REF]. For algae, F v /F m = 0.70  0.05 and F v /F m = 0.50  0.05 are observed during the exponential and stationary phases of growth, respectively. Based on these observations, a rough measurement of F v /F m was done in order to evaluate the global photosynthetic potential (PP) index of the phytoplankton. For algae, two situations were defined: F v /F m  0.65 (active growth, PP index: (+)), F v /F m  0.5 (stationary growth, PP index: (-)); between these two values, the PP index was (+/-). For cyanobacteria in active growth, F v /F m varied from 0.75 to 0.5 [START_REF] Campbell | Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation[END_REF]. Then, when the amount of cyanobacteria was more than 20 %, different thresholds were used. In that case, it was defined as follows:

(+) when F v /F m  0.5 and (-) when F v /F m  0.5.

The PP index was measured during the bloom event in the 'Cahaya Bahru' reservoir (Fig. 8b). It showed a change in the physiological status of the phytoplankton. At the beginning, the phytoplankton presented a PP index (+). Then, green algae produced a bloom.

It is likely that the second sampling corresponds to the beginning of the decline of the green algae population as the PP index was (+/-). This decline is tentatively attributed to nutrient shortage because of the large consumption of nutrients during the bloom and/or to zooplankton grazing pressure [START_REF] Oliver | Freshwater blooms[END_REF]. The PP index further decreased to (-), indicating a breakdown of biomass as illustrated by the decrease in Chl a concentration (Fig. 8a). It was followed by an increase to a PP index (+) presumably because the end of the monitoring period likely corresponded to the starting of a new green algae bloom (as seen in the corresponding increase in the Chl a concentration, Fig. 8a).

Conclusions

We have described a new multi-wavelength fluorometer, the PhytoSensor, and a mathematical modelling to measure in few minutes the chlorophyll biomass, and to evaluate the taxonomic composition and the photosynthetic potential of water samples containing mixed phytoplankton organisms. The device detects biomass as low as 2 µg.L -1 , discriminates between three groups (green and brown microalgae, and cyanobacteria) even in conditions where one is dominant, and monitors a change in biomass of one taxon which represents a small fraction (<10 %) of the total phytoplankton. A modelling, based on pigment and physiological status differences, allows the discrimination between blue and red cyanobacteria when cyanobacteria are dominant. Its main innovation results from the simultaneous combination of two independent measuring principles, the minimum level of fluorescence and the fluorescence induction kinetics, for a better identification of the taxonomic groups. Combining all parameters it was possible to monitor a bloom in a reservoir used as a drinking-water supply in Southeast Asia. The slopes of the linear fits correspond to the proportionality factors (Pf) under blue (•), green (▲) and red (■) lights listed in Table 1. Standard variation was not more than 5 % for blue and red lights and 10 % for green light. a The parameters are labelled as Pf for proportionality factor between Chl a concentration and fluorescence; b, g and r for blue, green and red lights respectively; and G, D, C for green algae (reference species: Chlorella sp.), diatoms (reference species: Phaeodactylum sp.)

Figures legends

representative of the brown microalgae, and cyanobacteria, respectively. For cyanobacteria, two spectral groups were distinguished: the blue cyanobateria, BC (reference species: Synechococcus sp.) which absorb preferentially in the orange-red range (absence of phycoerythrin) and the red cyanobacteria, RC (reference species: Pseudoanabaena sp.) which additionally absorb in the green range (presence of phycoerythrin).

b The values of Fr/Fg were used to refine the cyanobacteria evaluation procedure: when Fr/Fg < 5, the set of Pf RC was substituted to the set of Pf BC (see the text for details).

c Aged cyanobacteria designate cells which are in the stationary phase of growth for days.

Parésys, Rigart, Rousseau, Wong, Fan, Barbier and Lavaud T im e (s ) 8c

  sample compartment was isolated from ambient light by a cap. On the fourth side, chlorophyll fluorescence was detected by a miniature photomultiplier module (PM) (Hamamatsu H5701-50, together with an amplifier and a high-voltage supply) protected by an interference filter (Corion, S10 680 F, 680  10 nm). Each light source consisted of an array of six light emitting diodes (LED) and a specific interference filter combination. Blue light, 15 µE.m -2 .s -1 (maximum light intensity in the sample compartment): LED Everlight 383 UBC, band pass of 430  30 nm obtained with filters Maier SPX R 500 + Andover 450 FL07 + Andover 600 FL07. Green light, 65 µE.m -2 .s -1 : LED Marl Ultrabright 'pure green' plus a 540  15 nm filter Corion, XM 540. Red light, 250 µE.m -2 .s -1 : LED Hewlett Packard HLMP-DH08 plus a 625  20 nm filter Corion, XM 625.

Fig. 1 .

 1 Fig. 1. (a) Schematic representation of the PhytoSensor device top viewed. PM:

Fig. 2 .

 2 Fig. 2. (a) Schematic representation of the feed-back electronic loop that allows the regulation

Fig. 3 .

 3 Fig. 3. Chlorophyll fluorescence (in arbitrary units) induction kinetics during a red light excitation (50 µg Chl a.L -1 ) of (a) blue cyanobacteria (BC) (Synechococcus sp.), diatoms (D)

Fig. 4 .

 4 Fig. 4. Room temperature 680 nm fluorescence excitation spectra ('photosynthetic action

Fig. 5 .

 5 Fig. 5. Chlorophyll fluorescence emission under low light excitation (F o in arbitrary units) as a function of the Chl a concentration (in µg.L -1 ) for blue cyanobacteria (Synechococcus sp.).

Fig. 6 .

 6 Fig. 6. The three steps of the evaluation procedure. The modelling evaluates the proportions

Fig. 7 .

 7 Fig. 7. Particle number (per mL) (Δ), and Chl a concentration (in µg.L -1 ) as measured by the

Fig. 8 .

 8 Fig. 8. (a) Chl a concentration (in µg.L -1 ) of green algae (grey), cyanobacteria (dark) and

  Figure 1

  Figure 8

Table 1

 1 Proportionality factors between F o chlorophyll fluorescence emission and Chl a concentration of different species of green algae, diatoms and cyanobacteria. a

	Pfb	Pfg	Pfr	Fr/Fg b

.0 Red Cyanobacteria (RC), Pseudoanabaena sp. 1.3 16.3 20.5 1.3

	RC aged, Pseudoanabaena sp.	2.0	27.5 60.4 2.2

Table 2

 2 Chl a concentration and % of green algae (G) (Chlorella sp.), diatoms (D) (Phaeodactylum sp.) representative of the brown microalgae, and blue cyanobacteria (BC)

	(Synechococcus sp.) as measured by the PhytoSensor and the HPLC analysis from mixtures
	with different proportions.
		Chl a (µg. L -1 ) % G	% D	% BC
	Expected values	20	33	33	33
	PhytoSensor a	20,4  1,0	34  2	34  1	32  2
	HPLC analysis	18,5	31	41	28
	Expected values	19.0	50	25	25
	PhytoSensor a	17.4  0.3	57  2	27  2	26  3
	HPLC analysis	18.0	57	21	22
	Expected values	19.0	75	12.5	12.5
	PhytoSensor a	15.7  0.2	65  1	19  1	16  1
	HPLC analysis	17.2	75	11	14
	Expected values	21.0	25	50	25
	PhytoSensor	19.0  1.0	32  5	45  2	23  3
	HPLC analysis	18.0	32	45	23
	Expected values	20.3	12.5	75	12.5
	PhytoSensor	17.6  0.9	18  1	70  2	12  1
	HPLC analysis	19.8	13	77	10
	Expected values	20.0	25	25	50
	PhytoSensor	18.9  0.9	23  2	24  2	53  4
	HPLC analysis	18.7	31	27	42
	Expected values	18.1	12.5	12.5	75
	PhytoSensor	19.3  0.3	9  2	10  2	81  4
	HPLC analysis	20.2	17	14	69

a PhytoSensor data are mean value ( SD) of three measurements.

Parésys, Rigart, Rousseau, Wong, Fan,

Barbier and Lavaud Parésys, Rigart, Rousseau, Wong, Fan, Barbier and Lavaud

Acknowledgements

This work was supported by the Suez-ONDEO Services Society. We thank A. Korber for the artwork, A. Joder for maintenance of cultures, all the technical staff in Malaysia and China laboratories which provided valuable assistance, T. Levaillant for the coordination of the first series of field experiments, Pr. J.-C. Duval for experimental assistance, helpful discussions and help for the writing of the manuscript, and Drs. A.-L. Etienne and P. Kroth for critical reading and improvement of the manuscript.