Orens Pasqueron De Fommervault

Christophe Migon

D ' Fabrizio

Ortenzio

Maurizio Ribera D'alcala

Laurent Coppola

Temporal variability of nutrient concentrations in the northwestern Mediterranean Sea (DYFAMED time-series station)

Keywords: Northwestern Mediterranean, DYFAMED, time-series, nutrients

Nitrate, phosphate, and silicate concentration profiles were measured at monthly frequency at the DYFAMED time-series station (central Ligurian Sea) between 1991 and 2011. The resulting data set, which constitutes the longest open-ocean time-series in the Mediterranean Sea, underwent quality control. A reproducible climatological pattern was observed with an unprecedented resolution, confirming the typical seasonal cycle of mid-latitudes. In summer and autumn, when the water mass is well stratified, i.e. the mixed layer depth (MLD) is shallow, nutrient concentrations in surface are very low or under the detection limit. In winter, as a result of the MLD extent, nutrients are supplied to the surface layer. Then, nutrient concentrations progressively decrease during spring. MLD appears to play a key role in controlling nutrient availability in the surface layer, but a direct, quantitative relationship between MLD and nutrient concentrations is difficult to establish due to undersampling.

Regarding nutrient molar ratios (N:P, Si:N, and Si:P), results show anomalous values compared to those of other oceanic regions, presumably due to strong influence of external sources. As a consequence, nutrient molar ratios exhibit a seasonal pattern, with, in particular, an increase of the N:P ratio in condition of stratification. Over the period 1991-2011, the DYFAMED data set reveals decadal trends in nitrate and phosphate concentrations in deep waters (+0.23% and -0.62%, respectively) resulting in increasing N:P and Si:P ratios (+1.14% and +0.85% per year, respectively). Such a long-term variability is presumably related to changes in water mass and/or changes in external sources, even if is difficult to assess due to not enough concomitant data from atmospheric and riverine inputs.

Introduction

The distribution of nutrient concentrations is driven by a complex combination of biotic (i.e. phytoplankton production, excretion and remineralisation) and abiotic forcing (upwelling, thermohaline circulation, atmospheric and terrestrial inputs). Among the abiotic factors, the mixed layer depth (MLD, which indicates on the depth reached by turbulent mixing in a recent past; [START_REF] Brainerd | Surface mixed and mixing layer depths[END_REF][START_REF] Kara | Mixed layer depth variability over the global ocean[END_REF][START_REF] De Boyer Montegut | Mixed layer depth over the global ocean: An examination of pro-file data and a profile-based climatology[END_REF]) is probably the most important. MLD variability has important repercussions on nutrient distribution, as it is known to play a key role in controlling the nutrient re-distribution in the water column and, therefore, driving their availability to phytoplankton [START_REF] Mann | Dynamics of Marine Ecosystems: Biological-Physical Interactions in the Oceans[END_REF]. In temperate regions, primary production is critically controlled by nutrient availability, which, in turn, is strictly dependent on MLD dynamics [START_REF] Obata | Global verification of critical depth theory for phytoplankton bloom with climatological in situ temperature and satellite ocean color data[END_REF][START_REF] Wilson | Global climatological relationships between satellite biological and physical observations and upper ocean properties[END_REF].

Compared to other temperate seas, the Mediterranean Sea exhibits extremely low nutrient concentrations [START_REF] Mcgill | The relative supplies of phosphate, nitrate and silicate in the Mediterranean Sea. Rapport des Procès Verbaux des Réunions de la CIESM[END_REF][START_REF] Krom | Phosphorus limitation of primary productivity in the eastern Mediterranean Sea[END_REF], with a significant geographical variability characterised by a west-to-east gradient [START_REF] Pujo-Pay | Integrated survey of elemental stoichiometry (C, N, P) from the western to eastern Mediterranean Sea[END_REF][START_REF] Moutin | Influence of anticyclonic eddies on the biogeochemistry from the oligotrophic to the ultraoligotrophic Mediterranean (BOUM cruise)[END_REF]. At climatological scale, the gradient is often considered as a consequence of the anti-estuarine circulation of the basin [START_REF] Crispi | Nutrient cycling in Mediterranean basins: the role of the biological pump in the trophic regime[END_REF]. Few nutrients available in the Atlantic surface inflow entering the Mediterranean Sea are consumed along the way to the eastern basin, and exported to deep water [START_REF] Crise | The Mediterranean pelagic ecosystem response to physical forcing[END_REF][START_REF] Lazzari | Seasonal and inter-annual variability of plankton chlorophyll and primary production in the Mediterranean Sea: a modelling approach[END_REF]. A major part of these nutrients do not cross the Sicilian strait and are stored into the western basin. It results in an outgoing Mediterranean nutrient-enriched water flow at Gibraltar, and a loss of nutrients (10% for N and P, and up to 50% for Si; Durrieu de [START_REF] De Madron | Marine ecosystems' responses to climatic and anthropogenic forcings in the Mediterranean[END_REF]. A compensation from external sources is commonly postulated [START_REF] Béthoux | Nutrients in the Mediterranean Sea, mass balance and statistical analysis of concentrations with respect to environmental change[END_REF][START_REF] Ribera D'alcalà | Nutrient ratios and fluxes hint at overlooked processes in the Mediterranean Sea[END_REF].

Another specificity of the Mediterranean Sea is the anomalous value of nutrient molar ratios, compared to those of other oceanic regions [START_REF] Herut | Atmospheric input of nitrogen and phosphorus to the Southeast Mediterranean: sources, fluxes and possible impact[END_REF][START_REF] Kress | Spatial and seasonal evolution of dissolved oxygen and nutrients in the Southern Levantine Basin (Eastern Mediterranean Sea): Chemical characterization of the water masses and inferences on the N:P ratios[END_REF][START_REF] Pujo-Pay | Integrated survey of elemental stoichiometry (C, N, P) from the western to eastern Mediterranean Sea[END_REF][START_REF] Ribera D'alcalà | Nutrient ratios and fluxes hint at overlooked processes in the Mediterranean Sea[END_REF]. In particular, N:P ratios in the stocks are high, compared to the typical value of 16, but the Redfield model [START_REF] Redfield | On the proportions of organic derivations in seawaterand their relation to the composition of plankton[END_REF] may not be relevant for semi-enclosed seas such as the Mediterranean [START_REF] Takahashi | Redfield ratio based on chemical data from isopycnal surfaces[END_REF][START_REF] Geider | Redfield revisited: variability of C:N:P in marine microalgae and its biochemical basis[END_REF][START_REF] Wang | On the 'strict homeostasis' assumption in ecological stoichiometry[END_REF]. Basically, it is explained either by the strong influence of external sources, which are generally N-enriched, relative to P [START_REF] Krom | Nutrient budget for the Eastern Mediterranean: implications for P limitation[END_REF][START_REF] Bartoli | Atmospheric input of dissolved inorganic phosphorus and silicon to the coastal northwestern Mediterranean Sea: fluxes, variability and possible impact on phytoplankton dynamics[END_REF][START_REF] Markaki | Variability of atmospheric deposition of dissolved nitrogen and phosphorus in the Mediterranean and possible link to the anomalous seawater N/P ratio[END_REF][START_REF] Ludwig | River discharges of water and nutrients to the Mediterranean and Black Sea: Major drivers for ecosystem changes during past and future decades? Progr Oceanogr 80:199-217 trace the climatology of water masses and to validate incoming data from operational oceanography[END_REF], or to dinitrogen fixation [START_REF] Béthoux | Nutrients in the Mediterranean Sea, mass balance and statistical analysis of concentrations with respect to environmental change[END_REF][START_REF] Sachs | Oligotrophy and nitrogen fixation during eastern Mediterranean sapropel events[END_REF][START_REF] Pantoja | Stable isotope constraints on the nitrogen cycle of the Mediterranean Sea water column[END_REF], although this process might not be significant in the Mediterranean Sea [START_REF] Ibello | First basin-wide experimental results on N 2 fixation in the open Mediterranean Sea[END_REF][START_REF] Sandroni | Dry atmospheric deposition and diazotrophy as sources of nitrogen to northwestern Mediterranean oligotrophic surface waters[END_REF][START_REF] Yogev | Is dinitrogen fixation significant in the Levantine Basin, East Mediterranean sea?[END_REF].

Consequently to this high N:P ratio, it is commonly admitted that phytoplankton and heterotrophic bacteria in the western basin are limited by phosphate (PO 4 3-) during the stratification period (e.g., [START_REF] Thingstad | P limitation of heterotrophic bacteria and phytoplankton in the northwest Mediterranean[END_REF][START_REF] Migon | Phosphorus in rainwater: Partitioning, inputs and impact on the surface coastal ocean[END_REF][START_REF] Marty | Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the wester Mediterranean Sea at the DYFAMED time-series station (1991-1999)[END_REF], while phytoplankton growth in the euphotic layer is limited by N during the spring bloom period (e.g. [START_REF] Béthoux | Phosphorus and nitrogen behavior in the Mediterranean Sea[END_REF][START_REF] Marty | Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the wester Mediterranean Sea at the DYFAMED time-series station (1991-1999)[END_REF][START_REF] De Madron | Marine ecosystems' responses to climatic and anthropogenic forcings in the Mediterranean[END_REF].

From this point of view, the northwestern Mediterranean area plays a crucial role in the functioning of the whole basin. In winter, submitted to a strong physical forcing (due to the action of cold and dry winds, combined with low temperatures) surface waters are cooled and their evaporation is enhanced, increasing their salinity. This regularly initiates the process of convection, which conducts, sometimes, to dense water formation events [START_REF] Marty | Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the wester Mediterranean Sea at the DYFAMED time-series station (1991-1999)[END_REF][START_REF] Millot | Circulation in the Mediterranean Sea[END_REF][START_REF] Marty | Hydrological changes in the Ligurian Sea (NW Mediterranean DYFAMED site) during 1995-2007 and biologeochimical consequences[END_REF]. Strong MLD events drive the thermohaline circulation of the whole northwestern Mediterranean (MEDOC Group 1970), and also contribute to re-distribute nutrients within the water column. The role of intense convection events (and MLD in general) on the Mediterranean nutrient distribution is still debated. Modeling exercises [START_REF] Crise | A seasonal three-dimensional study of the nitrogen cycle in the mediterraneansea : Part i. model implementation and numerical results[END_REF][START_REF] Crise | The Mediterranean pelagic ecosystem response to physical forcing[END_REF]) identify deep convection events as the primary factor in determining the seasonal nutrient availability in the surface layer. Recently, autonomous platforms smoothed this statement by observing a nitrate increase two months before the deep convection event [START_REF] D'ortenzio | Observinf mixed layer depth, nitrate and chlorophyll concentrations in the northwestern Mediterranean: A combined satellite and NO 3 profilling floats experiment[END_REF]. Only based on a one-year sampling, the results obtained from autonomous platforms confirmed, however, that only a seasonal monitoring may shed light on the interactions between MLD and nutrient distribution in a complex area such as the northwestern Mediterranean.

However, despite several studies already carried out in the Mediterranean Sea (e.g. [START_REF] Béthoux | Nutrients in the Mediterranean Sea, mass balance and statistical analysis of concentrations with respect to environmental change[END_REF][START_REF] Kress | Spatial and seasonal evolution of dissolved oxygen and nutrients in the Southern Levantine Basin (Eastern Mediterranean Sea): Chemical characterization of the water masses and inferences on the N:P ratios[END_REF][START_REF] Marty | Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the wester Mediterranean Sea at the DYFAMED time-series station (1991-1999)[END_REF][START_REF] Ribera D'alcalà | Nutrient ratios and fluxes hint at overlooked processes in the Mediterranean Sea[END_REF][START_REF] Krom | Nutrient budget for the Eastern Mediterranean: implications for P limitation[END_REF][START_REF] De Madron | Marine ecosystems' responses to climatic and anthropogenic forcings in the Mediterranean[END_REF], seasonal and inter-annual dynamics of nutrient concentrations remain poorly known. In this context, measurements from the DYFAMED time-series sampling station (located in the northwestern Mediterranean area) constitute an invaluable data set to study evolving Mediterranean biogeochemistry. The present paper firstly proposes to address the climatological pattern of the seasonal cycle of nutrients and molar ratios. Our results were directly compared to the pioneer study of [START_REF] Marty | Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the wester Mediterranean Sea at the DYFAMED time-series station (1991-1999)[END_REF], who worked on part of the same time-series: we analysed more deeply the link between nutrient seasonal variability and abiotic forcing. In addition, the seasonal shift from Nlimitation during winter and spring to P-limitation during the oligotrophic period, observed by [START_REF] Marty | Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the wester Mediterranean Sea at the DYFAMED time-series station (1991-1999)[END_REF] was re-examined. In the second part of the paper, we also analysed longer time scales, by exploiting the 21-year data collected at DYFAMED. It is likely that rapid environmental, climatic and meteorological changes around the Mediterranean basin are capable of altering the stocks and the molar ratio of nutrients at decadal time scales [START_REF] Turley | The changing Mediterranean Sea -a sensitive ecosystem[END_REF][START_REF] Marty | Hydrological changes in the Ligurian Sea (NW Mediterranean DYFAMED site) during 1995-2007 and biologeochimical consequences[END_REF]. Due to relatively reduced dimensions of the basin (2.51 x 10 6 km 2 ) and very short residence time of Mediterranean waters (50-100 years; [START_REF] Millot | Circulation in the Mediterranean Sea[END_REF], compared to those of the global ocean (500-1000 years), the Mediterranean area could be then considered as a "sentinel" to identify the effect of climate change on ocean biogeochemistry [START_REF] Siokou-Frangou | Plankton in the open Mediterranean Sea: a review[END_REF]. In this paper, we addressed more specifically, and for the first time in the western Mediterranean, the decadal evolution of nutrient concentrations, in relation to environmental variables (MLD, external inputs etc.).

Experimental

Site description

The DYFAMED site is located in the central Ligurian Sea (28 nautical miles off the continental French coast, 2350m deep, 43°25'N, 7°52E; Fig. 1). Cruises were conducted every month, apart from impossibility due to bad meteorological conditions or ship maintenance. CTD casts were obtained on each cruise [START_REF] Marty | Hydrological changes in the Ligurian Sea (NW Mediterranean DYFAMED site) during 1995-2007 and biologeochimical consequences[END_REF]. Additional data were collected in 2004 during nine MELISSA (MEditerranée LImitationS SAisonnières) cruises, in January and February, and from April to October. The DYFAMED station is surrounded by the permanent geostrophic Ligurian frontal jet flow that results from the cyclonic circulation of the Northern Current [START_REF] Millot | Circulation in the Western Mediterranean Sea[END_REF]. This front creates a band (~ 30 km width, more than 250m depth) that presumably separates the sampling area from coastal lateral inputs by a strong horizontal density gradient [START_REF] Niewadomska | Submesoscale physicalbiogeochemical coupling across the Ligurian current (northwestern Mediterranean) using a bio-optical glider[END_REF]. The Northern Current also isolates the sampling area from direct riverine inputs, such inputs being in addition particularly poor in the Ligurian Sea [START_REF] Migon | Riverine and atmospheric inputs of heavy metals to the Ligurian Sea[END_REF]. Episodic, infrequent intrusions of waters from the Northern Current may occur in winter [START_REF] Millot | Circulation in the Western Mediterranean Sea[END_REF]. However, the site is believed minimally affected by lateral inputs. Acoustic Doppler Current Profiler (ADCP) measurements have shown that the DYFAMED station is sheltered from the Northern Current [START_REF] Andersen | One-month study in the open NW Mediterranean Sea (DYNAPROC experiment, May 1995): overview of the hydrobiogeochemical structures and effects of wind events[END_REF]. Three major water masses are evidenced in the area [START_REF] Béthoux | Nutrients in the Mediterranean Sea, mass balance and statistical analysis of concentrations with respect to environmental change[END_REF][START_REF] Millot | Circulation in the Western Mediterranean Sea[END_REF][START_REF] Marty | Hydrological changes in the Ligurian Sea (NW Mediterranean DYFAMED site) during 1995-2007 and biologeochimical consequences[END_REF]: the surface water (~ 0-200m-depth), strongly affected by local climatic conditions [START_REF] Schröder | Deep and intermediate water in the Western Mediterranean under the influence of the Eastern Mediterranean Transient[END_REF], the subjacent layer (approximately between 200m and 600m depth at the DYFAMED site) characterised by maximum temperature and salinity due to the intrusion of Levantine Intermediate Water [START_REF] Millot | Circulation in the Western Mediterranean Sea[END_REF][START_REF] Marty | Hydrological changes in the Ligurian Sea (NW Mediterranean DYFAMED site) during 1995-2007 and biologeochimical consequences[END_REF], and the deep layer characterised by low salinity, and low temperature.

Mixed layer data

Temperature and salinity profiles from 1991 to 2011 were post-calibrated (using the standard Sea-Bird Electronics method) and all spikes and questionable data were removed from the data set. The potential density was calculated using the Thermodynamic Equation Of Seawater -2010 (TEOS-10).

The intensity of mixing was assessed through the variation of the MLD. Here, the MLD was calculated as the depth where the difference of density from the surface reference, fixed at 10m-depth, is 0.03 kg m -3 (de Boyer [START_REF] De Boyer Montegut | Mixed layer depth over the global ocean: An examination of pro-file data and a profile-based climatology[END_REF][START_REF] D'ortenzio | Seasonal variability of the mixed layer depth in the Mediterranean Sea as derived from in situ profiles[END_REF]. Density profiles were calculated from temperature and salinity profiles and linearly interpolated at 1m-depth interval. If no observation was available at 10m, the shallowest measurement was used as the surface reference if it was above 20m, otherwise MLD was not computed.

Nutrient analysis

From 1991 to 2011, time-series nutrient concentrations have been measured at the DYFAMED site. Nutrient samples were collected using 12 clean 12L Niskin bottles (General Oceanics 1010X) mounted on a carrousel water sampler (SeaBird SBE32) with CTD sensors (Seabird SBE911). The bottles were generally closed at 12 depths (10, 30, 50 or Deep Chlorophyll Maximum, 80, 120, 200, 300, 500, 800, 1000, 1500 and 2000m), and samples were transferred to 60mL polyethylene flasks. These samples were poisoned with 100µL HgCl 2 (i.e., 20µg L -1 ; Kirkwood 1992), and stored in a dark room at 5°C until analysis (until 2009), or placed directly to a freezer (from 2009 to 2011). After [START_REF] Dore | Freezing as a method of sample conservation for the analysis of dissolved inorganic nutrients in seawater[END_REF], low inorganic nutrient concentrations (basically oligotrophic seawater) can be adequately maintained for several months if samples are frozen immediately after collection. Only low concentrations of phosphate may be affected by the freezing process. However, the consequence is to increase variability, but not to bias the result in one direction or the other.

Furthermore, the effect on phosphate concentration is small, only detectable using sensitive low-level techniques, and would be included in the normal analytical variability of standard techniques [START_REF] Dore | Freezing as a method of sample conservation for the analysis of dissolved inorganic nutrients in seawater[END_REF]. The same applies to nitrate concentrations. Samples with silicate concentrations >120 µM may also be biased by freezing, due to silicate polymerisation, but this does not apply to the concentration range of the present data set.

All nutrient samples were analysed by standard automated colourimetry system, using a Technicon AutoAnalyser II (from 1991 to 2008) and since 2009 with a Seal Analytical continuous flow AutoAnalyser III (AA3) at the Observatoire Oceanologique de Villefranchesur-Mer. Nitrate (NO 3 -) ions were analysed according to the protocol of [START_REF] Bendschneider | A new spectrophotometric method for the determination of nitrite in sea water[END_REF] with a detection limit of 0.05µM and 0.01µM (since 2009). Phosphate (PO 4 3-) ions were analysed according to the protocol of [START_REF] Murphy | A modified single solution method for determination of phosphate in natural waters[END_REF] with a detection limit of 0.02µM. Silicate (Si(OH) 4 ) ions were analysed according to the protocol of [START_REF] Strickland | A practical handbook of seawater analysis[END_REF] with a detection limit of 0.05µM and 0.02µM (since 2009). For the present work, the total number of raw data was 3579 (nitrate), 3290 (phosphate), and 3699 (silicate).

Quality control of nutrient data

Owing to the strong scattering of results, caused by various types of possible errors (uncertainty on measurements, approximations, change of operator, etc.), nutrient data underwent a quality control. As a first step, data were pooled per month to avoid biases due to seasonal variations. Then, data were pooled by depth class (Table 1), in order to get homogeneous data within each class (surface water was typically better sampled than deeper water). For each depth and each nutrient, a central trend and a scattering index were computed. The median has been selected as the central trend, because it is statistically more robust and little influenced by outliers compared with, e.g. the mean, and the median absolute deviation (MAD) was used to measure the scattering of data. For a given nutrient, and for each depth, MAD is computed as follows:

(1) where is the median, and the ensemble of measured concentrations within a given depth class. To render MAD a consistent estimator for the estimation of the standard deviation ( ), a scale factor b is defined as:

(2)

b is determined from the quantile function of the , and is usually taken to be 1.4826 for normally distributed data. From the above, 1.4826.MAD is approximately equal to the standard deviation. Then, for each nutrient, a median profile and confident intervals have been determined. Measurements are considered as wrong if

(3)

Measurements are considered as questionable if (4)

Measurements are considered as good if

(5)

After the quality control processing and considering only data above detection limit, 87%, 86%, and 90% of the data for nitrate, phosphate, and silicate, respectively, were considered as good data. It should also be mentioned that values available once the quality control process is applied are not necessarily correct, they are just considered as non-aberrant. This procedure may delete some low-frequency extreme events, but it result in a limited effect on the study of seasonal processes and decadal trends. In the following, only data that fulfill equation ( 5

Results and Discussion

MLD dynamics

At climatological scale, MLD exhibits the typical pattern of temperate seas (Fig. 3a). In autumn and winter, deepening of the MLD is observed, with maximum median values around 70m-depth in January and February. This period is characterised by strong scattering of MLD values (Fig. 3a), and MLD deeper than 100m-depth are often observed. In spring, there is a gradual increase of surface density, leading to a progressive stratification of the water column. than 300m (Fig. 4a). In particular, the MLD was very intense in winter 2006, with values greater than 2000m, which was observed for the first time at DYFAMED, and evidenced using modelling approach [START_REF] Heimbürger | Temporal variability of vertical export flux at the DYFAMED time-series station (Northwestern Mediterranean Sea)[END_REF]. Conversely, from 2006 to 2011, no significant events were measured at the DYFAMED station. Over this period, the MLD never exceeded 200m-depth.

The analysis of MLD time-series is particularly problematic when winter events are analysed.

Median values of MLD deduced from monthly cruises are underestimated because mixing events can be shorter than the temporal interval of the measurements. Moreover, cruise sampling are cancelled when extreme weather conditions occur (i.e., during convection). In the following, we will discuss the possible impact of these results, by comparing them with nutrient data. Despite this limitation, MLD data at DYFAMED provide enough information to improve our comprehension of nutrient dynamics, which will be described in the next section.

Seasonal variability of nutrient concentrations

Composite years built from 1991-2011 data for each nutrient (Fig. 3b-d) exhibit clear seasonal patterns in relation to physical processes (Fig 3a). Contrasted seasonal situations may be outlined in the surface layer, where maximum variability is generally observed [START_REF] Béthoux | Deep-water in the Western Mediterranean Sea, yearly climatic signature and enigmatic spreading[END_REF], in relation to nutrient dynamics. From December to February, when maximum values of MLD are observed, nutrients accumulated in depths are brought to the surface layer, and increasing concentrations along the first one hundred metres are observed. Median concentrations range around 2.70 µM (nitrate), 0.13 µM (phosphate), and 2.0 µM (silicate) (Fig. 3 and Table 2). In spring, concomitantly with a progressive stratification of the water column, one can observe a decrease in nutrient concentration in the surface layer. Median values of nitrate concentrations decrease from 2.70 µM in February to 0.28 µM in May (from 0.14 µM to 0.04 µM for phosphate). This decrease is less apparent for silicate (Fig. 3 and Table 2). Summer period typically starts in June-July. During this period, the water column is stratified (MLD is minimal), and depleted surface waters are isolated from underlying layers [START_REF] Marty | Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the wester Mediterranean Sea at the DYFAMED time-series station (1991-1999)[END_REF][START_REF] D'ortenzio | Life in the Mediterranean Sea: A look at habitat changes[END_REF]. Nutrient concentrations are always <1 µM along the 0-50m-depth water column, and close to (or under) the detection limit in very oligotrophic situations (Fig. 3 and Table 2). The thickness of the depleted surface layer progressively increases. During autumn, the MLD is, on average, still shallower than the depleted layer, and not yet important enough to allow the homogenisation of the water column (Fig. 3a). Depleted layers are roughly at their deepest level, reaching 70m-depth for nitrate concentrations, and 90m-depth for phosphate concentrations (Fig. 3). During this period, wind episodes may partly break the stratification of the water column and, therefore, allow the injection of nutrients in surface waters (DiTullio and Laws 1991; [START_REF] Andersen | One-month study in the open NW Mediterranean Sea (DYNAPROC experiment, May 1995): overview of the hydrobiogeochemical structures and effects of wind events[END_REF].

However, no nutrient concentration increase was observed in surface from the present data set. This is presumably due to the monthly frequency of the sampling, which does not allow to observe short scale variations before November, when destratification is actually observed, on average (Fig. 3). Nutrients are re-injected in surface water once the MLD exceeds the depth of the depleted layer, which occurs typically in winter (Fig. 3 and The relatively small variations observed among concentration profiles are more likely due to natural fluctuations (inter-annual variability, change in water mass, etc.), and hide the potential impact of seasonality on deep nutrient concentration. In fact, assuming a shift from an extreme summer condition (surface water depleted from 0 to 100m-depth) to a theoretically rectilinear profile in winter, the expected change in deep water would be around 0.3 µM for nitrate concentrations, which is generally below the standard deviation associated with intrinsic variability due to physical processes and the whole procedure to determine nutrient concentrations (Table 2). In addition, the readjustment of deep nutrient concentrations following these events might be so rapid that it is not possible to detect concentration variations through monthly sampling. A two-way ANOVA confirms that seasonality has no significant effect on nutrient concentrations in intermediate and deep water in the present data set (see Annex).

The MLD cycle largely constrains nutrient concentrations in surface layer but a direct relationship between MLD magnitude and nutrient concentrations may be complex and not linear (Fig. 4b-d and related to the cyclonic circulation of the northwestern Mediterranean that uplifts the nutrient deep reservoir [START_REF] Crise | The Mediterranean pelagic ecosystem response to physical forcing[END_REF]. However, this statement should be moderated, due to the monthly sampling. Indeed, observed nutrient concentrations in surface result from the past weeks of MLD dynamics and do not directly emerge from the MLD measured at the time of the sampling. The duration of winter mixing events should be taken into account, but this point cannot be here elucidated any further.

Seasonal variability of molar ratios

To avoid errors in the computation of molar ratios (calculated as the quotient of two elemental concentrations), essentially when nutrient concentrations are close to the analytical precision, the theoretical relative error was calculated from the partial derivative method:

(1)

where is the ratio between the two elements and , and (resp.

) the uncertainty on the measurement of (resp. ). Then, is the relative error on the ratio. Ratios with an error greater than 30% were therefore discarded. For example, results for N:P ratio showed that, in surface (< 50m-depth), the mean relative error value is around 75%, and can reach more than 130% in summer, when NO 3 -and PO 4 3-concentrations are low (assuming an uncertainty on nitrate and phosphate measurement in the order of 0.02 µM; Aminot and Kerouel 2007). In comparison, the maximum relative error under 200m-depth never exceeds 10%. This results in two types of bias: scattering of the data and convergence towards low values in surface (Fig. 5), those values being generally considered not reliable (e.g. [START_REF] Krom | Phosphorus limitation of primary productivity in the eastern Mediterranean Sea[END_REF][START_REF] Marty | Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the wester Mediterranean Sea at the DYFAMED time-series station (1991-1999)[END_REF]. The number of N:P ratio values considered as not reliable (error greater than 30%) represents 10.5% of the whole data set, and 75% of these questionable values lie above 50m-depth.

The DYFAMED data set was splitted into four calendar-based seasons: Winter (December-February), spring (March-May), summer (June-August), and autumn (September-November).

Seasonal median profiles were obtained by calculating median values into 17 depth classes (Table 1). All year long, ratios are constant in deep and intermediate layers, and the scattering of the data is low. Median values are around 20:1 for the N:P ratio, slightly below 1 for the Si:N ratio, and around 19 for the Si:P ratio (Fig. 6). As for nutrient concentrations, seasonal changes are detected in the surface water. In winter, when the process of vertical mixing occurs, the N:P ratio exhibits a quasi-rectilinear median profile, from the bottom to the surface. The relative abundances of nutrients in surface water mirror the nitrate-to-phosphate ratio in the nutritive material stored in depths. Strong variability is observed in the surface water (down to 200m-depth), with N:P values equivalently distributed around the median. It results in a T-shaped envelope of points, centred on 20:1 (Fig. 6a). In spring, the N:P ratio remains constant, as for winter situation. In summer, as soon as the stratification of the water column develops, the N:P ratio in surface evolves towards higher values (median value around 30, with possible maxima up to 70). Autumn may be viewed as the continuation of the summer period. During this period, the water column is still stratified, and N:P ratio values are maxima (Fig. 6a). [START_REF] Marty | Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the wester Mediterranean Sea at the DYFAMED time-series station (1991-1999)[END_REF] obtained similar results with respect to N:P ratio and inferred there is a Nlimitation during winter and spring (because all the N:P ratio values below 20 were observed during this period). We recommend caution when addressing the complex issue of the limiting factor since it depends on both phytoplankton physiology and resource competition [START_REF] Klausmeier | Optimal nitrogen-tophosphorus stoichiometry of phytoplankton[END_REF]). First, in winter, both nitrate and phosphate are abundant in the surface layer (Fig. 3b-d), suggesting that nutrients are probably not the limiting factor. Then, the T-shaped envelope of points observed in both winter and spring (Fig. 6a) is characteristic of ideal (i.e., non-fractioned) situation of nutrient co-variation (see figure 1 in [START_REF] Fanning | Nutrient provinces in the sea: concentrations, ratios, reaction rate ratios, and ideal covariation[END_REF].

This suggests that the net removal of nitrate and phosphate by biota occurs in proportions close to the molar ratio in the stock (around 20:1), with alternation between periods of Ndepletion, relative to P (right portion of the T) and periods of P-depletion, relative to N (left portion of the T). In any case, this suggests that N and P, overall, may play a similar role in the control of phytoplankton growth in blooming conditions. Therefore, N does not appear clearly as the limiting factor in winter and spring. By contrast, high values of N:P ratio observed in summer and spring suggest an incomplete removal of nitrate once all phosphate is consumed by phytoplankton (typical of P-limited oligotrophic configurations). With respect to Si:N and Si:P ratios, we observe increasing values from spring to autumn because surface water becomes strongly depleted in nitrate and phosphate compared to silicate (Fig. 3b-d). It is noteworthy that profiles of N:Si ratio and, more markedly, profiles of Si:P ratio display a permanent curvature in the intermediate layer. Indeed, silicate concentration profiles exhibit a different shape, compared with those of nitrate and phosphate: Si(OH) 4 concentrations increase progressively from surface to depth, whereas nitrate and phosphate concentration profiles are generally characterised by a strong gradient between the depleted layer and the enriched deep layer. The behaviour of Si is different from that of other nutrients insofar as its recycling is slow, because it essentially depends on the dissolution of diatom frustules [START_REF] Kamatani | Dissolution rates of silica from diatoms decomposing at various temperatures[END_REF][START_REF] Tréguer | Kinetics of dissolution of Antarctic diatom frustules and the biogeochemical cycle of silicon in the Southern Ocean[END_REF]). Even if our results suggest that, basically, Si does not play any role in the limitation of primary production, the profiles recorded on Fig. 6b and6c do not permit to discard the potential episodic control of diatom production by Si during the early bloom period, as suggested by [START_REF] Marty | Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the wester Mediterranean Sea at the DYFAMED time-series station (1991-1999)[END_REF].

Decadal trends in nutrient concentrations in deep layer

Decadal variability of nutrient concentrations and molar ratio was studied by considering only samples acquired at depth (below 800m), where the variability is the smallest [START_REF] Béthoux | Deep-water in the Western Mediterranean Sea, yearly climatic signature and enigmatic spreading[END_REF]. Trend analysis was conducted using the non-parametric Mann-Kendall test [START_REF] Mann | Nonparametric tests against trend[END_REF][START_REF] Kendall | Rank Correlation Measures[END_REF], which determines whether a time-series exhibits a monotonic pattern. To support the findings, regression analysis was also performed. According to both of these tests, the null hypothesis assumes that there is no trend, and this is tested against the alternative hypothesis that assumes that there is a trend. The non-parametric Pettitt test [START_REF] Pettitt | A non-parametric approach to the change-point problem[END_REF]) was applied to the de-trended data [START_REF] Vincent | Comments on 'Observed trends in indices of daily temperature extremes in Soiuth America 1960-2000[END_REF][START_REF] Bates | Trend estimation and change point detection in individual climatic series using flexible regression methods[END_REF]) to find a significant break in the data series. The null hypothesis of stationarity was tested against a shift in the average value. This test provides the indication of when a potential break point occurs. All tests were run at 5% significance level.

Over the 1991-2011 period, increase of nitrate concentration, and decrease of phosphate concentration were observed in deep waters (Fig. 7), and were statistically significant (pvalues <0.05; Table 3). The slopes of the regression line show an increase of 0.23% and a decrease of 0.62% per year on average for nitrate and phosphate concentrations, respectively.

By contrast, silicate concentrations do not exhibit any trend over the considered period (pvalue >0.05). The Pettitt test also shows that abrupt changes were detected in 2005-2006 in all time-series (i.e., nitrate, phosphate and silicate), characterised by a slight but significant increase in nutrient concentration (Fig. 7 and Table 3). A similar analysis was achieved for molar ratios (Fig. 7 and Table 3). According to the trends displayed for nutrient concentrations, we observed a significant increase by 1.14% per year for N:P deep molar ratio, which represents an increase of 4.2 in the value of N:P ratio over the 20-year period. In the same way, the increase of Si:P molar ratios (0.85%) is mainly due to the decrease of phosphate concentration. Si:N ratio does not show any trend, even if a slight increase was (1) The statistical analysis realised by [START_REF] Béthoux | Nutrients in the Mediterranean Sea, mass balance and statistical analysis of concentrations with respect to environmental change[END_REF] may not be directly comparable to our work. Historical data were acquired upon low frequency sampling and the spatial variability was not taken into account (e.g. gathering data from Ligurian and Alboran Seas).

(2) Changes in external sources (atmospheric and riverine inputs) may have occurred.

Particularly, an increase in nitrate flux in rivers was observed from the 1970s to the 1990s, before remaining stable in recent years [START_REF] Ludwig | River discharges of water and nutrients to the Mediterranean and Black Sea: Major drivers for ecosystem changes during past and future decades? Progr Oceanogr 80:199-217 trace the climatology of water masses and to validate incoming data from operational oceanography[END_REF]. Similarly, phosphate loads increased in the 1970s (two times the nitrate increase), but a strong decrease was observed in the 1990s thanks to the ban on phosphate in detergents, and/or the upgrade of waste water treatment plants [START_REF] Ludwig | River discharges of water and nutrients to the Mediterranean and Black Sea: Major drivers for ecosystem changes during past and future decades? Progr Oceanogr 80:199-217 trace the climatology of water masses and to validate incoming data from operational oceanography[END_REF]. Results observed at the DYFAMED time-series station might mirror this trend. Regarding changes in non-riverine emission sources (i.e., mainly atmospheric deposition), we have not enough post-1990s data at present time on the regional atmospheric deposition to conclude.

(3) Changes in water mass may have occurred. An increase in the frequency and the intensity of winter convection events was recorded from 2003 to 2006 (Fig. 4), and was attributed to a significant warming and salinification of the water column [START_REF] Millot | Large warming and salinification of the Mediterranean outflow due to changes in its composition[END_REF][START_REF] Schröder | Deep and intermediate water in the Western Mediterranean under the influence of the Eastern Mediterranean Transient[END_REF][START_REF] Skliris | Hydrological changes in the Mediterranean Sea in relation to changes in the freshwater budget: a numerical modelling study[END_REF], which supposedly permitted to trigger the diving process with the smallest heat loss [START_REF] Leaman | Hydrographic structure of the convection regime in the Gulf of Lions: winter 1987[END_REF]Schröder et al. 2010b). At the DYFAMED site, a regular increase of temperature and salinity was recorded in deep water (+0.005°C yr -1 and +0.0022 psu yr -1 during the 1995-2005 period), and in intermediate water [START_REF] Schröder | Deep and intermediate water in the Western Mediterranean under the influence of the Eastern Mediterranean Transient[END_REF][START_REF] Marty | Hydrological changes in the Ligurian Sea (NW Mediterranean DYFAMED site) during 1995-2007 and biologeochimical consequences[END_REF]. The propagation of the Eastern Mediterranean Transient has been suggested as a possible explanation for the increase in temperature and salinity observed in intermediate waters since this event strongly affected the water flux at the Sicilian strait with an enhanced salt export from the eastern Mediterranean Sea to the western Mediterranean Sea [START_REF] Klein | The large deep transient in the Eastern Mediterranean[END_REF]. A cumulated deficit in fresh water budget over years 2003/2004/2005, and a concomitant increase in surface salinity were also observed [START_REF] Stott | Human contribution to the European heatwawe of 2003[END_REF][START_REF] Marty | Hydrological changes in the Ligurian Sea (NW Mediterranean DYFAMED site) during 1995-2007 and biologeochimical consequences[END_REF]. This peculiar situation led to the intense winter convection observed in 2006 (Fig. 4). New warmer and saltier western Mediterranean deep water was formed [START_REF] Smith | Observations of new western Mediterranean deep water formation using ARGO floats 2004-2006[END_REF] as already observed in winter 2004-2005 in the Gulf of Lions and in the Catalan basin [START_REF] González-Pola | Observation of an abrupt disruption of the long-term warming trend at the Balearic Sea, western Mediterranean Sea, in summer 2005[END_REF][START_REF] Font | Sequence of hydrographic changes in NW Mediterranean deep water due to the exceptional winter of 2005[END_REF][START_REF] Schröder | Deep and intermediate water in the Western Mediterranean under the influence of the Eastern Mediterranean Transient[END_REF]Schröder et al. 2010b). This yielded the uplift of the old western Mediterranean deep water by several hundreds of metres [START_REF] Schröder | An extensive western Mediteranean deep water renewal between 2004 and 2006[END_REF][START_REF] Schröder | Long-term monitoring programme of the hydrological variability in the Mediterranean Sea: a first overview of the HYDROCHANGES Network[END_REF], which usually occurs roughly below 2000m-depth [START_REF] Van Haren | Rectilinear and circular inertial motions in the Western Mediteranean Sea[END_REF]). Thus, an increase in nutrient concentration over the 800m to 2000m-depth range that indicates an increase in water age [START_REF] Kress | Temporal evolution of physical and chemical characteristics of the water column in the Easternmost Levantine basin (Eastern Mediterranean Sea) from 2002 to 2010[END_REF]Schröder et al. 2010a), could be the consequence of the presence of the

Conclusions

For the first time, a quality-controlled 20-year data set of nutrient concentrations in the northwestern Mediterranean was presented. Such a rich data set, almost unique for the open Mediterranean Sea, was analysed from different perspectives. In this work, we discussed the variability of nutrient dynamics by evoking the seasonal patterns of the MLD, and we estimated the possible temporal evolutions of nutrient stocks. Phosphate controls primary production in conditions of stratification (summer and autumn oligotrophy), but our results also suggest that PO 4 3-and NO 3 -play a similar role in blooming conditions. This contradicts the reigning paradigm of limitation driven by N in such conditions (e.g. [START_REF] Marty | Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the wester Mediterranean Sea at the DYFAMED time-series station (1991-1999)[END_REF].

Aknowledgements

The authors acknowledge a grant provided by the ACRI-ST company and the French National Association for Research and Technology (ANRT). This work was also supported by the French MOOSE network (funded by ALLENVI, INSU, EuroSites). The authors are also grateful to the French "Equipement d'avenir" NAOS project (ANR J11R107-F). The authors also thank the three anonymous reviewers who helped considerably in improving this manuscript.

  ) are taken into account. Distribution of sample number per year and per month is given in Fig.2.Years 1991 and 2004 were well sampled due to the monthly double casts performed at the DYFAMED site(1991) and to additional data from the MELISSA project (2004). In 2009-2011, nutrient samples were stored in a freezer instead of using poison (HgCl 2 ), and analysed at the Marseilles Institute of Oceanography. This change in sample preservation could explain the lower quality of analyses, hence the lower number of data from this period, once the quality control was performed. At monthly scale, the sampling was fairly regular, August being the least regularly sampled (annual maintenance of the ship). Raw DYFAMED and MELISSA nutrient concentration data, and the associated controlled values, can be found at ftp://oceane.obs-vlfr.fr/pub/laurcopp/

  Consequently, the MLD becomes thinner and rarely exceeds 60m (median values around 30m in March, falling around 10m in May). During summer, MLD values and scattering are minimal, indicating low inter-annual variability. Our data confirm the climatological patterns already described in the past (Marty and Chiaverini 2010; Heimbürger et al. 2013). MLD time-series (Fig. 4a) also confirm a strong variability in winter, at inter-annual scale. In particular, years 1999, 2000, 2005 and 2006 were characterised by strong MLD, i.e., deeper

  ). It is clear that deep MLD, generally observed in winter and early spring, induces high nutrient concentrations (Box 3 on Fig.4b-d). On the contrary, shallow MLD are associated with very low nutrient concentrations (Box 1, summer situation). Intermediate MLD (ranging from 20 to 100m-depth; Box 2) are observed in autumn, spring and winter, and have different effects depending on the season. In autumn, whatever the depth reached by the MLD, nutrient concentrations remain low (MLD shallower than the depleted layer). However, it is striking that high nutrient concentrations can be observed in winter and spring even though MLD values are not exceptionally great. In other words, deep convection, which is often believed to trigger nutrient supply to the surface layer, is not necessarily needed to observe high nutrient concentrations. Such a situation (shallow MLD and high nutrients concentrations) was already observed in the past[START_REF] D'ortenzio | Observinf mixed layer depth, nitrate and chlorophyll concentrations in the northwestern Mediterranean: A combined satellite and NO 3 profilling floats experiment[END_REF] Manca et al. 2004),

  observed in 2005-2006. No tipping points were detected at significant level (p <0.05) in N:P and N:Si time-series, probably because all nutrient concentrations sharply increased in the same time, i.e., around 2005-2006. Compared with the historical study performed by Béthoux et al. (1998) (nitrate and phosphate increased by 0.56 and 0.53% per year, respectively, over the 1961-1994 period, while silicate concentrations remained stable), the present study shows a slowing down of nitrate concentration increase by a factor of two, and a reversal of the trend for phosphate concentration. Several reasons may account for the differences in nitrate and phosphate trends.

  new deep water. The abrupt change occurring in 2005-2006 (linked to the deep mixing event) is believed to strongly contribute to the increase of deep nitrate concentration over the 1991-2011 period. As well, the decrease in phosphate concentration over the 1991-2011 was probably reduced by this event. The increase in silicate concentration occurring in 2005-2006 resulting from the same event might counterbalance a gradual decrease over the 1991-2011 period and, therefore, hide any trend.

  Over longer time scales, an important change characterised by an increase in nutrient concentration in the 800 to 2000m-depth layer occurred in[2005][2006], presumably driven by a deep water uplift. This increase is believed the main cause in the increasing trend observed for nitrate concentration during the whole period, even if the contribution of external sources cannot be excluded. The same increase is superimposed upon a regular decrease in phosphate concentration. As a result of the strong decrease observed in nitrate concentration, N:P and Si:P ratios significantly increase. The trends observed suggest that the role of P as limiting factor of primary production may be currently increasing, as already suggested[START_REF] D'ortenzio | On the trophic regimes of the Mediterranean Sea: a satellite analysis[END_REF][START_REF] De Madron | Marine ecosystems' responses to climatic and anthropogenic forcings in the Mediterranean[END_REF]. The perpetuation of projects such as DYFAMED time-series (as well as similar projects, as HOT or BATS) is certainly a priority for future oceanography. However, our results demonstrate that several processes are difficult to characterise with the available temporal resolution of data. Some statements of this paper should be then moderated by taking into account the bias introduced by monthly sampling. Indeed, for the case of processes that occur over a short duration (e.g. winter convection), such periods necessarily gather several sampling configurations that do not strictly correspond to the process in question. Increasing the temporal resolution of ship-based sampling is costly and logistically very difficult. However, even in optimal conditions, with unlimited ship-time and resources, meteorological conditions should prevent a long-term high-frequency sampling. The only realistic alternative is a combination of ship-based operations and autonomous sampling. The estimation of nitrate concentrations from moorings and profiling floats has been already achieved in the past[START_REF] Johnson | Diel nitrate cycles observed with in situ sensors predict monthly and annual new production[END_REF][START_REF] Johnson | Long-term nitrate measurements in the ocean using the In Situ Ultraviolet Spectrophotometer: sensor integration into the APEX profiling float[END_REF], strongly improving our capacity to detect and understand the chemical dynamics of the ocean at unexplored temporal scales. A nitrate sensor will be added to the DYFAMED mooring in the next months, while a network of floats equipped with nitrate (plus florescence, coloured dissolved organic matter (CDOM), optical backscattering coefficient (bb) and photosynthetically available radiation (PAR)) sensors is already operational in the northwestern Mediterranean (NAOS project). The combination of all the components of the future DYFAMED observing system (ship, moorings, profiling floats) will strongly enhance our capability to understand the nutrient dynamics (including chemical limitation) of the northwestern Mediterranean. It will also provide pertinent data to confirm the present results.

  Figure captions

Fig. 1 :Fig. 2 :

 12 Fig. 1: Location of the DYFAMED time-series sampling site. The black arrows figure the

Fig. 3 :

 3 Fig. 3: Monthly box plot of the mixed layer depth (MLD) at DYFAMED (a). The central red mark is the median, and the edges of the box are the 25 th and 75 th percentiles. Composite

Fig. 4 :

 4 Fig. 4: MLD time-series over the 1991-2011 period (a). Scatter plot of mean concentration in

Fig. 5 :Fig. 7 :

 57 Fig.5: Distribution of N:P ratios in surface (0-50m), considering ratios with high (> 30%) and

Fig. 1

 1 Fig. 1

Fig. 2

 2 Fig.2

Fig. 3

 3 Fig.3

Fig. 4

 4 Fig.4

Fig. 5

 5 Fig.5

Fig. 6

 6 Fig. 6

Fig. 7

 7 Fig.7

Table 2 )

 2 . This annual cycle is characteristic of the northwestern Mediterranean, despite possible interannual variations. A two-way ANOVA indicated a significant influence of seasonality on nitrate, phosphate and silicate surface concentrations (P-value <0.01), relative to inter-annual variability. Detailed results of the ANOVA are given in Annex. Below 200m-depth, no clear seasonal variability can be observed (Table2). Whatever the nutrient, intermediate and deep However, as the DYFAMED zone is an area where deep water formation occurs, one would also expect seasonal changes in deep water concentration.

water appear insignificantly affected by seasonal variations. Median values of nutrient concentrations in intermediate and deep water (Table 2) are relatively constant whatever the season (around 8 µM for nitrate, 0.3 µM for phosphate, and 7 µM for silicate), and characterised by low deviation.

Table captions Table 1 :

 captions1 Distribution of available data according to depth. Reference depths and associated intervals are given (in db) for each nutrient.

	Table 2: Monthly median nutrient concentrations integrated over the surface layer (0-50m),
	the intermediate layer 200-600m, and the deep layer (>800m). The deviation is given by
	equation (2).

Table 3 :

 3 Statistical analysis for nutrients and nutrient ratios. P-values less than 0.05 indicates a strong presumption against null hypothesis, meaning that result obtained from tests can be considered as significant.

	Table 1
		depth classes	Nitrate phosphate silicate
	reference [db] Interval [db]	number of sampling
	5	0-10	198	252	326
	20	10-30	239	260	342
	40	30-50	328	277	342
	60	50-70	221	195	213
	80	70-90	207	183	199
	100	90-110	196	179	188
	130	110-150	326	294	310
	175	150-200	232	211	217
	250	200-300	156	138	148
	350	300-400	152	136	148
	450	400-500	162	143	157
	550	500-600	143	125	139
	700	600-800	279	245	272
	900	800-1000	174	158	166
	1150	1000-1300	163	141	149
	1550	1300-1800	229	201	215
	2000	>1800	174	152	168
		Total sampling	3579	3290	3699


Maria Kitsara
email: maria.kitsara@imb-cnm.csic.es

Pierre Joanne

Emmanuelle Solène

Ibtihel Ben Boitard

Barbara Dhiab

Philippe Poinard

Christian Menasché

Patricia Gagnieu

Onnik Forest

Yong Agbulut

Chen

Solène Emmanuelle Boitard

Ibtihel Ben Dhiab

Barbara Poinard

Philippe Menasché

Christian Gagnieu

Patricia Forest

Onnik Agbulut

Yong Chen

Fabrication of cardiac patch by using electrospun collagen fibers

Keywords: electrospinning, collagen scaffold, biocompatibility, tissue engineering, cardiac cell therapy

Introduction

Engineered scaffolds have been widely used as structural and functional supports on which cells are seeded for the generation of cell therapy products. In the field of cardiac therapy, this approach is challenging but holds a real promise for improving function of the chronically failing myocardium [START_REF] Menasché | Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience[END_REF]. Previously, a large number of investigations have been devoted to the culture of cardiac cells using scaffolds made of synthetic polymers. In particular, nanofibrous scaffolds obtained by electrospinning of poly-L-lactic acid (PLLA) and poly-(caprolactone) (PCL) could be produced for the formation of functional cardiac cell layers [START_REF] Castellano | A comparison of electrospun polymers reveals poly(3-hydroxybutyrate) fiber as a superior scaffold for cardiac repair[END_REF]. However, because of the lack of cell affinity these synthetic polymers are inherent less attractive than natural polymers -such as collagen, fibrinogen, elastin -for in vivo applications [START_REF] Sell | Electrospinning of collagen/biopolymers for regenerative medicine and cardiovascular tissue engineering[END_REF]. Among them, collagen constitutes one of the main proteins of the extracellular matrix, and this allows for a close simulation of the natural fibrillar structure of cardiac tissue [START_REF] Corda | Extracellular matrix and growth factors during heart growth[END_REF]. Accordingly, it is interesting to consider electrospun collagen scaffolds as supports and carriers of cardiac cells that can be implanted for repair of the failing myocardium [START_REF] Matthews | Electrospinning of Collagen Nanofibers[END_REF].

Different types of clinically approved collagen are now available but the ability of making fibrous scaffolds out of them has yet to be demonstrated. One of the issues is the crosslinking process, which uses chemical reagents to render the fibrous structures stable in the culture medium and after implantation. The most common crosslinkers for collagen fibers are glutaraldeyde -well known for its efficiency but somehow toxic -and combinations of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS)a system which is less toxic but often causes a film-like morphology after the immersion of the fibers in water [6] [7]. Alternatively, a citric acid (CA) based system has been recently applied and shown the requested biocompatibility [START_REF] Jiang | Water-stable electrospun collagen fibers from a non-toxic solvent and crosslinking system[END_REF].

In this work, we study the electrospinning parameters of clinically approved collagen in order to produce optimal nanofiber scaffolds for three-dimensional culture of cardiac cells. In particular, we were interested in creating a patch form of scaffold that can be used for therapy purposes. Our results show that the colonization of cardiac cells is critically dependent on the crosslink parameters and that our collagen scaffolds are fully compatible with the implantation requirements, thereby providing a way towards a scaffold based cardiac cell therapy.

Materials and Methods

Collagen solutions preparation and electrospinning

Different types of clinically approved collagen, including atelocollagen, acid fibrous, basic fibrous were provided by Biom'Up (Saint-Priest, France) in form of dry material after lyophilization. For electrospinning, the raw collagen materials were dissolved using a solvent system containing ethanol, water and a variety of salts [START_REF] Dong | Electrospinning of collagen nanofiber scaffolds from benign solvents[END_REF]. Specifically, the buffer solution is composed of the following salts dissolved in deionized water: potassium chloride, sodium chloride, potassium phosphate monobasic, sodium phosphate dibasic heptahydrate. The salts concentration is 20x in the collagen buffer solution. The final solvent system consists of buffer solution and ethanol in a ratio 1:1 v/v with a collagen concentration of 16 % w/v.

For the crosslinking of the collagen scaffolds a system containing CA, glycerol as extending agent and sodium hypophosphite (SHP) as catalyst was used [START_REF] Jiang | Water-stable electrospun collagen fibers from a non-toxic solvent and crosslinking system[END_REF]. The three crosslinking components were added to the collagen solution before the electrospinning. Different concentrations of CA and SHP were studied: 5% CA with 2.1% SHP and 10% CA with 4.2% SHP, keeping constant the glycerol amount to 3% (weight percentage based on the weight of collagen).

In electrospinning, the solution is fed through a thin needle opposite to a collecting plate and a high voltage is applied to form a jet of the solution that travels from the needle to the grounded collecting plate (fig. 1). In our study, the collagen solution was loaded to a syringe with a 23 gauge blunt needle and the flow rate was controlled via a pump at 1ml/h. By applying voltage of 13.5 kV and keeping a distance of 5cm between the needle tip and the grounded collector, fibers were deposited to the latter at room temperature (20±2 o C). After 8 minutes of continuous electrospinning, collagen scaffolds were obtained that could be easily peeled off from the collector (fig. 2).

Post-electrospinning treatment

After electrospinning, a crosslinking post-treatment was applied in order to make the scaffolds insoluble in cell culture medium and facilitate their handling. The carboxyl groups of CA reacted with the additional hydroxyl groups introduced by glycerol in order to form covalent bridges between the collagen fibers. The scaffolds were baked at 150 o C for three different periods (1.5h, 2h, 2.5h).

The crosslinked scaffolds were mounted onto handing devices for biologic assays. Cell crowns® holders were utilised for this purpose and then placed in 24-well plates for cell culture.

Characterization methods

The structure of the electrospun collagen scaffolds was studied after the steps of electrospinning and crosslinking treatment using scanning electron microscopy (SEM) and contact angle measurements.

The SEM measurements were performed using the e-LiNE (Raith). A thin gold layer of 5nm was deposited prior to measurements and images at different magnifications were obtained on at least three different areas in the same sample. The measurements were performed under extra high tension of 10kV.

The contact angle measuring instrument was the Drop Shape Analyzer -DSA 30 (Krüss). The sessile drop method was utilized and the measurements took place at room temperature (20 o C). The volume of the applied droplets of deionized water was 1.5 μl. The contact angle data were obtained by averaging over five measurements in different areas on the sample surface.

Cardiomyoblasts culture

The H9c2 cell line derived from embryonic rat heart tissue was used in this study. Cells were cultured in high glucose DMEM+Glutamax supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin. They were seeded on the collagen scaffold after passage using trypsin-EDTA. After two days of culture, scaffolds were washed with PBS and fixed with 1% paraformaldehyde solution. After 2 washing steps, they were incubated with 5% bovine serum albumin (BSA) during 1 hour. Then Alexa Fluor® 488 Phalloidin (Life technologies) was used to stain the cells. After 3 washes in PBS, collagen scaffolds were mounted with mowiol. Images were captured using a motorized confocal laser scanning microscope (LSM 700, Zeiss).

Epicardial grafts

All procedures were approved by our institutional Ethics Committee and complied with the European legislation (Directive 2010/63/EU) on animal care. Conditionally-invalidated serum response factor (SRF) mice (MHC-MerCreMer:Sf/Sf) were used as recipients [START_REF] Parlakian | Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart[END_REF]. The Cre-mediated excision of floxed SRF alleles in the heart was induced by daily intraperitoneal tamoxifen (28 g/g/day; Sigma-Aldrich, France) injections on 3 consecutive days with the day of the first injection counted as day 1. Three weeks after tamoxifen administration, the animals underwent a left lateral thoracotomy after intraperitoneal ketamine (100 mg/kg; Merial, France)-xylazin (10 mg/kg; Bayer, Germany) anaesthesia and tracheal ventilation. Analgesia was performed for 2 days after surgery with a 10 mg/kg subcutaneous injection of ketoprofen (Merial). Acellular collagen scaffolds were sutured on the left ventricle. All animals were sacrificed at the end of experiment by cervical dislocation. Hearts were immediately removed, fixed in Tissue-Tek (Sakura, USA) and frozen in liquid nitrogen-cooled isopentane until they were sliced into 8 to 12-µm thick cryosections using a cryostat. The sections were stained with HE (hematoxylin and eosin) as described by Dubowitz [START_REF] Dubowitz | Muscle Biopsy: A Practical Approach[END_REF]. Images were taken with an inverted microscope (Leica DMIL) equipped with a digital camera (Qicam, Qimaging).

Results and Discussion

Characterization of crosslinked scaffolds

SEM

Amongst the different collagen types, atelocollagen derived from native collagen by enzymatic extraction exhibited the best performance both in terms of dissolution to the benign solvent system (within few hours at room temperature) and of electrospinning ability. The acid fibrous collagen required one week to be dissolved and the basic fibrous collagen was not compatible with the used solvent system resulting to a partial dissolution after 3 weeks. All the types of collagen were tested for their ability to create fiber mats by electrospinning. Acid and basic fibrous types ended up to nanofibers with knots and it was not able to create a thick enough mat that could be unpeeled from the collector. These types of nanofibers might be used for the surface modification of cell culture substrates e.g. by directly depositing the electrospun fibers on the latter, but cannot be used as implantable scaffolds which is our intention. On the other hand, atelocollagen proved to be able to create uniform continuous fibers without knots (Fig. 3) that can be easily unpeeled from the collector (Fig. 2). Fig. 3a and 3b show the atelocollagen mesh-structured scaffold and demonstrate, at a higher magnification, the uniformity of the fibers with a diameter of 1±0.3μm. After the post-electrospinning crosslinking treatment, the samples were characterized before and after their immersion into deionized water in order to verify the efficacy of the method. Neither the duration of baking nor the concentration of the cross-linkers did affect the fibers morphology before the immersion to water.

Conversely, the morphology of the samples after the immersion in water varied in relation with the concentrations of CA and SHP. At low concentrations, loss of porosity was observed as shown in fig. 4a, whereas the morphology was significantly improved for 10% CA and 4.2% SHP (fig. 4b,c). CA and (c) 10% CA at higher magnification.

Contact angle measurements

Collagen scaffolds before and after the crosslinking treatment were then characterized. Figure 5a shows the surface of a non-crosslinked scaffold, five seconds after the deposition of water droplet, which spread immediately all over the scaffold, causing scaffold dissolution. For the measurements of the crosslinked scaffolds with 10% CA, the latter were immersed in water and then placed in cell crowns® holders. The water contact angles obtained with dry samples showed that the surfaces did not dissolve, but are hydrophilic with a contact angle of 50 ° just five seconds after the droplet deposition (fig. 5b). The water contact angles decreased down to 30 ° just 60 seconds after the droplet deposition, which further indicates the porosity of the samples (fig. 5c).

Biological validation of the scaffolds

In Vitro Studies: Cellularisation of collagen scaffolds

The electrospun collagen scaffolds were successfully used for culture of H9c2 cells. Figure 6 shows a confocal microscopic image after immunostaining for actin and nuclei which demonstrates the colonization of the 10 % CA scaffold. Clearly, the cells were spread all over the scaffold and show a nice structure of their actin filaments (the blue cells are more deeply sunk into the collagen than the others). The cells seem to spread more extensively as the baking time was longer. It should be noted that, by increasing the baking time, the stiffness should be enhanced, which could be more desirable in our case. Moreover, comparing the different concentrations of CA, it is obvious that the higher baking time (2.5h) led to a greater extent of cell colonization. Preliminary experiments with human pluripotent stem cell-derived cardiac progenitors have also been performed, showing a promising efficacy of the fabricated collagen scaffold. The next step will be the implantation of the cell-seeded scaffold in mice with failing myocardium with the hope that the scaffold may provide an optimal microenvironment to the cells, thereby allowing them to improve heart function, either by integrating structurally within the recipient tissue or, more likely, by activating endogenous reparative pathways.

In Vivo Studies: implantation of acellular collagen scaffolds

In this study, we used a double transgenic mouse model which allows the inactivation of SRF only in cardiomyocytes as tamoxifen-inducible Cre recombinase expression is under the control of the cardiomyocyte-specific alpha myosin heavy chain promoter [START_REF] Parlakian | Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart[END_REF]. Five days after tamoxifen injection there is a drop of SRF mRNA and protein levels. Then, mice exhibit a progressive dilated cardiomyopathy, leading to a non-ischaemic heart failure and death within 10 weeks. This model allows a timed control of the functional deterioration, thereby allowing a better comparison between treated and non-treated mutant animals.

Non-ischaemic heart failure can only be treated by drugs in contrast to ischaemic cardiomyopathies. If the treatment fails, the only remaining options are heart transplantation or permanent implantation of assist devices [START_REF] Clegg | Clinical and costeffectiveness of left ventricular assist devices as a bridge to heart transplantation for people with end-stage heart failure: a systematic review and economic evaluation[END_REF]. Our goal is to develop cell therapy as a new tool for the cure of non-ischaemic cardiomyopathies. The first issue to address is the biocompatibility of the nano-fabricated collagen scaffold. To this end, 10 conditionally-invalidated serum response factor (SRF) mice (aMHC-MerCreMer:Sf/Sf) were transplanted with the 10% CA collagen scaffold. The biocompatibility was found excellent, as the scaffold was detectable from 2 days (4 mice) to 2 weeks (6 mice) after the implantation, with no visible inflammation (fig. 7, A &B). It was slightly degraded over time as its thickness only decreased to 60% after two weeks of implantation (fig. 7,C). This decreased thickness also indicates that the inflammation is very low. Moreover, the porosity of the electrospun collagen scaffold did not significantly change between 2 and 14 days, indicating that the structure of the patch is not altered (fig. 7,D). It should be noted, that we also implanted 5 % CA electrospun collagen scaffolds in 12 mice. The results were very similar to the 10 % CA after 2 days of implantation. But after 2 weeks, we were not able to find the scaffolds in 62.5% of the implanted mice. This suggests that the 5% CA electrospun collagen scaffolds seem to be less resistant in vivo. Together, all these results demonstrate the good biocompatibility of the electrospun clinically-approved collagen scaffold in the context of dilated cardiomyopathy.

Conclusions

The present work has been designed to create a cardiac patch which can be used as a cell carrier for therapy purposes. Among different approaches, electrospinning appears to be the simplest but yet versatile method for the fabrication of fibrous scaffolds from a large variety of materials. Then, the challenge is to find the most suited material and processing parameters. Although the electrospinning techniques have been well documented, few of them applied to cardiac cell therapy which requires a full control of the porous size, the mechanical stiffness and the bio-reactivity or bio-degradability of the scaffold. As a first step of our approach, we produced nanofibers by electrospinning of a clinically proven collagen dissolved in a non-toxic solvent system in contrary to the widely used 1,1,1,3,3,3 hexafluoro-2-propanol [START_REF] Sell | Electrospinning of collagen/biopolymers for regenerative medicine and cardiovascular tissue engineering[END_REF]. In addition, we chose a citric acid-based system for cross-linking of the electrospun nanofibers, which makes our fabrication process compatible to the therapy purposes. After crosslinking, the collagen fibrous scaffold could be easily mounted on a standard device for in vitro studies. This should allow more systematic investigations to elucidate the dependence of cell migration and three-dimensional colonization as a function of pore size and thickness of the scaffold. The fabricated nanofiber scaffolds could also be used for in-vivo testing. Our preliminary results have shown the feasibility of implantation of this patch. Finally, the mechanical stiffness of the scaffold will have to be optimized in order to match that of the heart to be repaired.

Figure 1 .

 1 Figure 1. Schematic representation of the electrospinning apparatus.

Figure 2 .

 2 Figure 2. Images of the atelocollagen fibrous scaffold a) deposited onto aluminum collector, b) after the peeling-off.

Figure 3 .

 3 Figure 3. SEM images of atelocollagen fibers after electrospinning at different magnifications: (a) 1x and (b) 10x.

Figure 4 .

 4 Figure 4. SEM images of crosslinked collagen scaffolds after immersion in water (a) 5% CA, (b) 10% CA and (c) 10% CA at higher magnification.

Figure 5 .

 5 Figure 5. Images of water droplets on collagen scaffolds with 10% CA (a) uncrossliked, (b,c) crosslinked captured at different time after droplet deposition.

Figure 6 .

 6 Figure 6. Confocal images of H9c2 cardiomyoblasts on the electrospun collagen scaffold (10% CA) after 48h cell culture.

Figure 7 .

 7 Figure 7. Biocompatibility of the electrospun collagen scaffold. Hearts of aMHC-MerCreMer:Sf/Sf mice were explanted 2 days (A) or 14 days (B) after implantation. The mean thickness (C) and the porosity (D) of the grafted patches were analyzed.

Acknowledgements

This work was supported by the French National Research Agency (ANR-12-RPIB-0015) and by the Fondation de l'Avenir (ET2-658). P.J. was supported by a fellowship from the Fondation Lefoulon-Delalande and S.B. from the Conseil Régional d'Ile de France (DIM Biothérapies). M.K. would like to thank Ms. Anne-Laure Vayssade from Microfluidique, MEMs and Nanostructures Laboratory of ESPCI for allowing her using their contact angle measurement instrument.