Julie Jimenez
email: julie.jimenez@inrae.fr

Marco Grigatti

Elisa Boanini

Dominique Patureau

Nicolas Bernet

The impact of biogas digestate typology on nutrient recovery for plant growth: accessibility indicators for first fertilization prediction

Keywords: digestates, typology, fertilizers, characterization indicators, nutrient recovery

In recent years, anaerobic digestion of organic waste (OW) is rapidly appearing as a winning waste management strategy by producing energy and anaerobic digestates that can be used as fertilizers in agricultural soils. In this context, the management of the OW treatment process to maximize agrosystem sustainability satisfying the crop nutrient demands represents the main goal. To investigate these traits, two protocols to assess the plant availability of digestate nitrogen (N) and phosphorus (P) were evaluated. With this aim, the N and P availability was determined on 8 digestates and 2 types of digestate-based compost from different OW via sequential chemical extractions (SCE). In addition, the digestates were tested in soil incubations and in plant pot tests with Italian ryegrass and compared with chemical fertilizer and a non-amended control soil. The N extracted from digestates via SCE was related to soil N mineralization and plant N recovery. The C: N ratio had negative impact on mineralized N and its recovery in shoots ( = -. × + . , r²=0.67), whereas water extractable mineral N was positevely related to the root N apparent recovery fraction (N-ARF) with ( = × + . ! , r²=0.53). The shoot P-ARF was positively correlated with the inorganic water extractable fraction of P ("# $ = . ! × % & -$ -. × % & -$ + . '(, r²=0.71) whereas the root P-ARF was positively correlated with the less accessible fractions

Introduction

An increasing interest exists in improving the soil quality following the utilization of organic wastes (OW) within the environmentally friendly bio-refinery approach [START_REF] Alburquerque | Agricultural use of digestate for horticultural crop production and improvement of soil properties[END_REF][START_REF] Gissén | Comparing energy crops for biogas production -yields, energy input and costs in cultivation using digestate and mineral fertilization[END_REF]. In this respect, anaerobic digestion is a major building block in the circular bioeconomy.

Furthermore, both energy (ie. biomethane) and organic fertilizers (ie. digestate) replacing the chemical fertilizers provided to crops can be recovered from anaerobic digestion of OW. From an agronomic and economic point of view, digestate use as a fertilizer can be considered not only as a supplement for traditional organic fertilizers (i.e. slurry) but also as an alternative fertilizer, which under certain soil conditions, is more effective than using NPK fertilizer (Barlog et al., 2019). Indeed, anaerobic digestion is reported as a suitable treatment to easily recover P and N fertilizers from digestate [START_REF] Mazzini | Phosphorus speciation during anaerobic digestion and subsequent solid/liquid separation[END_REF]. This is due to organic matter and free organic nutrients biodegraded into mineral forms. However, agricultural reuse of treated OW as soil fertilizers is limited by environmental constraints related to the quality of organic matter, nutrient availability and safety issues. Furthermore, the chemical composition of digestate depends on the nature of feedstock and digestion process [START_REF] Alburquerque | Agricultural use of digestate for horticultural crop production and improvement of soil properties[END_REF][START_REF] Guilayn | Valorization of nonagricultural digestates: a review for achieving added-value products[END_REF], Barlog et al., 2019). Indeed, a statistical classification of 91 digestates based on their agronomic quality (i.e. C, N, P, total solids, organic matter) was applied on a large range of raw digestates by [START_REF] Guilayn | Valorization of nonagricultural digestates: a review for achieving added-value products[END_REF] and revealed 6 groups of different digestate typology. By analyzing the obtained digestates groups, two criteria impacted the classification: the TS concentration of the digestates associated with dry or wet anaerobic digestion process and the feedstock type, as following: liquid fibrous feedstock (crop residues silage, cattle slurry), liquid sewage sludge, liquid pig slurry, slurries co-digested with silage and green wastes (dry anaerobic digestion), municipal solid wastes (dry anaerobic digestion) and fibrous feedstock (dry anaerobic digestion of cattle manure and green waste). In this framework, mineral N can be readily available for crops but can be temporarily immobilized by microbial biomass during OW mineralization in soil [START_REF] Lashermes | Typology of exogenous organic matters based on chemical and biochemical composition to predict potential nitrogen mineralization[END_REF]. Therefore, it is important to know the OW mineralization N kinetics to optimize the N supply synchronisation with plant requirement in agricultural systems.

In this context, to better control and manage the quality of digestates, a better knowledge of the accessibility and availability of nutrients and organic matter would improve the fertilizing potential of these products. According to [START_REF] Möller | Effects of anaerobic digestion on digestate nutrient availability and crop growth: A review[END_REF], an accurate characterization of digestate nutrient content and organic matter (OM) composition, combined with experiments to assess the N mineralization and N immobilization processes after field spreading, would be essential for a better characterization of the driving factors driving N turnover in the soil.

Total content of nutrients in OW harldy predicts their fertilizer potential. [START_REF] Ahmad | Aging Effects of Organic and Inorganic Fertilizers on Phosphorus Fractionation in a Calcareous Sandy Loam Soil[END_REF] reported more than 80% of applied P is rapidly immobilized being unavailable for plant following adsorption/precipitation processes or conversion into organic form. To gain a better insight on this issue, many authors proposed to characterize the available P via P fractionation. [START_REF] He | Phosphorus Distribution in Sequentially Extracted Fractions of Biosolids, Poultry Litter, and Granulated Products[END_REF] reported that bioavailability of applied P depends on the presence of specific P forms and that labile P includes the sum of inorganic and organic P from H2O and NaHCO3 extracted fractions. In this regard, [START_REF] Grigatti | Phosphorous in digestate-based compost : chemical speciation and plant-availability[END_REF]2017;2019), performed a dedicated SCE on digestates and composts in order to measure the phosphorous potentially available for plants. The authors found good correlations between the water-and the sodium bicarbonate-extractable P from composts with short-and mediumterm plant P uptake. [START_REF] Lashermes | Typology of exogenous organic matters based on chemical and biochemical composition to predict potential nitrogen mineralization[END_REF] proposed an OW classification based on their chemical characteristics to predict N availability. However, only 2% of the 273 OW studied came from similar typology of digestates coming from fibrous feedtstock anaerobic digestion with low N availability (i.e. digestates was classified in a group with initial N concentration under 65 g kg -1 and C: N ratio around 15). However, according to [START_REF] Guilayn | Valorization of nonagricultural digestates: a review for achieving added-value products[END_REF], the N content from digestates varies from 20 g kg -1 to 4

Lashermes study with different typologies of digestate to classify their fertilizer potential. To do that, the first step would be to evaluate the impact of digestate typology on plant growth. Recently, a methodology proposed for organic matter characterization has been applied to a large range of OW in order to predict both bio-avalability and complexity/biodegradability of the organic matter [START_REF] Jimenez | A new organic matter fractionation methodology for organic wastes: Bioaccessibility and complexity characterization for treatment optimization[END_REF]. This technique has been successfully used for OM biodegradation kinetics modelling (anaerobic digestion, compost, soil) [START_REF] Jimenez | Methane production and fertilizing value of organic waste: organic matter characterization for a better prediction of valorization pathways[END_REF] and for organic N dynamics in anaerobic digestion [START_REF] Bareha | Characterization and prediction of organic nitrogen biodegradability during anaerobic digestion: A bioaccessibility approach[END_REF]2019). The basic idea was to find a consistent characterization method in order to describe the bioprocess kinetics and to model the whole treatment chain in terms of organic carbon fate, e.g. anaerobic digestion, compost and organic matter fate in soil. The methodology used is based on SCE to simulate organic matter bioaccessibility for microorganisms combined with threedimensional fluorescence spectroscopy to analyze the organic matter complexity. The challenge is now to transfer this technique to the characterization of nitrogen accessibility in order to predict its fate after land-spreading and its availability for plant growth.

Apart from the previously mentioned studies, there is no literature information on the development of indicators for both N and P plant availability from digestates. This is the reason this study aimed to propose indicators based on chemical extractions and characterization for N and P.

In this light, the objectives of this work were to determine the plant-available N and P fractions based on characterization of the accessibility study in order to: (i) assess the impact of typology of OW on the nutrient availability, and (ii) use these indicators to predict plant's nutrients recovery. This study, focussed on a wide range of anaerobic digestates from many types of OW. The results could be used for rapid diagnostic of the (N, P) fertilization potential of a digestate.

Materials and methods

Organic waste

In order to ensure a representative survey, the anaerobic digestates used in this work were selected on the basis of the results reported by [START_REF] Guilayn | Valorization of nonagricultural digestates: a review for achieving added-value products[END_REF]. The typology developed by [START_REF] Guilayn | Valorization of nonagricultural digestates: a review for achieving added-value products[END_REF] was used to select the digestate samples out of a large panel of digestates. For this study, 6 main groups were selected from a statistical study applied to the agronomic characteristics: sewage sludge, municipal waste, cow manure (dry -anaerobic digestion), pig manure (liquid anaerobic digestion), centralised (co-digestion), and crop residues. Table 1 presents the samples used with the type of OW used as feedstock and the conditions of the anaerobic digestion process. Post-treated digestates through phase separation (solid or liquid) and composting were also added. Indeed, phase separation is the most classical digestate post-treatment [START_REF] Alburquerque | Agricultural use of digestate for horticultural crop production and improvement of soil properties[END_REF], leading to two products of different quality (i.e. solid phase as soil amendment and liquid phase as fertilizer). Besides, two digestate-based types of compost (FFMSW_2 and Sludge_2) were used since composting is widely adopted treatment for the solid phase of urban OW digestate to meet the European fertilizing regulation parameters (European Parliament and Council of the European Union, 2016). The organic waste samples were freeze-dried and ground (ø 1mm), in order to reduce the particle size effect in the soil incubations and plant growth experiments.

Analytical measurements

Freeze-dried ground samples were further ball milled and analysed for the main physico-chemical parameters. The moisture was determined at 105 ± 2°C until constant weight (24-48h), the volatile solid (VS) was determined on the total solids (TS) at 550 °C for 4 h. The total carbon (TC) and total nitrogen (TN) were determined via an elemental analyser (FlashSmart, Thermo Fisher Scientific). The total nutrient and trace elements were determined by ICP (Inductively Coupled Plasma-OES, Spectro Arcos, Ametek) on ≈250 mg of samples after microwave assisted digestion with 65% HNO3 + 37% HCl. All the analyses were done in duplicates.

Based on [START_REF] Jimenez | A new organic matter fractionation methodology for organic wastes: Bioaccessibility and complexity characterization for treatment optimization[END_REF], the sequential chemical extractions of organic matter were applied on the freeze-dried and grounded samples (0.5 g). An orbital shaker was used for the extractions steps as following described:30 mL of 0.01M CaCl2 (twice, 1h, 30°C, 300 rpm) ; 0.01M (NaOH+NaCl) (4 times, 15 min, 30 °C, 300 rpm); 0.1M HCl (once, 1h, 30°C, 300 rpm), 0.1M NaOH (4 times, 1h, 30 °C, 300 rpm) and 72% H2SO4 (twice, 3h, 30°C, 300 rpm). The fractions names are respectively: Soluble extracted from Particulate Organic Matter (SPOM), Readily Extractible Organic Matter (REOM), Slowly Extractible Organic Matter (SEOM) and Poorly Extractible Organic Matter (PEOM). The not extracted fraction is called Non Extractible Organic Matter (NEOM). Each extraction was done in two replicates, centrifuged and the supernatant collected and filtered. The recovered pellets were submitted to the subsequent extraction. Ammonium and total nitrogen were measured in the supernatants using HachLange® kits based on colorimetry methods (LCK 303, 2-47 mg N L -1 , and LCK 238, 5-40 mg L -1 respectively).

The organic products were submitted to P fractionation via SCE according to the method of [START_REF] Dou | Laboratory procedures for characterizing manure phosphorus[END_REF]. Freeze-dried and ball-milled products were extracted for 24 h with deionized water (H2O) in an end-over-end shaker, and then centrifuged. The supernatants were passed through a Whatman #42 filter, and the recovered pellets were extracted with 0.5 M NaHCO3 (pH 8.5) for 24 h. The same procedure was repeated with 0.1N NaOH and 1N HCl. The residual pellets were treated with a mixture of 96% H2SO4 and 30% H2O2 via hot acid digestion at 360 °C. Inorganic P (Pi) in the extracts was determined via the molybdenum blue method of Murphy and Riley (1962). The P recovered in each fraction [water extractable P (H2O-P), bicarbonate extractable P (NaHCO3-P), alkali extractable P (NaOH-P), acid extractable P (HCl-P), Residual-P] was calculated as follows: where Pi;fraction x is the inorganic P determined in each fraction (H2O, NaHCO3, NaOH, HCl, Residual), and Ptot OW is the total-P determined in the different organic waste via ICP after microwave-assisted acid digestion.

The total recovery was calculated as the sum of all fractions (H2O-P + NaHCO3-P + NaOH-P + HCl-P + Residual-P) by using the Equation2: represents the sum of the single P recovery values (Pi) in each fraction (H2O-P + NaHCO3-P + NaOH-P + HCl-P + Residual-P), and Ptot OW is the total P determined in the different products via ICP after microwave-assisted acid digestion. Organic P was calculated as the difference between Ptot and Pi in the first four fractions.

According to [START_REF] He | Phosphorus Distribution in Sequentially Extracted Fractions of Biosolids, Poultry Litter, and Granulated Products[END_REF], H2O-P, NaHCO3-P, NaOH-P and HCl-P are respectively water-soluble, bioavailable, potential bioavailable (Fe/Al bound) and Ca-bound P.

X-ray diffraction

Samples submitted to X-ray diffraction analysis were carefully ground and placed in a flat stage sample holder. Data were collected by means of a PANalytical X'Pert PRO powder diffractometer equipped with a fast X'Celerator detector. Cu Kα radiation was used (40 mA, 40 kV). The 2θ range was from 5° to 60° with a step size of 0.1° and time/step of 100 s. Data were processed for phase identification using a HighScore Plus software package (PANalytical).

Soil incubation

Soil

The soil used for the incubation and pot trial was collected from the top layer in a field in the Po Valley (Bologna, Italy). This soil showed the following characteristics: pH (H2O 1:2.5), 7.90; particlesize distribution; 184 mg kg -1 , sand, 425 mg kg -1 , silt, 391 mg kg -1 clay; total CaCO3, 85 g kg -1 ; total organic carbon (TOC), 10.2 g kg -1 ; total Kjeldahl nitrogen (TKN), 1.60 g kg -1 ; C:N, 8.3; exchangeable K, 330 mg kg -1 as K2O, CEC 27.2 meq. 100 g -1 . The total (extractable in aqua regia + HF) Al, Fe and P were 35661, 22224 and 808 mg kg -1 respectively. The NH4-oxalate (pH 3) extractable Al and Fe were 764 and 2158 mg kg -1 respectively, while the Na dithionite-citrate extractable Al and Fe were 281 and 2462 mg kg -1 respectively.

Incubation tests

Soil incubation was conducted in 500 ml plastic vessels with perforated plastic cap, on 250 g of soil (TS basis). The soil was rewetted at 60 % of the water holding capacity (WHC) and pre-incubated for 4 weeks at 25 °C in the dark. Water content was kept at 60% of WHC by weighing the vessels every 2-3 days and by adjusting with water drops if needed. After this period, the organic products (i.e. digestates) were added to the soil at 170 kg N ha -1 , defined as the maximum N load per year authorized by the European Nitrates Directive (1991) and accurately mixed by hand. Chemical references used as positive control (Ctrl + ) were added by distributing N (as NH4NO3) at the same concentration of 170 kg N ha -1 , and in addition P and K were added at 117 and 147 kg ha -1 (as KH2PO4). On the basis of N loading the P added to the soil with the different organic product was on average 20.45 +/-2.5 mg P kg soil -1 . Calculation of the amount of digestate mass added is described by the Equations 3 to 5 considering 0.3 m of soil depth (d) and a bulk density of 1.33 g/cm 3 for 1 ha of soil.

= = 5 YZ[\]^_^\ ×7 YZ[\]^_^\ ×`a YZ[\]^_^\ 5 ]bZc Equation 4
From the Equation 4, digestate amount is calculated as:

m e;6f9ghgf = e:9;9×5 ]bZc 7 YZ[\]^_^\ ×`a YZ[\]^_^\ Equation 5
mdigestate is the digestate mass (kg) Ndigestate is the N concentration of the digestate (gN.kgTS-1) TSdigestate is the Total solids content of the digestate (% of fresh matter) msoil is the considered soil mass in the test (kg) Three replicates for each treatment were assessed in a completely randomized block design, in addition to an unfertilized treatment control (Ctrl -). Two parallel soil sampling series were done to follow the P and N evolution during incubation. Olsen-P, was determined according to [START_REF] Watanabe | Test of an ascorbic acid method for determining phosphorus in water and NaHCO3 extracts from soils[END_REF] at days: 0, 14, 28, 56, 84.The Olsen-P data were used to calculate the relative percentage extractable (RPE) P, used to normalise the P extractability for each treatment relative to Olsen-P obtained for Ctrl + [START_REF] Grigatti | Organic wastes as alternative sources of phosphorus for plant nutrition in a calcareous soil[END_REF]. The mineral N course in soil was assessed by extracting soil samples (1g TS basis) at days 0, 14, 28, 56 and 84 with 1M KCl for 30 min on an end-over-and end shaker. The solution was filtered over a Whatman #42 filter and ammonium and nitrates were determined using Hach Lange® kits (LCK 304, 0.015-2 mg N.L -1 and LCK 339, 0.23-13.5 mg N L -1 respectively).

Plant pot trials

Based on Grigatti et al. (2014 ;[START_REF] Gerke | Phytate (Inositol Hexakisphosphate) in Soil and Phosphate Acquisition from Inositol Phosphates by Higher Plants[END_REF], the digestate samples were added to the ground soil at 170 mg N kg -1 and thoroughly mixed by hand in 2 liter plastic pots (ø 140 mm × h 150 mm). These were filled with 1 liter of inert material (agricultural light expanded clay), and 1 kg of each different treated soil in 3 replicates. Pots were seeded with 0.8 g of seeds of Italian ryegrass (Lolium multiflorum subsp.

Italicum), cv. Sprint, covered with a thin layer of sand to prevent drying, watered and placed in a growth chamber at 16-18 h day-night photoperiod at 13-23 °C (±3 °C) day-night temperature, the light was ensured by 6 Philips Master Tld 58 W-840 tubes. Besides the organic products, an unfertilized control (Ctrl -), and a chemical reference (at the same rate used for soil incubation) was added (Ctrl + ).

The same treatments and dosis performed in soil incubation test were applied. The use of fast growing species as ryegrass in controlled conditions (moisture; temperature; light) leads to a multiple harvest approach giving the opportunity to the best description of apparent N and P utilization kinetics in the time frame of a growing season (Gunnarson et al., 2010;[START_REF] Schiemenz | Biomass ashes and their phosphorus fertilizing effect on different crops[END_REF][START_REF] Tampio | Agronomic characteristics of five different urban waste digestates[END_REF].

After emergence, plants were regularly watered with tap water to keep soil at 60 % Water Holding Capacity (WHC). At each harvest, ryegrass plants were cut 2 cm above ground and collected 3 times: at 28, 56 and 84 days after sowing. The plant biomass was then dried in a forced air oven at 60 °C for 3 days and weighed, to determine dry weight (DW) per pot. Dry biomass was also ball-milled for subsequent analysis. At the last harvest, the roots were divided from the soil with a combined watersieving separation, and the root biomass was then dried as above, weighed and milled. On plant tissue (shoots and roots), the total P content was determined by means of ICP after (HNO3, H2O2,) microwave assisted digestion. The total N content was determined by elementar analysis as the OW samples. Apparent plant N and P utilization efficiency was calculated on the basis of the Apparent Recovery Fraction (ARF) approach (Gunnarsonn et al., 2010), according to the Equation 6:

ARF_X (%) = ∑ j kl +mn on+ pnq / /j kl +mn r os / j +ttnt ! /u • Equation 6
10 in which X uptake treatment (tn) is the total nitrogen or phosphorus uptake (mg pot -1 ) of a fertilizer treatment at time t (i = cut 1-3.); X uptake ctrl - ti is the total nitrogen or phosphorus uptake (mg pot -1 ) of the unfertilized control at time t (i = cut 1-3); X added is the total nitrogen or phosphorus added to the pot (mg pot -1 ).

Chemical nutrient equivalent coefficient keq is defined as the equivalent chemical nutrient dosis (i.e.

positive control) percentage needed to reach similar crop yield and is calculated according the Equation 7.

k fx X = z{|_z {| •^€c• Equation 7

Statistical analysis

All the data from the plant growth experiment were analyzed by means of Kruskal-Wallis non parametric test. Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA)

were also performed on all the data using FactomineR package from the R software. Partial Least Square Regression (PLSR) was performed for variables prediction using the SIMCA® software. For PCA analysis, data from [START_REF] Grigatti | Organic wastes as alternative sources of phosphorus for plant nutrition in a calcareous soil[END_REF] were included as far as similar experiments were done on P speciation and plant pot tests. The three samples were D1, D2 and BD, which are respectively digestate from thermophilic wastewater sludge digestion, digestate from mesophilic winery sludge treatment and digestate from mesophilic bovine slurry and energy crops treatment. Table 1 presents these samples, the conditions of the anaerobic digestion process and the associated feedstocks.

Results and discussion

Digestates characterization

The main physico-chemical characteristics of the studied digestates are reported in the Table 2. A high variability of the parameters analysed was apparent. Indeed, organic matter ranged between 40 and and 90% (TS basis), the TKN varied between 15 and 45 g kg -1 while P ranged between 4 and 20 g kg - samples were the digestates of FFMSW and its compost (between 15 and 17 g N kg -1 ). The richest P samples were the sludge digestate (Sludge_1) and its compost (Sludge_2), along with the solid phase of the centralised digestate (Centr_1) and digestate D2 (sludge from winery processing) with a P content ranging between 18 and 20 g kg -1 . The FFMSW_1 digestate and its compost FFMSW_2 besides the silage straw digestate (Agri_3) were the poorest P samples (4 g P kg -1 ). PCA and HCA analyses were performed on the samples characteristics for a whole sight of the digestates profiles thus allowing the comparison with the typology of [START_REF] Guilayn | Valorization of nonagricultural digestates: a review for achieving added-value products[END_REF]. The results are presented in the Supplementary Material (Figure A.1). The PCA was applied on nine parameters (i.e from TS to K in Table 2). The first two components recovered ≈ 80% variance. The first component (PC1) was mainly related to the TS, negatively related to OM and K.

PC1 clustered dried (i.e. FFMSW_1, composts Sludge_2 and FFMSW_2) and liquid AD digestates (i.e. all the others). The second component (PC2) was formed by P, which was negatively related to the C: N ratio related to fibrous composition. The sludge digestate samples were associated with P concentration variable whereas Agri_3 (wheat straw digestate) was mainly associated to C: N ratio variable. Furthermore, Sludge_1 and 2 were the samples containing the highest metals concentrations (Fe, Al, Pb, Cu, Mn, Mo, and Cd).

These results were in agreement with the raw digestate typology found by [START_REF] Guilayn | Valorization of nonagricultural digestates: a review for achieving added-value products[END_REF].

Indeed, Agri_1 and Agri_3 characteristics were in agreement with the characteristics associated to the fibrous feedstock digestate group after dried digestion (i.e. cattle manure and crop residues). Agri_2 characteristics were consistent with those of the pig slurry digestate group from the typology.

Sludge_1 characteristics were consistent with those of the sludge digestate group and FFMSW_1 characteristics fit with the municipal solid wastes digestate group characteristics. BW_1 characteristics were mainly close to the biowaste and municipal solids waste digestate composition after dry digestion. However, NH4 concentration and NH4: TN ratio had similar values than the manure co-digestion (i.e. NH4: TN ratio > 70%). This is probably due to the high ratio of agroindustrial waste in the feedstock used in addition of biowastes. Only digestate from the wet digestion of fibrous substrate was not taken into account (silage) in this study. Composts Sludge_2 and FFMSW_2 and Centr_1 and 2 obtained after phase separation were not compared with the typology as they were not raw digestates.

Attention should be paid to the results for TKN and N-NH + 4 concentration obtained by the raw sample analysis and to the freeze-dried sample extraction analysis. Regarding some digestates, highly significant positive differences were obtained between raw and freeze-dried samples analyses (from 40% for Centr_2 to 61% for Agri_2 of TKN loss). This result was due to ammonia volatilization during freeze-drying operation. The loss of ammonia could have an impact on soil incubation and plant N recovery results. Considering the 8 others digestates, no significant differences appeared and a linear relation was obtained between raw sample analysis and freeze-dried analysis (i.e. TKN_raw = 0.9443.TKN_freeze-dried, R² = 0.8832). Furthermore, the N typology of Agri_2 became similar as Agri_1 and 3 (fibrous feedstock involving manures). NH4: TN ratio evolved from 76% to 39%. This ratio decreased also for Centr_2 from 46% to 9% after freeze-drying. Consequently, Agri_2 and Centr_2 results were used in statistical tests by using the freeze-dried analysis. N-ARF recovery will be discussed accordingly. However, concerning liquid digestates with high ammonia content as pig slurry digestates, the best option would be to perform soil incubation and plant pot trials with fresh matter as Rigsby et al. ( 2013), de la Fuente et al. (2013). A higher mass of soil would be required to maintain the soil WHC with the liquid digestate moisture, according Equation 5.

This first characterization approach showed that the digestates selected were representative of a large range of digestates (i.e. municipal waste, sludge, manure, crop residue, bio-wastes, centralised).

X-ray powder diffraction

The X-ray powder diffraction has been widely use for the study of the crystalline P species in anaerobic digestate and compost [START_REF] Li | Characterization of phosphorus species distribution in waste activated sludge after anaerobic digestion and chemical precipitation with Fe3+ and Mg2+[END_REF][START_REF] Grigatti | Phosphorous extractability and ryegrass availability from bio-waste composts in a calcareous soil[END_REF]2019). In this light the XRD profiles from the tested products can give a valuable insight to their P extractability. The XRD patterns from the digestates are presented in Figure 1. The XRD patterns of samples Agri_1, Agri_2 and Agri_3 were similar

showing the presence of crystalline inorganic phases, namely quartz (PDF no. 01-087-2096), calcite (PDF no. 01-085-1108) and sylvine (KCl, PDF no. PDF no.41-1476).

However, these three samples differed significantly in their amorphous phase content, which was definitively higher in Agri_3, followed by Agri_1, whereas sample Agri_2 was mostly constituted of crystalline material. Furthermore, Agri_2 presents also struvite peaks (PDF no. 15-0762), that could be detected on account of the higher crystallinity of this sample. Amongst the analyzed samples, Sludge_1

was the most crystalline one, with no significant presence of amorphous material. The Sludge_1 pattern showed the presence of calcite and quartz as main constituting phases, together with a low amount of vivianite (Fe3(PO4)2(H2O)8, PDF no. 01-075-1186). [START_REF] Li | Phosphorus speciation in sewage sludge and the sludge-derived biochar by a combination of experimental methods and theoretical simulation[END_REF] found also that quartz was one of the major phases for sewage sludge. Sludge_2 was somewhat similar to Sludge_1, but less crystalline so that only calcite and quartz were detected (in this case quartz is more abundant than calcite). FFMSW_1 and FFMSW_2 were very similar both regarding the amount of amorphous phase which is present together with the fine crystalline fraction of the materials. Regarding the qualitative detection of crystalline phases, it was possible to identify quartz (the most abundant), calcite, together with low amounts of kaolinite (Al2(Si2O5) (OH)4 , PDF no.01-080-0885) and sylvite. BW_1, Centr_1 and Centr_2 patterns all displayed the presence of quartz, calcite and struvite as crystalline components, but significantly differed for the amount of amorphous material that was relatively low in Centr_2, and higher and similar in BW_1 and Centr_1. The relationship between the XRD outocomes and the P extractability are dicussed in the following section.

Sequential chemical extractions of Nitrogen and Phosphorous

The N fractionation protocol used in this work was developed by [START_REF] Jimenez | A new organic matter fractionation methodology for organic wastes: Bioaccessibility and complexity characterization for treatment optimization[END_REF], and has been validated to describe OM and N evolution during biological degradation processes [START_REF] Jimenez | Methane production and fertilizing value of organic waste: organic matter characterization for a better prediction of valorization pathways[END_REF][START_REF] Bareha | Characterization and prediction of organic nitrogen biodegradability during anaerobic digestion: A bioaccessibility approach[END_REF]. The P fractionation has been adapted from [START_REF] Hedley | Changes in inorganic and organic soil phosphorous fraction induced by cultivation practises and by laboratory incubations[END_REF], using methodology by [START_REF] Grigatti | Phosphorous in digestate-based compost : chemical speciation and plant-availability[END_REF][START_REF] Grigatti | Phosphorous extractability and ryegrass availability from bio-waste composts in a calcareous soil[END_REF][START_REF] Grigatti | Organic wastes as alternative sources of phosphorus for plant nutrition in a calcareous soil[END_REF]. These authors showed the link between labile-P from organic waste samples and the plant P-uptake. The N and P speciations have been assessed

applying both fractionation protocols to the selected digestates (Figure 2 a and b respectively). The results showed that the digestates performed different N and P speciations. This was related to the digestate typology and the nature of the feedstock for anaerobic digestion, according to [START_REF] Guilayn | Valorization of nonagricultural digestates: a review for achieving added-value products[END_REF]. The observed variability in N and P speciation showed the significant effect of the feedstock on the tests discussed in section 3.2. In the study of N accessibility (Figure 2 a), mineral nitrogen was assessed in the water extractable fraction SPOM. The non-extractable organic nitrogen varied between 25 and 48% except for Agri_3 with only 5% N in the NEOM fraction.

HCA was applied on the N speciation data (not shown). Four groups were found, ordered by N accessibility basis as follows: (i) Group 1: High NH4-N SPOM samples: Centr_1 and BW_1. This result is consistent since these samples had poor TS; (ii) Group 2: High organic SPOM and REOM:

Agri_2 and Sludge_1. SPOM and REOM are mainly composed of accessible and readily extractable proteins; (iii) Group 3: High organic SEOM: Agri_3. SEOM is mainly composed of complex proteins and humic-like substances; (iv) Group 4: High PEOM organic N content samples: Agri_1, FFMSW_1, Centr_2, FFMSW_2 and Sludge_2. PEOM extraction targets holocellulose-like compounds found in fibrous digestates as Agri_1, FFMSW_1 and 2. Phase separation concentrates OM and fibers in the solid phase [START_REF] Guilayn | Valorization of nonagricultural digestates: a review for achieving added-value products[END_REF] as Centr_2. Finally, the compost Sludge_2 was also clustered in this group because of its co-composting with green wastes.

P was extracted in each fraction as reported in Figure 2 b. Phosphorus fractionation showed that inorganic P content was higher than organic P in all the fractions, as reported in the studies of [START_REF] Grigatti | Phosphorous in digestate-based compost : chemical speciation and plant-availability[END_REF][START_REF] Grigatti | Phosphorous extractability and ryegrass availability from bio-waste composts in a calcareous soil[END_REF][START_REF] Grigatti | Organic wastes as alternative sources of phosphorus for plant nutrition in a calcareous soil[END_REF] on digestates and composts, and as observed by [START_REF] He | Phosphorus Distribution in Sequentially Extracted Fractions of Biosolids, Poultry Litter, and Granulated Products[END_REF] on poultry litter and dried wastewater sludge. [START_REF] Mazzini | Phosphorus speciation during anaerobic digestion and subsequent solid/liquid separation[END_REF] showed that 78 to 93% of TP was inorganic following the SCE from six types of animal slurries digestates. The authors showed relevant NaOH (5-43% of organic P) and HCl (2-25% of organic P) extractable organic P, relating this to inorganic P microbial immobilization during anaerobic digestion for microorganism growth. Indeed, soluble inorganic P would be transformed into organic P compounds such as phosphates monoesters or DNA.

This biological organic-P may be rapidly mineralized once in soil [START_REF] He | Phosphorus Distribution in Sequentially Extracted Fractions of Biosolids, Poultry Litter, and Granulated Products[END_REF]. In this context, NaOH and H2O were the most organic-P rich fractions from the samples investigated in this work.

Furthermore, Agri_1, 2 and 3, and FFMSW_1 mainly showed NaOH extractable organic P. Agri_3 contained the highest organic P content (≈40%), and 1.5 to 4-folds higer than other samples. In water Agri_2 and 3 showed 60% of organic P, higher than BD (49%).

The results obtained by [START_REF] He | Phosphorus Distribution in Sequentially Extracted Fractions of Biosolids, Poultry Litter, and Granulated Products[END_REF] on poultry litter showed that a large part of organic P was in the NaOH fraction and was mainly related to phytate-like. [START_REF] Mazzini | Phosphorus speciation during anaerobic digestion and subsequent solid/liquid separation[END_REF] showed similar results on crop residues digestates. The authors showed also that organic P were mainly extracted in NaOH fraction of 6 agro-wastes digestates as shown by Agri_1 and 3. Organic P was observed in HCl fraction from Sludge_1 and 2 in this study. [START_REF] He | Phosphorus Distribution in Sequentially Extracted Fractions of Biosolids, Poultry Litter, and Granulated Products[END_REF], observed also organic P in HCl fractions related to nonhydrolysable fractions in the dried wastewater sludge. The NaHCO3 and the HCl fractions showed to be the most inorganic P rich factions from the samples tested in this work thus fitting to available P and Cabounded P. The labile-P fraction (H2O + NaHCO3) was found at the highest level in the agricultural residue digestates (Agri_1, 2 and 3). Similar observations on bovine manure and energy crops digestate (BD) were made by [START_REF] Grigatti | Organic wastes as alternative sources of phosphorus for plant nutrition in a calcareous soil[END_REF]. Then the biowaste and agro-food industries digestates (BW_1, Centr_1 and 2) had also high labile-P. The FFMSW samples showed intermediate P accessibility. The sludge samples were characterized by a high NaOH extractable P, related to metal-bounded P. Indeed, this result was consistent with both Al and Fe content (≈7 and ≈37 g kg -1 ). [START_REF] Li | Phosphorus speciation in sewage sludge and the sludge-derived biochar by a combination of experimental methods and theoretical simulation[END_REF], showed mainly NaOH extractable Al, while Fe was NaOH soluble being also extracted by HCl in sewage sludge, thus showing Fe-bounded P was partially occluded. Amongst the others samples, compost Sludge_2

showed important poorly available HCl-P, being very similar to FFMSW_2, these results were consistent with the P fractionation of composts showed by [START_REF] Grigatti | Phosphorous in digestate-based compost : chemical speciation and plant-availability[END_REF][START_REF] Grigatti | Phosphorous extractability and ryegrass availability from bio-waste composts in a calcareous soil[END_REF][START_REF] Grigatti | Organic wastes as alternative sources of phosphorus for plant nutrition in a calcareous soil[END_REF].

Finally, HCA showed six groups (clustering not shown). Groups were ordered according to the chemical availability level (i.e. Labile-P> NaOH-P> HCl-P) as follow: (i) Very high P accessibility related to high H2O-P fractions: digestates from liquid feedstock (no manure), biowastes and winery (BW_1, D1 and D2). These samples were characterized by amorphous P and crystalline P (quartz and struvite); (ii) High P accessibility associated with organic H2O-P and NaHCO3-P: digestates from liquid manure (BD and Agri_2). These samples were characterized by crystalline P (struvite); (iii) Moderate P accessibility (intermediary P speciation): digestates from organic fraction from municipal wastes digestates and centralised digestates (FFMSW_1, FFMSW_2, Centr_1 and Centr_2). These samples were characterized by an amorphous phase and mainly quartz and calcite as crystalline phases; (iv) Poor-P accessibility associated with organic NaHCO3-P and NaOH-P, associated with digestates from fibrous substrates as cow manure with crops residues and wheat straw (Agri_1 and Agri_3). These samples were characterized by a high amorphous phase; (v) Very poor P accessibility associated with inorganic NaOH-P and low fraction of available P: digestate from solid phase of sludge (Sludge_1). This sample was characterized by a crystalline P phase (mainly quartz and calcite); (vi) The poorest accessible P associated with inorganic HCl-P was compost of sludge digestate (Sludge_2). This sample was characterized by less crystalline P than Sludge_1 with calcite and quartz.

For some groups, the feedstock type seemed to have an impact on the P accessibility of digestates (solid phase of sludge, liquid manure, liquid feedstocks, fibrous feedstock). Another observation was that the amorphous and crystalline characteristics also seemed to be associated with some groups: fibrous digestates were mainly composed of amorphous phase and contained organic P in NaHCO3 and NaOH.

The labile P fractions had in common amorphous P and struvite as crystalline P. The most crystalline Prich fractions were found in the solid phase of sludge digestates before and after composting, containing low available P and high sparingly soluble HCl-P.

The eight tested digestates represented a wide range of inherent characteristics and accessibility patterns for both N and P. In this framework, the soil and plant test results can give a deeper insight to these issues as discussed in the following section.

Soil incubation and plant pot trials

Table 4 shows the cumulative amount of biomass harvested in ryegrass plant pot tests. Any significant differences between treatments for tissue and root (Kruskal-Wallis test p=0.89 and 0.23 respectively).

Nevertheless, it appears that poor biomass was obtained in Agri_2 treatment (pig manure digestate), and in FFMSW_2 treatment (FFMSW digestate compost), close to the negative control. The best results were obtained in Centr_1 and BW_1 (solid phase of a centralised digestate and biowaste digestate). The total plant biomass (shoot+ root) was the highest (g pot -1 ) in Centr_1 (4.11) ≥ Sludge_2

Nitrogen fate

The N mineralization data from soil incubation (Figure 3) showed the net cumulated mineral N (i.e.

NH4 + NO3 mass) during soil incubation (Figure 3a) and the net cumulated mineralization rate of organic N (Figure 3b).

Concerning the available mineral N (N-min), BW_1, Centr_2, Sludge_1, Sludge_2, Agri_2 and FFMSW_1 achieved the highest cumulated values after 84 days of incubation. On the contrary, Centr_1, FFMSW_2, Agri_3 and Agri_1 performed poorly cumulated N-min. These latter were more fibrous samples, with high C: N ratios.

The cumulated organic N mineralization rate was calculated (Figure 3b). Two groups of treatments appeared and were classified in the same order than the cumulated N-min: (i) positive mineralization rate associated with the Sludge_1, Centr_2, BW_1, Agri_2, Sludge_2 and FFMSW_1 treatments and

(ii) negative mineralization rate associated with Centr_1, FFMSW_2, Agri_1 and Agri_3. Sludge_1

showed the highest N mineralization rate (30%) suggesting that this digestate was rich in hydrolysable proteins. Organic N from the liquid digestates Centr_2 and Agri_2 showed respectively 21% and 18% of mineralized organic N, following Sludge_1 (30%). This result is consistent with Guilayn et al.

(2019) who reported that the highest organic N came from sludge digestate and pig-slurry digestate group. Sludge_2 and FFMSW_1 performed lower N mineralization rates (11%). Similar results were observed in liquid or solid digestates by Rigsby et al. (2013). They showed that the solid phase from municipal solid wastes digestate (C: N ratio of 11) had negative N mineralization rate whatever the soil composition used (sandy, silty or clay). On the contrary, Rigsby et al. (2013) showed that the liquid digestates from slurry (C: N ratio of 4), had 16% to 40% N mineralization while food waste digestate (C: N ratio of 14) reached 30% N mineralization.

The negative mineralization rates group was associated to an immobilization of mineral nitrogen by the soil microorganisms during their growth [START_REF] De La Fuente | Soil C and N mineralisation and agricultural value of the products of an anaerobic digestion system[END_REF]. This was related to the increased microbial activity following the OW addition to the soil. Indeed, nitrogen is the main limiting nutrient for plant growth, especially for a fast growing, highly demanding species such as Italian ryegrass (Grigatti et al., 2011). These results are in agreement with [START_REF] Cavalli | CO2 emissions and mineral nitrogen dynamics following application to soil of undigested liquid cattle manure and digestates[END_REF] which reported immobilization for high C: N ratio anaerobic digestates. The authors showed the low C: N ratio (5 to 7) associated to the raw digestate and its liquid phase (cattle slurry/mais silage) induced net N mineralization (≈ 30%). On the contrary, the cellulose-and volatile fatty acids-rich solid phase (C:

N ratio, 20) induced lower net N mineralization (9-16%). Indeed, negative correlations were found between the N mineralization rate and C: N ratio (r= -0.73, p = 0.029) as observed by [START_REF] Morvan | Biochemical composition and kinetics of C and N mineralization of animal wastes: a typological approach[END_REF] on animal manure wastes.

Considering the impact of process treatment of digestates, composting appeared to negatively affect N mineralization. N mineralization rate from Sludge_2 (11%) was lower than Sludge_1 (30%) and FFMSW_1 (11%) had highest mineralized values than FFMSW_2 (-2%). Phase separation impacted also organic N mineralization as shown by [START_REF] Cavalli | CO2 emissions and mineral nitrogen dynamics following application to soil of undigested liquid cattle manure and digestates[END_REF]. Solid phase (Centr_1) reached negative values of minerlization whereas Centr_2 reached 21% of mineralized N.

Plant growth experiments were affected by fertilizer treatment for N-ARF (Kruskal-Wallis, p = 0.026) only for shoot plant tissues. Cumulated N recovery by plant tissues was plotted in the Figure 4. The chemical treatment showed the greatest shoot N recovery (70%) with a total of 76% of N-ARF. N uptake recovery was mainly observed in shoots (64% to 87% of the total recovery). Root N-ARF was measured between 7% and 36% of the total N-ARF, except for Agri_1 and Agri_3 treatments where negative to zero values were obtained.

Sludge_1 and Sludge_2 achieved the best N-ARF amongst the digestates treatments (16.5 and 15.1%).

These results were consistent with the Sludge_1 treatment yielding the highest N-min available for plant growth. Centr_2 (13.7%), BW_1 (13.2%) and Centr_1 (11.6%) treatments showed intermediate total N recovery. The FFMSW_1, FFMSW_2 treatments followed with 8.2% and 6.4% respectively.

Finally, a last group formed by Agri_1 and Agri_3 was observed with low N-ARF of 1.8% and 2.6% respectively. In soil incubation tests, Agri_1 and 3 showed N immobilization. Accordingly to these outcomes, the plant N-ARF was negative or close to 0 and close to the unfertilized soil treatment.

Considering the digestate treatments impact, composting (i.e. FFMSW_2 and Sludge_ 2) lowered the fertilizing N value of its associated digestate (i.e. FFMSW_1 and Sludge_1 respectively). Phase separation impacted also the N fertilizing potential as shown by [START_REF] Tambone | Solid and liquid fractionation of digestate: Mass balance, chemical characterization, and agronomic and environmental value[END_REF]. Centr_2 treatment induced higher N mineralization rates and N-ARF than Centr_1 treatment. According to N speciation and characterization, the solid phase Centr_1 contained more fibers than the liquid phase Centr_2.

PCA and HCA were performed to find correlations between the N availability data, digestate characteristics, mineralized N percentages and N-ARF from shoots and roots as reported in Figure A.2 a (supplementary material). No significant correlation was observed between the total N content, the mineralization rate and the N-ARF. However, a strong correlation was obtained between the C: N ratio and N-ARF from shoots-N (r=-0.82, p=0.004) as in the mineralization N tests without plants. This was described by a linear equation (Equation 8) based on the data of this study and validated by data from digestates and composts studied in [START_REF] Grigatti | Phosphorous in digestate-based compost : chemical speciation and plant-availability[END_REF]. Similarly, [START_REF] Decoopman | Valeur azote des digestats de methanisation[END_REF] found a correlation between C: N ratio of agricultural digestates and plant N-ARF measured in field experiments on cereals.

Shoots 7 = -0.0085 × 7 + 0.1782, r Ž = 0.6718 Equation 8

The fiber component of digestates had a negative impact on mineralised N and N recovery by plant tissues. Similarly, the fiber fraction PEOM of digestates was negatively correlated with cumulated mineralized N (-0.58, p=0.08) and shoots-N although to a lesser extent (r=-0.58, p = 0.44). Moreover, cumulative mineralized N and shoots-N ARF were positively correlated (r=0.72, p=0.02) which was consistent with the availability of N for plant growth. Concerning roots, there was a positive correlation between roots N-ARF and nitrates content (r=0.72, p=0.017), SPOM_NH4 (r= 0.61, p=0.06) whereas SPOM_org and roots_N ARF were negatively correlated (r=-0.59, p=0.07). A linear equation (Equation 9) was found between Roots N-ARF and water extracted mineral N (i.e.

W @?•''•K_"A" = NO3 -+ SPOM_NH4).

Roots 7 = 5E -× N 9:<™š<f5;› + 0.0138, r Ž = 0.5313 Equation 9

According to [START_REF] Gunnarsson | Use efficiency of nitrogen from biodigested plant material by ryegrass[END_REF], plants respond to N availability with a different root/shoot nutrient allocations and plant growth rate could be influenced by the ammonia/nitrates ratio in soil solution due to the different N sources. Authors observed an increase in root biomass as a result of the availability of a high amount of ammonia. C: N ratio is associated to the fibrous level of an OW and seemed to be discriminant enough to predict N-ARF and organic N mineralization rate. Digestates with C: N ratios ≥15 were not suitable as N fertilisers whereas digestates with lower C: N ratios had a high potential of N fertilizer.

Accessibility fractionation of N allowed a consistent digestate classification. Furthermore, interesting correlation between roots-ARF N and water extractable nitrates and ammonia were found.

Phosphorous fate

The Olsen-P course during the soil incubation is shown by Figure 5a. The Olsen-P evolution in soil depends on several phenomena: (i) the physico-chemical conditions of the soil can trap some weakly bound P, (ii) the organic part of P can be mineralized and (iii) P can be taken up by soil microorganisms for their growth.

The control soil had the lowest Olsen-P content throughout the incubation (12-14 mg kg soil -1 ), whereas the compared treatments exhibited different Olsen-P courses in time. All the tested samples showed an available P depletion during the first two weeks. The chemical control (Ctrl + ) performed the fastest depletion. Indeed, the chemical P treatment (phosphate salt) can be rapidly fixed with Ca components in calcareous soil [START_REF] Alburquerque | Agricultural use of digestate for horticultural crop production and improvement of soil properties[END_REF]. In this context, the presence of organic matter and the different P forms from digestates can have positive interactions with soil, performing lower fixation and higher efficiency in comparison to chemical P sources [START_REF] Alburquerque | Agricultural use of digestate for horticultural crop production and improvement of soil properties[END_REF].

In some treatments, an Olsen-P increase appeared between 14 and 28 days (Agri_2, Centr_1, BW_1 and FFMSW_1) probably due to organic P mineralization before another decrease until 56 days. This latter decrease led to a lower quantity of available P for plant growth. The RPE obtained was between 50 and 84%. The RPE of the Centr_2 treatment was the highest with 84% vs. the chemical P treatment, followed by Sludge_2 (74%). FFMSW_1, Agri_2 and Agri_3 were the samples where the P fixation was the highest (RPE of 60% in average). The other digestates were intermediate between the two groups. FFMSW_1, Agri_2 and Agri_3 contained high fractions of water P.

The pot tests showed the treatments affected the plant P tissue (p-value = 0.021). In this context the control soil performed the worst at the first cut (1.93 mg.pot -1 ), showing very poor performance during the plant pot test reaching 4.55 mg pot -1 on average (Figure 5b and5c). The shoot-P ARF is shown in Figure 4a and b. P-ARF from Agri_2 and Agri_3 was negative from the beginning. This result was consistent with the soil incubation observations. This can be linked with high fixation of P in soil as suggested by [START_REF] Ahmad | Aging Effects of Organic and Inorganic Fertilizers on Phosphorus Fractionation in a Calcareous Sandy Loam Soil[END_REF]. In the second cut (day 56), the cumulative shoots P-ARF decreased in all the treatments. Only FFMSW_1 and Centr_2 showed opposite outcomes. Then, between the second and the third cut (day 56; 84), shoot-P ARF further increased. In the end the whole P-ARF (%) was: Centr_1 (5.78)≈ FFMSW(5.31)≥BW_1 (4.35)>Centr_2(3.78)>Sludge_1 (2.72)≥Agri_1

(2.44)≥Sludge_2 (2)>FFMSW_2 (1.6)>Agri_2 (0.6)≈Agri_3 (0.5%).

FFMSW_2, Agri_2 and 3 showed lower shoots P-ARF than the chemical treatment (1.81%). That means they were not suitable for P fertilization on calcareous soil.

The root-P content was unaffected by the treatment. However, the root P-ARF (%) was: Agri_1

(2.46)> Agri_3 (2.04)> FFMSW_1 (1.18) = Sludge_1 (1.17) = Sludge_2 (1.16)> Centr_1 (1.04)> BW_1 (0.3)> Centr_2 (0.00)> FFMSW_2 (-0.08)>Agri_2 (-0.21). Shoots-P and roots-P recovery potential were different depending on the treatment, except for Agri_2 and FFMSW_2 which had the lowest ARF P recovery for both.

availability. They reported that P concentrations on shoots were correlated with N concentrations in shoots. Indeed, a significant correlation was observed in this study between total N-ARF and total P-ARF (r = 0.8152, p = 0.0022) with a linear relationship (ARF_N/ARF_P = 7.7535, r² = 0.6626) thus proving N and P recovery by plants were linked.

PCA and HCA analysis were performed on both shoots and roots P uptake and P speciation for the 8 digestates studied beside the three digestates from [START_REF] Grigatti | Organic wastes as alternative sources of phosphorus for plant nutrition in a calcareous soil[END_REF] Agri_1 has high P total recovery in both shoots and roots as BD, but Agri_3 shows a high recovery only in roots. It seems that the fibrous characteristic enhances P recovery above all in in plants roots through the organic fraction of NaOH-P.

The groups obtained were similar to digestate typology groups, except for Agri_2 and BD which were not in a same group. When doing statistical treatments on the shoots results and P speciation for the 8 digestates used in addition to the 3 digestates used by [START_REF] Grigatti | Organic wastes as alternative sources of phosphorus for plant nutrition in a calcareous soil[END_REF], some correlations were found. Water-P fraction was positively correlated with shoots (r=0.49, p=0.09). This result was also observed by [START_REF] Grigatti | Phosphorous extractability and ryegrass availability from bio-waste composts in a calcareous soil[END_REF] for composts. As reported by Ahmad et al., 2018, available P consists in the exchangeable and soluble P corresponding to water and Na-bounded P fractions (H2O+ NaHCO3) whereas occluded P consists into P bound with metals (Fe, Al) and Ca. However, a simple linear model did not fit between inorganic H2O-P fraction (H2O-Pi) and shoots P-ARF. PLS model was used showing that two main significant variables were needed to predict shoots P-ARF (Equation 10). H2O-Pi fraction fertilisation according to the soil composition [START_REF] Alburquerque | Agricultural use of digestate for horticultural crop production and improvement of soil properties[END_REF] and not only based on overall P analysis. Indeed, this was the case of sludge digestates Sludge_1 and 2 which had the highest P contents (same level of Centr_1) but their treatments obtained moderate P-ARF. From P results, first guidelines can be given according to the digestate feedstock typology for the studied calcareous soil and ryegrass. Pig manure digestate (Agri_2) seemed not suitable for P fertilization. Fibrous digestates rich on wheat straw Agri_1 and 3 had very low P fertilizing potential whereas FFMSW_1 presented higher potential as the solid phase Centr_1. The compost FFMSW_2 had also low P fertilizing potential and the liquid phase Centr_2 was intermediate. Sludge 1 and 2 had intermediary results and biowaste digestate BW_1 showed high N and P fertilizing potential.

Conclusions

This study focused on different typologies of digestates classified according to their process and to their feedstock. In this context, both P and N speciations showed a wide accessibility range according to feedstocks charateristics. The chemical accessibility indicators described the nutrient availability for plants and allowed the digestates classification on N and P fertilizing potential basis. The N and P speciation impacted the results from incubations with bare soil as well as the apparent coefficients of the use of N and P by the plant for its growth. Depending on the tissue collected (shoot or root), the speciation variables having a significant impact were different for P and N, for the type of calcacerous soil used. First models were found to predict P recovery in shoots and roots using P speciation.

Furthermore, C: N ratio value was significant and could be used for shoots N-ARF prediction wheras mineral water extracted N could be used for roots N-ARF prediction. Thus, a more detailed knowledge of the digestates would allow more adequate control of fertilization. Moreover, composting and phase separation impacted the nutrient recovery and can be used as an actuator to propose different organic fertilizers type. In terms of perspectives, field trials on contrasted soils qualities for crops with contrasting nutrient needs should be carried out in order to offer a guide to fertilization by type of digestate. Finally, N and P speciation studied could be used in dynamic models to improve soil and plant model predictions for digestate use in agriculture.

( 3 .

 3 75) ≥ BW_1 (3.72) ≥ FFMSW_1 (3.70) ≥ Ctrl + (3.65) ≥ Sludge_1 (3.48) ≥ Agri_3 (3.38) ≥ Centr_2 (3.23) ≥ Agri__1 (3.18) ≥ Agri_2 (3.02) ≥ FFMSW_2 (2.93) ≥ Ctrl -(2.90).

  associated with fibrous digestate with high C: N (>25), high SEOM_N and PEOM_N and low shoots-N ARF. This type of digestate is not suitable as N fertilizer; (ii) Group 2: FFMSW_2 and Agri_1 15<C/N<13, intermediary protein-like and fibrous substrate digestates with high organic SPOM_N and PEOM_N for Agri_1 as compost FFMSW_2. They recovered very low roots N-ARF and intermediate level of shoots N-ARF; (iii) Group 3: Centr_1, FFMSW_1 and BW_1 municipal solids and biowastes digestates, with C: N ratios of 15 in average, high ammonium and nitrate levels in the labile fraction and high levels of total N recovery; (iv) Group 4: The protein-like digestates (Sludge_1, Centr_2, Agri_2) which have a poor C: N (<10), poor PEOM_N, high SPOM_Norg content and the highest total N recovery. Sludge_2 was classified in the Group 4 because of its lower C: N ratio (9.7) and its high N mineralization level.

Figure 1 :

 1 Figure 1: X-ray diffraction of the digestates. The main peaks of different crystalline phases are identified by symbols: struvite; quartz; calcite; vivanite; * sylvine;

Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 2345 Figure 2: SCE fractions of TKN (a) and TP (b) obtained for all the digestates, according chemical accessibility. Error bars, SE (n=2) 750

  , as shown by Figure A.2 b inSupplementary Material. Six groups were found according HCA analysis. The results showed that the P-ARF was classified not only according to the digestate's nature but also according to their P accessibility. The groups found were: (i) group 1 : Sludge_2, compost of solid phase of sludge digestate, HCl rich with an intermediate level of P recovered in shoots and roots; (ii) group 2 :

	Sludge_1, solid phase of sludge digestate (inorganic NaHCO3 and NaOH fractions rich), with an
	intermediate level of P recovered in shoots and roots; (iii) groups 3-4 : Centr_1, FFMSW_1,
	FFMSW_2, Centr_2, Agri_2 (intermediate to low total P-ARF); (iv) groups 5-7 : BW_1, D1, D2 and
	BD (high total P-ARF and highest inorganic H2O-P); (v) group 6 : Agri_1, Agri_3, fibrous digestates.

Table 1 :

 1 Definition and origins of digestate samples

	Sample	Type	Scale	Anaerobic	Digester	Post-	Substrate	Reference
	Name			Digestion	Hydraulic	treatment
				conditions:	retention
				type (dry or	time
				liquid), feed
				mode (batch or
				continuous) and
				temperature
	Agri_1	digestate	Operating scale	Dry batch	60 days	-	Cow manure
			(farmer)	mesophilic
	Agri_2	digestate	Operating scale	Liquid continuous	60 days	-	Pig manure co-
			(farmer)	mesophilic			digested with
							energetic crop and
							vegetable residues
	Agri_3	digestate	Lab scale	Dry batch	100 days	-	wheat straw silage
				mesophilic
	Sludge_1	Solid	Operating scale	Liquid continuous	20 days	Press filter	wastewater
		phase	(private agency)	mesophilic			treatment sludge
		from
		digestate
	Sludge_2	compost	Operating scale	Liquid continuous	20 days	Press filter	digestate of
		of solid	(private agency)	mesophilic		and	wastewater
		digestate				composting	treatment sludge
							(1/3) and green
							wastes (2/3)	This study
	FFMSW_1	digestate	Operating scale	Dry continous	20 days	-	Fermentable fraction
			(private agency)	thermophilic			from municipal solid
							waste (FFMW)
	FFMSW_2	compost	Operating scale	Dry continuous	20 days	Compostin	96%FFMSW, 50%
		of	(private agency)	thermophilic 20		g	green waste, 14%
		digestate					agro-industrial
							wastes
	BW_1	digestate	Operating scale	Liquid continous	60 days	-	Biowastes (60%)
			(governmental)	mesophilic			from supermarkets
							co-digested with
							agro-industrial
							wastes (28%) and
							crop residues (12%)
	Centr_1	Solid	Operating scale	Liquid continous	45 days	screw press	Oil (20%), crop
		phase of	(governmental)	mesophilic			residues (10%),
		digestate					agro_industrial
	Centr_2	Liquid	Operating scale	Liquid continous	45 days	screw press	wastes (55%),
		phase of	(governmental)	mesophilic			sewage sludge
		digestate					(10%), biowaste
							(5%)
	D1	Digestate	Operating scale	Liquid continous	na	-	Wastewater
				thermophilic			treatment sludge	Grigatti et
	D2	Digestate	Operating scale	Liquid continous	na	-	Wine sludge	al. (2019)
				mesophilic
	BD	digestate	Operating scale	Liquid continous	na	-	Bovine slurry and
				thermophilic			energy crops

Table 2 :

 2 Physico-chemical characteristics of the investigated digestates

	Parameters Units	Agri_1 Agri_2 Agri_3 Sludge_1 Sludge_2 FFMSW_1 FFMSW_2 BW_1	Centr_1 Centr_2 D1	D2	BD
	TS	%	17.1%	4.4%	14%	22.4%	59.1%	19.7%	53.3%	24.9%	26.4%	6% 3.94% 3.09% 5.05%
	VS	%TS	70.2%	70.1%	87.0%	51.7%	48.5%	48.7%	41.8%	79.0%	82.2%	60.0% 58.2% 59.4% 68.3%
	TOC	g kg -1	382.90	404.71	454.83	283.35	258.01	279.30	232.55	438.64	438.17	320.53 344.2 397.8 515.0
	COD	g.kg -1	1104.00 1400.56 1235.50	679.00	726.00	712.09	536.00 1291.00 1245.00 1497.58	nd*	nd	Nd
	TKN**	g.kg -1	25.85	115.00	nd	40.36	26.40	22.20	13.61	29.79	23.33	75.67	nd	nd	nd
	TKN***	g.kg -1	27.98	44.53	17.72	43.06	26.61	17.78	15.39	30.89	28.62	45.47	47.8	44.9	43.0
	C/N		13.68	3.52	25.67	6.58	9.70	15.71	15.11	14.20	15.31	7.05	7.2	8.9	12.0
	NH4 + -N**	g.kg -1	6.28	87.85	2.99	10.80	0.92	10.5	0.04	27.68	6.81	34.74	nd	nd	nd
	P	g.kg -1	5.36	10.37	4.00	20.44	16.15	4.21	4.23	10.79	6.93	17.98	7.1	18.4	6.2
	K	g.kg -1	21.22	27.41	12.98	2.15	4.90	11.49	8.22	7.25	4.86	16.10
	S	g.kg -1	3.59	6.23	1.63	6.23	5.33	2.22	5.00	2.16	6.27	7.58		nd*
	Al	g.kg -1	2.31	3.23	0.65	6.66	12.56	8.68	2.73	8.46	2.68	9.33
	Ca	g.kg -1	18.53	23.08	13.44	69.08	41.25	37.05	29.72	42.31	17.56	27.08	10	37	11
	Fe	g.kg -1	1.58	2.81	0.94	42.60	37.48	5.79	5.06	7.89	11.54	12.07	1.9	8.6	1.0
	Ca/P		0.29	0.27	0.24	2.08	2.32	1.38	1.19	0.73	1.67	0.67	1.4	2.0	1.7
	Mg	g.kg -1	5.49	6.28	1.56	3.52	3.72	3.24	4.29	3.47	1.98	5.13	2.8	11.6	5.2
	References						This study						Grigatti et al. (2019)

.The most N-rich samples (44-48 g kg -1 ) were the liquid phase of the centralised digestate (Centr_2), the pig manure digestate (Agri_2) and the sludge digestates (Sludge_1, D1 and D2). The poorest

Acknowledgements

This project is supported by the Agropolis Fondation under the reference ID 1502-302 through the « Investissements d'avenir » program (Labex Agro:ANR-10-LABX-0001-01) and is part of the EUfunded project AgreenskillsPlus. Authors acknowledge Antoine Haddon for his useful advices.

impacted positively shoots P-ARF, as expected, whereas organic H2O-P fraction (H2O-Po) had a negative impact. Therefore, available form of P for shoots growth was associated to H2O-Pi fraction, as expected.

H2O-Po fraction is accessible but has to be mineralized to be available. Shoots = 0.1153 × H Ž O P ; -0.2777 × H Ž O P : + 0.0249, r²=0.71 Equation 10No correlations were found between ARF results and TP or phosphates concentrations (r=-0.12, p=0.74, r=-0.26, p=0.46 respectively) meaning that the TP concentration is not enough to predict P uptake by plants.

Organic NaHCO3-P fraction (NaHCO3-Po) was positively correlated with roots (r=0.62, p=0.025 with D1, D2 and BD). Organic NaOH-P fraction (NaOH-Po) was also positively correlated with roots (r=0.79, p=0.006). A PLS regression model was found between roots P-ARF and inorganic and organic P content in NaHCO3-P and NaOH-P fractions (Equation 11).

Roots = 0.0955 × NaHCO R P : + 0.0955 × NaOH P : -0.0584 × NaHCO R P ; + 0.0128, r²=0.8641 Equation 11Negative impact of inorganic labile NaHCO R was found whereas organic P forms impacted positively the roots P-ARF. As previously mentioned, phytate and others phosphates esters can be extracted in the NaOH-P fraction [START_REF] He | Phosphorus Distribution in Sequentially Extracted Fractions of Biosolids, Poultry Litter, and Granulated Products[END_REF]. Roots can exude phosphatases able to hydrolyse phytate and organic P forms able to be absorbed by roots [START_REF] Lambers | Root Structure and Functioning for Efficient Acquisition of Phosphorus: Matching Morphological and Physiological Traits[END_REF][START_REF] Gerke | Phytate (Inositol Hexakisphosphate) in Soil and Phosphate Acquisition from Inositol Phosphates by Higher Plants[END_REF].

Interestingly, roots and shoots P recoveries were correlated with different P fractions. Finally, chemical nutrient equivalents were calculated for all the treatments. The obtained values were above 100% except for Agri_2 (35%) and Agri_1 if only shoots were considered. That means that all the studied digestates (except from pig slurry) can substitute chemical P needs.

Similarly to N results, phase separation impacted the P fertilizing potential.


Dr Matthieu Frasca
email: matthieu.frasca@u-bordeaux.fr

Camille Sabathe

Suzette Delaloge

Angeline Galvin

Anne Patsouris

Christelle Levy

Marie Mouret-Reynier

Isabelle Desmoulins

Laurence Vanlemmens

Bachelot T 10

Goncalves A 11

Perotin V 12

Uwer L 13

Frenel Js 14

J M Ferrero

Bouleuc C 15

Eymard Jc 16

Dieras V 17

Leheurteur M 18

Petit T 20

Dalenc F 21

Jaffre A 22

Chevrot M 23

Courtinard C 23

Palliative care delivery according to age in 12,000 women with metastatic breast cancer: Analysis in the multicentre ESME-MBC cohort 2008-2016

Keywords: Palliative care, Palliative medicine, Age, Inpatient care, Metastatic breast cancer

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

With over 2 million new cases in 2018, breast cancer is the most commonly occurring cancer in women and the second most common cancer overall [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]. Almost a third of breast cancers relapse with a distant metastatic lesion, and approximately 5% of breast cancer patients are already metastatic at the time of diagnosis (de novo metastatic) [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]. Patients with metastatic breast cancer (MBC) often require palliative care integrated into the routine comprehensive cancer care [START_REF] Gobbini | Time trends of overall survival among metastatic breast cancer patients in the real-life ESME cohort[END_REF][START_REF] Cardoso | 4th ESO-ESMO International Consensus Guidelines for Advanced Breast Cancer (ABC 4)dagger[END_REF]. Palliative care is an interdisciplinary and patient-centred approach that is aimed at improving the quality of life of patients facing life-threatening illnesses; it involves the treatment of physical, psychosocial, and spiritual problems, providing an advanced care planning and an extra layer of support [START_REF] Sepulveda | Palliative Care: the World Health Organization's global perspective[END_REF]. Many clinical trials and guidelines support the integration of palliative care approaches in patients with advanced cancers [START_REF] Ferrell | Integration of Palliative Care Into Standard Oncology Care: ASCO Clinical Practice Guideline Update Summary[END_REF][START_REF] Temel | Early palliative care for patients with metastatic non-small-cell lung cancer[END_REF](7). Governmental programs have regularly supported the development of inpatient palliative care (IPC) [START_REF] Van Beek | Comparison of legislation, regulations and national health strategies for palliative care in seven European countries (Results from the Europall Research Group): a descriptive study[END_REF][START_REF] Poulalhon | Deaths in France: Characteristics, place of death, hospitalisations and use of palliative care during the year before death[END_REF]. However, data regarding the efficiency of IPC delivery among cancer patients in Europe are limited [START_REF] Frasca | Palliative care versus hospice care in cancer patients. A systematic review[END_REF].

The survival of patients with metastatic breast cancer (MBC) differs among the three main tumoural subtypes, which are hormone receptor-positive (HR+), human epidermal growth factor receptor 2positive (HER2+), and triple-negative breast cancer (HR-/HER2-) [START_REF] Gobbini | Time trends of overall survival among metastatic breast cancer patients in the real-life ESME cohort[END_REF]. A recent study reported that older patients who are diagnosed with metastatic breast cancer have a poorer prognosis than young patients, except in case of triple-negative breast cancer [START_REF] Deluche | Contemporary outcomes of metastatic breast cancer among 22,000 women from the multicentre ESME cohort 2008-2016[END_REF]. Age is also a leading cause of comorbidities and disabilities, posing therapeutic challenges in cancer patients [START_REF] Ferrat | Four Distinct Health Profiles in Older Patients With Cancer: Latent Class Analysis of the Prospective ELCAPA Cohort[END_REF]. The combination of chronic conditions increases the need for IPC delivery in older cancer patients. However, an increasing number of studies have reported a low number of referrals for palliative care in older cancer patients [START_REF] Adsersen | Is admittance to specialised palliative care among cancer patients related to sex, age and cancer diagnosis? A nation-wide study from the Danish Palliative Care Database (DPD)[END_REF][START_REF] Janah | Access to palliative care for cancer patients between diagnosis and death: a national cohort study[END_REF]. The majority of studies have analysed global cancer populations [START_REF] Frasca | Palliative care versus hospice care in cancer patients. A systematic review[END_REF] and failed to compare patients with similar cancer-related needs. The comparison of IPC delivery in young and older patients with similar cancers is essential for MBC patients due to the variable prognosis of the different tumoural subtypes.

Most studies on palliative care delivery have analysed deceased populations, which entails a high risk of selection bias [START_REF] Frasca | Palliative care versus hospice care in cancer patients. A systematic review[END_REF][START_REF] Walshe | Patterns of access to community palliative care services: a literature review[END_REF]. The MBC cohort of the UNICANCER ESME (Epidemio-Strategy-Medical-Economical) programme includes MBC patients primarily treated within the national network of comprehensive cancer centres. Such real-world data could provide a better understanding of the needs of certain patient subgroups as well as the true impact of health care delivery, from early-to late-stage disease. Additionally, most studies have not taken into account the risk of death before palliative care [START_REF] Adsersen | Is admittance to specialised palliative care among cancer patients related to sex, age and cancer diagnosis? A nation-wide study from the Danish Palliative Care Database (DPD)[END_REF][START_REF] Osagiede | Palliative Care Use Among Patients With Solid Cancer Tumors: A National Cancer Data Base Study[END_REF]. Death can introduce bias in the study and should be methodologically considered as a competing risk [START_REF] Grand | Regression models for expected length of stay[END_REF].

The aim of this study was to assess whether age is an independent factor of the cumulative incidence function of IPC delivery in the ESME-MBC cohort (2008 -2018), taking into account the competing risk of death.

Data sources and methods

ESME MBC data platform

The ESME-MBC cohort is a population-based registry, which includes patient data form all ongoing individual national cohorts. We collected real-life retrospective data from consecutive MBC patients treated in 18 French Comprehensive Cancer Centres (FCCC) between 01 January, 2008, and 31 December, 2016. Eligible patients were at least 18-year-old individuals diagnosed with MBC. The selection date corresponds to the date of consultation, surgical operation, or treatment for the management of a histologically proven MSC in one of the participating centres. Our analyses included all ESME patients with available hospitalisation data. Patient information was acquired from medical records and multidisciplinary team meeting reports [START_REF] Gobbini | Time trends of overall survival among metastatic breast cancer patients in the real-life ESME cohort[END_REF]. For the present study, data were collected until the cut-off date (11 October, 2018). This study was authorised by the French data protection authority (registration ID 1704113 and authorisation No. DE-2013-117, NCT03275311).

Definitions

The primary outcome of this study was the IPC delivery, which was identified using the ICD-10 palliative care coding (Z51.5) based on inpatient hospital stays in all French acute care units. This code indicates patients with a non-curable disease, who had received palliative care in beds in a medical ward, regrouped palliative care beds in a ward, or beds in a palliative care unit. The event of interest was the first inpatient stay at the hospital after MBC diagnosis with a Z51.5 code. We considered death as a competing event, potentially preventing the occurrence of IPC delivery.

Duration of IPC was defined as the time between the first IPC delivery and the date of death or lost to follow-up.

Age at MBC diagnosis (< 65 years, ≥ 65 years) was considered the main explanatory factor. The threshold of 65 years is often used to define older patients, and this cut-off was also recommended for breast cancer patients [START_REF] Brain | HER2-targeted treatment for older patients with breast cancer: An expert position paper from the International Society of Geriatric Oncology[END_REF][START_REF] Scotte | Addressing the quality of life needs of older patients with cancer: a SIOG consensus paper and practical guide[END_REF][START_REF] Wildiers | International Society of Geriatric Oncology consensus on geriatric assessment in older patients with cancer[END_REF]. Covariates were the primary tumour subtype (HER2+ or HER2-/HR+ or missing, HER2-/HR-), the metastatic-free interval (MFI: de novo, relapse 6-24 months, relapse ≥ 24 months), the presence of multiple metastatic sites, the presence of visceral metastasis, the centre activity (based on the patient admission number from 2008 to 2016: < 1400, ≥ 1400), and the period of selection (2008-2011; 2012-2016) (e-Table 1). For the ESME-MBC cohort, HER2 and HR status were obtained from existing results about metastatic tissue sampling, if available. In cases where this information was not available, HER2 and HR status were based on the last sampling in early disease.

De novo metastatic disease was defined as patients who had metastases at the time of primary tumour diagnosis or within 6 months (2).

Statistical analysis

The estimation of the risk function of IPC used Aalen-Johansen estimator to take into account the competing risk of death. Kaplan-Meier method was used to analyse patient survival. Our analysis first estimated pseudo values of 2-year and 8-year CIF of IPC for each patient (function "pseudoCI", R package "pseudo"). The pseudo-values of CIF were used to describe the contribution of each patient's characteristics to the CIF of IPC, taking into account the competing risk of death [START_REF] Klein | SAS and R functions to compute pseudo-values for censored data regression[END_REF]. In a second step, linear regression models were used to estimate the mean changes in pseudo-values associated to age at metastatic diagnosis (two models: 2-year and 8-year CIF of IPC). All analyses were adjusted to the tumour subtypes, disease-free interval, number of metastatic sites, presence of visceral metastases, centre activity, and period of selection. Univariate analysis verified the interactions between age and tumour subtype or centre activity. Analyses were performed using the R software, version 3.6.

Results

Patient characteristics, tumour features, and patient survival

The ESME-MBC cohort included 22463 MBC patients diagnosed between 2008 and 2016 in the 18 participating centres. The characteristics of ESME-MBC 2008-2016 cohort have been previously described [START_REF] Deluche | Contemporary outcomes of metastatic breast cancer among 22,000 women from the multicentre ESME cohort 2008-2016[END_REF]. Hospitalisation data were available for 12471 patients. Based on our selection, we excluded 96 patients who had received IPC before admission; hence, our analyses included 12375 patients (Figure 1). The cohort used in our analyses was comparable to the global ESME-MBC 2008-2016 cohort (e-Table 2). The median age of the patients at the time of primary tumour diagnosis was 54 years [Q1-Q3: 45-84], while the median age of the patients at the time of MBC diagnosis was 61 years [Q1-Q3: 51-71] (65+ years group: 5093, 41.2%). Performance status comparison indicated an ambulatory status (score 0-2) for 7353 patients (59.4%). The primary tumours were classified as triplenegative (HER2-/HR-) in 1555 patients (12.6%). De novo MBC was diagnosed in 3541 patients (28.6%), while 8463 patients (68.4%) exhibited tumour relapse 24 months or more after the diagnosis of the primary tumour. Multiple metastatic sites were observed in 23.0% (2836) of patients and visceral involvement occurred in 56.4% (6984) of patients. High-level recruiting centres treated 34.6% (4279) of patients (Table 1). The median overall survival of the analysed cohort was 41.5 months (95% confidence interval [CI], 40.5 -42.4) (Figure 2). As expected, the patients aged 65+ had a decreased survival (<65 yo, 43.5 months; 95% CI, 42.

IPC delivery in the whole cohort

The median follow-up was 41.5 months (95% CI, 40.5 -42.5). During the study period, 2121 patients of the analysed cohort (29.9%) received IPC (27.6% in deceased sub-group, n=6727). The CIF of IPC in the analysed cohort was 10.3% (95% CI, 10.2 -10.4) at 2 years and 24.8% (95% CI, 24.7 -24.8) at 8 years (Figure 4). Initiation of IPC occurred during the first two years after MBC diagnosis in half of the patients (Table 2). For the subgroup of patients who died after IPC (1859), the median time to death after IPC was 20 days (95% CI, 9 -43).

Figure 4: Cumulative incidence of IPC delivery in patients of the ESME-MBC cohort 2008-2016 (n=12375).

Aalen-Johansen method. <0.01 1 Adjusted to the inclusion period 2 For the subgroup of patients with all the reference characteristics (<65 yo, non-triple negative subtype, De Novo MBC, single and non-visceral metastatic disease, included in low-level recruiting centres). 3 Interaction age* centre recruitment: p=0.02

Discussion

The present nationwide study involved analyses using real-world data, providing a more precise insight into the efficiency of IPC delivery in a large cohort of MBC patients with a long-term follow-up.

Our results revealed that IPC is infrequently used in clinical practice, being provided to approximately a quarter of MBC patients and mainly in the month before death. Age is a factor affecting IPC delivery, but this effect depends on tumour subtype and centre activity. Specific subgroups of MBC patients particularly required short-term IPC after diagnosis, for example, young HER2-/HR-patients and older patients with other tumour subtypes. On the other hand, the oldest patients treated in low-level recruiting centres received less long-term IPC than the youngest. As these findings were derived from real-world data, they could be extrapolated to the whole French MBC population [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]. The median OS reported here is higher than that which previous studies, based on the ESME-CSM cohort, have reported (39.5 months), which might be due to the more recent period of inclusion [START_REF] Brain | HER2-targeted treatment for older patients with breast cancer: An expert position paper from the International Society of Geriatric Oncology[END_REF][START_REF] Caswell-Jin | Change in Survival in Metastatic Breast Cancer with Treatment Advances: Meta-Analysis and Systematic Review[END_REF].

Herein, we showed that, over a period of 10 years, the rate of IPC delivery in French MBC patients was higher than in other countries (18.2%) [START_REF] Bramley | The economic burden of endof-life care in metastatic breast cancer[END_REF]. The fact that French palliative medicine services deliver earlier interventions than reported hospice facilities might partially explain this difference. We found that the rate of IPC delivery among MBC patients was lower than that studies based on deceased subgroups have reported in French global cancer population (52%), women with genital cancers (64.6%) or patients with metastatic lung diseases (70.6%) [START_REF] Poulalhon | Deaths in France: Characteristics, place of death, hospitalisations and use of palliative care during the year before death[END_REF][START_REF] Janah | Access to palliative care for cancer patients between diagnosis and death: a national cohort study[END_REF][START_REF] Goldwasser | Timing of palliative care needs reporting and aggressiveness of care near the end of life in metastatic lung cancer: A national registry-based study[END_REF]. This first finding on alive MBC patients highlights the specific needs of MBC patients. The exclusion of IPC delivered outside the FCCC might also partially explain this lower delivery. Regarding IPC integration in clinical practice, we found that IPC was more frequently delivered during the first two years after MBC diagnosis, probably driven by critically ill patients at diagnosis. We also highlighted that the majority of patients received their first IPC during terminal disease. Admittedly, our analyses focused on interdisciplinary interventions excluding palliative care consultations. Although increasing evidence suggests the benefit of early interdisciplinary palliative care in patients with advanced cancer, implementing such practices in clinical routine remains challenging, and requires a deep understanding of the needs and a cautious care planning. Furthermore, our study highlighted the influence of age on the short-term IPC delivery in MBC patients, depending on the tumour subtype. The short-term rate of IPC was higher in young triplenegative MBC patients compared to the oldest, which is consistent with the higher tumour grades' proportion and the lower survival in this subgroup [START_REF] Liedtke | The prognostic impact of age in patients with triple-negative breast cancer[END_REF]. This result might also reflect the lower rate of disease-targeting interventions in older patients with a highly progressive MBC leading to a lower referral to the IPC facilities of cancer centres. On the other hand, older patients with other tumour subtypes received more short-term IPC. This finding is consistent with a recent study that has reported that age was associated with decreased survival in patients with HR+/HER2-and HER2+ subtypes [START_REF] Deluche | Contemporary outcomes of metastatic breast cancer among 22,000 women from the multicentre ESME cohort 2008-2016[END_REF]. The identification of age-related comorbidities might lead to a more frequent referral to palliative care teams [START_REF] Soubeyran | Predictors of early death risk in older patients treated with first-line chemotherapy for cancer[END_REF][START_REF] Frasca | Alterations in comprehensive geriatric assessment decrease survival of elderly patients with cancer[END_REF]. According to these results, clinical trials assessing the short-term impact of age on palliative care delivery in MBC patients should take into account the impact of the different tumour subtypes.

Our analyses also highlighted the link between age and long-term IPC delivery in MBC patients, depending on the centre activity. Eight years after diagnosis, IPC delivery was less frequent in older patients than in young patients in centres with low patient admission number, while in centres with high patient admission number, IPC delivery was comparable. This finding might be explained by a higher rate of referral of older patients to more suitable institutions for follow-up, as well as a lower rate of implementation of IPC and/or geriatrics facilities outside large centres [START_REF] Gobbini | Time trends of overall survival among metastatic breast cancer patients in the real-life ESME cohort[END_REF]. These results suggest an increasing implementation of palliative care services in FCCC, aimed at providing access to IPC to more patients.

Our study provides the oncology community with a novel insight into the efficiency of IPC delivery in MBC patients. To our knowledge, only one study has assessed the economic burden of end-of-life care in MBC patients [START_REF] Bramley | The economic burden of endof-life care in metastatic breast cancer[END_REF]. Another retrospective study has also investigated the 2-year IPC delivery in a French global cancer population [START_REF] Janah | Access to palliative care for cancer patients between diagnosis and death: a national cohort study[END_REF]. The ESME platforms provided the opportunity for analyses involving high-quality, real-life data. The follow-up ensured an adequate analysis of the long-term effects. Moreover, the large number of patients included improved the detection of time-varying effects and differences among the patient subgroups, including the usually under-represented older patients.

Most of the current knowledge is based on studies in deceased cancer patients, assuming the requirement of palliative care. Some cancers may not be the primary cause of care or death, especially in older patients, and may not lead to specialised palliative care delivery. Finally, our analyses took into account the competing risk of death; this strategy improved the accuracy of the results for patients with increased risk of mortality. Contrary to Fine and Gray models, using pseudoobservations enabled us to interpret the estimates quantitatively.

On the other hand, our study failed to consider non-interdisciplinary IPC, such as hospital-based palliative care consultation. The study outcomes also excluded IPC delivery outside the FCCC.

However, our analyses focused on the first IPC delivery, which is often performed by FCCC teams. In addition, some potential confounding factors, including psycho-social data, were not available. Finally, it remains uncertain whether the ESME cohort is representative of all MBC patients, as it only includes patients who received treatment.

Conclusions

In conclusion, our findings provide further insight into the current short-term and long-term medical needs of palliative care. IPC is an emergent but growing practice for MBC patients, yet it is mostly delivered to patients with a terminal disease. Specific subgroups of MBC patients particularly require such interventions, including young patients with triple-negative breast cancer, older patients with other tumour subtypes, and patients treated in large cancer centres. Our study highlighted the challenge for older MBC patients diagnosed outside large FCCC. We believe that taking into account age at the time of MBC diagnosis as a factor of IPC delivery will allow for better design of future interventions. Our findings support better integration of IPC in clinical practice and harmonisation of palliative care accessibility. Further studies are required to include palliative care interventions delivered outside the FCCC, based on national medico-administrative data.

  2 -45.2 vs 65+ yo, 38.4 months; 95% CI, 36.7 -40.2) (Figures 3).

Figure 1 :

 1 Figure 1: Flow chart depicting the selection of the study population from the ESME-MBC cohort 2008-2016.

187 3 4 191 Figure 2 :

 31912 Figure 2: Overall survival of patients in the ESME-MBC cohort 2008-2016 (n = 12375). Kaplan-Meier method.

Figure 3 :

 3 Figure 3: Overall survival of patients in the ESME-MBC cohort 2008-2016 (n = 12375) according to the age of the patients. Kaplan-Meier method.

4 '

 4 De novo' if the metastatic condition was detected at the time of the primary disease diagnosis or within the following 180 days. IPC: Inpatient Palliative Care; MBC: Metastatic Breast Cancer; HER2-: Human Epidermal growth factor receptor 2-negative; HR-: Hormonal Receptor-negative; MFI: Metastatic-Free Interval

Table 1 : Characteristics of the analysed patients of the ESME-MBC cohort 2008-2016 (n=12375).

 1

	184
	Characteristics	Analysis population (n = 12375) N % Med [Q1-Q3]
	Age (years) at diagnosis of the primary tumour		54.0 [45.0-64.0]
	Age (years) at MBC diagnosis		61.0 [51.0-71.0]
	Age groups at the MBC diagnosis
	< 65 years	7282	(58.8)
	≥ 65 years	5093	(41.2)
	Performance status
	Score 0 to 2	7353	(59.4)
	Score 3 to 4	474	(3.8)
	Missing	4548	(36.8)
	Tumour sub-types
	HER2+	2208	(17.8)
	HER2-/HR+	7791	(63.0)
	HER2-/HR-	1555	(12.6)
	Missing	821	(6.6)
	MFI (months) (n=8834) 1		70.7 [32.4-135.6]
	De novo versus relapsed
	De Novo MBC 2	3541	(28.6)
	Relapsed MBC: MFI 6 months -24 months	1464	(11.8)
	Relapsed MBC: MFI ≥ 24 months	7370	(59.6)
	Number of metastatic sites 3
	One	6615	(53.5)
	At least two	5760	(46.5)
	Type of metastases 4
	At least one visceral (excluding brain metastases)	6574	(51.3)
	At least one brain metastases	372	(3.0)
	Non-visceral metastases only (skin and lymph nodes)	1517	(12.3)
	Bone-only metastases	3290	(26.6)
	Others	622	(5.1)
	Visceral metastasis
	No	6574	(53.1)
	Yes	5801	(46.9)
	Centre activity
	< 1400 admissions	8096	(65.4)
	≥ 1400 admissions	4279	(34.6)

MBC: Metastatic Breast Cancer; MFI: Metastatic-Free Interval 185 1 MFI as continuous variable is estimated only among relapsed MBC patients. 186 2 'De novo' if the metastatic condition was detected at the time of the primary disease diagnosis or within the following 180 days.

Table 2 : IPC delivery for each year of follow-up. ESME-MBC cohort 2008-2016 (n=12375).

 2

	Follow-up (Months)	N	%	IPC initiation (n=2121) Cumulative N Cumulative %
	12	700	33.0	700	33.0
	24	503	23.8	1203	56.8
	36	378	17.8	1581	74.6
	48	232	10.9	1813	85.5
	60	166	7.8	1979	93.3
	72	85	4.0	2064	97.3
	84	38	1.8	2102	99.1
	96	15	0.7	2117	99.8
	108	4	0.2	2121	100.0
	IPC: Inpatient Palliative Care

Table 4 : Characteristics associated with 8-year cumulative incidence function of IPC delivery after diagnosis of MBC in ESME-MBC cohort 2008-2016 (n = 12375). Linear regressions with pseudo- observations of cumulative incidence function as outcome.

 4

	Characteristics

Acknowledgements

We thank all patients who allowed us to build the present ESME real-life cohort. We thank the 18 French Comprehensive Cancer Centres for providing the data and each ESME local coordinator for managing the project at the local level. Moreover, we thank the ESME Scientific Committee members for their ongoing support. 18 Participating French Comprehensive Cancer Centres (FCCC): I. Curie, Paris/Saint-Cloud, G. Roussy, Villejuif, I. Cancérologie de l'Ouest, Angers/ Nantes, C. F. Baclesse, Caen, ICM Montpellier, C. L. Bérard, Lyon, C. G-F Leclerc, Dijon, C. H. Becquerel, Rouen; I. C. Regaud, Toulouse; C. A. Lacassagne, Nice; Institut de Cancérologie de Lorraine, Nancy; C. E. Marquis, Rennes; I. Paoli-Calmettes, Marseille; C. J. Perrin, Clermont Ferrand; I. Bergonié, Bordeaux; C. P. Strauss, Strasbourg; I. J. Godinot, Reims; C. O. Lambret, Lille. ESME central coordinating staff:

IPC delivery according to age

The age at admission was associated with both 2-year and 8-year CIF of IPC. The association between age and the 2-year CIF of IPC was dependent on the tumour subtype, whereas the effect of age on the 8-year CIF was dependent on the centre activity. At 2 years, among patients with a triplenegative MBC, those aged under 65 years had a 5% higher CIF of IPC than those aged 65+ (65+/<65: β = -0.05; 95% CI, -0.08 to -0.01), after adjusting to MFI, number of metastatic sites, visceral involvement, centre activity, and period. At the same time, among patients with other tumour subtypes, patients aged 65+ had a 2% higher CIF of IPC than younger patients (65+/<65: β=0.02; 95% CI, 0.01 -0.03), after adjusting to same covariates. At 8 years, among patients treated in low-level recruiting centres, IPC was less frequent in patients aged 65+ years compared with younger patients (65+/<65: β=-0.03; 95% CI, -0.06 to -0.01) after adjusting to tumour subtype, MFI, number of metastatic sites, visceral involvement, and period. At the same time, age is not associated to a reduced 8-year CIF of IPC in high-level recruiting centres (Table 3 andTable 4). <0.01 1 Adjusted to the inclusion period 2 For the subgroup of patients with all the reference characteristics (<65 yo, non-triple negative subtype, De Novo MBC, single and non-visceral metastatic disease, included in low-level recruiting centres). 3 Interaction age*tumour subtype: p<0.01