Noémie Janot

Marc F Benedetti

Pascal E Reiller
email: pascal.reiller@cea.fr.

Colloidal α-Al 2 O 3, Europium(III) and Humic Substances Interactions: A Macroscopic and Spectroscopic Study

Keywords: Humic acid, Europium, Alumina, Luminescence, Sorption, Ternary complex BRIEFS

Influences of humic acid and pH on retention and speciation of Eu(III) in contact with colloidal α-Al 2 O 3 are analyzed by macroscopic batch experiments and time-resolved laser luminescence spectroscopy.

Introduction

Natural organic matter (NOM) interactions with mineral colloids in soils partly control the speciation and migration of heavy metals as well as nanoparticles in the environment, and notably of lanthanides and actinides [START_REF] Mccarthy | Mobilization of Transuranic Radionuclides from Disposal Trenches by Natural Organic Matter[END_REF][START_REF] Mccarthy | Lanthanide Field Tracers Demonstrate Enhanced Transport of Transuranic Radionuclides by Natural Organic Matter[END_REF] . The modeling of ternary systems consisting of metal-NOM-mineral surface has often led to discrepancies. Particularly, the additivity of the binary systems was not fulfilled [START_REF] Christl | Interaction of Copper and Fulvic Acid at the Hematite-Water Interface[END_REF][START_REF] Vermeer | Metal Ion Adsorption to Complexes of Humic Acid and Metal Oxides: Deviations from the Additivity Rule[END_REF] . In some examples, successful modeling exercises were proposed for fulvic acids [START_REF] Weng | Cu2+ and Ca2+adsorption to Goethite in the Presence of Fulvic Acids[END_REF][START_REF] Heidmann | Sorption of Cu and Pb to Kaolinite-Fulvic Acid Colloids: Assessment of Sorbent Interactions[END_REF] . These samples can be assumed to be more or less similar to simple organic molecules [START_REF] Schulthess | Competitive Adsorption of Aqueous Carbonic and Acetic Acids by an Aluminum Oxide[END_REF] . Fulvic acids (FA) sorption is stable or decreases with increasing ionic strength [START_REF] Schlautman | Adsorption of Aquatic Humic Substances on Colloidal-Size Aluminum Oxide Particles: Influence of Solution Chemistry[END_REF][START_REF] Filius | Adsorption of Fulvic Acid on Goethite[END_REF][START_REF] Weng | Adsorption of Humic Substances on Goethite: Comparison between Humic Acids and Fulvic Acids †[END_REF] , whereas sorption of humic acids (HA) onto oxides increases with ionic strength [START_REF] Schlautman | Adsorption of Aquatic Humic Substances on Colloidal-Size Aluminum Oxide Particles: Influence of Solution Chemistry[END_REF][START_REF] Weng | Adsorption of Humic Substances on Goethite: Comparison between Humic Acids and Fulvic Acids †[END_REF][START_REF] Murphy | Interaction of Hydrophobic Organic Compounds with Mineral-Bound Humic Substances[END_REF] . Hence, it is still difficult to propose interpretations of HA effects on the modeling of ternary systems in a wide parametric space [START_REF] Vermeer | Metal Ion Adsorption to Complexes of Humic Acid and Metal Oxides: Deviations from the Additivity Rule[END_REF][START_REF] Lippold | Effect of Humic Matter on Metal Adsorption onto Clay Materials: Testing the Linear Additive Model[END_REF] . Moreover, heterogeneous composition and nature of HA [START_REF] Maccarthy | THE PRINCIPLES OF HUMIC SUBSTANCES[END_REF][START_REF] Rice | Fractal Nature of Humic Materials[END_REF] , and constant modification of HA aggregates with physico-chemical conditions of the solution [START_REF] Conte | Conformational Arrangement of Dissolved Humic Substances. Influence of Solution Composition on Association of Humic Molecules[END_REF] or due to sorption [START_REF] Hur | Molecular Weight Fractionation of Humic Substances by Adsorption onto Minerals[END_REF][START_REF] Claret | Fractionation of Suwannee River Fulvic Acid and Aldrich Humic Acid on α-Al 2 O 3 : Spectroscopic Evidence[END_REF] do not ease the modeling of these systems. The accurate understanding and description of the HAcontaining systems are still under discussion. A better description of these systems is still needed to improve the understanding of the behavior of metals and nanoparticles in the environment.

One way to obtain both macroscopic and spectroscopic information on Ln/An(III) sorption onto mineral surfaces is through time-resolved laser-induced luminescence spectroscopy (TRLS). Due to their luminescence properties, one can have a direct insight on speciation of the ions in solution, in contact with humic substances [START_REF] Thomason | Fluorescence Studies of Metal-Humic Complexes with the Use of Lanthanide Ion Probe Spectroscopy[END_REF][START_REF] Kumke | Fluorescence Quenching and Luminescence Sensitization in Complexes of Tb 3+ and Eu 3+ with Humic Substances[END_REF][START_REF] Brevet | Spectral and Temporal Luminescent Properties of Eu(III) in Humic Substance Solutions from Different Origins[END_REF][START_REF] Marang | Spectroscopic Characterization of the Competitive Binding of Eu(III), Ca(II), and Cu(II) to a Sedimentary Originated Humic Acid[END_REF][START_REF] Reiller | Bi-Exponential Decay of Eu(III) Complexed by Suwannee River Humic Substances: Spectroscopic Evidence of Two Different Excited Species[END_REF][START_REF] Marang | Combining Spectroscopic and Potentiometric Approaches to Characterize Competitive Binding to Humic Substances[END_REF] as well as sorbed onto mineral surfaces [START_REF] Huittinen | Sorption of Cm(III) and Gd(III) onto Gibbsite, α-Al(OH)3: A Batch and TRLFS Study[END_REF][START_REF] Rabung | 1021/es102592a Part 1: Batch Sorption and Time-Resolved Laser Fluorescence Spectroscopy Experiments[END_REF] or in ternary systems [START_REF] Tan | Sorption of Eu(III) on Humic Acid or Fulvic Acid Bound to Hydrous Alumina Studied by SEM-EDS, XPS, TRLFS, and Batch Techniques[END_REF] . Measurements can be made at low metal ion concentration, to get as close as possible to environmental concentrations. TRLS has been used in either combination of the ternary systems, but up to now no direct comparison has been done between the two binary systems and the corresponding ternary system in a whole range of pH.

The aim of this study was to investigate the influence of Aldrich humic acid on the behavior and the speciation of a lanthanide, Europium(III), as a chemical analog of An(III), in presence or absence of α-Al 2 O 3 . Aluminum oxides are not the more frequent mineral surfaces found in the environment, but their aluminol surface sites are part of aluminosilicates reactive sites, very abundant in natural systems. Besides, its transparency for light allows spectroscopic measurements with mineral in solution, contrary to iron oxides [START_REF] Huittinen | Sorption of Cm(III) and Gd(III) onto Gibbsite, α-Al(OH)3: A Batch and TRLFS Study[END_REF] . Macroscopic and spectroscopic experiments have been carried out to see the influence of pH on the evolution of the different systems.

Experimental Section

Materials Commercial Aldrich humic acid was purified (PAHA) according to ref 27. Stock suspension at 1 g/L has been prepared by diluting PAHA in NaOH (pH around 10) in order to completely dissolve the sample. The solution was then stored in the dark under N 2 . Proton exchange capacity was determined by potentiometry and found at 6.12 eq/kg [START_REF] Janot | Using Spectrophotometric Titrations To Characterize Humic Acid Reactivity at Environmental Concentrations[END_REF] . Alumina (α-Al 2 O 3 ) was purchased from Interchim (pure 99.99%, size fraction 200-500 nm). The solid was washed thrice with carbonate-free NaOH and thrice with Millipore water before drying and storage at room temperature under N 2 atmosphere according to ref 29. The specific surface area measured by N 2 -BET method was 15 m²/g. Point of zero charge was determined by acid-base titrations and found at pH = 9.4 (data not shown). Europium(III) stock solution (10 -3 S1). First solid α-Al 2 O 3 , then PAHA and Eu(III) stock solutions and finally freshly degazed electrolyte solutions were weighed, with no specific contact time applied between additions. To minimize carbonation of the systems, nitrogen was swept on the top of the solution before closing the tubes. Ionic strength was fixed at 0.1 mol/L NaClO 4 . The pH values of the solutions were adjusted by addition of fresh 0.1 mol/L HClO 4 and NaOH solutions. They were measured using one combined glass electrode (Radiometer Analytical) connected to a Radiometer Analytical pH Meter. To prevent KClO 4 precipitation in the electrode frit, the electrode filling solution was modified with NaCl at 0.1 mol/L. The pH electrode was calibrated using buffer solutions (pH 4.01, 7.01, and 10.00). The solutions were applied to three dimensional stirring for 3 days of equilibration at room temperature before pH measurements and analyses.

After equilibration, subsamples (2.4 ml) were taken from the centrifugation tubes and analyzed in quartz cuvettes (10 mm). Preliminary experiments showed no significant differences between aliquots taken from one same tube. Eu(III) laser-induced properties were studied in contact with either PAHA, or α-Al 2 O 3 , or both components using TRLS. The excitation laser beam was generated by a 355 nm tripled output of a Continuum Nd-YAG laser, coupled to an optical parametric oscillator system (Panther II, Continuum, USA). The time-resolved luminescence signal is collected at 90° and focused into an Acton spectrometer (slit 1 mm) equipped with 600 lines/mm grating. For the time-resolved detection, the luminescence signal was collected during a gate width L = 300 µs, at an initial gate delay D = 10 µs after the excitation by a laser flash. To increase the signal to noise ratio, 1000 accumulations were performed for each spectrum. To avoid the aggregation and settling of the samples occurring in α-Al 2 O 3 -containing systems, these samples were manually shaken between two measurements. All luminescence measurements were performed at ambient temperature (21°C ± 2). The excitation wavelength was set at λ exc = 393.8 nm. The observed luminescence corresponds to the transitions of the 5 D 0 excited state to the ground 7 F j manifold [START_REF] Bünzli | Luminescent Probes[END_REF] . They are the 5 D 0 → 7 F 0 forbidden for magnetic and electric reasons (λ max ≈ 580 nm), the 5 D 0 → 7 F 1 magnetic dipole transition (λ max ≈ 593 nm), and the 5 D 0 → 7 F 2 "hypersensitive" transition (λ max ≈ 615 nm), [START_REF] Judd | Optical Absorption Intensities of Rare-Earth Ions[END_REF][START_REF] Ofelt | Intensities of Crystal Spectra of Rare-Earth Ions[END_REF] (SI Figures S1 andS2).

To characterize adsorbed amounts of PAHA and Eu(III) onto α-Al 2 O 3 , after equilibration the solutions were ultra-centrifuged at 60 000 rpm during 2 h. Concentration of PAHA in the supernatant was measured by a Shimadzu TOC-VCSH analyzer, calibrated in concentration of PAHA between 0 and 50 mg HA /L. The quantification of non-sorbed Eu(III) was made using TRLS using the standard addition method [START_REF] Berthoud | Direct Determination of Traces of Lanthanide Ions in Aqueous Solutions by Laser-Induced Time-Resolved Spectrofluorimetry[END_REF] , diluting an aliquot of the supernatant in K 2 CO 3 3 mol/L. This method allow determination of Eu(III) concentration down to 10 -9 mol/L.

Results and Discussion

Macroscopic Adsorption Experiments Figure 1 shows the influences of pH and Eu(III) on PAHA adsorption onto α-Al 2 O 3 . The humic acid sorption is typically decreasing when pH increases and α-Al 2 O 3 surface becomes less positively charged, as for other oxides [START_REF] Schlautman | Adsorption of Aquatic Humic Substances on Colloidal-Size Aluminum Oxide Particles: Influence of Solution Chemistry[END_REF][START_REF] Reiller | Retention Behaviour of Humic Substances onto Mineral Surfaces and Consequences upon Thorium (IV) Mobility: Case of Iron Oxides[END_REF][START_REF] Varadachari | Complexation of Humic Substances with Oxides of Iron and Aluminum[END_REF] . Adsorption is nevertheless significant throughout the pH range, going from 1.24 mg HA /m² at pH 4.0 (i.e. 76% of initial amount of PAHA) to 0.61 mg HA /m² at pH 8.8 (i.e. 37%).

The presence of Eu(III) seems to homogeneously increase the retention of the humic substance onto α-Al 2 O 3 surface throughout the pH range studied. For the ternary system, PAHA sorption goes from 1.60 mg HA /m² at pH 3.9 (i.e. 88%) to 0.69 mg HA /m² at pH 9.6 (i.e. 38%).

Schlautman and Morgan reported an increase of HA sorption onto γ-Al 2 O 3 in presence of Ca 2+ between pH 4 and 10 for different coverage rates (from 0.1 to 5.6 mg HA /m²) in 0.1 mol/L NaCl, but did not observe this increase in their Ca/FA/alumina system [START_REF] Schlautman | Adsorption of Aquatic Humic Substances on Colloidal-Size Aluminum Oxide Particles: Influence of Solution Chemistry[END_REF] . Krepelova et al. also noticed this increase in sorption of HA in a U(VI)/HA/kaolinite system (initial ratio of 0.32 mg HA /m²), but only at 0.01 mol/L NaClO 4 and not at 0.1 mol/L 37 . The same trend was reported by Christl and Kretzschmar when studying fulvic acid sorption in a Cu/FA/hematite system in 0.01 NaNO 3 media (initial ratio of 0.68 mg FA /m²), but not at 0.03 and 0.1 mol/L 3 . Weng et al. observed an increase of FA sorption in a Cu/FA/goethite system at 0.1 mol/L NaNO 3 for two initial FA concentrations, i.e 0.13 and 0.80 mg FA /m² [START_REF] Weng | Cu2+ and Ca2+adsorption to Goethite in the Presence of Fulvic Acids[END_REF] . This latter value is closer to our organic matter-tomineral ratio, i.e. 1.86 mg HA /m². However, the direct comparison between HA and FA may not be straightforward regarding the influence of ionic strength on FA and HA sorption [START_REF] Schlautman | Adsorption of Aquatic Humic Substances on Colloidal-Size Aluminum Oxide Particles: Influence of Solution Chemistry[END_REF][START_REF] Filius | Adsorption of Fulvic Acid on Goethite[END_REF][START_REF] Weng | Adsorption of Humic Substances on Goethite: Comparison between Humic Acids and Fulvic Acids †[END_REF][START_REF] Murphy | Interaction of Hydrophobic Organic Compounds with Mineral-Bound Humic Substances[END_REF] .This increase of HS adsorption in ternary systems is often interpreted as the formation of a metal bridge between the negatively charge surface and humic binding sites, which is unlikely at pH 4, i.e. before the sorption edge of Eu(III). Schlautman and Morgan attribute this increase at low to neutral pH to hydrophobic bonding, polyvalent ions screening the repulsive charges of the humic molecule at the mineral surface and thus enhancing HA adsorption [START_REF] Schlautman | Adsorption of Aquatic Humic Substances on Colloidal-Size Aluminum Oxide Particles: Influence of Solution Chemistry[END_REF] . One could also propose the variation of humic conformation induced by ionic strength [START_REF] Avena | Volume and Structure of Humic Acids Studied by Viscometry PH and Electrolyte Concentration Effects[END_REF][START_REF] Rice | Applications of Light and X-Ray Scattering to Characterize the Fractal Properties of Soil Organic Matter[END_REF] or agglomeration induced by 4/12 dx.doi.org/10.1021/es102592a complexation of Ln(III) [START_REF] Caceci | Investigation of Humic Acid Samples from Different Sources by Photon Correlation Spectroscopy[END_REF][START_REF] Plaschke | Combined AFM and STXM in Situ Study of the Influence of Eu(III) on the Agglomeration of Humic Acid[END_REF] . The ratios between the different components, as well as the metal charge, may also play a major role.

Figure 2 shows the influence of pH and PAHA on Eu(III) adsorption onto α-Al 2 O 3 . In the binary Eu(III)/α-Al 2 O 3 system, the adsorption curve shows the expected sorption-edge around pH 7 for Ln/An(III) [START_REF] Alliot | Effect of Aqueous Acetic, Oxalic and Carbonic Acids on the Adsorption of Americium onto α-Alumina[END_REF][START_REF] Fairhurst | The Influence of Humic Acid on Europium-Mineral Interactions[END_REF][START_REF] Fairhurst | The Influence of Humic Acid on the Adsorption of Europium onto Inorganic Colloids as a Function of PH[END_REF][START_REF] Righetto | Competitive Actinide Interactions in Colloidal Humic Acid-Mineral Oxide Systems[END_REF] , increasing from 3.5 × 10 -6 mmol Eu /m² retained at pH 4.4, that is, 5% of initial amount of Eu(III), to 6.4 × 10 -6 mmol Eu /m² at pH 9.8 (i.e. ≈100%). Inorganic speciation of Eu(III) using data from Hummel et al. 45 is shown in SI Figure S3a. Eu 3+ is predominant up to pH 7, and then hydrolyzed species become the predominant ones. Eu(III) adsorption increases just before its first hydrolysis, as previously observed by Bradbury and Baeyens onto illite [START_REF] Bradbury | Sorption Modelling on Illite Part I: Titration Measurements and the Sorption of Ni, Co, Eu and Sn[END_REF] . They have shown that surface complexation of metals is closely related to their first hydrolysis constants [START_REF] Bradbury | Sorption Modelling on Illite. Part II: Actinide Sorption and Linear Free Energy Relationships[END_REF] . As expected, presence of HA changes Eu(III) behavior: at pH < 6, PAHA enhances Eu(III) retention onto the surface [START_REF] Fairhurst | The Influence of Humic Acid on Europium-Mineral Interactions[END_REF][START_REF] Fairhurst | The Influence of Humic Acid on the Adsorption of Europium onto Inorganic Colloids as a Function of PH[END_REF] . Around their first acidity constants, PAHA molecules, negatively charged, adsorb onto the positively charged oxide surface at low pH values, and Eu(III)-PAHA complexes adsorb onto the surface. The Eu(III)-PAHA speciation under our experimental conditions determined by ECOSAT [START_REF] Keizer | a Computer Program for the Calculation of Chemical Speciation and Transport in Soil-Water Systems[END_REF] software and generic data from Milne [START_REF] Milne | Generic NICA-Donnan Model Parameters for Metal-Ion Binding by Humic Substances[END_REF] is shown in SI Figure S3b.

At pH greater than 7, the presence of PAHA is slightly decreasing Eu adsorption compared to the binary system, as previously observed in other metal-oxide systems [START_REF] Tan | Sorption of Eu(III) on Humic Acid or Fulvic Acid Bound to Hydrous Alumina Studied by SEM-EDS, XPS, TRLFS, and Batch Techniques[END_REF][START_REF] Reiller | Retention Behaviour of Humic Substances onto Mineral Surfaces and Consequences upon Thorium (IV) Mobility: Case of Iron Oxides[END_REF][START_REF] Fairhurst | The Influence of Humic Acid on Europium-Mineral Interactions[END_REF][START_REF] Fairhurst | The Influence of Humic Acid on the Adsorption of Europium onto Inorganic Colloids as a Function of PH[END_REF][START_REF] Reiller | Influence of Addition Order and Contact Time on Thorium(IV) Retention by Hematite in the Presence of Humic Acids[END_REF] . This is commonly thought to be due to the progressive decrease of HA sorption with increasing pH and the resulting competition for Eu(III) between adsorbed HA and HA in solution. At pH ≥ 8, the adsorption of Eu(III) onto the surface increases slightly again, resulting from the competition of Eu(III) between α-Al 2 O 3 surface, surface-adsorbed PAHA and dissolved PAHA. Error bars correspond to 2σ. Eu(III) luminescence spectra and decay times in the different systems were analyzed by TRLS at different pH. The peak shapes and asymmetry ratios, i.e. 5 D 0 → 7 F 2 to 5 D 0 → 7 F 1 ( 7 F 2 / 7 F 1 ), provide information on Eu(III) speciation [START_REF] Bünzli | Luminescent Probes[END_REF] . The calculation of ratios avoids us the necessity to take into account light attenuation for comparing the different systems, as this information is independent from the total luminescence recorded. The evolution of these ratios depending on pH is shown in Figure 3 for the different systems. Luminescence decay times have been measured with delay steps of 10 to 25 µs. They are plotted against pH for the different systems in Figure 4. They have been adjusted to the decay of 5 D 0 → 7 F 1 transition area (between 585 and 600 nm) [START_REF] Kumke | Fluorescence Quenching and Luminescence Sensitization in Complexes of Tb 3+ and Eu 3+ with Humic Substances[END_REF][START_REF] Brevet | Spectral and Temporal Luminescent Properties of Eu(III) in Humic Substance Solutions from Different Origins[END_REF][START_REF] Reiller | Bi-Exponential Decay of Eu(III) Complexed by Suwannee River Humic Substances: Spectroscopic Evidence of Two Different Excited Species[END_REF] , as it is less sensitive to solution conditions [START_REF] Judd | Optical Absorption Intensities of Rare-Earth Ions[END_REF][START_REF] Ofelt | Intensities of Crystal Spectra of Rare-Earth Ions[END_REF] .

5/12

Binary Systems. As seen in Figure 3, the 7 F 2 / 7 F 1 ratio in Eu(III)/α-Al 2 O 3 system increases from 0.6 at pH 4.4 to 2.0 at pH 8.8. When pH increases, the possibility for Eu(III) to form surface complexes on α-Al 2 O 3 is also increasing, as seen in macroscopic data. The 5 D 0 → 7 F 0 transition appears for pH > 5.5, indicating a loss of symmetry in the ion environment above this pH value, which corresponds to the beginning of Eu(III) adsorption (Figure 2). Mono-exponential luminescence decay time () of Eu(III) ions in binary Eu(III)/α-Al 2 O 3 system increases with pH, from = 120 ± 3 µs at pH 4.4 to = 277 ± 5 µs at pH 8.8 (Figure 4). A greater luminescence decay time means that there is a higher probability for an excited Eu ion to emit a photon than to lose energy through other nonradiative pathways, i.e. quenching by water molecules, -OH groups, and -NH groups. The increase of is interpreted as the substitution of water molecules in the first coordination sphere of the luminescent ion [START_REF] Horrocks | Lanthanide Ion Probes of Structure in Biology. Laser-Induced Luminescence Decay Constants Provide a Direct Measure of the Number of Metal-Coordinated Water Molecules[END_REF][START_REF] Supkowski | On the Determination of the Number of Water Molecules, q, Coordinated to Europium(III) Ions in Solution from Luminescence Decay Lifetimes[END_REF] . Here, the increase of τ from 120 µs to 277 µs would correspond to the 6/12 Nevertheless, this number must be taken carefully, as previous studies have shown that the relationship between luminescence decay time and the number of surrounding water molecules may depend on the system studied [START_REF] Takahashi | Formation of Outer-and Inner-Sphere Complexes of Lanthanide Elements at Montmorillonite-Water Interface[END_REF] . One should better consider the more complete relationship determined otherwise [START_REF] Horrocks | Lanthanide Ion Probes of Structure in Biology. Laser-Induced Luminescence Decay Constants Provide a Direct Measure of the Number of Metal-Coordinated Water Molecules[END_REF][START_REF] Supkowski | On the Determination of the Number of Water Molecules, q, Coordinated to Europium(III) Ions in Solution from Luminescence Decay Lifetimes[END_REF] , which requires the measure of the decay time in D 2 O in all the three systems, which was out of the scope of this study.

In Eu(III)/PAHA systems, 7 F 2 / 7 F 1 increases from 2.6 at pH 4.1 to 4.6 at pH 8.5 (Figure 3). There is apparently a continuous evolution of the chemical environment of Eu(III) throughout the pH range. This could be linked to the increase of Eu(III) binding to PAHA with increasing pH and the evolution of the type of bonding of Eu(III) within the humic structure [START_REF] Morgenstern | The Formation of Mixed-Hydroxo Complexes of Cm(III) and Am(III) with Humic Acid in the Neutral PH Range[END_REF] . The evolution parallels the Eu(III)/α-Al 2 O 3 system at a different magnitude, with 7 F 2 / 7 F 1 > 1 at all pH. In presence of PAHA, the 5 D 0 → 7 F 0 transition is always present (as in SI Figure S2a at pH 4.1). Together with the fact that the 5 D 0 → 7 F 2 peak is the most prominent one at all pH, this means that Eu(III) is always bound to the humic acid.

A typical bi-exponential luminescence decay is recorded throughout the pH range, with a short luminescence decay time ( 1 < 70 µs) and a longer one (120 <  2 < 250 µs), as previously seen [START_REF] Kumke | Fluorescence Quenching and Luminescence Sensitization in Complexes of Tb 3+ and Eu 3+ with Humic Substances[END_REF][START_REF] Brevet | Spectral and Temporal Luminescent Properties of Eu(III) in Humic Substance Solutions from Different Origins[END_REF][START_REF] Marang | Spectroscopic Characterization of the Competitive Binding of Eu(III), Ca(II), and Cu(II) to a Sedimentary Originated Humic Acid[END_REF][START_REF] Reiller | Bi-Exponential Decay of Eu(III) Complexed by Suwannee River Humic Substances: Spectroscopic Evidence of Two Different Excited Species[END_REF] (Figure 4). The first decay time is shorter than free aqueous Eu 3+ (τ ≈ 114 µs) [START_REF] Horrocks | Lanthanide Ion Probes of Structure in Biology. Laser-Induced Luminescence Decay Constants Provide a Direct Measure of the Number of Metal-Coordinated Water Molecules[END_REF] and independent of pH, which suggests that this phenomenon barely depends on the chemical environment of Eu(III) and may be due to a fast exchange between Eu(III) and humic acid [START_REF] Reiller | Bi-Exponential Decay of Eu(III) Complexed by Suwannee River Humic Substances: Spectroscopic Evidence of Two Different Excited Species[END_REF][START_REF] Freyer | Formation of Cm Humate Complexes in Aqueous Solution at PHc 3 to 5.5: The Role of Fast Interchange[END_REF] Ternary System. A comparable ternary system was studied by Tan et al. by TRLS at pH 6.3 on hydrous alumina (mixture of bayerite, boehmite, and diaspore) and Chinese soil HA/FA [START_REF] Tan | Sorption of Eu(III) on Humic Acid or Fulvic Acid Bound to Hydrous Alumina Studied by SEM-EDS, XPS, TRLFS, and Batch Techniques[END_REF] . The direct comparison of the 7 F 2 / 7 F 1 ratios may not be done [START_REF] Brevet | Spectral and Temporal Luminescent Properties of Eu(III) in Humic Substance Solutions from Different Origins[END_REF] , but the trends can be compared. Recalculated 7 F 2 / 7 F 1 from Tan et al. [START_REF] Tan | Sorption of Eu(III) on Humic Acid or Fulvic Acid Bound to Hydrous Alumina Studied by SEM-EDS, XPS, TRLFS, and Batch Techniques[END_REF] are equal to 2.0 in their Eu/hydrous alumina system and 5.0 in their Eu/HA system. In their ternary system, this value is equal to 4.8, which is similar to the Eu/HA binary system. Therefore, no change in Eu(III) environment is expected between the HAcontaining systems.

Here this behavior is probed for a wider pH range.

The 7 F 2 / 7 F 1 ratios of ternary Eu(III)/PAHA/α-Al 2 O 3 system are much more important than in Eu(III)/α-Al 2 O 3 system, and are slightly increasing from 3.0 at pH 4.3 to 4.0 at pH 7.6 (Figure 3). Above pH 7.6, the peak ratio is constant, but there is a shift from 615 to 617 nm of 5 D 0 → 7 F 2 maxima accompanied with a change in peak shape (SI Figure S4), indicating a further change in Eu(III) chemical environment. At pH < 7, the 7 F 2 / 7 F 1 ratios of binary Eu(III)/PAHA or ternary Eu(III)/PAHA/α-Al 2 O 3 systems are very close, as it was for Tan et al. at pH 6.3 [START_REF] Tan | Sorption of Eu(III) on Humic Acid or Fulvic Acid Bound to Hydrous Alumina Studied by SEM-EDS, XPS, TRLFS, and Batch Techniques[END_REF] . It seems that Eu(III) has a very similar environment in both systems, whether it is in binary Eu(III)/PAHA or sorbed with PAHA onto α-Al 2 O 3 in ternary system. This would suggest that throughout this pH range Eu(III) is directly linked to sorbed humic aggregates and has a limited access to the aluminol surface sites. Conversely, Tan et al. [START_REF] Tan | Sorption of Eu(III) on Humic Acid or Fulvic Acid Bound to Hydrous Alumina Studied by SEM-EDS, XPS, TRLFS, and Batch Techniques[END_REF] reported based on XPS that Eu(III) was fixed to both components in the ternary system. This is not apparent here, as it was not in their TRLS spectra.

As shown in Figure 4, there is a bi-exponential decay in the ternary system (see SI Figure S5), which first decay time is directly comparable to the one in Eu(III)/PAHA system. This fast deexcitation is another direct indication that Eu(III) is strongly linked to sorbed humic aggregates.

These results also suggest that sorbed Eu(III) in the ternary system is always bound to the sorbed humic acid entities, even at high pH when humic acid sorption is the lowest (Figure 1).

The values of the longer decay time  2 , from 382 ± 55 µs at pH 4.4 to 308 ± 25 µs at pH 8.0, are more important for the ternary system than for both binary systems, i.e. between 120 and 212 µs. This is not directly comparable to the results from Tan et al. [START_REF] Tan | Sorption of Eu(III) on Humic Acid or Fulvic Acid Bound to Hydrous Alumina Studied by SEM-EDS, XPS, TRLFS, and Batch Techniques[END_REF] who observed a decrease in the decay time between the binary Eu/hydrous alumina, i.e. 219 µs, and ternary, i.e. 163 µs; and an increase between the binary Eu/HA, i.e. 111 µs, and ternary. These authors did not report the typical bi-exponential decay in the presence of HA, maybe because of too long delay step (viz. 15 µs for Eu/HA, and 35 µs for Eu/HA/hydrous alumina), which already had precluded the determination of bi-exponential decay in past studies [START_REF] Plancque | Europium Speciation by Time-Resolved Laser-Induced Fluorescence[END_REF] . Nevertheless, the increase of luminescence decay time between the binary Eu/HA and ternary Eu/HA/hydrous alumina system was observed both in Tan et al. [START_REF] Tan | Sorption of Eu(III) on Humic Acid or Fulvic Acid Bound to Hydrous Alumina Studied by SEM-EDS, XPS, TRLFS, and Batch Techniques[END_REF] and in this study. One can also compare the results on the basis of the mass of HA per surface of oxide in both systems. Tan et al. [START_REF] Tan | Sorption of Eu(III) on Humic Acid or Fulvic Acid Bound to Hydrous Alumina Studied by SEM-EDS, XPS, TRLFS, and Batch Techniques[END_REF] used 0.02 mg HA /m² (10 mg HA /L for 4.4 g/L hydrous alumina with specific area of 105 m²/g) when we were using 1.86 mg HA /m². The lesser amount of mineral surface could be an explanation for these differences, as well as for the interaction with alumina evidenced by XPS [START_REF] Tan | Sorption of Eu(III) on Humic Acid or Fulvic Acid Bound to Hydrous Alumina Studied by SEM-EDS, XPS, TRLFS, and Batch Techniques[END_REF] .

Assuming that τ 1 is due to a fast exchange mechanism and τ 2 is due to complexation environment, according to reference 52 it would mean that there is only 2 water molecules remaining in the first coordination sphere of Eu(III) in Eu(III)/PAHA/α-Al 2 O 3 system at pH 4.4, when there would be 7 in the Eu(III)/PAHA system at the very same pH. Knowing the reserves on the applicability of this relationship to this particular case we cannot take this value for granted.

At low pH, the spectrum in the ternary system is almost the same as the spectrum of Eu-PAHA complex, and very different from the one of Eu(III) in contact with α-Al 2 O 3 (SI Figure S2a). The spectroscopic and macroscopic data lead us to think that, at pH < 7, Eu-PAHA complexes are sorbed onto α-Al 2 O 3 surface. The increase in τ 2 may then be due to a more constrained environment of Eu(III), but of the same symmetry, in the sorbed humic structure, which may decrease the probability of the Eu(III) ions to lose energy through quenching moieties.

When pH is increasing, the slower decay time τ 2 decreases from 382 µs at pH 4.4 to 185 µs at pH 9.6, which means that there is a greater probability of Eu(III) to be submitted to quenching process when PAHA is less sorbed onto the surface, or that Eu is progressively more exposed to quenching groups. One can also consider the fractionation of humic acid during sorption. After the pH-edge there is a small decrease in Eu(III) sorption in the ternary system (Figure 2). This is in fact reflecting the progressive change in the structure of Eu(III)/PAHA sorbed onto the surface and the competition of Eu(III) with dissolved PAHA. At pH greater than 8.5, the 5 D 0 → 7 F 1 transition of the binary Eu(III)/α-Al 2 O 3 and the ternary systems are the same (SI Figure S2b), giving indication that alumina has a direct influence on Eu(III) environment. Above pH 8, influence of the mineral surface and possible ternary complexes should be taken into account when trying to model such a system.

Figure 1 .

 1 Figure 1. Surface concentration of PAHA on α-Al 2 O 3 depending on pH, with () or without (◊) Eu(III). C(Alumina) = 1 g/L, C(PAHA) initial = 26.5 ± 2 mg/L, [Eu(III)] initial = 10 -6 mol/L, I = 0.1 mol/L NaClO 4 . Error bars correspond to 2σ.

Figure 2 .

 2 Figure 2. Surface concentration of Eu(III) on α Al -2 O 3 depending on pH, with (•) or without (□) PAHA. C(Alumina) = 1 g/L, C(PAHA) initial = 28 mg/L, [Eu(III)] initial = 10 -6 mol/L, I = 0.1 mol/L NaClO 4 .Error bars correspond to 2σ.

Figure 3 .

 3 Figure 3. Evolution of 5 D 0 → 7 F 2 / 5 D 0 → 7 F 1 ratio depending on pH: C(α Al -2 O 3 ) = 1 g/L, C(PAHA) initial = 28 mg/L, [Eu(III)] initial = 10 -6 mol/L, I = 0.1 mol/L NaClO 4 .

Figure 4 .

 4 Figure 4. Evolution of Eu(III) luminescence decay times in contact with PAHA, α Al -2 O 3 or both compounds depending on pH; C(Alumina) = 1 g/L, C(PAHA) initial = 28 mg/L, [Eu(III)] initial = 10 -6 mol/L, I = 0.1 mol/L NaClO 4 ; Error bars correspond to 2σ.

  mol/L) was obtained from the dissolution of Eu 2 O 3 (Johnson Matthey, 99.99%) in HClO 4 . All solutions were prepared using freshly boiled milli-Q water.

	Methods Batch contact experiments were
	conducted in 10.4 ml Beckman centrifugation
	tubes (355603). The different concentrations of
	compounds were 1 g/L of α-Al 2 O 3 , 10 -6 mol/L of
	Eu(III) and 28 mg/L of PAHA, which assured a
	complete complexation of Eu(III) by PAHA (see
	refs 23 and 30 confirmed by Supporting
	Information (SI) Figure

  . When pH increases,  2 continuously increases, from 137 ± 2 µs at pH 4.4 to 246 ± 42 µs at pH 8.5, which means that water molecules are progressively excluded from the coordination sphere of Eu(III) when pH increases.

							τ Eu/Al2O3
		400					τ2 Eu/PAHA τ1 Eu/PAHA
							τ2 Eu/PAHA/Al2O3
	Luminescence decay time (µs)	100 150 200 250 300 350					τ1 Eu/PAHA/Al2O3 τ Eu
		50
		0
		4	5	6	7	8	9	10
					pH
					7/12

Acknowledgments This work was financed through the MRISQ (DEN/DISN/AvC) project of CEA. This is IPGP contribution 3089.

Supporting Information Available

Five figures show Eu(III) luminescence spectra in contact with different concentration of PAHA at pH ≈ 4, Eu(III) luminescence spectra in the different systems at pH ≈ 4.2 and 8.7, inorganic and humic Eu(III) speciation in solution as a function of pH, evolution of Eu(III) luminescence spectra in the ternary system at three different pH, and luminescence decay of Eu(III) in the ternary system at pH 4.4. This material is available free of charge via the Internet at http://pubs.acs.org.


Jorge Cubo

Mariana V A Sena

Paul Aubier

Guillaume Houee

Penelope Claisse

Mathieu G Faure-Brac

Ronan Allain

Rafael C L P Andrade

Juliana M Sayao

Gustavo R Oliveira

Were Notosuchia (Pseudosuchia, Crocodylomorpha) warm-blooded? A palaeohistological analysis suggests ectothermy

Keywords:

Most Notosuchia were terrestrial active predators. A few of them were semiaquatic, or were insectivorous, omnivorous or herbivorous. A question relative to their thermometabolism remains to be answered: Were Notosuchia warm-blooded? Here we use quantitative bone palaeohistology to answer this question. Two variables were used as proxies to infer the thermometabolism: resting metabolic rate and red blood cell dimensions. Resting metabolic rate was inferred using relative primary osteon area and osteocyte size, shape and density. Blood cell dimensions were inferred using harmonic mean canal diameter and minimum canal diameter. All inferences were performed using phylogenetic eigenvector maps. Both sets of analyses suggest that the seven species of Notosuchia sampled in this study were ectotherms. Considering that extant Neosuchia (their sister group) are also ectotherms, and that archosaurs were primitively endotherms, parsimony suggests that endothermy may have been lost at the node Metasuchia (Notosuchia -Neosuchia) by the Lower Jurassic. Semiaquatic taxa such as Pepesuchus may have had thermoregulatory strategies similar to those of recent crocodylians, whereas the terrestrial taxa (Araripesuchus, Armadillosuchus, Iberosuchus, Mariliasuchus, Stratiotosuchus) may have been thermoregulators similar to active predatory varanids. Thermal inertia may have contributed to maintaining a stable temperature in large notosuchians such as Baurusuchus.

INTRODUCTION

Notosuchia is a group of extinct crocodyliforms that lived from the Middle Jurassic (Razanandrongobe sakalavae, Dal Sasso et al., 2017) to the Middle Miocene (Sebecus, [START_REF] Buffetaut | Discovery of crocodilian Sebecus in Miocene of eastern Peru[END_REF][START_REF] Busbey | New material of Sebecus cf. huilensis (Crocodilia: Sebecosuchidae) from the Miocene La Venta Formation of Colombia[END_REF]. Some groups became extinct after the crisis at the end of the Cretaceous (Peirosauridae, Uruguaysuchidae), whereas others survived until the Miocene (Sebecosuchia). Notosuchians present a diverse array of body size, and inhabit different environments. Some small to medium-sized taxa (e.g., Araripesuchus, [START_REF] Sereno | Cretaceous Crocodyliforms from the Sahara[END_REF], and some large ones (e.g., Baurusuchus, Pol et al., 2014) are terrestrial, whereas other forms are semiaquatic (e.g., Kaprosuchus, Mahajangasuchus, Stolokrosuchus, Pepesuchus, Riff et al., 2012;[START_REF] Orme | A new notosuchian from the Late Cretaceous of Brazil and the phylogeny of advanced notosuchians[END_REF][START_REF] Grigg | Biology and evolution of crocodylians[END_REF][START_REF] Wilberg | Evolutionary structure and timing of major habitat shifts in Crocodylomorpha[END_REF]. Moreover, these crocodyliforms were diversified in terms of diet: carnivorous (e.g., Notosuchus and baurusuchids), insectivorous or omnivorous (e.g., Araripesuchus, Candidodon, Mariliasuchus), omnivorous (e.g., Armadillosuchus), and herbivorous (e.g., Simosuchus, Pakasuchus, Chimaerasuchus) [START_REF] Marinho | An armadillo-like sphagesaurid crocodyliform from the Late Cretaceous of Brazil[END_REF][START_REF] Godoy | An additional baurusuchid from the cretaceous of Brazil with evidence of interspecific predation among Crocodyliformes[END_REF][START_REF] Melstrom | Repeated evolution of herbivorous crocodyliforms during the age of dinosaurs[END_REF]. A question of their palaeobiology remains unanswered: were notosuchians warm-blooded? Endotherms (e.g. birds and mammals) have high metabolic rates (tachymetabolism) and generate enough internal heat to enable physiological regulation of body temperature, while ectotherms (e.g. most other vertebrates) have low metabolic rates (bradymetabolism) and must thermoregulate behaviourally. Archosaurs are considered as being primitively endotherms. This hypothesis is based on cardiovascular [START_REF] Seymour | Evidence for endothermic ancestors of crocodiles at the stem of archosaur evolution[END_REF], respiratory [START_REF] Farmer | Unidirectional airflow in the lungs of alligators[END_REF] and osteohistological [START_REF] Legendre | Palaeohistological evidence for ancestral high metabolic rate in archosaurs[END_REF] evidence. Considering that the outgroups Calyptosuchus wellesi (Aetosauria) and Postosuchus kirkpatricki (Rauisuchidae) were found endotherms [START_REF] Cubo | Bone histology of Azendohsaurus laaroussii: Implications for the evolution of thermometabolism in Archosauromorpha[END_REF], and that many notosuchians are active terrestrial predators, the null hypothesis suggests that Notosuchia may have inherited endothermy whereas Neosuchia, their sister group, may have lost this condition when they became secondarily aquatic during the Lower Jurassic. The last hypothesis is congruent with the fact that the thermic conductivity in water is higher than in the air (0.59 versus 0.024 W/(m . K); [START_REF] Vogel | Living in a physical world V. Maintaining temperature[END_REF]. The heat capacity (J/K) and the density (Kg / L) of water are also higher than those of the air. Therefore, it is more costly to maintain a high body temperature in the water than in the air. Considering that most Neosuchia are sit-andwait predators and have lower energy budgets than active aquatic endothermic predators like dolphins and penguins, natural selection may have favoured the loss of endothermy at the node Neosuchia. Qualitative histological analyses performed to date in Notosuchia are not conclusive. [START_REF] Cubo | Bone histology of Iberosuchus macrodon (Sebecosuchia, Crocodylomorpha)[END_REF] found an isolated femoral bone tissue of Iberosuchus macrodon that can be interpreted as either radial fibrolamellar bone tissue formed at extremely high growth rates and suggesting endothermy, or as compacted spongiosa formed at lower growth rates typical of ectotherms. [START_REF] Sena | Bone microanatomy of Pepesuchus deiseae (Mesoeucrocodylia, Peirosauridae) reveals a mature individual from the Upper Cretaceous of Brazil[END_REF] found in the appendicular bones of Pepesuchus deiseae bone tissues formed at moderate growth rates. Here we use quantitative bone palaeohistology and the phylogenetic comparative method to elucidate what was the thermometabolic condition of these amazing archosaurs.

MATERIAL AND METHODS

MATERIAL

We included seven species of Notosuchia: -Iberosuchus macrodon Antunes 1975. Two partial femora (IPS4930 and IPS4932) from the Palaeocene of La Boixedat (Spain). Institut Català de Paleontologia (ICP) (Sabadell, Spain). Histological descriptions of these bones can be found in [START_REF] Cubo | Bone histology of Iberosuchus macrodon (Sebecosuchia, Crocodylomorpha)[END_REF].

-Mariliasuchus amarali Carvalho & Bertini 1999. A right humerus (UFRPE 5311) from the Turonian-Maastrichtian of the Bauru Group (Brazil), Universidade Federal Rural de Pernambuco (UFRPE) (Recife, Brazil).

-Stratiotosuchus maxhechti Campos et al. 2001. A femur (MCT1714-R) and a tibia (DGM 1477-R) from the Campanian-Maastrichtian of the Bauru Group (Brazil), Museu de Ciências da Terra (MCT), under a temporary loan to Universidade Federal de Uberlândia (UFU).

-Pepesuchus deiseae [START_REF] Campos | On a new peirosaurid crocodyliform from the Upper Cretaceous, Bauru Group, southeastern Brazil[END_REF]. A right tibia (MN 7466-V) from the Campanian-Maastrichtian of the Bauru Group (Brazil), Museu Nacional do Rio de Janeiro (Brazil).

The analyses aimed at inferring resting metabolic rates in fossils include 18 species of extant tetrapods. Resting metabolic rates were measured by [START_REF] Montes | Relationships between bone growth rate, body mass and resting metabolic rate in growing amniotes: a phylogenetic approach[END_REF] using repirometry, with the exception of values for Capreolus capreolus, Oryctolagus cuniculus and Lepus europaeus, that were taken from [START_REF] Olivier | First palaeohistological inference of resting metabolic rate in an extinct synapsid, Moghreberia nmachouensis (Therapsida: Anomodontia)[END_REF]. Analyses performed to infer red blood cell size in fossils include 14 species of extant tetrapods. Red blood cell sizes were taken from [START_REF] Huttenlocker | Bone microvasculature tracks red blood cell size diminution in Triassic mammal and dinosaur forerunners[END_REF], who measured them by imaging the stained blood smears.

OSTEOHISTOLOGICAL FEATURES

Bone histological features used to construct paleobiological inference models were: relative primary osteon area (RPOA, described by [START_REF] Fleischle | Quantitative histological models suggest endothermy in plesiosaurs[END_REF], as primary osteon density), osteocyte size, shape and density (described by [START_REF] Cubo | Paleohistological estimation of bone growth rate in extinct archosaurs[END_REF] and vascular canal diameter [START_REF] Huttenlocker | Bone microvasculature tracks red blood cell size diminution in Triassic mammal and dinosaur forerunners[END_REF]. Values for the sample of Notosuchia were quantified in this study (supplementary file 1) and values for the sample of extant taxa were taken from Faure-Brac & Cubo (2020) for the femoral RPOA, from [START_REF] Legendre | Palaeohistological evidence for ancestral high metabolic rate in archosaurs[END_REF] and Olivier et al. (2016) for humeral, femoral and tibiae osteocyte size, shape and density, and from [START_REF] Huttenlocker | Bone microvasculature tracks red blood cell size diminution in Triassic mammal and dinosaur forerunners[END_REF] for femoral canal's harmonic mean and minimum diameter.

RPOA and osteocyte size, shape and density were used to infer resting metabolic rate (RMR in ml O2 h -1 g -0.67 ) in Notosuchia. Considering that all sampled extant tetrapods were juveniles in the phase of active growth [START_REF] Legendre | Palaeohistological evidence for ancestral high metabolic rate in archosaurs[END_REF], these variables were quantified in the deep cortex of Notosuchia analysed. By deep cortex we mean in primary bone near endosteal bone. The reason is that the deep cortex of adults was formed when they were juveniles. For Iberosuchus, quantifications were performed on IPS4930 as it is younger than IPS4932. Harmonic mean canal diameter and minimum canal diameter were used to infer red blood cell width (µm) and area (µm 2 ) in Notosuchia. The variable "canal diameter" corresponds to the diameter of vascular spaces in bone tissue. Values for the sampled extant tetrapods were taken from [START_REF] Huttenlocker | Bone microvasculature tracks red blood cell size diminution in Triassic mammal and dinosaur forerunners[END_REF], who analysed adult specimens. Therefore, we quantified the vascular canal diameters in the outer cortex of the sampled Notosuchia, and we used Iberosuchus IPS4932 because it is older than IPS4930.

PHYLOGENY AND PHYLOGENETIC COMPARATIVE METHODS

For extant taxa, we used phylogenies compiled by [START_REF] Legendre | Palaeohistological evidence for ancestral high metabolic rate in archosaurs[END_REF] We used phylogenetic eigenvector maps (PEMs) and the 'MPSEM' package [START_REF] Guenard | Phylogenetic eigenvector maps: a framework to model and predict species traits[END_REF] in R (R Development Core Team, 2016) to infer our dependent (response) variable using a set of explanatory variables. We checked the normality of residuals of the explanatory variables (the differences between the values fitted by the PEM model and the actual values) using Shapiro-Wilk tests. If a distribution of residuals was significantly different from normality, the corresponding raw variable needs to be transformed. Finally, we performed leave-one-out cross-validation tests.

For this, we estimated values of the dependent variable of extant taxa (for which these values are known) using the inference procedure of the 'MPSEM' package [START_REF] Guenard | Phylogenetic eigenvector maps: a framework to model and predict species traits[END_REF], and compared these inferred values to the empirical ones using a PGLS regression and the 'caper' package [START_REF] Orme | A new notosuchian from the Late Cretaceous of Brazil and the phylogeny of advanced notosuchians[END_REF] in R (R Development Core Team, 2016).

RESULTS

RESTING METABOLIC RATE INFERENCES PERFORMED USING RPOA AND OSTEOCYTE DENSITY, AREA AND SHAPE

The datasets used to perform analyses are available in supplementary file 1. We used PEMs to infer the mass-independent resting metabolic rates (RMRs) of Notosuchia using RMRs values quantified in a sample of extant tetrapods, histological features quantified in both Notosuchia and extant tetrapods, and the phylogeny. The dependent (response) variable is RMR, and the explanatory variables are the phylogeny and osteohistological features: RPOA and osteocyte density, area and shape. The response variable (RMR) was transformed using natural logarithms because raw values are skewed. With a single exception, the distributions of residuals of the explanatory variables (the differences between the values fitted by the PEM models and the actual values) were not significantly different from normality (supplementary file 2). Therefore, no transformation of explanatory variables was needed. For each bone we chose the model that maximizes the R 2 value and, in case of tie, the model that minimizes the AIC value.

The selected models were (supplementary file 2): phylogeny + osteocyte shape for the humerus (supplementary figure 1), phylogeny + osteocyte area for the femur (Fig. 1), and phylogeny + osteocyte shape for the tibia (supplementary figure 2). Inferences and 95% confidence intervals are shown in Table 1. Additionally, we inferred values of the dependent variable of extant taxa (for which these values are known) using leave-oneout cross-validation tests. For the sample of extant taxa, regressions of inferred values to empirical values performed using PGLS were highly significant: R 2 =0.994, p-value = 2.2e-16 for the humerus; R 2 =0.990, p-value = 2.2e-16 for the femur; R 2 =0.977, p-value = 1.521e-10 for the tibia.

RED BLOOD CELL SIZE INFERENCES PERFORMED USING VASCULAR CANAL DIAMETER

The datasets used to perform analyses is available in supplementary file 1. We used PEMs to infer red blood cell (RBC) dimensions of Notosuchia using RBC values quantified in a sample of extant tetrapods, vascular canal diameters quantified in both Notosuchia and extant tetrapods, and the phylogeny. The dependent (response) variables were RBCwidth and RBCarea, and the explanatory variables are femoral harmonic mean canal diameter and femoral minimum canal diameter. The distributions of residuals of the explanatory variables (the differences between the values fitted by the PEM model and the actual values) were not significantly different from normality (supplementary file 2). Therefore, no transformation of explanatory variables was needed. For each dependent variable we chose the model that maximizes the R 2 value and, in case of tie, the model that minimizes the AIC value. The selected models are (supplementary file 2): phylogeny + harmonic mean canal diameter to infer red blood cells width (Fig. 2); and phylogeny + minimum canal diameter to infer red blood cells area (supplementary figure 3). Inferences and 95% confidence intervals are shown in Table 2. Additionally, we inferred values of the dependent variable of extant taxon (for which these values are known) using leave-one-out cross-validation tests. For the sample of extant taxa, regressions of inferred values to empirical values performed using PGLS were significant: R 2 =0.891, p-value=3.981e-07 for red blood cell width and R 2 = 0.425, p-value=0.01154 for red blood cell area.

DISCUSSION METHODOLOGICAL CONSIDERATIONS

We used two variables as proxies to infer endothermy: resting metabolic rate [START_REF] White | The scaling and temperature dependence of vertebrate metabolism[END_REF][START_REF] Ho | Temperature, metabolic power and the evolution of endothermy[END_REF] and red blood cell dimensions [START_REF] Snyder | Red blood cells: Centerpiece in the evolution of the vertebrate circulatory system[END_REF][START_REF] Huttenlocker | Bone microvasculature tracks red blood cell size diminution in Triassic mammal and dinosaur forerunners[END_REF]. For a range of body masses from 50 to 1000g, the resting metabolic rate of birds and mammals exceed those of ectotherms by a factor of, respectively, 15 and 12 (Clarke & Portner, 2010).

Furthermore, birds and mammals are characterized by smaller vascular canal minimum sizes than ectotherms [START_REF] Huttenlocker | Bone microvasculature tracks red blood cell size diminution in Triassic mammal and dinosaur forerunners[END_REF]. It has been shown that smaller capillaries produce higher diffusive gas exchange and higher resistance to blood flow [START_REF] Snyder | Red blood cells: Centerpiece in the evolution of the vertebrate circulatory system[END_REF]. In endotherms, red blood cells undergo cell deformation during capillary flow [START_REF] Snyder | Red blood cells: Centerpiece in the evolution of the vertebrate circulatory system[END_REF]. Therefore, in these organisms, the presence of small capillaries increases resistance even if there are more capillaries in a given volume of tissue. The presence of smaller vascular canal minimum sizes in endotherms (birds and mammals) can thus be associated with their high oxygen uptake and their four chambered heart, allowing high systemic blood pressures [START_REF] Snyder | Red blood cells: Centerpiece in the evolution of the vertebrate circulatory system[END_REF][START_REF] Huttenlocker | Bone microvasculature tracks red blood cell size diminution in Triassic mammal and dinosaur forerunners[END_REF]. Resting metabolic rate, red blood cell dimensions, and the thermometabolism (endothermy or ectothermy), are known in our sample of extant tetrapods. They are inferred in the sample of Notosuchia using a model that includes osteohistological features and the phylogeny. The interesting point is that we performed two separate sets of analyses, using two independent samples of extant tetrapods, to infer two variables used as proxies of endothermy (resting metabolic rate, red blood cell dimensions), and we obtained the same inferences in Notosuchia (see below).

We can wonder about the effect of the number of extinct taxa (for which we performed inferences), relative to the number of extant ones used to construct the inference models. Results are the same irrespective of the relative number of extinct taxa included in the model: as an example, Stratiotosuchus maxhechti was inferred as being ectotherm using a model based on the bone histology of the femur (five extinct taxa) and using a model based on the bone histology of the tibia (two extinct taxa).

Finally, we checked the reliability of our inferences performing leave-one-out crossvalidation tests: we estimated values of the dependent variable of extant taxa (for which these values are known) using the inference procedure, and performed regressions of the inferred values to the measured (actual) values. All regressions were significant, suggesting that the inferences performed for Notosuchia are reliable.

PALEOBIOLOGICAL INFERENCES

The question to be answered is: were Notosuchia warm-blooded? Considering that archosaurs were primitively endotherms [START_REF] Seymour | Evidence for endothermic ancestors of crocodiles at the stem of archosaur evolution[END_REF][START_REF] Farmer | Unidirectional airflow in the lungs of alligators[END_REF][START_REF] Legendre | Palaeohistological evidence for ancestral high metabolic rate in archosaurs[END_REF], that the outgroups of Notosuchia analysed to date (Calyptosuchus wellesi and Postosuchus kirkpatricki) were found endotherms [START_REF] Cubo | Bone histology of Azendohsaurus laaroussii: Implications for the evolution of thermometabolism in Archosauromorpha[END_REF], and that Notosuchia includes a wide range of active terrestrial predator species [START_REF] Carvalho | Uberabasuchus terrificus sp. nov., a new Crocodylomorpha from the Bauru Basin (Upper Cretaceous), Brazil[END_REF]2007;[START_REF] Carvalho | Baurusuchus salgadoensis, a new Crocodylomorpha from the Bauru Basin (Cretaceous), Brazil[END_REF][START_REF] Nascimento | A new Baurusuchidae from the Upper Cretaceous of Brazil[END_REF][START_REF] Godoy | An additional baurusuchid from the cretaceous of Brazil with evidence of interspecific predation among Crocodyliformes[END_REF], the null hypothesis suggests that Notosuchia were endotherms. This hypothesis has been refuted by our results. Both sets of analyses, those aimed at inferring resting metabolic rates and those performed to estimate red blood cell dimensions, show that the seven species of Notosuchia sampled in this study may have been ectotherms. Considering that extant Neosuchia (their sister group) are also ectotherms, parsimony suggests that endothermy may have been lost at the node Metasuchia (Notosuchia -Neosuchia) by the Lower Jurassic (Fig. 3). Among Notosuchia, semiaquatic taxa such as Pepesuchus may have had thermoregulatory strategies akin to those of recent crocodylians (e.g., control of heat flow through the skin by basking, and opening the mouth to promote cooling by evaporation; [START_REF] Grigg | Biology and evolution of crocodylians[END_REF], whereas the terrestrial species (Araripesuchus, Armadillosuchus, Iberosuchus, Mariliasuchus, Stratiotosuchus) may have been thermoregulators similar to active predatory varanids. Varanid lizards have high aerobic capacities and can sustain higher activity levels than other lizards [START_REF] Pough | The Advantages of ectothermy for tetrapods[END_REF][START_REF] Mendyk | On the thermal husbandry of monitor lizards[END_REF]. These terrestrial notosuchians may have occupied sunlit areas for basking or while foraging, and they possibly entered into burrows [START_REF] Marinho | An armadillo-like sphagesaurid crocodyliform from the Late Cretaceous of Brazil[END_REF][START_REF] Carvalho | Climate's role in the distribution of the Cretaceous terrestrial Crocodyliformes throughout Gondwana[END_REF] for lowering body temperature when necessary. [START_REF] Clarac | The crocodylian skull and osteoderms: A functional exaptation to ectothermy?[END_REF] showed that, in crocodylians, «osteoderms collect the external heat during the basking periods as they become significantly warmer than the surrounding skin. The presence of osteoderms in terrestrial Notosuchia (e.g., Iberosuchus) probably involved in heat intake during basking suggests that these organisms relied on external heat to perform high levels of activity inferred by functional morphology analyses. In the large sebecosuchians (e.g., ;[START_REF] Orme | A new notosuchian from the Late Cretaceous of Brazil and the phylogeny of advanced notosuchians[END_REF] the body temperature should have been more or less stable throughout the day because of thermic inertia. This feature made them more independent from external heating, which may have allowed extra time for hunting. The lifestyle of a few taxa is still debated. This is the case of Mariliasuchus. [START_REF] Nobre | Postcranial skeleton of Mariliasuchus amarali Carvalho and Bertini, 1999 (Mesoeucrocodylia) from the Bauru Basin, Upper Cretaceous of Brazil[END_REF] observed that Mariliasuchus amarali shares postcranial features with recent eusuchians, and concluded that this taxon probably had a sprawling type posture and an amphibian lifestyle. However Mariliasuchus amarali also presents cranial features which corresponds to a terrestrial habit, like a short and high skull, lateral orbits, frontal external nares, and long and robust limbs, indicating a quadrupedal posture [START_REF] Vasconcellos | Estágios de desenvolvimento de Mariliasuchus amarali, Crocodyliformes Mesoeucrocodylia da Formacão Adamantina, Cretáceo Superior da Bacia Bauru, Brasil[END_REF][START_REF] Andrade | Morphological and anatomical observations about Mariliasuchus amarali and Notosuchus terrestris (Mesoeucrocodylia) and their relationships with other South American notosuchians[END_REF]. Besides some histological and microanatomic features were found in Mariliasuchus' thin sections (unpublished data) which corroborates its terrestrial habit, such as the absence of bone specializations, tubular ribs and long bones showing a compact cortical bone and a free marrow cavity or a loose spongiosa filling in medullary region [START_REF] Houssaye | Adaptive Patterns in Aquatic Amniote Bone Microanatomy-More Complex than Previously Thought[END_REF].

Baurusuchus

Moreover Mariliasuchus amarali is found in deposits of the Adamantina Formation from Bauru group which are interpreted as semi-arid environments with sand sheets crossed by ephemeral river systems surrounding an interior desert inside Paraná basin during Late Cretaceous from the Gondwana [START_REF] Fernandes | Evolution and palaeoenvironment of the Bauru Basin (Upper Cretaceous, Brazil)[END_REF]. For all these reasons we considered that Mariliasuchus had a terrestrial lifestyle. Future research on the palaeophysiology, the lifestyle, the diet and on the general paleobiology of Notosuchia are necessary to fully understand their ecological role and the causes of their extinction Phylogeny for extant taxa has been compiled by [START_REF] Legendre | Palaeohistological evidence for ancestral high metabolic rate in archosaurs[END_REF] and [START_REF] Olivier | First palaeohistological inference of resting metabolic rate in an extinct synapsid, Moghreberia nmachouensis (Therapsida: Anomodontia)[END_REF], and for extinct one has been taken from [START_REF] Geroto | New material of Pepesuchus (Crocodyliformes; Mesoeucrocodylia) from the Bauru Group: implications about its phylogeny and the age of the Adamantina Formation[END_REF].

Figure 2. Red blood cell width inferred for a sample of Notosuchia using palaeohistology and phylogenetic eigenvector maps. We used a model that included phylogeny + harmonic mean canal diameter for the femur as explanatory factors. Blue squares indicate ectothermy and red squares endothermy. For fossil taxa, segments represent the 95% confidence interval of the inferences. Abbreviations: AIC, Akaike information criterion. Phylogeny for extant taxa has been compiled by [START_REF] Huttenlocker | Bone microvasculature tracks red blood cell size diminution in Triassic mammal and dinosaur forerunners[END_REF], and for extinct one has been taken from [START_REF] Geroto | New material of Pepesuchus (Crocodyliformes; Mesoeucrocodylia) from the Bauru Group: implications about its phylogeny and the age of the Adamantina Formation[END_REF]. Inferences of extinct taxa have been performed in this study, with the exception of Azendohsaurus laaroussii, Calyptosuchus wellesi and Postosuchus kirkpatricki, that were performed by [START_REF] Cubo | Bone histology of Azendohsaurus laaroussii: Implications for the evolution of thermometabolism in Archosauromorpha[END_REF]. Phylogeny for extant taxa has been compiled by [START_REF] Legendre | Palaeohistological evidence for ancestral high metabolic rate in archosaurs[END_REF] and [START_REF] Olivier | First palaeohistological inference of resting metabolic rate in an extinct synapsid, Moghreberia nmachouensis (Therapsida: Anomodontia)[END_REF], and for extinct one has been taken from

Bones

Taxa Inferred RMR (ml O2 h -1 g -0.67 ) 1. Inferences of resting metabolic rate (ml O2 h -1 g -0.67 ) of the sample of Notosuchia analysed in this study using phylogenetic eigenvector maps models that included the following predictors: phylogeny + osteocyte shape for the humerus, phylogeny + osteocyte area for the femur, and phylogeny + osteocyte shape for the tibia.

Abbreviations: CI, confidence interval.

-

  Araripesuchus wegeneri Buffetaut 1981. A partial femur (MNHN.F.GDF660) from the Aptian of Gadoufaoua (Niger), Museum national d'Histoire naturelle (MNHN) (Paris, France). -Armadillosuchus arrudai Marinho & Carvalho 2009. A partial femur (LPRP-USP 0774) from the Turonian-Maastrichtian of the Bauru Group (Brazil), Universidade de São Paulo (USP) (Ribeirão Preto, Brazil). -Baurusuchus sp. Price 1945. A partial femur (LPRP-USP 0634 C) from the Turonian-Maastrichtian of the Bauru Group (Brazil), USP.

  and Faure-Brac & Cubo (2020) for figures 1, 3, and supplementary figures 1 and 2, and by Huttenlocker & Farmer (2017) for figure 2 and supplementary figure 3. Several phylogenetic analyses have been published for Notosuchia. Pol et al. (2014) found the following topology for the species of our sample: (Araripesuchus (Mariliasuchus (Baurusuchus -Stratiosuchus -Iberosuchus))). Kellner et al. (2014) proposed the same topology for these species. In contrast, Geroto & Bertini (2019) proposed a different topology for the species of our sample: ((Iberosuchus -Pepesuchus) (Araripesuchus ((Mariliasuchus -Armadillosuchus)(Stratiosuchus -Baurusuchus)))). We followed the more recent one (that published by Geroto & Bertini, 2019). Please note that in the topologies published by Pol et al. (2014) and Kellner et al. (2014), all seven species of our sample are included in the node Notosuchia, whereas in the topology used in this study (that published by Geroto & Bertini, 2019), Iberosuchus and Pepesuchus are not included in this node.

FIGURE CAPTIONS Figure 1 .

 CAPTIONS1 FIGURE CAPTIONS

Figure 3 .

 3 Figure 3. Optimization of the presence of endothermy onto a phylogeny of tetrapods.

Table 2 .

 2 Inferences of red blood cells dimensions of the sample of Notosuchia analyzed in this study using a PEM model. The model to infer red blood cells width (µm) included phylogeny + harmonic mean canal diameter as predictors. The model to infer red blood cells area (µm 2 ) included phylogeny + minimum canal diameter. Abbreviations: CI, confidence interval; RBC, red blood cells.

	Taxa	Inferred RBC	Inferred	Lower limit	Upper limit
		variables	values	95% CI	95% CI
	Araripesuchus wegeneri	RBCwidth (µm)	11.343	9.699	12.988
		RBCarea (µm 2 )	149.990	131.047	168.934
	Armadillosuchus arrudai	RBCwidth (µm)	11.200	9.541	12.859
		RBCarea (µm 2 )	152.124	130.900	173.348
	Baurusuchus sp.	RBCwidth (µm)	11.136	9.492	12.780
		RBCarea (µm 2 )	148.022	127.322	168.722
	Iberosuchus macrodon	RBCwidth (µm)	12.577	10.391	14.764
		RBCarea (µm 2 )	155.884	132.297	179.470
	Stratiotosuchus maxhechti	RBCwidth (µm)	12.533	10.403	14.663
		RBCarea (µm 2 )	161.011	137.690	184.332

[START_REF] Geroto | New material of Pepesuchus (Crocodyliformes; Mesoeucrocodylia) from the Bauru Group: implications about its phylogeny and the age of the Adamantina Formation[END_REF]. Red branches: endothermy. Blue branches: ectothermy.

ACKNOWLEDGEMENTS

We thank Roger Seymour (The University of Adelaide), Holly Woodward (Oklahoma State University) and an anonymous reviewer for helpful comments that improved the quality of the manuscript. We gratefully thank Dr. Max Langer (Universidade de São Paulo), Dr. Douglas Riff (Universidade Federal de Uberlândia), and Dr. Felipe Chinaglia Montefeltro (Universidade Estadual Paulista) and Alexander W. A. Kellner (Museu Nacional -UFRJ) for making available the thin-sections images of Baurusuchus sp., Armadillosuchus arrudai and Stratiotosuchus maxhecthi. We thank the curator of the archosaur collection of the Muséum national d'Histoire naturelle (Paris, France) for allowing us to perform thin sections of a partial femur (GDF660) of Araripesuchus wegeneri, and to Hayat Lamrous (Sorbonne Université) for performing these sections. We thank the curators of the Paleovertebrate Sector of the Museu Nacional (MN/UFRJ) which provides the Pepesuchus deiseae and Mariliasuchus amarali and Stratiotosuchus maxhecthi's fossils. Some of the specimens used here were prepared in the Paleohistology lab at Des Moines University (Medical and Health Sciences Institution). The authors sincerely thank Des Moines University and, especially, Dr. Sarah Werning for having Dr. Rafael Andrade as an exchange student, and the opportunity to use their facilities