Guillaume Fleury
email: guillaume.fleury@iphc.cnrs.fr

Mirella Del

Remi Barillon

Effect of mineral surface properties (alumina, kaolinite) on the sorptive fractionation mechanisms of soil fulvic acids: molecular-scale ESI-MS studies

Keywords: humic substances, kaolinite, alumina, fractionation, mass spectrometry

We addressed the effects of mineral surface properties (kaolinite versus Al-oxide) on the sorption-driven fractionation of a soil fulvic acid (FA) at acidic pH, mainly by means of ESI(-)-FTMS analysis of initial and supernatant solutions of FA sorption batch experiments. The MS data provided clear molecular-scale evidence of distinct mechanisms and molecular parameters controlling the FA fractionation upon its sorption on clay and oxide surfaces, respectively.

Identification of sorbing and not-sorbing FA compounds in kaolinite-solution systems revealed a weak fractionation amongst members of -CO 2 series of aliphatics or not-condensed aromatics (NCAs) at pH 3.8, and almost no sorption of poorly-oxygenated polycyclic aromatic compounds (PACs) and NCAs. This first molecular-scale description of a FA fractionation in a clay-solution system suggests that H-bonding with low affinity sites (aluminol/silanol) on the basal planes of the clay particles is the main mechanism of sorption.

Due to the predominance of such weak and poorly-selective mechanism, the sorption of aliphatic and NCA molecules bearing oxygenated functionalities was prevented at pH 5, due to dissolved Al competing successfully for their coordination.

In contrast, a strong FA fractionation was observed onto alumina, with a preferential retention of PACs and highly-oxygenated aliphatics and NCAs. The major part of the poorly oxygenated aliphatics was left in solution. The sorption degree of NCAs and aliphatics was strongly correlated with molecular acidity. For PACs and poorly-oxygenated NCAs, the sorption was driven by reactions of surface ligand exchange (for the most oxygenated compounds) or by hydrophobic interactions (for the least oxygenated compounds).

INTRODUCTION

Humic substances (HSs) like fulvic acids (FAs) are complex heterogeneous mixtures of thousands of biochemically stable polymeric substances [START_REF] Aiken | Humic substances in soil, sediment, and water: geochemistry, isolation and characterization[END_REF] which are ubiquitous in all terrestrial surface systems and which account for a great part of organic carbon in soils. HSs show a variety in the composition, structure and reactivity of their constituents and functional groups [START_REF] Swift | Molecular weight, size, shape, and charge characteristics of humic substances: Some basic considerations[END_REF][START_REF] Plancque | Molecular structure of fulvic acids by electrospray with quadrupole time-of-flight mass spectrometry[END_REF]. It has long been known that HSs have the ability to interact with metals [START_REF] Dobbs | Characterization of metal binding sites in fulvic acids by lanthanide ion probe spectroscopy[END_REF][START_REF] Tipping | Structural and sorption characteristics of adsorbed humic acid on clay minerals[END_REF][START_REF] Milne | Generic NICA-Donnan model parameters for metal-ion binding by humic substances[END_REF]Janot et al., 2013), organic pollutants [START_REF] Rebhun | Dissolved humic substances for remediation of sites contaminated by organic pollutants. Binding-desorption model predictions[END_REF] and surfaces of (nano) particles of clays and Al-or Fe-oxyhydroxides [START_REF] Ochs | Coordinative and hydrophobic interaction of humic substances with hydrophilic Al203 and hydrophobic mercury surfaces[END_REF][START_REF] Schlautman | Adsorption of aquatic humic substances on colloidal-size aluminum oxide particles: Influence of solution chemistry[END_REF][START_REF] Feng | Chemical and mineralogical controls on humic acid sorption to clay mineral surfaces[END_REF]Janot et al., 2013), with the sorption of HSs resulting in its chemical fractionation between minerals and solution [START_REF] Meier | Fractionation of aquatic natural organic matter upon sorption to goethite and kaolinite[END_REF][START_REF] Zhou | Size fractionation upon adsorption of fulvic acid on goethite: equilibrium and kinetic studies[END_REF][START_REF] Reiller | Sorption of Aldrich humic acid onto hematite: Insights into fractionation phenomena by electrospray ionization with quadrupole time-of-flight mass spectrometry[END_REF]. Sorption-driven fractionation of HSs is expected to influence surface characteristics (reactivity, sorption capacity, charge) of minerals, dispersion behavior of suspended nanoparticles which are potential pollutants' carriers, and metal binding capacity of dissolved organic matter in many geochemical systems [START_REF] Ochs | Coordinative and hydrophobic interaction of humic substances with hydrophilic Al203 and hydrophobic mercury surfaces[END_REF][START_REF] Schlautman | Adsorption of aquatic humic substances on colloidal-size aluminum oxide particles: Influence of solution chemistry[END_REF][START_REF] Yang | Investigation of Eu(III) immobi iz ion on γ-Al2O3 surfaces by combining batch technique and EXAFS analyses: Role of contact time and humic acid[END_REF].

Understanding of the fractionation of HS during the sorption at surfaces of mineral particles is thus of interest in many environmental issues including sorption / mobility of trace metal elements in soils and aquatic systems. It has some implications on the evaluation of the impact on health of the release of pollutants in the ecosystems and on the safety assessments of remediation processes of polluted soils or radioactive waste repositories in geological formations [START_REF] Loffredo | Fate of anthropogenic organic pollutants in soils with emphasis on adsorption/desorption processes of endocrine disruptor compounds[END_REF][START_REF] Bryan | The effects of humic substances on the transport of radionuclides: Recent improvements in the prediction of behaviour and the understanding of mechanisms[END_REF].

Building a comprehensive description of the coupled cycles of organic matter and pollutants in natural systems requires some basic knowledge of the fractionation trends exhibited by HSs during their sorption at mineral surfaces relevant to soils and waters, as well as a thorough understanding of the mechanisms and molecular parameters driving the fractionation -which is still lacking-. Speaking in a general way, several mechanisms of sorption of HSs onto minerals were proposed, including surface ligand exchange, specific adsorption, electrostatic interactions, hydrophobic effects, H-bonding and metal-ion bridging [START_REF] Ochs | Coordinative and hydrophobic interaction of humic substances with hydrophilic Al203 and hydrophobic mercury surfaces[END_REF][START_REF] Schlautman | Adsorption of aquatic humic substances on colloidal-size aluminum oxide particles: Influence of solution chemistry[END_REF]. In many studies, HSs were considered as bulk materials bearing polar functionalities and spectroscopic/modeling evidence was given that carboxyl functional groups of HS [START_REF] Fu | Interaction of humic substances and hematite: FTIR study[END_REF][START_REF] Ghosh | Sorption and fractionation of a peat derived humic acid by kaolinite, montmorillonite, and goethite[END_REF] are involved in the macroscopic sorption of HS at surfaces of iron or aluminum oxide minerals and that the contribution of phenol groups increases with pH [START_REF] Filius | Modeling the binding of fulvic acid by goethite: the speciation of adsorbed FA molecules[END_REF]Claret et al., 2008). In the last two decades, there has also been a growing interest in getting a detailed understanding of the behavior of HSs during their sorption at (nano) mineral -solution interfaces. Many spectroscopic evidences were provided that a strong sorptive fractionation takes place between the diverse classes of organic compounds of a humic substance (e.g. [START_REF] Meier | Fractionation of aquatic natural organic matter upon sorption to goethite and kaolinite[END_REF]Claret et al., 2008;[START_REF] Ghosh | Sorption and fractionation of a peat derived humic acid by kaolinite, montmorillonite, and goethite[END_REF]. It was reported that the hydrophobic fractions of FAs and/or the organic molecules with high contents in aromatic moieties activated by oxygenated functionalities were preferentially sorbed -as compared to aliphatic fractions-on the surfaces of metallic oxide minerals [START_REF] Meier | Fractionation of aquatic natural organic matter upon sorption to goethite and kaolinite[END_REF][START_REF] Zhou | Size fractionation upon adsorption of fulvic acid on goethite: equilibrium and kinetic studies[END_REF]Kaiser, 2003;Claret et al., 2008). Moreover, several studies combining size-exclusion-chromatography and spectroscopy analysis have revealed a size fractionation of fulvic or humic acids during their sorption at several mineral surfaces (e.g. [START_REF] Hur | Effects of pH and phosphate on the adsorptive fractionation of purified Aldrich humic acid on kaolinite and hematite[END_REF], which was explained by structural trends in the molecular weight fractions (degree of aromaticity, functionalities…) and the underlying sorption processes. Since recently, the development of an advanced technique, namely ultra-high resolution Fourier transform mass spectrometry (FTMS), has offered unique opportunities for identifying the molecules constitutive of a HA or FA (e.g. Kujawinski et al., 2002;D'Andrilli et al., 2010) and for approaching the issue of the HS-metallic oxidesolution interactions at the molecular scale [START_REF] Reiller | Sorption of Aldrich humic acid onto hematite: Insights into fractionation phenomena by electrospray ionization with quadrupole time-of-flight mass spectrometry[END_REF][START_REF] Galindo | Molecular Level Description of the Sorptive Fractionation of a Fulvic Acid on Aluminum Oxide Using Electrospray Ionization Fourier Transform Mass Spectrometry[END_REF][START_REF] Galindo | Chemical fractionation of a terrestrial humic acid upon sorption on alumina by high resolution mass spectrometry[END_REF]. Published FTMS data for the characterization of HSs [START_REF] Plancque | Molecular structure of fulvic acids by electrospray with quadrupole time-of-flight mass spectrometry[END_REF][START_REF] Stenson | Ionization and Fragmentation of Humic Substances in Electrospray Ionization Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry[END_REF][START_REF] Sutton | Molecular Structure in Soil Humic Substances: The New View[END_REF] have supported the model proposed by [START_REF] Wershaw | Model for humus in soils and sediments[END_REF] and by [START_REF] Engebretson | Micro-organization in dissolved humic acids[END_REF], and recently revived by [START_REF] Piccolo | The supramolecular structure of humic substances[END_REF] and [START_REF] Plancque | Molecular structure of fulvic acids by electrospray with quadrupole time-of-flight mass spectrometry[END_REF], which describes an HS as a supramolecular association of small organic entities ("building blocks" of ca. 500 Da for FAs) assembled via weak bonds. This model has led to view an HS as a complex mixture of thousands of polycarboxylates of distinct molecular weight, degree of aromaticity or acidity, and chemical reactivity.

Recently, the ESI-FTMS technique was applied by our research team to the identification of sorbing (and not sorbing) molecules of an aquatic fulvic acid [START_REF] Galindo | Molecular Level Description of the Sorptive Fractionation of a Fulvic Acid on Aluminum Oxide Using Electrospray Ionization Fourier Transform Mass Spectrometry[END_REF], and of a terrestrial humic acid [START_REF] Galindo | Chemical fractionation of a terrestrial humic acid upon sorption on alumina by high resolution mass spectrometry[END_REF] during experiments of HS-alumina-solution interactions. A striking result was an inverse correlation existing between the degree of sorption of a molecule within a CH2 series and its number of CH2 groups, and a positive correlation between the degree of sorption and the number of CO2 groups in a COO series, for homologous series containing up to nine members. In agreement with previous experimental work by [START_REF] Evanko | Influence of structural features on sorption of NOManalogue organic acids to goethite[END_REF] on simple organic acids and modelling work by [START_REF] Janot | Characterization of humic id ivi y modi i ions du o dso p ion on o α-Al2O3[END_REF] suggesting that enhanced sorption with increasing number of carboxyl groups was due to changes in compound acidity, the correlations observed for FA by ESI-FTMS provided for the first time a molecular scale evidence that the sorptive fractionation of complex mixtures of thousands of organic compounds as fulvic acids was controlled by molecule acidity, which was remarkable. These ESI-FTMS findings also strongly supported that the FA subunits, held together by hydrophobic forces and hydrogen bonding, were disrupted during sorption because of strong FA-alumina surface interactions via surface ligand exchange mechanism, which was consistent with the concept of supramolecular assembly for HS [START_REF] Piccolo | The supramolecular structure of humic substances[END_REF][START_REF] Piccolo | Effects of sample properties and mass spectroscopic parameters on electrospray ionization mass spectra of size-fractions from a soil humic acid[END_REF]. Hydrophobic interactions were moreover found to be a mechanism cooperating to the sorption onto alumina of the poorly-oxygenated polycyclic aromatic compounds (PACs) of a terrestrial HA, with the degree of molecule hydrophobicity influencing the degree of molecule sorption.

In contrast to the metallic oxide systems, very few information is available on the sorption-driven fractionation of HSs onto clay minerals and no molecular scale description of the process has been reported so far. Unlike for iron or aluminum (hydr)oxides, the aliphatic fractions of a humic acid were found to be more prone to sorption on the surface of kaolinite and montmorillonite than the aromatic fractions (Wang and Xing, 2005;[START_REF] Ghosh | Sorption and fractionation of a peat derived humic acid by kaolinite, montmorillonite, and goethite[END_REF]. Such contrasting results between metallic oxides and clays have pointed the need to acquire detailed knowledge of the molecular characteristics and the sorption mechanisms driving HS sorptive fractionation in clay mineralsolution systems, which are relevant to many geochemical systems. In particular, such knowledge is critically needed for developing a comprehensive understanding of the sorptive fractionation of FAs in the various types of soils (rich in metal oxi-hydroxides or in clays).

In this study, we addressed the effects of mineral surface properties, i.e., clay versus aluminum oxide, on the molecular scale fractionation of a terrestrial FA during its sorption at mineral surface by mean of complementary techniques, traditional batch experiments, electro-kinetic measurements and electrospray ionization FTMS. The systems under investigation included an extracted FA (PPH) and kaolinite or alumina as a model sorbent taken as being representative of Al (hydr)oxides or clays in soils, respectively. First, batch sorption experiments and on-line electrophoretic mobility measurements were performed in PPH-containing mineral suspensions for studying the pH-dependency of sorption of PPH onto alumina and kaolinite and for gaining insights into surface charges of PPH-loaded particles. The use of a LTQ Orbitrap XL hybrid mass spectrometer allowed deriving the exact elemental composition of the molecules of PPH present in the solution before and after contact with the metal oxide or the clay mineral, for experiments performed under acidic and middle acidic solutions (pH 3.5 and 5.0, respectively). The molecular approach was used to retrieve the distinct orders of relative affinity of the FA molecules for the surface of a clay and a metallic oxide, respectively, and to explore the relations existing between chemical characteristics of the PPH molecules (such as number of CO2 functionalities, degree of aromaticity or acidity…) and their degree of relative sorption onto mineral. The molecular-scale results presented here represent a significant contribution to our understanding of the fractionation trends of a terrestrial FA during its sorption onto a clay mineral, and of the sorption mechanisms involved, and highlight the strong effects of mineral surface properties on the fractionation behavior of terrestrial fulvic acids.

EXPERIMENTAL SECTION

Materials

The fulvic acid denoted as "PPH" was extracted from a soil sample collected in a beech forest developed on sandstone, near La Petite-Pierre, Bas-Rhin, France. The extraction was carried out by using the International Humic Substances Society (IHSS, St. Paul, MN, USA) standard method for isolation of soil fulvic acids. This method, excerpted from [START_REF] Sparks | Organic Matter Characterization[END_REF], uses XAD-8 resin adsorption to isolate such acids. Due to the chemical properties of the resin, the fulvic acids used here only contain hydrophobic organic compounds.

Stock solutions of PPH fulvic acid were prepared and stored frozen in the dark until used.

Alumina colloids were Alfa Aesar's α-Al 2 O 3 crystallites (chemical purity: 99.95%, surface area: 7.6 m 2 /g) with a particle size of 280 nm as determined by dynamic light scattering ([α-Al 2 O 3 ] = 2.5 g.L -1 , particle size determined in % of intensity, w 50% = 90 nm, polydispersity index = 0.12). The kaolinite sample used was the standard sample KGa-1b provided by The Clay Minerals Society. This sample was characterized by [START_REF] Pruett | Sampling and analysis of KGa-1 B well-crystallized kaolin source clay[END_REF], who reported a particle distribution of 57.8% < 2µm and 32.0% < 0.5µm, as well as a surface area of 11.7m².g -1 . This value for surface area of kaolinite has been used as such and no attempt was made to determine again this value.

All solutions were prepared using reagent grade chemicals and ultra-pure water (purity > 18 MΩ.cm).

Experimental setup

Experiments of FA sorption at acidic pH and 298K on alumina and kaolinite were carried out for analysis of the supernatants by ESI(-)-FTMS and study of the mineral surface characteristics on FA sorptive fractionation. The experiments were carried out at pH 3.8 ± 0.1 and 4.9 ± 0.1 for both minerals.

Alumina and kaolinite suspensions were prepared in individual PEHD tubes with solid-to-solution ratios of 25 and 15 g.L -1 , respectively. These values of solid-to-solution ratios were chosen so that the surface areas of alumina and kaolinite used in the experiments fall in the same range of values (ca. 170 and 150 m 2 /L, respectively). Efforts were made to adjust the concentration of PPH added in the individual tubes, so that the values of FA-to-mineral ratios (r) of all experiments fall within a narrow range (r 5mg C/g, or more precisely, r 0.6 and 0.8 mgC/m 2 of mineral for sorption of FAs on kaolinite and alumina, respectively). No attempt was made to fix the ionic strength of the initial experimental solutions, as background salts were previously observed to alter considerably the ESI-MS response. After shaking of the tubes for 24 hours at 298K, the final pH values of the suspensions were measured. The suspensions were then centrifuged at 8000 rpm during 2.5 hours for solution-colloid separation and the supernatants were collected. Aliquots were taken for measurements of dissolved organic carbon, DOC, using a Shimadzu TOC -V CPH analyzer, and of dissolved aluminum content using Inductively Coupled Plasma Mass Spectrometry (quantification limit: 5.3µg/L). All the remaining supernatant solutions were adjusted (using a 0.01M HCl solution) to a same pH value of 3.8 ± 0.1 prior to ESI-MS analysis. Such an acidification procedure allows a direct comparison of the datasets obtained for the different experiments (as pH is known to affect ionization of compounds during ESI-MS analysis). An exception was however made for the supernatant of the PPH-kaolinite sorption experiment at pH 5.0, which was analyzed as collected because the ESI(-)MS spectra obtained for the acidified supernatant sub-sample were found to be of very poor quality due to the amount of salt introduced in the sample to obtain a pH of 3.8. It is to be noted that the ESI-MS data obtained from an analysis at pH 5.0 cannot be straightforwardly compared to those obtained from analysis at pH 3.8. Complementary series of batch experiments were performed to study the macroscopic sorption of FA as a function of pH and to perform on-line electro-kinetic measurements of the FA-containing suspensions of kaolinite or alumina. Same procedure as that described above was used, except that an aliquot of sample was taken before centrifugation for particle electrophoretic mobility (EM) measurements, which were carried out using dynamic light scattering with a Malvern Zetasizer Nano ZS instrument. Values reported for EM correspond to mean values calculated on the basis of three replicates of a same measurement. Sorption isotherms and EM values in the pH range 3.5-7 were determined for the alumina -PPH systems (r = 5-10 mgC/g). For the kaolinite -PPH system, both the effect of pH and ratio r on the percent of FA sorption and EM of particles were investigated (r value up to 50 mgC/g).

ESI-MS instrumentation and data analysis

Instrumentation and acquisition parameters

A Thermo Scientific LTQ Orbitrap hybrid mass spectrometer was used for the analysis in ESI negative ionization mode of native FA solutions and supernatants. Negative ion ESI appears to be the most appropriate ionization mode for the analysis of anionic compounds such as fulvic acids, which are already partly preionized in solution. Ultra-pure water was used as the electrospray solvent as other solvents have been found to alter the FA properties [START_REF] Piccolo | Chromatographic and spectrophotometric properties of dissolved humic substances compared with macromolecular polymers[END_REF]. The samples were infused directly into the ESI source with a spray voltage of 3.5 kV at a flow rate of 10 μL.min -1 , and were analyzed in negative ion mode. Nitrogen was used as the drying and spraying gas. For each acquisition, 100 scans (2s/scan) were co-added using Xcalibur software. The Orbitrap MS was externally calibrated in negative ion mode on the 50-2000 m/z range using a LTQ/FT-Hybrid ESI Calmix solution, for a mass accuracy better than 3 ppm. Instrument operating parameters (capillary voltage and temperature, tube lens voltage) were optimized one after the other for an optimal transmission of higher m/z ions. It was found that modifying the instrumental parameters did not affect important characteristics of spectra such as ion distribution, charge state or mass spacing patterns. The source capillary was finally held at -50 V and 275 °C, and a voltage of -240 V was applied to the tube lens.

All ESI(-) mass spectra were recorded in the ranges 120-400 m/z and 400-798 m/z in order to improve the mass accuracy in the region of low masses as well as the signal-to-noise ratio (S/N) for compounds having the highest values of m/z in samples.

Elemental formula determination

Elemental formulae were assigned to the measured m/z values using the Xcalibur software. The following criteria were chosen for assignment of molecular formulas to the peaks detected on the mass spectra:

 Signal-to-noise ratio of peak > 4,  Mass accuracy better than 3 ppm,  Application of the following ranges: 14 N  1; 16 O  50; 12 C  200; 1 H  600 (N, O, C and H being respectively the number of Nitrogen, Oxygen, Carbon and Hydrogen atoms).

 0,2C  H  2C + 3 Double Bond Equivalent
Elemental analyses showed that the PPH sample contains low amounts of Nitrogen (< 3% w/w). Sulfur and Phosphorus contents were assumed negligible (<1% w/w). Odd m/z values correspond to even mass, and relate to chemical formulae with even number of nitrogen atoms. As the nitrogen content in the sample does not exceed a few percent, we assumed that compounds peaking at odd m/z primarily contain C, H and O atoms. All possible formula attributable to a given odd m/z value were thus calculated by considering only C, H and O atoms. Due to low nitrogen content in HS, species peaking at even m/z were considered to contain at most one N atom, in addition to C, H and O atoms. Ions corresponding to 13 C isotopes and elemental formulae that did not match the above criteria were not taken into account.

Van krevelen diagrams

The Van Krevelen (VK) diagram plots the H/C ratio vs. the O/C ratio of a molecule and is an effective graphical method for displaying complex data from NOM analysis by ultrahigh-resolution mass spectrometry [START_REF] Kim | Graphical Method for Analysis of Ultrahigh-Resolution Broadband Mass Spectra of Natural Organic Matter, the Van Krevelen Diagram[END_REF]. On the VK diagrams, the identified compounds are classified into three categories on basis of their value of Aromaticity Index (A.I.) proposed by [START_REF] Koch | From mass to structure: an aromaticity index for high-resolution mass data of natural organic matter[END_REF]. The authors proposed a modified formula for calculating A.I., considering that half of the oxygen is σ-bound: They defined two threshold values of AI to distinguish between three categories of compounds, i.e., between compounds having an unambiguous polycyclic aromatic structure (PACs, having 0.67 < A.I.), a notcondensed aromatic structure (NCAs, having 0.5 < A.I. < 0.67), and a more or less pronounced aliphatic character (A.I. < 0.5).

Definition of I

In order to explore the mechanisms of the sorptive fractionation of FAs, we used the parameter I defined by [START_REF] Galindo | Molecular Level Description of the Sorptive Fractionation of a Fulvic Acid on Aluminum Oxide Using Electrospray Ionization Fourier Transform Mass Spectrometry[END_REF]. The value of I for a given ion is equal to the ratio of the normalized peak intensity of this ion in the supernatant (after sorption) on the normalized peak intensity of the ion in the initial solution (before sorption), with the normalization being made to the sum of the intensities of all the ions (Total ion current, TIC) of the considered solution. Trends in the values of I for compounds in a FA supply information on trends existing in the orders of relative affinity of compounds for the mineral surface: the lower the value of I of a FA ion, the higher its relative affinity (by comparison with that of the other detected FA ions) for the surface of the mineral. Ions with I = 0 are totally sorbed at the surface of mineral, and ions with I ≠ 0 are distributed between the surface and the solution.

It is well known that the intensity of an ion on a mass spectrum not only depends on its concentration but also on its ionization efficiency and on the matrix, i.e. on the number, the quantity and the structure of the other ions present in the sample. A simple dilution of initial FA solution can thus possibly have an effect on the relative peak intensities of the ions in the solution. As bulk concentration of PPH is lower in the supernatants collected at the end of the sorption experiments than in the native PPH solution (due to PPH sorption at the surface of the minerals), it is necessary to determine the effect of such a sorption-driven decrease in solution concentration on the I values of the FA ions and to differentiate it from the effect on I of the sorptive fractionation process itself. To this end, we compared the ESI(-)MS spectra recorded for initial PPH solution at pH 3.8 and at pH 4.9 used in the experiments to those obtained for PPH native solutions diluted by a factor 2, and we calculated the "I values" of the FA ions resulting from a pure dilution of samples. Dilution by a factor of 2 was chosen because approximately 50% of total organic carbon was found to be removed from solution during the experiments of PPH sorption onto alumina and kaolinite. It was found (cf. Fig. A1 in the Appendix) that all PPH ions display I values equal to 0.85 ± 0.15 due to dilution effect only (except for the 400-798 m/z range at pH 3.8 where the values were equal to 1.05 ± 0.15). Applying these results of the dilution tests to the interpretation of the I values of ions derived from the sorption experiments, it can be considered that a value of I lower than 0.7 (or 0.9 for the range 400-798 m/z range at pH 3.8) indicate a rather good sorption of an ion on the mineral surface, while ions showing values comprised between 0.7 and 1 (or 0.9 and 1.2 for the range 400-798 m/z range at pH 3.8) are poorly sorbed or not sorbed at all, and ions showing values higher than 1 (or 1.2) are not sorbed at all during the experiment.

RESULTS

Macroscopic description of the FA-mineral systems

Fig. 1 shows the percentage of organic carbon of PPH sorbed onto alumina or kaolinite as a function of pH, for different FA/mineral ratios (r). The results are as follows.

Regarding alumina, first, the sorption isotherm obtained for PPH indicate a slight pH-dependency of the sorption, i.e., a slight decrease of the percentage of sorption with increasing pH. Such a decrease with increasing pH value is consistent with an increase of the rate of deprotonation of both the reactive groups of the FA molecules and the aluminol sites on the surface of alumina. It has been widely reported that the carboxyl and phenol functional groups of FAs exhibit wide distributions of pK values, which are mostly lower than 4 for carboxyl groups (e.g. [START_REF] Ritchie | Proton-binding study of standard and reference fulvic acids, humic acids, and natural organic matter[END_REF] and higher than 8 for phenol moieties (e.g. [START_REF] Gu | Adsorption and desorption of natural organic matter on iron oxide: mechanisms and models[END_REF]. Moreover, the IEP of the alumina particles used here was previously found to be equal to 8.5 (Del [START_REF] Nero | u ivi y o α-Al2O3 and mechanisms of phosphate sorption: In situ ATR-IR sp os opy nd ζ po n i studies[END_REF]. An increase of the deprotonation with pH thus has the effect of diminishing electrostatic attractions between aluminol sites on the mineral surface and FA molecules, which is unfavorable to a close approach of the organic molecules to the vicinity of the surface. Thus, such features may lead to a slight decrease of the percentage of macroscopic sorption with pH observable in Fig. 1. The results of EM measurements performed on the systems investigated are presented on Fig. 2. The data show a sign reversal of the surface charge of the suspended alumina particles in the presence of PPH, evidencing that the FA-alumina interactions are not of electrostatic nature only but involve the formation of inner-sphere fulvate complexes on the surface of alumina. Chemical sorption is indeed the only sorption process capable of inducing such a sign reversal [START_REF] Fairhurst | The influence of humic acid on europium-mineral interactions[END_REF].

Fig. 1 shows that PPH has a lower overall affinity for the kaolinite surface than for the alumina surface (cf. data at r= 5mgC/g). There also appeared that the percentage of PPH sorption decreases with an increase of the PPH / kaolinite ratio (cf. data at r= 5 and 55 mgC/g). Such a feature indicates that multiple sites on the mineral surface were involved in the sorption of the PPH molecules and/or several types of surface complexes of very distinct values of formation constant were formed. The former hypothesis is consistent with results of many previously published studies reporting the existence of weak surface sites at basal planes of kaolinite particles and of strong sites (high-affinity sites) at edges and/or defects of kaolinite [START_REF] Sverjensky | Thoretical prediction of single-site surface-protonation equilibrium constants for oxides and silicates in water[END_REF]. The high-affinity sites existing in limited amounts onto the kaolinite particles might be saturated in our experiments at low surface loadings of FA. High-affinity sites have long been reported to be protonated under acidic and neutral pH conditions whereas the early deprotonation of basal aluminol sites [START_REF] Wieland | Dissolution kinetics of kaolinite in acidic aqueous solutions at 25°C[END_REF]) is (partly) responsible for an overall negative surface charge of kaolinite (e.g. data on Fig. 2 obtained in the absence of FA). EM measurements reported in Fig. 2 shows that the sorption of the PPH molecules imparts negative charges to the kaolinite surface, which is consistent with involvement of the highaffinity sites in the sorption.

In summary, surface properties of mineral have an effect on the macroscopic behavior of sorption of FA at acidic/near neutral pH, which highlights a need for further understanding their effect on the fractionation of FA.

ESI(-)-FTMS spectra of initial solution of PPH and chemical formula assignments

The full mass spectrum recorded in the mass range 120-2000m/z for the native solution of PPH, using the negative ionization mode, showed a mono-modal distribution of peaks lying between 120-800m/z (cf. Fig. A2 in the Appendix). The presence on our mass spectra of peaks at m/z<800 only is globally consistent with previously published ESI-MS spectra of fulvic acids, which exhibit compounds of molecular weight no higher than a few kDa [START_REF] Piccolo | The supramolecular structure of humic substances[END_REF][START_REF] Sutton | Molecular Structure in Soil Humic Substances: The New View[END_REF][START_REF] Reemtsma | Determination of molecular formulas of natural organic matter molecules by (ultra-) high-resolution mass spectrometry[END_REF]. Several authors have underlined that their mass spectrometry data support the view of HS as being a supramolecular assembly, which can be dissociated during ionization in the ESI probe, in particular when high voltages are applied to the transfer optics [START_REF] Piccolo | Effects of sample properties and mass spectroscopic parameters on electrospray ionization mass spectra of size-fractions from a soil humic acid[END_REF]. Fig. 3 shows the ESI(-) mass spectra recorded for the native solution of PPH in the ranges 120-400 m/z and 400-798 m/z. Circa 1725 and 1190 peaks were detected with S/N > 4 in the aforementioned mass ranges, respectively. The spectra display peaks of high intensity at odd-integral values of m/z and peaks of lower intensity at even-integral values of m/z, with the latter peaks having been reported in previously published studies to correspond to either the 13 C-containing isotopes of the odd m/z ions or to components containing one nitrogen atom [START_REF] Brown | Effect of Experimental Parameters on the ESI FT-ICR Mass Spectrum of Fulvic Acid[END_REF][START_REF] Leenheer | Molecular Resolution and Fragmentation of Fulvic Acid by Electrospray Ionization/Multistage Tandem Mass Spectrometry[END_REF][START_REF] Stenson | Ionization and Fragmentation of Humic Substances in Electrospray Ionization Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry[END_REF][START_REF] Kim | Graphical Method for Analysis of Ultrahigh-Resolution Broadband Mass Spectra of Natural Organic Matter, the Van Krevelen Diagram[END_REF]. No spacing equal to (1.0034/n)m/z (with n = 2, 3…) was detected between the monoisotopic peak and the corresponding peak for the one 13 C-containing isotope, showing that no multiply charged species were generated in the ESI probe during analysis of PPH. The PPH mass spectra display a groups [START_REF] Stenson | Exact Masses and Chemical Formulas of Individual Suwannee River Fulvic Acids from Ultrahigh Resolution Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectra[END_REF][START_REF] Reemtsma | Differences in the molecular composition of fulvic acid size fractions detected by size-exclusion chromatography-on line Fourier transform ion cyclotron resonance (FTICR-) mass spectrometry[END_REF][START_REF] Hertkorn | Natural Organic Matter and the Event Horizon of Mass Spectrometry[END_REF], as reported previously for spectra of DOM analyzed by using FT-ICR MS [START_REF] Stenson | Ionization and Fragmentation of Humic Substances in Electrospray Ionization Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry[END_REF][START_REF] Stenson | Exact Masses and Chemical Formulas of Individual Suwannee River Fulvic Acids from Ultrahigh Resolution Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectra[END_REF][START_REF] Reemtsma | Differences in the molecular composition of fulvic acid size fractions detected by size-exclusion chromatography-on line Fourier transform ion cyclotron resonance (FTICR-) mass spectrometry[END_REF][START_REF] Hertkorn | Natural Organic Matter and the Event Horizon of Mass Spectrometry[END_REF]. Homologous -CH 2 series identified for PPH by Kendrick mass analysis were found to contain up to 14 molecules, which suggests that the -CH 2 group is an important constitutive element in the structure of FAs (e.g. Fig. A3 in the Appendix). Numerous homologous series were observed and accounted for the large number of different basic structures in PPH, similar to what was reported for DOM samples (Kujawinski, Hatcher, et al., 2002). i.e., polycyclic aromatic compounds (PACs), not-condensed aromatics (NCAs), and compounds of aliphatic character. Such VK diagrams with a wide distribution of compounds is somehow contrasting with most of those previously published for soil humic substances [START_REF] Rice | Statistical evaluation of the elemental composition of humic substances[END_REF]D'Andrilli et al., 2015) showing data-points gathered in more restricted areas of O/C. However, there are observed in Fig. 4b

Molecular-scale study of the sorptive fractionation of PPH

Influence of mineral surface characteristics

At first examination, ESI(-) spectra provide strong evidence that PPH fractionated during the sorption process.

Fig. 3b and Fig. 3c report spectra of supernatants collected in PPH sorption experiments which were conducted at pH 3.8 ± 0.1 (and PPH-to-solid ratio of 5mgC/g) and which led to 63% and 40% sorption of organic carbon onto α-alumina and kaolinite, respectively (Fig. 1). The total ion currents recorded for the mass spectra of the supernatants were lower than those recorded for the initial solution. Moreover, peak intensities were lower in the mass spectra of the supernatants than in those of PPH. Circa 19% and 11% of compounds detected with S/N > 4 on the PPH mass spectra were not detected on the supernatants' spectra, indicating that the compounds were totally sorbed on the surfaces of alumina and kaolinite during the experiments, respectively. On the contrary, some ions were detected on the mass spectra of the supernatants only. This phenomenon has already been reported by [START_REF] Reiller | Sorption of Aldrich humic acid onto hematite: Insights into fractionation phenomena by electrospray ionization with quadrupole time-of-flight mass spectrometry[END_REF] and [START_REF] Galindo | Chemical fractionation of a terrestrial humic acid upon sorption on alumina by high resolution mass spectrometry[END_REF] in their ESI MS studies of sorption of HA onto hematite and HSs onto alumina, respectively. Actually, the intensity of an ion on a mass spectrum not only depends on its concentration but also on its ionization efficiency and on the matrix, i.e. on the number, the quantity and the structure of the other ions present in the sample. A possible explanation for the detection of some ions in the supernatants' spectra only is that ions undetectable in the native solutions of PPH because of matrix complexity might be ionized more efficiently in the conditions of the supernatants' matrixes due to combined effects of their low sorption onto minerals and of a release of the ionization charge upon preferential sorption of certain PPH compounds. In summary, important features of the spectra were that the extent of the lowering of peak intensities from initial solution to supernatants was varying considerably with the considered ion -going from a peak disappearance to a lowering higher than that of the TIC-, and with the type of mineral. Hence, the degree of sorption of a PPH compound was dependent on both the type of compound and the characteristics of mineral surface.

VK diagrams in Fig. 5 provide information on the order of relative affinity of PPH molecules (sorted as a function of their values of I) for the surfaces of alumina and kaolinite. A main result is that PPH is strongly fractionated between mineral surfaces and solution. Some compounds of low values of I (0<I<0.7 in Figs. 5a,c and 0<I<0.9 in Fig. 5b,d

Relative affinity order for mineral surfaces of PPH compounds in -CO 2 and -CH 2 series

Relations existing between the sorption degree of a molecule in a -CO 2 series and its number of CO 2 groups, as representative for the number of carboxyl functionalities, or between the sorption degree of a molecule in -CH 2 series and its number of -CH 2 groups, as representative for length/number of aliphatic chains, were investigated to gain further information on the mechanisms involved in the sorption of PPH onto alumina and kaolinite. -CO 2 series or -CH 2 series are made up by compounds whose chemical formula differ only by their number of CO 2 or CH 2 , respectively. Figs. 6a,b report, for alumina-solution systems at pH 3.9, the evolution of I as a function of number of carbon atoms (n C ) of PPH compounds in -CO 2 series of aliphatic compounds or oxygenated NCAs compounds (O/C>0.2), and in -CO 2 series of PACs or poorly-oxygenated NCAs, respectively. Figs. 6c,d refer to the results obtained for the -CH 2 series. A striking result is the difference in the n C -I relations existing between the two sets of series. For the aliphatic compounds or the NCAs with O/C>0.2 (Fig. 6a), the -CO 2 series show an inverse correlation between the value of I and the number of carbon atoms, n C. This indicates that the degree of sorption of a molecule in the series is directly related to its number of carboxyl groups. For the -CH 2 series, a sharp increase of I with n C was observable (Fig. 6c) indicating that the affinity for surface of a molecule within a series decreases with an increasing of the length/number of aliphatic chains. Such remarkable and monotonous n C -I relations, which were observed for all the -CO 2 /-CH 2 series investigated, suggest that a single mechanism controls the degree of sorption onto alumina of aliphatic compounds and oxygenated NCAs. The fact that distinct molecules of a same -CO 2 /-CH 2 series display very different degrees of sorption -some being totally sorbed (I = 0) and others being not sorbed at all (I > 1.2)-clearly indicate that the supramolecular structure of the FA existing in solution is greatly modified during sorption. Probably, the sorption implies strong forces which compete efficiently against the weak cohesion forces of the supramolecular assemblies, leading to different sorption behaviors for the distinct FA building blocks at the surface of alumina. Regarding the PACs and the poorly-oxygenated NCAs, more complex features were observed. The degree of sorption of a molecule within a -CH 2 series was found to be only weakly correlated with its number of CH 2 groups (only a small increase of I with n c is observable in Fig. 6d). In contrast, -CO 2 series showed a noticeable and not monotonous evolution of I with n C . Indeed, bell-shaped curves were observed: the last members of the -CO 2 series showed a decrease of I with an increase of n C while the first members of the series showed the opposite (Fig. 6b). Hence, PACs and NCA compounds with O/C<0.2 show different sorption trends according to their molecular O/C ratio, which suggests that within a -CO 2 series of compounds two different mechanisms of sorption (at least) operate, one favoring the retention of molecules containing some oxygen and another favoring the retention of molecules depleted in oxygen. Figs. 7a,b show, for kaolinite-solution systems at pH 3.7, plots of I versus n c for -CO 2 series of aliphatic compounds or oxygenated NCAs compounds (O/C>0.2) and for -CO 2 series of PACs or poorly-oxygenated NCAs, respectively. The same series as those described above for alumina were considered for kaolinite. The most striking result is that the values of I for -CO 2 series of aliphatics or highly oxygenated NCAs (Fig. 7a) are in a very limited range, showing that all compounds in a series have almost a similar relative affinity for the surface of kaolinite. Such a trend in kaolinite-solution systems is also visible for the -CO 2 series of PACs and oxygen-depleted NCA molecules (Fig. 7b). These results contrast markedly with those reported above for the surface of alumina, for which values of I varied largely amongst compounds of a -CO 2 series (cf. Figs. 6a,b). It is also to be noted that, in the PPH-kaolinite system, none of the -CO 2 series investigated showed well-sorbed molecules with values of I lower than 0.5. All these results confirm that the clay surface is less selective than the Al-oxide surface and that molecular characteristics such as number of -CO 2 groups and/or number/length of aliphatic chains are not determining parameters regulating the sorptive fractionation of PPH onto kaolinite.

Effect of pH on the sorptive fractionation of PPH

Regarding the sorption onto alumina, it appears that the relative affinities of PPH molecules for the surface do not change significantly in the acidic pH range 3-5, as shown by comparing VK diagrams obtained for the system at pH = 4.8 (Figs. 8a,b) and at pH = 3.9 (cf. Figs. 5a,b). Correlations observed at pH 4.8 between the degree of molecule sorption and molecular parameters within -CO 2 or -CH 2 series (data not shown) were similar to those described for the pH 3.9 (cf. paragraph 3.3.2).

Regarding the sorption onto kaolinite, it is first to be noted that fewer compounds are represented on the VK diagrams reporting the relative affinities of PPH molecules for surface at pH 5.0 (Figs. 8c,d) than at pH 3.7 (Figs. 5c,d). This is due to the fact that supernatants for the kaolinite-PPH-solution systems had to be analyzed at pH 5.0 (cf. section 2.2), which leads to an alteration of the MS response (for illustrative purposes, compare VK diagrams of native PPH solution analyzed at pH 3.8 in Fig. 4 and at pH 5.0 in Fig. A4 in the Appendix).

Despite fewer compounds are detected for the native solution of PPH at pH 5.0 than at pH 3.8, some key trends in the pH-dependency of the sorption of PPH onto kaolinite can still be determined. Some highly oxygenated aliphatics and NCAs are poorly sorbed at pH 5.0 (while they display a good affinity for the surface at pH 3.7). This suggests the existence of a process that competes against sorption at pH 5.0 and tends to keep these highly oxygenated molecules in solution. Interestingly, the final concentration of dissolved aluminum in kaolinite-PPH suspensions at pH 5.0 was found to be ca. four times higher than in kaolinite suspensions without PPH (cf. Fig. A5 in the Appendix). This supports the formation in kaolinite-PPH systems of solution complexes implying the most oxygenated aliphatic and NCA compounds and the dissolved aluminum originating from the dissolution of kaolinite.

DISCUSSION

A main finding is that the trends in the sorptive fractionation of PPH in kaolinite-solution systems are very different than those observed for alumina, in the present work or in previously published studies of molecularscale alumina-HS-interactions [START_REF] Galindo | Molecular Level Description of the Sorptive Fractionation of a Fulvic Acid on Aluminum Oxide Using Electrospray Ionization Fourier Transform Mass Spectrometry[END_REF][START_REF] Galindo | Chemical fractionation of a terrestrial humic acid upon sorption on alumina by high resolution mass spectrometry[END_REF]. Such a finding provides strong evidence that the mineral surface properties are a key parameter governing the sorptive fractionation of HS.

All the results obtained at the molecular scale for the kaolinite-solution system suggest that the sorption of PPH onto kaolinite is mostly the result of a weakly selective mechanism. For example, the dispersion of the totally sorbed PPH compounds over a wide region of VK diagram and the closeness of the I values of the molecules in -CO 2 series well exemplify that the number of CO 2 groups in a molecule and, more generally, molecular acidity, have a slight influence on the degree of sorption of NCAs and aliphatics onto kaolinite.

These features rule out a substantial contribution to PPH sorption of a surface ligand exchange mechanism between the hydroxyl groups existing on the kaolinite surface and the carboxyl groups of PPH. Though, it has been widely reported that high affinity aluminol sites on singly coordinated oxygens at edges and steps of kaolinite surface have chemical properties close to the surface sites of alumina (e.g. [START_REF] Kummert | The surface complexation of organic acids on hydrous y-Al2O3[END_REF]. These sites probably have a strong affinity for the highly oxygenated compounds of PPH but are not numerous enough to have a measurable effect on the overall sorptive fractionation pattern of PPH.

Based upon the structure of fulvic acid molecules, which have numerous functional groups with hydrogens bound to heteroatoms (N, O), hydrogen bonding seems to be a good candidate because such a mechanism is not as selective as surface ligand exchange. Hydrogen bonding well accounts for the lack of preferential sorption observed amongst all aliphatics and NCA molecules of O/C > 0.2 (Fig. 5d). Sorption of most of PPH compounds, including the hydrogenated compounds, may thus imply aluminol or silanol groups existing on the doubly coordinated oxygens located on the basal planes of kaolinite, which are poorly selective sites expected to be involved in weak sorption mechanisms such as hydrogen bonding. Probably, such weak sorption mechanisms do not compete efficiently against the cohesion forces of the supramolecular FA assemblies. The supramolecular structure would thus be globally preserved during sorption, accounting for the similar affinities of the different FA compounds for the surface of kaolinite. Implication of these low affinity sites not only accounts for a weak fractionation amongst members of -CO 2 series of aliphatics or NCAs during their sorption onto kaolinite, but also for the very low degree of sorption of PACs. No positive n c -I correlation were observed in kaolinite-solution systems for -CO 2 series of PACs with low O/C ratios, suggesting a poor contribution of hydrophobic interactions between PACs and kaolinite surface. Possibly, the rather limited degree of sorption onto kaolinite of the PACs bearing oxygenated functionalities (due to low amounts of high affinity surface aluminol sites) resulted in few amounts of hydrophobic domains available, in the first layer of PPH molecules sorbed at the clay surface, for further interactions with hydrophobic moieties of NCAs and PACs of low O/C present in solution. Moreover, the first layer of sorbed compounds is only weakly bonded on the basal planes of the kaolinite surface, preventing a strong attach of poorly-oxygenated PACs on the surface. Implication of the low affinity sites on the basal planes of kaolinite also accounts for the pH-dependency of the sorption of the highly oxygenated aliphatic and NCA compounds of PPH. A decrease of the degree of sorption of these molecules at pH 5.0 results from their complexation with aluminum in solution competing against their sorption at the surface of the mineral. Indeed, an increase in pH value induces the deprotonation of both the aluminol/silanol groups on the basal planes of kaolinite, as well as of the carboxyl groups of PPH.

Such a deprotonation has the effect of preventing hydrogen bonding of PPH molecules on the kaolinite surface sites, and of decreasing the degree of sorption of the highly oxygenated aliphatic and NCA compounds. Moreover, the deprotonation of the carboxyl groups of these molecules also favors the formation of FA-aluminum complexes in solution. The pH-dependency of the sorption of highly oxygenated aliphatic and NCA compounds in PPH is thus the result of both these processes.

The molecular-scale findings on the sorptive fractionation trends of PPH during sorption at the clay surface are quite contrasting with those observed for the alumina-solution-PPH systems, for which molecules showing the highest affinity for the mineral surface are the PACs and the highly oxygenated compounds amongst aliphatics or NCAs. For the latter compounds, strong evidence is provided that a single mechanism controls their sorption at the alumina surface, as shown, for example, by the remarkable positive correlations existing for PPH compounds in CO 2 series between the degree of sorption of a molecule and its molecular O/C ratio.

Highly-oxygenated NCA and aliphatic compounds carry multiple carboxyl and phenol groups which are well known to be involved in ligand exchange reactions (specific adsorption) with surface aluminol groups, e.g. [START_REF] Schlautman | Adsorption of aquatic humic substances on colloidal-size aluminum oxide particles: Influence of solution chemistry[END_REF]. The positive correlation observed between affinity for the surface of alumina and number of carboxyl groups of a PPH compound in a -CO 2 series even includes molecules containing up to 5 groups or more (e.g. Fig. 6). As discussed in previously published papers by our team [START_REF] Galindo | Molecular Level Description of the Sorptive Fractionation of a Fulvic Acid on Aluminum Oxide Using Electrospray Ionization Fourier Transform Mass Spectrometry[END_REF], it is unlikely that more than three of carboxyl groups of a molecule are involved simultaneously in multiple bonding with aluminol groups at the surface of alumina. The concomitant increase of affinity for alumina surface and nCO 2 of compounds in CO 2 -series of the types highly-oxygenated NCAs or aliphatics is thus due likely to a change in molecule acidity. Such an interpretation was already proposed for simple organic acids by [START_REF] Evanko | Influence of structural features on sorption of NOManalogue organic acids to goethite[END_REF], who suggested that enhanced sorption with increasing number of carboxyl groups of acids was due to changes in the acidity of compound rather than to multiple bonding, and was supported by the results of [START_REF] Janot | Characterization of humic id ivi y modi i ions du o dso p ion on o α-Al2O3[END_REF]. The interpretation is also supported by our results

showing that a lengthening of aliphatic chains of a molecule within a -CH 2 series, which is expected to decrease the molecule acidity due to donor inductive effect, decreases the affinity for the surface of alumina.

Regarding the NCAs with O/C < 0.2 and the PACs, the bell shape of the curves reporting affinity of molecules in CO 2 -series for alumina surface vs. number of carboxyl groups indicate that different mechanisms are responsible for the sorption of molecules of lowest O/C ratios and highest O/C ratios, respectively. For the latter molecules, acidity is the key parameter controlling the degree of sorption (same types of n C -I inverse correlation as those observed for the aliphatics), and surface ligand exchange is the mechanism driving the sorption. For the former compounds, the positive n c -I correlation observed rules out acidity as a parameter governing sorption. The existence of this correlation for compounds having many hydrophobic moieties (e.g. aromatic structures) and few oxygenated functional groups provides strong evidence that their degree of hydrophobicity determines their affinity for the surface of the oxide. A possible explanation is as follows. As evidenced by the impact of molecule acidity on the degree of sorption of a FA molecule onto alumina, -either for aliphatics, NCAs and PACs-, the supramolecular structure of FA appears to be not preserved during sorption, allowing : (i) the sorption of the most highly acidic FA compounds to be sorbed via surface ligand exchange involving their hydrophilic moieties and (ii) the release into solution of PACs with very low O/C and their subsequent sorption via interaction between their hydrophobic moieties and those of the strongly sorbed PACs (which form a first sorption layer onto alumina). Another possible interpretation is that the electric fields induced by the surface charge of alumina influence the dielectric constant of water [START_REF] Booth | The Dielectric Constant of Water and the Saturation Effect[END_REF]. Water at the surface of alumina would thus have a lower apparent dielectric constant, which favors the adsorption of hydrophobic molecules. Molecular-scale evidence that the degree of hydrophobicity governs the degree of sorption of poorly-oxygenated PACs and NCAs onto a metallic oxide has been published recently for a peat humic acid [START_REF] Galindo | Chemical fractionation of a terrestrial humic acid upon sorption on alumina by high resolution mass spectrometry[END_REF] but has never been so far demonstrated for fulvic acids.

The molecular-scale results presented here for the sorption of PPH are useful for rationalizing the contrasting results of spectroscopic studies published on the sorption of bulk FAs at the surfaces of metallic (hydr)oxides and clays, respectively. [START_REF] Ghosh | Sorption and fractionation of a peat derived humic acid by kaolinite, montmorillonite, and goethite[END_REF] studied by spectroscopy techniques the fractionation behavior of HS on minerals with different surface properties. They reported that aliphatic fractions of HS are more prone to sorption at the surface of clay minerals than the highly aromatic fractions, while carboxylic functional groups play a significant role in the sorption of HA on goethite. Spectroscopic studies have long reported preferential sorption of aromatic structures bearing oxygenated functionalities of HS at the surface of metallic (hydr)oxides [START_REF] Meier | Fractionation of aquatic natural organic matter upon sorption to goethite and kaolinite[END_REF][START_REF] Zhou | Size fractionation upon adsorption of fulvic acid on goethite: equilibrium and kinetic studies[END_REF]Kaiser, 2003;Claret et al., 2008). For example, Kaiser showed for HS-solution-goethite systems a preferential removal from solution of aromatic and carboxyl C, whereas alkyl-C accumulated in the solution. Claret et al. (2008) reported a preferential sorption at the surface of alumina of the hydrophobic fractions of FA and/or of the FA compounds showing high contents in aromatic moieties and functional groups, as compared to aliphatic fractions. In the present study, on the one hand, we provide molecular-scale evidence that most of the acidic, aliphatic compounds of a FA display a greater affinity for the kaolinite surface than PACs because the most effective mechanism of sorption of FA molecules at such a clay surface is a weak sorption via H-bonds at the aluminol / silanol sites on the doubly coordinated oxygens located on the basal planes of the clay. On the other hand, the present molecular-scale study also confirms unequivocally that clear relationships exist between molecular parameters such as acidity or hydrophobicity of a molecule and its degree of sorption in a metallic oxide-solution system. The sorptive fractionation of FAs in such systems, in contrast to clay systems, is due to a competition between protons and metallic centers at the surface of the mineral for coordination of FA molecules, and to the subsequent sorption of poorly-oxygenated PACs by hydrophobic forces involving hydrophobic moieties in the first layer of sorbed FA molecules on the oxide surface.

CONCLUSION

The results of the present study on the fractionation behavior of a soil FA onto alumina and kaolinite provide valuable molecular-scale insights on the relationships existing between the chemical characteristics of the molecules and their distribution between aqueous and mineral phases, as well as on the distinct mechanisms involved in the fractionation of a FA during its sorption onto minerals of different surface properties. A main finding is that the molecular-scale fractionation of FA in kaolinite suspensions is highly contrasting with that observed here for alumina or with those reported in previously published studies for aluminum oxide [START_REF] Galindo | Molecular Level Description of the Sorptive Fractionation of a Fulvic Acid on Aluminum Oxide Using Electrospray Ionization Fourier Transform Mass Spectrometry[END_REF][START_REF] Galindo | Chemical fractionation of a terrestrial humic acid upon sorption on alumina by high resolution mass spectrometry[END_REF]. Such a finding highlights that the key parameter controlling the sorptive fractionation trends of FAs is the nature, i.e. oxides vs. clays, and the surface properties of sorbing minerals.

The data presented here confirm previous work by our team [START_REF] Galindo | Molecular Level Description of the Sorptive Fractionation of a Fulvic Acid on Aluminum Oxide Using Electrospray Ionization Fourier Transform Mass Spectrometry[END_REF] indicating that acidity of FA molecules is a main parameter determining the relative degree of sorption onto Al-oxides of FA molecules, with the highly acidic molecules bearing oxygenated functionalities being sorbed preferentially via surface ligand exchange involving the high-affinity aluminol surface sites. Our study also reveals that degree of hydrophobicity of PACs and NCAs of low O/C ratio is another key molecular parameter influencing the degree of sorption of these FA compounds, which are able to interact with hydrophobic domains of organic molecules (probably mainly PACs) strongly retained at the Al oxide surface within a first layer of specifically-sorbed organic molecules. The present study establishes that, whatever the FA composition and in a range of acidic pH values, surface ligand exchange and hydrophobic interactions are the main (cooperative) mechanisms leading to the preferential retention on metal oxides of most of PACs, of NCAs with high O/C ratios (>0.5) and of highly-oxygenated aliphatic compounds (O/C>0.7), whereas hydrogenated and less oxygenated NCAs and aliphatics are left in the solution.

The first molecular-scale description presented here of the fractionation of a FA in a clay mineral-solution system reveals that, unlike for metallic oxides, molecular acidity has no substantial effect on the degree of sorption of a FA molecule onto a clay, and that surface ligand exchange is not the mechanism driving the sorptive fractionation of FA in such systems. Instead, the main mechanism of sorption is hydrogen bonding of FA molecules at the low affinity sites (aluminol or silanol) present on the basal planes of the clay, as evidenced here by the weak sorptive fractionation existing amongst members of -CO 2 homologous series of aliphatics or NCAs during their sorption onto kaolinite at low pH. In contrast, at slightly acidic pH (5), a fractionation occurs to a certain extent between molecules within a same class of FA compounds (for aliphatics as well as NCAs), due to a decreasing with pH of the degree of sorption of highly acidic molecules bearing oxygenated functionalities. Such a fractionation is explained by the fact that dissolved Al has the ability to compete successfully for the coordination of such reactive organic molecules against a weak sorption process such as H-bonding. Finally, the predominance of a weak and poorly-selective mechanism of sorption of FA molecules like H-bonding indirectly accounts for a very low degree of sorption of PACs or of low O/C NCAs onto kaolinite. Absence of preferential and specific sorption at surface of kaolinite of aromatic structures activated by oxygenated functionalities prevents the formation of a first layer of strongly-sorbed molecules exhibiting hydrophobic domains which would be available for further sorption of FA compounds via hydrophobic interactions.

The results of the present molecular scale studies have several implications on our knowledge of storage of natural organic matter in soils as well as on the cycle of metals. It is to be expected that clayey soils trap lesser amounts of condensed aromatics than metal oxide-rich soils and may retain non-selectively most of FA compounds of the types of aliphatics or not-condensed aromatics at acidic pH, via weak sorption mechanisms.

In turn, solutions percolating through clayey soils, if being slightly acidic, would contain a multiplicity of the FA organic acids bearing multiple oxygenated functionalities, owing to the high ability of these FA molecules for binding dissolved metals and their only weak sorption at clay surfaces. Such a feature would promote the transport of metal pollutants with soil solutions. Consequently, (long term) metal sorption capacities of clayey soils are expected to be rather low compared to that of soils rich in Al oxi-hydroxides.

Fig. 1 :Fig. 2 :

 12 Fig. 1: Sorption isotherms of organic carbon obtained for different PPH-to-mineral ratios in alumina or

  0156 amu repeating unit due to the existence of homologous series of species differing by -CH 2

Fig. 3 :

 3 Fig. 3: Negative ion ESI mass spectra in the ranges 120-400 m/z and 400-798 m/z of PPH native solution (a),

Fig. 4 :

 4 Fig. 4: VK diagram showing elemental formulae assigned to ESI(-) FTMS peaks detected at pH = 3.8 on the

Fig. 5 :

 5 Fig. 5b,d) are unambiguously not sorbed at all. Compounds of intermediate values of I are only poorly sorbed

Fig. 6 :

 6 Fig. 6: Evolution of value of I of PPH compounds as a function of number of carbon atoms (n C ) for aliphatics and NCAs with O/C>0.2 in -CO 2 (a) and -CH 2 (c) series, and for NCAs depleted in oxygen and PACs in -CO 2 (b) and -CH 2 (d) series, in alumina-solution systems at pH 3.9.

Fig. 7 :

 7 Fig. 7: Evolution of the values of I within -CO 2 series of aliphatics or NCAs with O/C>0.2 (a), and -CO 2 series of NCAs depleted in oxygen and PACs (b), after sorption of PPH onto kaolinite at pH 3.7.

Fig. 8 :

 8 Fig. 8: VK diagrams for PPH compounds sorted as a function of their relative affinity for the surface of

ACKNOWLEDGEMENTS

We thank IPHC and the Alsace Region for their financial support. We gratefully acknowledge the reviewers for their helpful comments, and for improvement of the manuscript.


Géraldine Picot-Colbeaux

Quentin Guillemoto

Charlotte Thierion

Aubéry Wissocq

Loïc Thomas

Jonathan Durand

Axel Aurouet

Denis Neyens

Marie Pettenati

Identifying SAT efficiency by modelling its specific aquifer environment influenced by natural and anthropogenic activities -exemple of the costal SAT

come

Identifying SAT efficiency by modelling its specific aquifer environment influenced by natural and anthropogenic activities -exemple of the costal SAT of Agon-Coutainville (France)

Picot-Colbeaux G. 1 , Guillemoto Q. 1,2 , Thiérion C. 3 , Wissocq A. 3 , Thomas L. 3 , Durand J. 3 , Aurouet A. 3 , Neyens D. 4 , Pettenati M. 1 , 1 French Geological Survey (BRGM, France); 2 Sorbonne University (France), 3 AnteaGroup (France), 4 IMAGEAU (France)

Most of Managed Aquifer Recharge (MAR) sites in France allow to preserve groundwater, to support an over-exploited groundwater table as well as to prevent saline intrusion, to reduce environmental pollution or to complete water treatment, through soil aquifer treatment (SAT). Groundwater modelling can provide the possibility to preview de feasibility of MAR or SAT system in its regional context and give a tool to identify and optimise the choice for its implementation. The application of a model for testing the influence of projects and management actions on site conditions may provide a framework for stakeholders to screen and select appropriate strategies for the management of water resources and environment. Further on, model allow long-term predictions when it calculated forecasting effects resulting from the natural specific environment as climate change or human-related changes in water use (Rossetto et al., 2018;Criollo et al., 2018;Ringleb et al., 2016;Kloppmann et al. 2012).

On the coastal environment of Agon-Coutainville (Normandy, France), SAT efficiency is questioned by natural and anthropogenic activities that could modify groundwater in flow velocities and quality. This SAT is as part of the full-scale operational wastewater treatment plant (WWTP) and which is sustainably integrated within the municipal wastewater treatment line during more than 14 years along the English Channel coast. The secondary treated wastewater (STWW) of ~1600m 3 /day is infiltrated alternatively into three natural reed bed areas of 35000 m 2 before reaching the sand dune aquifer. The direct discharge of STWW to the sea is thus avoided to guaranty the sustainability of the shellfish production and preserve the touristic economy along the coast (Picot-Colbeaux et al., 2021).

To forecast SAT efficiency in a such hydrosystem for which major natural and anthropogenic forcing factors change in time and space, an hydrogeological model included the SAT system is provided. First, a conceptual model is carried out based on the analysis of a significant amount of information that has been collected on five observation wells and on WWTP in order to identify the main factors driving this SAT system (Guillemoto et al. 2021). These data include geology, land-use, LIDAR digital elevation model, rainfall, potential evapotranspiration, streamflow, monitored groundwater levels and water quality, sea tides, as well as SAT functioning (STWW flow and quality, basin geometry and dynamic of infiltration). Second, a hydrodynamic and hydrodispersive numerical model for sustainable groundwater management is carried out on transient state based on the conceptual model and the data analysed. The model integrates SAT dynamic, atmospheric recharge, saline intrusion and river flow. Finally, the numerical model is calibrated with groundwater levels and groundwater quality before being used for simulating future natural and anthropogenic conditions. At each selected time step, the model calculates maps of the groundwater level and quality (solute concentration) that complete punctual groundwater observations, and then, it provides water balance and mass balance that is powerful to determine the efficiency of the SAT in producing fresh water on the surrounding brackish groundwater.


Preface

This special issue contains selected papers from the 18th European Symposium of Computer Aided Process Engineering (ESCAPE-18) which took place in Lyon, France, 1-4 June 2008. ESCAPE-18 was the 667th event of the European Federation of Chemical Engineering (EFCE) under responsibility of its CAPE Working Party. CAPE refers to computer aided methods, algorithms and techniques related to process and product engineering. The ESCAPE series brings the latest innovations and achievements by leading professionals from the industrial and academic communities. It serves as a forum for engineers, scientists, researchers, managers and students from academia and industry to present and discuss progress being made in the area of CAPE.

The special theme for ESCAPE-18 was "CAPE for the Users!". CAPE systems are to be put in the hands of end users who need functionality and assistance beyond the core scientific and technological capacities of the systems. User-friendliness, on-line or web-based advice, decision support, knowledge management and organisational issues are important points that must be taken into account when deploying a CAPE system. These issues were addressed in a special track including industrial case studies illustrating CAPE methods and tools. The other six main topics covered the usual scope of ESCAPE conferences, from off-line synthesis and design to on-line systems, including numerical and computational methods, integrated and multiscale modeling, societal aspects and CAPE education.

ESCAPE-18 included a rich programme of five plenary lectures and six keynote talks from leaders in science and industry, 101 contributions presented in 26 oral sessions and two poster sessions for other 247 contributions. After the symposium 32 corresponding authors were invited to submit an extended version of their contribution. All submitted papers were reviewed according to the standards of Computers and Chemical Engineering. Finally 22 papers have been accepted for publication in this special issue.

The content of this issue is organised along the main topics of ESCAPE-18, starting with two papers from the special theme "CAPE for the Users!": the opening plenary talk by Ba ñares-Alcantara on policy design and a technical paper by Morbach et al. on the design of OntoCAPE, an ontology for the domain of CAPE.

The next six papers relate to optimization, control and diagnosis of processes, starting with a plenary talk by Bonvin and Chachuat on real time optimisation strategies. The next paper on chance constrained optimization under uncertainty, by Arellano-Garcia and Wozny, covers work conducted during Arellano-Garcia's PhD Thesis. He was the winner of the EFCE Excellence Award 2008 in Recognition of an Outstanding PhD Thesis on CAPE. His PhD Thesis, completed at Technical University Berlin under the supervision of Pr. Wozny, received the best evaluation results in terms of innovativeness, breadth and depth of the study, quality of publications, industrial relevance, and international dimension. The next two papers, Collazos et al. and Ferreira and Zhang, deal with optimal control approaches. The section continues with a paper from Chao and Wang on genetic programming as a tool to extract knowledge from process historical data. Then follows a paper by Olivier-Maget et al. on model-based fault diagnosis in the case of hybrid systems.

The next series of six articles deals with various issues in process design and scheduling with a strong focus on batch processes. The first one by Denes et al. presents feasibility studies of a new double column system for batch distillation. Then, two papers on batch scheduling are given: Gimenez et al. give the second part of a series of two articles on a network based continuous representation which can be applied to maintenance and a few other activities, and to rescheduling when some of the schedule is already fixed; Subbiah et al. also extend previous work done with timed automata for the problem of tardiness minimization. The next paper is of industrial origin, with two authors from the Akzo-Nobel company, and deals with optimal choice of solvents and operating conditions in the production of free radical solution copolymers. The last two papers in this section address crystallization processes: the paper by Nagy, who won the best oral presentation award, presents a robust optimal control scheme, which takes parametric uncertainties into account to provide decreased variability of the shape of crystal size distribution; the paper by Lakerveld et al. shows a conceptual design approach using functional tasks -which represent fundamental physical events -in order to get a better control over crystalline product quality.

The theme integrated and multiscale modeling and simulation is represented by two papers. In his contribution, Fermeglia presents a hierarchical procedure for bridging the gap between atomistic and macroscopic modeling passing through mesoscopic. The concept of multiscale modeling is perfectly illustrated through applications of industrial interest. Van Dam et al. assess different modeling paradigms -equation-based models and agent-based modelsand give recommendations for their use for supply chain management.

How to push away the limits of numerical solving strategies? Manca et al. discuss the feasibility and the methods for solving very large and sparse systems of nonlinear algebraic equations. Zavala and Biegler demonstrate that a full-discretization approach coupled to a sparsity-exploiting interior point solver result in a fast strategy to solve both the NMPC (Nonlinear Model Predictive Control) and MHE (Moving Horizon Estimator) problems. They apply their strategy on low-density polyethylene tubular reactors. The last papers concern societal and educational issues. Bojarski et al. address the optimization of Supply Chain (SC) planning and design considering economical and environmental issues. A Life Cycle Assessment (LCA) approach is envisaged to incorporate the environmental aspects of the model. Professor Luis Puigjaner, co-author of this paper, received the long term achievements (LTA) award from the CAPE Working Party during ESCAPE-18. This CAPE award for long term achievements is given in recognition of a collection of outstanding achievements over an extended period of time to CAPE methods, tools and/or applications for the advancement of science, engineering and technology. The best poster presentation award was won by Estrada et al. for the poster entitled: "Developing a Lake Eutrophication Model and Determining Biogeochemical Parameters: a Large Scale Parameter Estimation Problem". The extended version of their paper is published in this special issue. Ayoub et al. propose a design and evaluation methodology for Biomass Utilization Networks (B-NETs) planning in local areas. Finally the last paper deals with the most important challenge for the future of CAPE community: Education. We thank professors Cameron and Lewin to share their vision of important issues in the area of CAPE education in the face of contemporary changes in knowledge development, information and communication technology advances and the practice of professional engineering.

As Guest Editors of this Special Issue, we are grateful to the numerous reviewers for their efficient and fruitful collaboration and their insightful comments and recommendations which have contributed a lot to the improvement of the overall quality of the papers. We also wish to thank Elsevier and professor Rafiqul Gani for enabling the publication of this special issue in due time.

We hope that this special issue will serve as a reference to the scientific and industrial community and will contribute to the progress in computer aided process and product engineering.

  doi:10.1016/j.compchemeng.2009.05.011

  INPT -ENSIACET, University of Toulouse, 118, route de Narbonne, 31077 Toulouse Cedex 04, France

	Bertrand Braunschweig *
	IFP, 1 & 4 avenue de Bois-Préau, 92852 Rueil
	Malmaison Cédex, France
	Xavier Joulia
	Process Systems Engineering (PSE) Department,
	Chemical Engineering Laboratory (LGC, UMR CNRS
	5503),


Quentin Guillemoto

Nicolas Devau

Géraldine Picot-Colbeaux

Danièle Valdes

Frédéric Mathurin

Marie Pettenati

Denis Neyens

Jean-Marie Mouchel

Wolfram Kloppmann

Identification of the main natural and anthropic variations affecting costal SAT systems -example of the Agon-Coutainville SAT (France)

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

Identification of the main natural and anthropic variations affecting costal SAT systems -example of the Agon-Coutainville SAT (France).

Guillemoto Q. 1,2 , Devau 1 N., Picot-Colbeaux G. 1 , Valdes D. 2 , Mathurin F 1 , Pettenati M. 1 , Neyens D. 3 , Mouchel J-M. 2 , Kloppmann W. 1 1 French Geological Survey (BRGM, France); 2 Paris Sorbonne University (France) ; 3 IMAGEAU (France) Managed aquifer recharge (MAR) systems are often applied to coastal area to limit seawater intrusion and improve water quality for irrigations purpose (Tzoraki et al., 2018) or drinking water purpose (Van Houtte and Verbauwhede, 2012). These MAR systems are integrated into the hydrosystem in which groundwater flows may be subject to variations caused by meteorological events and tides, but also by human activities such as secondary treated wastewater (STWW) discharges from wastewater treatment plants (WWTP). These natural and anthropic activities could influence groundwater flow velocities and hence the efficiency of the MAR system, notably when it is combined to Soil Aquifer Treatment (SAT).

In this study, we have identified the environmental and human forcing factors influencing the SAT system implemented in Agon-Coutainville (Normandy, France), which is part of the full-scale operational WWTP sustainably integrated within the municipal wastewater treatment line since 14 years along the English Channel coast. The STWW discharge of ~1600m 3 /day is infiltrated alternatively into three natural reed bed areas of 35000 m 2 before reaching the sand dune aquifer. The direct discharge of STWW to the sea is avoided to guaranty the sustainability of the shellfish production and preserve the touristic economy along the coast (Picot-Colbeaux et al., 2021).

To identify major natural and anthropogenic forcing factors driving this SAT system, time series analyses were carried out on environmental data such as sea tides, natural recharge estimated by Potential Evapo-Transpiration (PET) and rainfall records, STWW flow discharge in the three infiltrations pounds. The same analyses were carried out on groundwater level and electrical conductivity monitored in several observation wells. Then, these results were compared/correlated to give evidence of the impact of each factor in this SAT system.

The results show that all groundwater levels and electric conductivity are influenced by natural recharge but also locally by sea tides and STWW flows. A spatial information of the main forcing effects on groundwater is highlighted. Annual variations of natural recharge affect all the observation wells. STWW daily flow effects are identified on the closest piezometers of the infiltrations pounds. Relationship between STWW flow and natural recharge is also identified especially during winter periods showing that a large part of parasitic water was drained through sewer system, which have increased STWW flows in the SAT system. For tides, important monthly and/or diurnal effects are identified on the infiltration pound closest to seashore.

These results demonstrate that several environmental forcing factors affect the studied MAR/SAT system, potentially jeopardizing its efficiency. We hypothesized that changes in ; water level from natural recharge and STWW flow could modify vadose zone thickness, affecting groundwater flows and geochemical processes, as well for seawater intrusion.


M Laugier

N Giraud

B Goin

P Moissonnier

E Viguier

Biomechanical comparison of Polyaxial Locking System versus Dynamic Compression Plate constructs in canine cadaveric scapula

Keywords: compression plate, locking plate, polyaxial, shoulder arthrodesis, dog * significative difference one-way ANOVA F=15.1,

post hoc Tukey test p<0.

Introduction

Arthrodesis of the shoulder joint consists in immobilizing both scapula and humerus in a physiologic position with a plate and screws construct in order to achieve solid bone fusion. This palliative surgical procedure is commonly used to manage severe degenerative, traumatic and congenital shoulder pathology [START_REF] Fitzpatrick | Shoulder Arthrodesis in14 Dogs[END_REF]. Its results are good to excellent in 87.5 % of cases and limb function is often considered acceptable [START_REF] Pucheu | Surgical treatment of shoulder instability[END_REF]. However, construct failure is a frequent complication of canine shoulder arthrodesis and is often related to an insufficient bone anchorage of screws in the scapula [START_REF] Fitzpatrick | Shoulder Arthrodesis in14 Dogs[END_REF]. The scapular plate location is along the basis of the scapular spine, craniolaterally where the bone is significatively thicker according to a morphometric study [START_REF] Ocal | A morphometric study on the cross-sections of the scapular spine in dogs[END_REF]. Given the plate contouring and screw angulation needed, the most commonly used plates are Dynamic Compression Plate (DCP) and Low Compression DCP (LC-DCP). While some authors advocate the use of a second plate located caudolaterally in order to strengthen the construct [START_REF] Fitzpatrick | Shoulder Arthrodesis in14 Dogs[END_REF][START_REF] Kalff | Proximal focal humeral deficiency in a large breed dog[END_REF][START_REF] Oxley | Bilateral shoulder arthrodesis in a Pekinese using three-dimensional printed patientspecific osteotomy and reduction guides[END_REF], we wondered if we could improve its strength with a recently introduced model of Polyaxial Locking System (PLS) allowing free orientation and locking of the screws up to 15° angulation. The aim of this study was to compare the scapular stability of DCP and PLS constructs. We hypothesized that the PLS with a 15° sagittal divergence of implantation of the locking screws would yield a higher linear stiffness and pullout strength than the DCP construct under wire traction tests inspired by [START_REF] Acquaviva | Biomechanical testing of locking and nonlocking plates in the canine scapula[END_REF].

Methods

Sample preparation

Nine pairs of scapulae were harvested from adult dogs between 25 and 35 kg. Dogs were of similar size and died from reasons unrelated to this study. After careful removal of the soft tissues, macroscopic examination was performed to rule out any abnormality. An osteotomy of the scapular neck allowed us to remove irregular periarticular bone processes.

Implant design

We implanted all scapulae with 3 self-taping screws per plate which is the minimum number in the scapula according to the standard surgical technique. We used 3D printed drilling guides to ensure a predetermined angulation of the screw and a 45° angulation of the plate with the scapular spine & supraspinous fossa. We made two groups of scapulae, in both groups we had scapulae of the 9 dogs. In the first group, we used DCP 3.5 mm and positioned the screws perpendicularly to the plate. The second group was implanted with PLS 3.5 mm (PLS®, Aesculap B. Braun Vet Care; Tuttlingen, Germany). The middle screw was perpendicular to the plate while the two other screws were angulated 15 degrees away from the middle screw. Then, the implanted samples were included in polyurethane resin.

Biomechanical testing

Traction was exerted through a wire rope on the first free distal hole in the plate. The eighteen tests were performed using a traction system (AGS-X Shimadzu, Japan) with a pretest of 20 mm/s traction until the strength reached 10 N, straightening the system. The test consisted then in a 1 mm/min traction to failure. Failure was considered when the strength decrease was more than 10 % of maximum measured.

Data acquisition and processing

During tests, acquisition of data was carried out using TrapeziumX software (Shimadzu, Japan). Linear stiffness was assessed by calculating the slope of the low displacement curve in its linear interval for each pullout test. Failure mode, load of failure and linear stiffness were compared. Results were statistically analyzed using R software (R.app 3.6.2, GUI1.70; R Foundation for Statistical Computing). Significative difference was set at p < 0.05.

Results and discussion

We observed three different mode of failure (Figure 1): screw pull through (3/9 DCP; 1/9 PLS), plate bending (2/9 DCP; 6/9 PLS) and scapular bone bending or fracture (4/9 DCP; 2/9 PLS). There was no significative difference between DCP and PLS (Fisher's exact tests). No significative difference was found in load to failure and linear stiffness (Table 1) between DCP and PLS construct (one-way ANOVA, p=0.60 and p=0.27). We also found no significative difference in linear stiffness depending on the failure mode (one-way ANOVA, p=0.76). The failure load was significatively different between scapula bending/fracture and the others failure modes (one-way ANOVA F=15.1, followed by post hoc Tukey test p<0.001).

Subjective differences in mode of failure could be explained by the differences between locking and compression plate. PLS plates have a more elaborate design making easier the contouring while preserving the locking mechanisms but this could lead to more plate bending given the good anchorage of the screws.

In this study Scapula bending or fracture happened more often in DCP and for thin scapulae. However, this mode of failure is uncommon in clinical cases and might be the consequence of a shoulder abduction when both humerus and scapula are implanted. Even if the load at failure is significatively different, it is considered high enough not to be a relevant clinical concern.

Conclusions

We did not find any significative difference in pull out strength and linear stiffness between 3.5mm DCP and PLS constructs in our ex vivo model. At high values of strength, we observed more plate bending with PLS while we observed more screw pullout and scapula defects with DCP. Canine scapular PLS construct seems to offer a stability at least as good as with DCP when implanted in the context of shoulder arthrodesis. More investigations including cyclic testing are required before concluding on the clinical relevance in the dog.

Figure 1 .

 1 Figure 1. Modes of failure: (A) DCP pull through, (B) PLS plate bending, (C) PLS scapula bending.

Acknowledgements

We thank BBraun for providing the osteosynthesis material used during our experiments and Roland Roume for his contribution in printing 3D drill guides.