Mathieu Le Meur
email: mathieu.le-meur@univ-lorraine.fr

Laurence Mansuy-Huault

Catherine Lorgeoux

Allan Bauer

Renaud Gley

Delphine Vantelon

Emmanuelle Montargès -Pelletier

Spatial and temporal variations of Particulate Organic Matter from Moselle River and tributaries: A multimolecular investigation

Keywords: Suspended Particulate Matter, Moselle River, Particulate Organic Matter, Molecular Characterization

The spatial and temporal composition of organic matter (OM) was investigated in the Moselle watershed (Lorraine, France). The spatial composition was studied using different stations along the river and four tributaries. In addition, one anchor station was used in order to better understand the temporal OM composition during different hydrologic regimes and seasons.

The first part of the organic characterization consisted of the extraction of the lipidic OM with an automatic extractor. The extracts were analyzed for their lipidic fatty acids (Lip-FAs), polycyclic aromatic hydrocarbons (PAHs) and sterols using gas chromatography-mass spectrometry (GC-MS). The insoluble fraction was also analyzed with thermochemolysis-GC-MS (TMH-GC-MS) using tetramethylammonium hydroxide (TMAH) and lignin phenols, permethylated deoxy aldonic acids and refractory fatty acids (Ref-FAs) were analyzed. The results showed that one station, (Florange station) on the small Fensch stream tributary displayed variations in the composition of the molecular OM with higher microbial markers (C 16:1 , C 18:1w7 , C 18:1w9 , coprostanol) inputted to the highly industrialized and urbanized areas in this small sub-catchment. The other stations showed rather similar organic composition (Lip-FAs, Ref-FAs, sterols, lignin compounds, PAHs) due to the size of the catchment (10,000 Km 2 ) or the integrative state of the suspended particulate matter (SPM). The regime flow involved a decrease in SPM OM content as well as a decrease of the palmitoleic acid/palmitic acid (C 16:1 /C 16:0 ) ratio and an increase of (stigmastanol+sitostanol+stigmasterol+sitosterol)/(cholesterol+cholestenol) (C 29 /C 27(ST) ) ratio revealing the presence of more allochthonous OM. The increase in regime flow was also correlated with a growing contribution of more degraded OM in the SPM that could be related to the leaching of subsurface soils from the watershed or to the remobilization of surface sediments. Seasonal variations were also observed, with a stronger contribution of autochthonous production during low flow level in summer. This work shows the importance of OM characterization in SPM in order to better assess to the spatial and temporal OM variations in the water column.

Introduction

Rivers transport large amounts of organic matter (OM), from the terrestrial environment to the seas. In the river, OM can be transported in both dissolved (DOM or dissolved organic carbon, DOC) and particulate forms (POC or POM). It is estimated that 0.4 Gt/yr is exported by rivers to the oceans and the amount in particulate form is estimated to 0.17 Gt [START_REF] Ludwig | Predicting the oceanic input of organic carbon by continental erosion[END_REF][START_REF] Cole | Plumbing the global carbon cycle: integrating inland waters into the terrestrial carbon budget[END_REF]. The OM in aquatic systems is composed of a wide variety of complex compounds, resulting from the diversity of the natural processes of synthesis and degradation. One method used to classify OM is to separate the allochthonous OM from the autochthonous OM [START_REF] Filella | Freshwaters: which NOM matters?[END_REF]. The autochthonous OM is formed exclusively in the water column and is composed of the residues of phytoplankton, algae and aquatic bacteria [START_REF] Wetzel | Limnology: Lake and river ecosystems[END_REF]. In natural aquatic media or in aquatic media with low anthropogenic input, the allochthonous OM results mainly from the degradation of higher plants by bacteria and fungi and is transferred from soils to rivers by rainwater [START_REF] Filella | Freshwaters: which NOM matters?[END_REF]. However, in most European rivers, the allochthonous OM might also result from human activity and could originate from domestic or industrial wastewater, via urban surface run-off (Micić et al., 2011). In rivers with high anthropogenic pressure, the industrial input and domestic input bring organic and mineral micropollutants from which polycyclic aromatic hydrocarbons (PAHs) are an important and commonly studied family [START_REF] Jeanneau | Impact of highly contaminated river on a more important hydrologic system: changes in organic markers[END_REF][START_REF] Qiao | Oxygenated, nitrated, methyl and parent polycyclic aromatic hydrocarbons in rivers of Haihe River system, China: occurrence, possible formation, and source and fate in a water-shortage area[END_REF]. Because the PAHs are sorbed on suspended particulate matter (SPM) and due to their low water solubility, they are rather well preserved in aquatic media [START_REF] Punning | Historical changes in the concentrations of polycyclic aromatic hydrocarbons (PAHs) in lake Peipsi sediments[END_REF]. Then, they constitute relevant organic markers, particularly for an anthropogenic input.

One way to investigate the composition of OM in aquatic media and distinguish between the sources is the use of pyrolysis in combination with gas chromatography and mass spectrometry (Py-GC-MS). This technique, based on separation, has the advantage to identify the pyrolysis products of molecular markers, such as lignins, carbohydrates and fatty acids (FAs), permitting characterization of different organic contributions to soils, sediments, suspended matter and water [START_REF] Grasset | TMAH-preparative thermochemolysis for the characterization of organic matter in densimetric fractions of a Mediterranean forest soil[END_REF][START_REF] Micic | Natural, anthropogenic and fossil organic matter in river sediments and suspended particulate matter: a multi-molecular approach[END_REF][START_REF] Bertrand | Recent vegetation history from a swampy environment to a pond based on macromolecular organic matter (lignin and fatty acids) and pollen sedimentary records[END_REF][START_REF] Jeanneau | Sources and transfer mechanisms of dissolved organic matter during storm and inter-storm conditions in a lowland headcatchment: constraints from high-frequency molecular data[END_REF]. A classical analysis for deciphering the different inputs of OM to the water column consists of measurements of molecular-level lignin, either by extraction using cupric acid oxidation or by thermochemolysis-GC-MS (TMH-GC-MS) (e.g. [START_REF] Hernes | The role of hydrologic regimes on dissolved organic carbon composition in an agricultural watershed[END_REF][START_REF] Frazier | Characterization of organic matter from natural waters using tetramethylammonium hydroxide thermochemolysis GC-MS[END_REF]. The characteristics of these compounds can inform on the land use, and nature of crops or on the OM degradation and can also permit the differentiation between allochthonous and autochthonous sources [START_REF] Dalzell | Flood pulse influences on terrestrial organic matter export from an agricultural watershed[END_REF][START_REF] Hernes | The role of hydrologic regimes on dissolved organic carbon composition in an agricultural watershed[END_REF].

In particular cases, especially for small watersheds (a few km 2 ), such studies of lignin can decipher contributions from soil erosion and from river bank erosion [START_REF] Jeanneau | Sources and transfer mechanisms of dissolved organic matter during storm and inter-storm conditions in a lowland headcatchment: constraints from high-frequency molecular data[END_REF].

Most studies of lignin phenols concern DOM and some others deal with the molecular analysis of lignin compounds within suspended matter (POM; [START_REF] Ward | The compositional evolution of dissolved and particulate organic matter along the lower Amazon River -Obidos to the ocean[END_REF][START_REF] Bianchi | Temporal variability in terrestrially-derived sources of particulate organic carbon in the lower Mississippi River and its upper tributaries[END_REF]. Other compounds can also be studied in order to unravel the composition of the OM. For example, carbohydrates such as fucose can be used to reveal a microbial contribution as fucose is rarely encountered in vascular plant [START_REF] Jeanneau | Sources and transfer mechanisms of dissolved organic matter during storm and inter-storm conditions in a lowland headcatchment: constraints from high-frequency molecular data[END_REF][START_REF] Grasset | TMAH-preparative thermochemolysis for the characterization of organic matter in densimetric fractions of a Mediterranean forest soil[END_REF]. The analysis of the lipid extract by way of GC-MS is a second means of performing source apportionment. [START_REF] Boëchat | Land-use impacts on fatty acid profiles of suspended particulate organic matter along a large tropical river[END_REF] used lipidic fatty acids (Lip-FAs) in order to evaluate the influence of human activity and land use on OM composition. They found that, downstream of large cities, the Lip-FAs distribution was dominated by palmitic (C 16:0 ) and stearic (C 18:0 ) acids. These compounds were described as FAs from treated and raw sewage [START_REF] Quéméneur | Fatty acids and sterols in domestic wastewaters[END_REF][START_REF] Réveillé | Characterization of sewage sludgederived organic matter: lipids and humic acids[END_REF]. In a more extended study of POM composition, [START_REF] Medeiros | Flow discharge influences on input and transport of particulate and sedimentary organic carbon along a small temperate river[END_REF] showed the variation along the Mullica River (NJ, USA). Upstream, terrestrial markers from higher plants were predominant and sharply decreased downstream and were replaced by microbial markers. During high flow, significant amounts of upstream markers were found downstream, revealing the downstream transport of terrestrial material. Most studies concerning molecular scale investigation of OM in rivers focus on DOM. However, in aquatic media, POM acts as a carrier of pollutants, such as metals [START_REF] Bibby | Trace metal adsorption onto urban stream suspended particulate matter (Auckland region, New Zealand)[END_REF][START_REF] Hua | Comparison of trace metal adsorption onto different solid materials and their chemical components in a natural aquatic environment[END_REF] or organic chemicals [START_REF] Ni | Riverine inputs of total organic carbon and suspended particulate matter from the Pearl River Delta to the coastal ocean off South China[END_REF], especially hydrophobic contaminants [START_REF] Luo | Impacts of particulate organic carbon and dissolved organic carbon on removal of polycyclic aromatic hydrocarbons, organochlorine pesticides, and nonyphenols in a wetland[END_REF]. POM also regulates aquatic metabolism [START_REF] Cole | Freshwater in flux[END_REF] and can act as a ligand for particle aggregation [START_REF] Lartiges | Composition, structure and size distribution of suspended particulates from the Rhine River[END_REF]. The few studies about POM generally focus on one single molecular group and most of the time deal with spatial considerations rather than temporal variations. Furthermore, the multi-molecular investigation of OM composition is performed mainly on small streams. For example, [START_REF] Medeiros | Multi-biomarker characterization of sedimentary organic carbon in small rivers draining the Northwestern United States[END_REF] studied rivers with seasonal mean discharge ranging between 0.5 and 38 m 3 /s. This work aims at better understand the spatial and temporal variations of molecular OM in SPM from a relatively large watershed (11,500 km 2 ). The spatial OM composition was investigated using different stations along the Moselle River and four tributaries. The different stations have different land use and the goal was to better understand the effects of land use on OM composition. The temporal variations were investigated using an anchor station sampled several times during different seasons and regime flow. The aims were to test the influence of different hydrologic and climatic conditions on the molecular OM composition in SPM. This work complements a previous study [START_REF] Le Meur | Characterization of suspended particulate matter in the Moselle River (Lorraine, France): evolution along the course of the river and in different hydrologic regimes[END_REF] where the SPM was fully characterized in terms of mineralogy and grain size distribution.

Mineralogical markers demonstrated that SPM were rather homogeneous along the Moselle River, with no drastic variations from one station to another, and suggested that soil erosion was the main source of particles. Hydrological conditions were shown to play a stronger influence on elemental composition of SPM.

Material and methods

Study site

The study is located in the Moselle catchment in the northeastern France (Fig. 1; see also Le [START_REF] Le Meur | Characterization of suspended particulate matter in the Moselle River (Lorraine, France): evolution along the course of the river and in different hydrologic regimes[END_REF]. The mean annual temperature is 10°C and the mean annual precipitation is 775 mm. The Moselle watershed land use is heterogeneous with urban, cultivated and natural areas (Fig. 1 and Table 1). In order to evaluate the land use effect on the POM composition, we selected 10 stations along the Moselle River and four of its tributaries. These stations were selected on the basis of the land use. Fig. 1 and Table 1 show the urbanized parts of the Moselle catchment, using the Corine Land Cover layer (Corine Land Cover, 2006) and more details about the stations are provided in Le [START_REF] Le Meur | Characterization of suspended particulate matter in the Moselle River (Lorraine, France): evolution along the course of the river and in different hydrologic regimes[END_REF]. Among the 10 stations, Frouard (FRO) was selected as the anchor station to follow the water flow (Appendix A1) and seasonal variations in SPM properties. This station was selected due to the proximity to the laboratory and the easy access to the river. On the Moselle River, the urbanization area is rather uniform (Table 1), with values ranging from 4.2-4.9% from the most upstream station Flavigny (FLA) and the most downstream station Thionville (THI). Among the stations, we considered that the station on the Fensch River (FLO in Florange) was a reference for a highly urbanized stream, due to the high proportion of urbanized areas (33%). The high level of contamination of Fensch water was reported in previous studies [START_REF] Jeanneau | Impact of highly contaminated river on a more important hydrologic system: changes in organic markers[END_REF][START_REF] Jeanneau | Quantitative multimolecular marker approach to investigate the spatial variability of the transfer of pollution from the Fensch River to the Moselle River (France)[END_REF][START_REF] Montarges-Pelletier | The junction of Fensch and Moselle rivers, France; mineralogy and composition of river materials[END_REF] and is due to the past industrial activity on one part and to the occurrence of direct wastewater input on the other.

Sampling procedure

SPM samples were collected between October 2012 and March 2015 (Table 2). For the collection of SPM, river water was pumped and sent to a continuous flow field centrifuge (CEPA Z-41 20000 RPM, equivalent 17000 g). The flow rate was set to 600 l/h. In order to collect high amounts of SPM (between 7 and 70 g of dried sample, depending on water turbidity), the centrifuge was run between 2 and 4 h for a given sampling campaign. The performance was checked during the whole sampling operation by measuring turbidity and grain size distribution of the raw water entering the centrifuge bowl and the exiting waters.

The size cut-off of retained particles was approximate 3 µm and was checked with laser diffraction analysis of the centrifuge input and output at different times during the sampling procedure (Laser diffractometer SYMPATEC). SPM samples were gently recovered from the Teflon plates covering the internal surface of the centrifuge bowl, and immediately frozen and freeze dried. The SPM content of the water column (mg/l) was also determined by filtering river water (100 to 1000 ml) through 0.22 µm Millipore © nitrocellulose membranes (weighed before use). Filters were oven dried (40 °C) overnight. SPM content was determined from the difference in weight. Three filter membranes were prepared for each water sample. The samples were selectively measured on the basis of the land use properties at the station or the regime flow during the sampling.

Thermal analysis, OM content, Thermogravimetry (TG) and differential scanning calorimetry (DSC)

Thermal analysis was performed using differential scanning calorimetry (TG-DSC; Setaram Labsys Evo 1600) at the SOLEIL facilities (Gif sur Yvette, France). Samples (30 mg) were placed in an alumina crucible with an identical and empty crucible used as the reference, and heated from ambient to 1000 °C at 5 °C/min under an oxidizing atmosphere (flow rate 20 ml/min) of synthetic air (20% O 2 and N 2 balance). The data heat flow was corrected from the data of the standard measurement (measurement without sample).

Organic molecular characterization

Lipidic OM was extracted using dichloromethane (DCM) with an automatic extractor (Dionex ASE 350) at 100 °C and 10 6 Pa. In order to avoid spectrometer damage due to the presence of elemental S, it was removed by the addition of metallic Cu. The insoluble fraction was analyzed using TMH-GC-MS with tetramethylammonium hydroxide (TMAH). This method is suitable in order to investigate the molecular OM and, in particular, the tracers utilized to separate terrestrial versus aquatic inputs (Frasier et al., 2003;[START_REF] Jeanneau | Sources and transfer mechanisms of dissolved organic matter during storm and inter-storm conditions in a lowland headcatchment: constraints from high-frequency molecular data[END_REF]. The solvent-extracted SPM was mixed with solid TMAH in excess (1:5 % wt. ;[START_REF] Hatcher | Flash pyrolysis and in situ methylation of humic acids from soils[END_REF]). An aliquot (10-15 mg) was placed into a quartz tube and TMH-GC-MS (Hewlett Packard 6890-Hewlett Packard 5972) analysis carried out using a split/splitless injector. On-line flash pyrolysis at 620 °C was performed with a CDS 2000 Pyroprobe for 15 s. Pyrolysis products were separated on a DB 5-MS 60 m x 0.25 mm x 0.1 µm column.

Samples were injected in splitless mode at 300 °C. The oven was cooled to 0 °C (held 0.5 min) by injecting liquid CO 2 and was programmed to 40 °C (held 10 min) at 45 °C/min, then to 315 °C (held 10.5 min) at 5 °C/min. Pyrolysis products were identified from GC retention times and mass spectra from comparison with the Wiley mass spectral library. Assignments were also carried out from comparison with published mass spectra of pyrolysis products [START_REF] Challinor | Characterization of wood by pyrolysis derivatization-gas chromatography/mass spectrometry[END_REF][START_REF] Mason | A comparative study of the molecular composition of a grassland soil with adjacent unforested and afforested moorland ecosystems[END_REF]. A semi-quantification was applied for each sample. The peak area of the selected m/z value for each compound was integrated and corrected via a correction factor. The proportion of each compound class was calculated by dividing the sum of the areas of the compounds in the class by the sum of the peak areas of all analyzed compounds multiplied by 100. Eight lignin phenols were selected to describe the lignin composition, and six permethylated deoxy aldonic acids (glucose, mannose, galactose, xylose, rhamnose and fucose) to describe oses. The distribution of the Fatty acids in the refractory fraction (Ref-FAs) was also used (from n-C 6 to n-C 26 ).

The extracts were also analyzed with GC-MS for Lip-FAs, PAHs and sterols. Only the PAHs were quantified using internal standards and were analyzed separately. Four perdeuterated PAHs were added to each extract (naphthalene D 8 , acenaphthene D 10 , chrysene D 12 , perylene D 12 ). In addition, four oxygenated-PAHs (O-PAHs) and nitrogenous-PAHs (N-PACs) were added (quinolone D7, anthraquinone D8, dibenzofuran D, 9Hand fluorenone D8). PAHs are expressed in µg/g dry SPM. PAHs quantification was performed using a Shimadsu-2010 plus-QP2010 Ultra. This apparatus is equipped with a DB 5-MS column (60 m x 0.25 mm x 0.1 µm) and operated in selected ion monitoring (SIM) mode. Samples were injected in split mode (split ratio 1:5) at 300 °C. The GC oven temperature was programmed from 70 °C (held 2 min) to 130 °C at 15 °C/min, then from 130 °C to 315 °C (held 2 min) at 4 °C/min. Sterols and Lip-FAs were analyzed qualitatively after derivatization using N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS). Sterols and Lip-FAs were characterized using an Agilent GC7820/MS75. This apparatus was equipped with a DB 5-MS column (60 m x 0.25 mm x 0.25 µm) and operated in selected ion monitoring (SIM) mode. Samples were injected in split mode (split ratio 1:5) at 300 °C. The GC oven temperature was programmed from 70 °C (held 2 min) to 130 °C at 15 °C/min, then from 130 °C to 315 °C (held 2 min) at 4 °C/min.

Results

Thermal analysis, OM content, Thermogravimetry (TG) and differential scanning calorimetry (DSC)

The OM content of SPM was estimated from differential scanning calorimetry curves and thermal gravimetry curves (Appendix A2). In the 180-460 °C range, weight loss is essentially due to the exothermic combustion of OM. The content of OM varied from 6% to 12.6%, except for SPM from the FLO station which contained 22% (Appendix A2). The values were plotted against water discharge (Fig. 2) and showed a slight decrease with increase of water level. The decrease could be explained by the relative increase in lithogenic contribution to the SPM (see also [START_REF] Le Meur | Characterization of suspended particulate matter in the Moselle River (Lorraine, France): evolution along the course of the river and in different hydrologic regimes[END_REF]. The Total Organic Carbon (TOC) was also measured and the detailed results are presented in Le [START_REF] Le Meur | Characterization of suspended particulate matter in the Moselle River (Lorraine, France): evolution along the course of the river and in different hydrologic regimes[END_REF]. TOC values at FRO station also showed relative decrease with increase of the regime flow except for the highest regime flow (Appendix A3).

Macromolecular investigations

Fatty acids in the refractory fraction (Ref-FAs)

Fig. 3 displays the fatty acids distribution in the refractory fraction (Ref-FAs) for four samples collected in the same time period (November 2012), with similar regime flow. From upstream to downstream, the selected stations were Flavigny (FLA), Frouard (FRO) and Pont-à-Mousson (PAM). The distribution obtained for FLO sample (our urban reference) was also plotted. The Fig. 3 shows that the distributions in these SPM samples were similar from one sample to another. Indeed, n-C 6 to n-C 26 alkanoic acids were observed in all the samples and showed a strong even/odd carbon number predominance. The composition was clearly dominated by palmitic acid (C 16:0 ) and stearic acid (C 18:0 ). However, slight variation could be observed from one station to another. At the downstream station PAM, palmitic acid and stearic acid appeared relatively more abundant, whereas their respective contributions appeared weakened for the most upstream station at Flavigny. SPM sample collected in Florange (FLO, Fensch River), displayed a relatively higher contribution for C 16:1 , C 18:1ω7 and C 18:1ω9 (oleic acid) (Fig. 3). Due to the highly urbanized context, and the occurrence of several dozen examples of direct wastewater input to the Fensch River1 , the Florange sample was expected to display a drastically different fingerprint. However, the distribution in Fig. 3 shows an FA profile rather close to those of the Moselle SPM. The main differences occurred for C 16:1 , C 18:1ω7 and C 18:1ω9 . Sewage water or domestic water can be fingerprinted by the presence of branched iso (i) and anteiso (a) C 15:0 -acids and also C 16:1 [START_REF] Jardé | Detection of manure-derived organic compounds in rivers draining agricultural areas of intensive manure spreading[END_REF].

Only C 16:1 could be observed with a higher contribution reflecting the bacterial contribution in FLO sample. The FRO station was sampled several times with different regime flows and seasons. The FA distribution displayed the predominance of palmitic and stearic acids similar to other stations. The SPM sampled during the summer low flow season (FRO jun13 35 m 3 .s -1 and FRO jul13 15 m 3 .s -1 ) showed higher contribution of C 16:1 (Table 3) .

Permethylated deoxy aldonic acids derived from carbohydrates

Series of permethylated deoxy aldonic acids in the pyrolysis products of all samples from the Moselle catchment were assigned on the basis of the predominant m/z 129 fragment in the mass spectra. These compounds are derived from carbohydrates [START_REF] Fabbri | Characterization of soil humin by pyrolysis (/methylation)-gas chromatography/mass spectrometry: structural relationships with humic acids[END_REF][START_REF] Mason | A comparative study of the molecular composition of a grassland soil with adjacent unforested and afforested moorland ecosystems[END_REF] and are formed from the degradation of the glucose, galactose, xylose, rhamnose and fucose during the pyrolysis with TMAH. In all the samples, the most abundant compound was glucose, followed by galactose, xylose, rhamnose and fucose. These five permethylated deoxy aldonic acids are present in different proportions in the living organisms and can be used to differentiate sources [START_REF] Grasset | TMAH-preparative thermochemolysis for the characterization of organic matter in densimetric fractions of a Mediterranean forest soil[END_REF]. Glucose is present in most of living organisms and at a higher level in vascular plants [START_REF] Grasset | TMAH-preparative thermochemolysis for the characterization of organic matter in densimetric fractions of a Mediterranean forest soil[END_REF]. Mannose is present in higher proportion in gymnosperms than angiosperms [START_REF] Cowie | Carbohydrate sources in a coastal marine-environment[END_REF]. Conversely, xylose is produced in higher proportion by angiosperms than by gymnosperms [START_REF] Wicks | Carbohydrates signatures of aquatic macrophytes and their dissolved degradation products as determined by a sensitive high-performance ion chromatography method[END_REF]. Fucose is rarely observed in vascular plants but is present in higher proportion in some bacteria [START_REF] Cowie | Carbohydrate sources in a coastal marine-environment[END_REF]. The deoxy C 6 /C 5 ratio (rhamnose+fucose)/(xylose arabinose) was defined in order to detect any variation in carbohydrate variations with regime flow or seasons and with different stations (Table 3).

Concerning spatial variability, the ratio did not show a clear trend. At FRO, we observed a slight increase during the summer low flow period. [START_REF] Jeanneau | Sources and transfer mechanisms of dissolved organic matter during storm and inter-storm conditions in a lowland headcatchment: constraints from high-frequency molecular data[END_REF] observed evolution of the ratio with DOC. They showed that DOC was clearly higher than observed for our SPM.

They also showed that DOC was between 0.4 and 0.2 in the soils, which is close to the ratio in our SPM.

Lignin-derived compounds

Like the Ref-FAs and polysaccharides, the thermochemolysis of the SPM produced a series of lignin-derived methylated monomers. These phenol compounds were semi-quantified and were sorted in three groups, vanillyl, syringyl and cinnamyl. From these compounds we can separate the different plant tissues. Gymnosperm woody tissue contains mainly V units (80%) with C units whereas angiosperm woody tissues are characterized by V an S units in the same proportion with C units [START_REF] Clifford | A new rapid technique for the characterization of lignin in vascular plants: thermochemolysis with tetramethylammonium hydroxide (TMAH)[END_REF]Challinor, 2001). Non woody tissues (grass, leaves, needles, stems) have V, C and S units in the same proportion. Several studies using CuO oxidation showed that the use of ratios such as C/V and S/V can permit the identification of terrestrial inputs [START_REF] Hedges | The lignin geochemistry of marine sediments from the southern Washington coast[END_REF]Hedge et al., 1982). However, [START_REF] Clifford | A new rapid technique for the characterization of lignin in vascular plants: thermochemolysis with tetramethylammonium hydroxide (TMAH)[END_REF] also showed that the TMAH thermochemolysis can provide similar results without quantification using robust ratios. The ratio cinnamyl/vanillyl (C/V) was calculated following [START_REF] Hedges | The lignin geochemistry of marine sediments from the southern Washington coast[END_REF]. C/V is consistently higher in macrophyte and grass-derived plant tissue relative to woody tissue, making it a useful ratio for differentiating between the different vegetation types in a watershed. Table 3 shows C/V for all the SPM samples in the Moselle watershed. For the samples Flavigny, Frouard NOV12 and Pont-à-Mousson NOV12, which were sampled in the same range of regime flow, C/V was higher at Flavigny (0.58) than at Frouard (0.44). The value at Pont-à-Mousson was 0.49. The Richemont station showed high C/V ratio (1.12). The values of C/V ratio also showed a slight decrease with increase in regime flow for Frouard samples except for FRO nov13 (C/V = 0.68). Supplementary ratios were used to investigate the oxidative diagenesis of lignin. Indeed, the degradation of lignin compounds can be inferred from an increase in total acid phenols relative to the total aldehyde phenols (syringic acid to syringaldehyde (Ad/Al(s)) and vanillic acid to vanillin (Ad/Al(v)) ratios). The work of [START_REF] Hedges | Lignin geochemistry of a Late Quaternary sediment core from Lake Washington[END_REF] showed that high values of these ratios can be explained by a degradation of lignin. Ad/Al (v) in the watershed varied between 1.36 and 2.96 (Table 3), indicating different states of lignin degradation. In the Moselle River, for SPM collected at Frouard, Ad/Al (v) slightly increased with water discharge, suggesting a contribution from more degraded lignin during flood events. Looking at the spatial variability, the SPM collected from the Madon and Meurthe Rivers, respectively at the PSV and BOU stations, displayed lower values for the ratio, suggesting that the lignin degradation could be influenced by the Strahler order of the stream and/or by the land use.

Lipid fraction

Fatty acids in the lipid fraction (Lip-FAs)

Fig. 4 shows the fatty acid distributions in the lipid fraction (Lip-FAs) for samples collected in November 2012 (FLA, FRO and PAM), and also for FLO. The distributions in the selected SPM samples were very similar to those in the refractory fraction. In this fraction again, the FAs were dominated by palmitic acid (C 16:0 ) and stearic acid (C 18:0 ). However, we could observe n-C 6 to n-C 32 alkanoic acids with a strong even predominance. Slight variation in distribution could also be observed from one station to another. C 18:1w7 was observed for the three stations on Moselle River but was not detected at FLO station. For the FLA and PAM stations, the C 20:0 to C 32:0 compounds were more represented than for the FRO and FLO stations. In addition, the Terrigenous/Aquatic Ratio (TAR) (C 28 +C 26 +C 24 )/(C 18 +C 16 +C 14 ) was higher for PAM (0.23) and FLA (0.24) than for FRO (0.01) and FLO (0.03), reflecting the higher contribution of terrestrial compounds at FLA and PAM. FLA station also displayed higher C 16:1 contribution (Table 4). FRO jul13 also shows higher C 16:1 /C 16:0 ratio.

Sterol composition

The sterol family was composed of 5β-Cholestan-3β-ol (coprostanol), choles-5-en-3β-ol (cholesterol), 24-ethylcholest-5-en-3β-ol (sitosterol), 24-ethyl-5βcholestan-3α-ol (24ethylepicoprostanol), 24-ethyl-5β-cholestan3β-ol (24-ethylcoprostanol), 24-ethylcholesta-5.22E-dien-3β-ol (stigmasterol), 24-Methylcholest-5-en-3β-ol (campesterol), 24-ethyl-5α-cholestan-3β-ol (sitostanol), 24-ethyl-5α-cholesta-22E-dien-3β-ol (stigmastanol), 24-methyl-5α-cholestan-3β-ol (campestanol), 5α-cholestan-3β-ol (cholestanol), 5β-cholestan-3α-ol (epicoprostanol). Fig. 5 shows the sterol distribution in the Moselle and Fensch samples.

Among the twelve sterols in the SPM samples, coprostanol and cholesterol dominated.

Coprostanol comes mainly from the degradation of cholesterol by bacterial activity in the digestive system of sheep, cattle, pigs, cats and humans [START_REF] Leeming | Using faecal sterols from humans and animals to distinguish faecal pollution in receiving waters[END_REF] and is the main sterol in human feces (60% of the steroids, [START_REF] Macdonald | Degradation of steroids in the human guts[END_REF] and so is often used as a waste water treatment plant (WWTP) marker [START_REF] Hatcher | Sewage contamination in the New York Bight. Coprostanol as an indicator[END_REF]. Cholesterol is a ubiquitous compound and can be encountered in both plant and animal kingdoms.

Different ratios can be used to identify any contamination in river waters. For example, values of coprostanol/cholesterol (Cop/Chol) > 0.5 are characteristic of human feces [START_REF] Grimalt | Assessment of fecal sterols and ketones as indicators of urban sewage inputs to coastal waters[END_REF][START_REF] Fattore | Sterols in sediment samples from venice Lagoon, Italy[END_REF][START_REF] Reeves | Faecal sterols as indicators of sewage contamination in estuarine sediments to the Tay Estuary, Scotland: an extended baseline survey[END_REF]. In the Moselle catchment, the values obtained varied between 0.2 and 2.94. In most cases, the value was > 0.5, indicating the high contribution of coprostanol (Table 4). During summer and low flow regime (FRO JUN13, FRO JUL13, 35 m 3 .s -1 and 15 m 3 .s -1 respectively), Cop/Chol ratio was low, showing a strong terrestrial and/or an autochthonous contribution of sterols. Cop/Chol increased with flow regime, indicating that waste water was directly discharged into the Moselle River or the lower autochthonous OM contribution. Only two samples showed values of 0.2. FLO station displayed the highest Cop/Chol ratio (2.9). This value could be related to the WWTP located upstream of the sampling site on the Fensch River. The ratio was also high at BOU (2.4), PAM (2.9) and FRO (2.58 for November 12). BOU and PAM stations are downstream of the WWTP of Nancy and Pont-à-Mousson cities respectively.

The

(stigmastanol+sitostanol+stigmasterol+sitosterol)/(cholesterol+cholestenol) ratio (C 29 /C 27 (ST) )can also be used in order to evaluate the proportion of allochthonous and autochthonous OM [START_REF] Nishimura | The occurrence of stanols in various living organisms and the behavior of sterols in contemporary sediments[END_REF]. Higher plants synthesize a C 29 sterol, βsitosterol [START_REF] Huang | Sterols as source indicators of organic materials in sediments[END_REF]. This compound can also be encountered in phytoplankton and notably in diatoms [START_REF] Volkman | A review of sterol markers for marine and terrigenous organic matter[END_REF][START_REF] Volkman | Sterols in microorganisms[END_REF]. In contrast, C 27 sterols have essentially an aquatic origin [START_REF] Huang | Sterols as source indicators of organic materials in sediments[END_REF]. The values of C 29 /C 27 (ST) increased with regime flow, indicating higher C 29 contribution or lower C 27 contribution (Table 4) and inform on the higher autochthonous OM during high flow regime. The high flow regimes SPM were sampled during the winter. In these winter samples, few diatoms were observed [START_REF] Le Meur | Characterization of suspended particulate matter in the Moselle River (Lorraine, France): evolution along the course of the river and in different hydrologic regimes[END_REF]. In addition, brassicasterol and 24-methylenecholesterol were not observed in our samples. These sterols are typical sterols encountered in diatoms.

The C 29 should have a terrestrial origin in our SPM during the winter storm event samples or at least the decrease of autochthonous OM contribution.

PAHs composition

The 16 PAHs listed by the US Environmental Protection Agency (EPA) were quantified and some ratios calculated to provide an overview of the PAHs contamination through the catchment (Table 5). The sum of 8 PAHs concentrations in the SPM from Moselle catchment ranged between 2.3 and 7.7 µg/g. All the samples displayed lower 8 PAHs concentrations than the regulatory value of 8.649 µg/g defined by the European legislation (recommended concentration to evaluate the quality of sediments and SPM in continental waters). The PAHs in SPM were dominated by heavy aromatic hydrocarbons (HMW-PAHs) from fluoranthene (Flt) to benzo(ghi)perylene (Bghi) (Table 5 and supplementary information 4). Indeed, the contribution of 4 to 6 ring PAHs accounted for 69 to 91% of the total PAHs concentration.

The dominant PAHs at all the stations was fluoranthene. The sum of 16 PAHs quantified in the SPM did not show clear spatial trends in the Moselle catchment. No clear increase in PAHs content could be observed for samples collected in the vicinity of highly urbanized areas (FLO station for example) and also for FRO samples collected during different regime flow (2.9 to 5.1 µg/g). Several ratios were tested to evaluate their variation in the catchment.

The An/178 ratio (anthracene/(anthracene+phenanthrene)) [START_REF] Budzinski | Evaluation of sediment contamination by polycyclic aromatic hydrocarbons in the Gironde estuary[END_REF]), the BaA/228 ratio (benzo(a)anthracene/(benzo(a)anthracene+chrysene)) and the Fl/202 ratio (fluoranthene/(fluoranthene+pyrene)) [START_REF] Yunker | PAHs in the Fraser River basin: a critical appraisal of PAH ratios as indicators of PAH source and composition[END_REF] are usually used to distinguish between petrogenic and pyrogenic inputs. Through the Moselle catchment BaA/228 varied between 0.53 and 0.58 (except for MIL 0.40 and FRO oct13 1.00) and was constant spatially and temporally. Fl/202 ratio also showed constancy, with values ranging from 0.54 to 0.59, except for FRO oct13 for which the value was 0.43 and for MIL (0.75). An/178 showed wider variation, with values ranging from 0.29 to 0.55. However, the variations did not show clear trend. The three ratios were rather stable along the Moselle catchment and with regime flow and highlighted the idea that PAH content in the SPM did not vary spatially and temporally.

Thus, the quantification of PAHs in SPM is a useful tool for evaluating the anthropogenic pollution and for comparing catchments with a surface area of similar order. The Oxygenated-PACs (O-PACs) and nitrogenous-PACs (N-PACs) were also quantified in the SPM and the results are presented in the supplementary data (Appendix A4) as there was no clear trend between the stations, regime flow or seasons.

Discussion

Spatial Variability in OM composition, influence of land use

One of the goals of this study was to evaluate the sensitivity of OM markers in order to trace variations along the Moselle River. The Moselle watershed is characterized by the evolution of land use through the catchment (Table 1). Consequently, the OM markers were expected to vary through the catchment. One station, FLO station, showed variations of the molecular OM composition. This station showed higher relative contribution of C 16:1 , C 18:1w7 , C 18:1w9 for the Ref-FAs and C 16:1 is one of the compounds characteristic of sewage water (Jarde et al., 2007).

Other compounds such as coprostanol was also higher. Such higher contribution was expected, as this station on the Fensch River is characterized by high anthropogenic pressure in this small watershed [START_REF] Jeanneau | Impact of highly contaminated river on a more important hydrologic system: changes in organic markers[END_REF][START_REF] Jeanneau | Quantitative multimolecular marker approach to investigate the spatial variability of the transfer of pollution from the Fensch River to the Moselle River (France)[END_REF][START_REF] Montarges-Pelletier | The junction of Fensch and Moselle rivers, France; mineralogy and composition of river materials[END_REF], 2014).

The mineral composition at Florange station is also impacted by the anthropogenic pressure with, in particular, the presence in high proportion of iron oxyhydroxides and trace metals such as lead and Zinc [START_REF] Le Meur | Characterization of suspended particulate matter in the Moselle River (Lorraine, France): evolution along the course of the river and in different hydrologic regimes[END_REF]. The FLA station, which is the most upstream station sampled, also showed a significant contribution of anthropogenic markers such as C 16:1 and coprostanol besides the low urban area (6%). A domestic water contribution was also expected at this station, located in the Flavigny city. However, the Lip-FAs showed a high contribution from C 24:0 to C 30:0 and TAR ratio was also low. These observations reflect a relatively high terrestrial contribution as these compounds are believed to be components of higher plants wax [START_REF] Rieley | The biogeochemistry of Ellesmere Lake, UK -I: source correlation of leaf wax inputs to the sedimentary lipid record[END_REF][START_REF] Meyers | Applications of organic geochemistry to paleolimnological reconstructions: a summary of examples from the Laurentian Great Lakes[END_REF].

The family of compounds corresponding to lignin derivatives also did not show a clear evolution along the course of the Moselle except for Richemont station. SPM from RIC station displayed C/V values higher than for the other samples (Table 3). This higher ratio could be related to the higher proportion of cultivated areas for the Orne watershed (67%).

Some other studies have reported variation of C/V for the Mississippi River [START_REF] Bianchi | Temporal variability in terrestrially-derived sources of particulate organic carbon in the lower Mississippi River and its upper tributaries[END_REF] and the Amazon River [START_REF] Ward | The compositional evolution of dissolved and particulate organic matter along the lower Amazon River -Obidos to the ocean[END_REF]. However, in these large rivers, the quantity of exported OM is high and explains the variation in lignin composition. In order to decipher any variation in C/V through the Moselle watershed, it would be necessary to take into account the regime flow of the river and also report on the OM productivity and exportation.

The degradation state of lignin, observed through the Ad/Al (v) ratio showed more preserved lignin in the Madon and Meurthe Rivers. The lignin degradation could thus be affected by the Strahler order of the stream or the land use.

Other compounds such as PAHs did not show a clear trend in the Moselle catchment, with relatively low variation in the sum of the 8 PAHs and low variation in the different ratios calculated (An/178; BaA/228; Fl/202). This relative homogeneity could be a result of the global atmospheric pollution and its deposition as particles in the river. For Tianjin River, the absence of a trend between upstream and downstream stations was also reported by [START_REF] Shi | Contamination of rivers in Tianjin, China by polycyclic aromatic hydrocarbons[END_REF]. They found better spatial relationships in sediments with greater contamination close to industrialized areas. SPM from Tianjin River were also enriched in HMW PAHs (32% of total PAHs; [START_REF] Shi | Contamination of rivers in Tianjin, China by polycyclic aromatic hydrocarbons[END_REF]. Because they are more hydrophobic, HMW PAHs would more likely enter in the river in particulate form from soil run off or atmospheric deposition [START_REF] Countway | Polycyclic aromatic hydrocarbon (PAH) distributions and associations with organic matter in surface waters of the York River, VA Estuary[END_REF].

The characteristics of POM did not vary through the catchment and only smaller subcatchments (e.g. Fensch) showed variation in relation with the extensive industrialization and urbanization. This similarity in composition could be the result of the size of the Moselle catchment, as it drains > 10,000 km 2 . The other explanation could come from the POM itself that appears to be more integrative than DOM. The mineral characterization of Moselle SPM also showed a relative homogeneity, with variations only encountered at Florange and Richemont [START_REF] Le Meur | Characterization of suspended particulate matter in the Moselle River (Lorraine, France): evolution along the course of the river and in different hydrologic regimes[END_REF].

Temporal variability of OM composition, influence of season and hydrological conditions

The other aim of this study was to decipher any variation in the OM composition with seasonality (mainly driven by temperature) and/or regime flow of the Moselle River. During low flow regimes at the FRO station, where the temperature was high, the samples were characterized by a higher deoxy C6/C5 ratio which indicates microbial contribution [START_REF] Rumpel | Gas chromatographic analysis of monosaccharides in a forest soil profile: Analysis by gas chromatography after trifluoroacetic acid hydrolysis and reductionacetylation[END_REF]. In addition, the number of diatoms during summer low flow regime is high [START_REF] Le Meur | Characterization of suspended particulate matter in the Moselle River (Lorraine, France): evolution along the course of the river and in different hydrologic regimes[END_REF]. The C 16:1 /C 16:0 ratio is also high and monounsaturated FAs are major components of freshwater algae and bacteria [START_REF] Cranwell | Monocarboxylic acids in lake sediments: Indicators, derived from terrestrial and aquatic biota, of paleoenvironmental trophic levels[END_REF][START_REF] Cranwell | Lipids of aquatic sediments and sedimenting particulates[END_REF][START_REF] Volkman | Novel unsaturated straight-chain C37-C39 methyl and ethyl ketones in marine sediments and a coccolithophore Emiliana Huxleyi[END_REF]. In addition the total OM content is high and the OM is relatively unaltered (low Ad/Al (v) ratio). All these markers encountered in the extract and in the macromolecular OM are in accordance with an enhancement of autochthonous OM during the summer low flow level with high temperatures.

The increase in regime flow, sampled during the winter storm period involved a decrease in the markers cited above, which can be assigned to a greater contribution of allochthonous OM and/or by rapid degradation of OM. A clear decrease in POM content was also observed (Fig.

2

). This can be assigned on one hand to the decrease in autochthonous OM as its production is high in warm seasons and its contribution to POM dominates at low flow periods. On the other hand, during high flow periods, allochthonous POM dominates. It can be assumed, that intense rain events provoke the contribution from subsurface soil horizons (a few cm), containing a lower amount of OM, or displaying higher degree of mineralization of OM (also see Le [START_REF] Le Meur | Characterization of suspended particulate matter in the Moselle River (Lorraine, France): evolution along the course of the river and in different hydrologic regimes[END_REF]. In addition, C/V ratio and Ad/Al (v) indicated more degraded lignin during high flow level (Fig. 6). Another explanation could be the remobilization of sediments in the water column. Other studies have shown variation in DOM composition, with less degraded lignin mobilized during high flow level in small catchments (few km 2 ; [START_REF] Dalzell | Flood pulse influences on terrestrial organic matter export from an agricultural watershed[END_REF][START_REF] Hernes | The role of hydrologic regimes on dissolved organic carbon composition in an agricultural watershed[END_REF][START_REF] Jeanneau | Sources and transfer mechanisms of dissolved organic matter during storm and inter-storm conditions in a lowland headcatchment: constraints from high-frequency molecular data[END_REF]. In their review, [START_REF] Marin-Spiotta | Paradigm shifts in soil organic matter research affect interpretations of aquatic carbon cycling: transcending disciplinary and ecosystem boundaries[END_REF] show that, during low flow, the DOM in the water column resembles deep OM from soil,

Acknowledgments

This study was performed in the context of the Zone Atelier Moselle (ZAM), which belongs to the French LTER network, and was financially supported by the French CNRS-INEE and Region Lorraine (CPER). The authors thank E. Rotureau and F. Fraysse for their contributions to manuscript reading. In addition, the authors would like to thank SARM-CRPG for chemical analyses. The authors also would like to thanks the two anonymous reviewers for their constructive comments.

Figure captions

Fig. 1. Moselle River catchment showing the main rivers (blue); urbanized areas (grey), cultivated areas (yellow), and forest (green) (Corine Land Cover, 2006). This figure also shows sampling stations (red points). On Moselle River, from upstream to downstream: Flavigny (FLA), Frouard (FRO), Millery (MIL), Pont à Mousson (PAM), Argancy (ARG), Thionville (THI), on Madon River, Pont-Saint-Vincent (PSV), on Meurthe River, Bouxières aux dames (BOU), on Orne River, Richemont (RIC), and on Fensch River, Florange (FLO).

  whereas, during high flow, the DOM resembles surface soil DOM, suggesting different pathways during high and low water flow. Our results suggest that POM transfer from soil to river in a large catchment would not follow the same path as DOM. As for the spatial distribution, the PAHs content of SPM (Σ16PAHs and Σ8PAHs) does not show a clear trend with regime flow or with temperatures.5 ConclusionsMolecular analyses of POM were performed in the Moselle catchment at different stations and during different flow regimes and seasons. Due to contrasted land use from upstream to downstream the Moselle watershed, SPM OM composition was expected to evolve. However, the OM composition was rather similar at the Moselle, Meurthe and Madon stations. Only one station, the FLO station, displayed variations in the POM chemistry with higher relative contribution of C 16:1 and coprostanol which are indicators of microbial contribution and sewage water. The Meurthe and Madon tributaries showed more preserved lignin through the Ad/Al (v) ratio. This observation suggest that the Strahler order of the stream or the land use can affect the lignin degradation. The PAHs showed slight variation through the catchment, highlighting the fact that a catchment of this size is integrative. This similarity can also come from the integrative status of the POM itself. SPM OM composition was also followed as a function of flow regime and seasons at FRO station. The increase of the limb of the hydrograph resulted in a decrease of OM content in SPM. In addition, the different lignin indicators (C/V and Ad/Al (v) ratios) revealed the presence of more degraded lignin at FRO station during high flow regime. These results could be explained by the higher contribution of allochthonous OM coming from the subsurface soil leaching or by the surface sediments remobilization. Seasonal variations were also observed with higher total OM content in SPM and high deoxy C6/C5 and C 16:1 /C 16:0 ratios during low flow level in summer. These markers traduce the enhancement of autochthonous OM at FRO station during low flow level in summer These results showed relative similarity of OM composition in SPM through the Moselle catchment with the exception of FLO station. However, we expected more contrasted OM composition because of the heterogeneous land use. The use of other markers such as Zn isotopic measurements could permit to better separate the different sources of SPM in the watershed (SPM from urban, forest or farm areas). In addition, the regime flow sampling in this study was realized during different days and additional sampling during one storm event should be performed in order to better characterize the OM composition variations in relation with regime flow.

Fig. 2 .

 2 Fig. 2. Evolution of OM content of Moselle SPM (estimated from DSC and TG curves (see Supplementary material A2); black squares: all SPM samples; grey diamonds: SPM samples from Frouard station.

Fig. 3 .

 3 Fig. 3. Composition of Ref-FAs in Moselle SPM for samples collected in November 2012 from four different stations; Flavigny (FLA), Frouard (FRO), Pont-a-Mousson (PAM) and for Florange sample (FLO).

Fig. 4 .

 4 Fig. 4. Composition of Lip-FAs in Moselle SPM for samples collected in November 2012 from four stations: Flavigny (FLA), Pont-a-Mousson (PAM) Frouard (FRO) and Florange sample (FLO).

Fig. 5 .

 5 Fig. 5. Composition of sterols in Moselle SPM for samples collected from four stations: Flavigny (FLA), Pont-à-Mousson (PAM), Frouard (FRO) and for Florange (FLO).

Fig. 6 .

 6 Fig.6. SPM weight loss (%), C/V and Ad/Al (v) ratios vs. water discharge (m 3 /s).

Table 1

 1 A. Background data for the different stations(Corine Land Cover, 2006) Water data for Moselle catchment(Hydro Eau France, 2015). River water discharge data taken from the hydrometric station in Toul for FLA and FRO, in Pulligny for PSV, in Custine for MIL, PAM and ARG, in Rosselange for RIC, in Knutange for FLO, and in Hagondange for THI; Ma refers to Madon River, Me to Meurthe River, O to Orne River, and F to Fensch River. b SPM concentration extrapolated from turbidity values

	Code	Town	Sample site	Watershed surface area (km 2 )	GPS coordinates	Urbanized area (%) a	Cultivated area (%)	Forest (%)
	FLA Flavigny	Residential area	2013	6°11'19.5''E 48°34'11.7''N	6.0	38.3	55.3
	PSV b Pont Saint Vincent	Agricultural catchment	1036	6°06'21.6''E 48°36'02.1''N	3.0	77.2	19.8
	FRO	Frouard	Upstream of junction with Moselle	3768	6°08'11.8''E 48°46'30.4''N	6.1	49.1	43.6
	BOU b Bouxières aux dames	Downstream of Nancy city	3055	6°08'58.5''E 48°45'22.7''N	6.7	43.7	49.3
	MIL	Millery	Residential area	6830	6°07'37.0''E 48°48'53.1''N	6.2	47.1	46.5
	PAM	Pont à Mousson	Downstream the city limits	7209	6°02'39.0''E 48°56'06.1''N	6.1	47.6	45.9
	ARG Argancy	Downstream of Metz city	9213	6°11'59.9''E 49°08'38.6''N	6.0	52.5	40.9
	RIC b Richemont	Downstream of city limit	1276	6°10'23.5''E 49°16'48.6''N	6.0	67.0	26.5
	FLO b Florange Industrial catchment	59	6°08'05.4''E 49°19'55.2''N	33.0	22.2	44.9
	THI Thionville	In Thionville city	10882	6°10'26.4''E 49°21'30.6''N	6.7	53.9	38.7

a b Station on a Moselle tributary: PSV, Madon: BOU, Meurthe; RIC, Orne; FLO, Fensch.

.Table

2

a

Table 3

 3 Data for the different samples from Moselle River watershed: regime flow (Q, m 3 .s -1 ); C 16:1 /C 16/0 ) ratio; Deoxy C6/C5 ratio, C/V ratio; Ad/Al (v) ratio Coprostanol/Cholesterol, C29/C27 (ST) and C 16:1 /C 16:0 from peak area of each product for the different Moselle catchment SPM samples in the lipidic fraction

				Deoxy
	Station	Q (m 3 /s)	C 16:1 /C 16:0	C6/C5	C/V	Ad/Al (v)
	FLA	71	0.22	0.52	0.58	2.12
	PSV* nov12	7	0.15	0.46	0.49	1.45
	PSV* dec12	28	0.10	0.42	0.33	1.59
	FRO jul13	15	0.36	0.57	0.50	1.38
	FRO jun13	35	0.17	0.48	0.65	1.77
	FRO dec13	36	0.24	-	0.42	1.44
	FRO oct14	44	0.46	-	0.51	1.87
	FRO nov12	82	0.16	0.55	0.44	1.86
	FRO feb14	124	0.14	0.55	0.40	1.86
	FRO nov13	198	0.61	-	0.68	2.96
	FRO dec12	397	0.06	0.54	0.36	2.04
	BOU	35	0.60	0.65	0.47	1.72
	MIL	136	0.44	0.65	0.52	1.77
	PAM nov12	81	0.10	0.55	0.49	2.24
	PAM fev13	475	0.08	0.45	0.92	2.7
	ARG	171	0.25	0.54	0.42	1.36
	FLO	1	0.36	0.56	0.38	1.87
	RIC	8	0.63	0.77	1.12	2.5
	THI	200	0.34	0.54	0.34	1.76

. Table

4

Personal communication from A. Fall, in charge of Hydrology department of the CAVF (Fensch Valley cities network, communauté d'agglomération du Val de Fensch)

SPM organic markers from the lipidic and the refractory fractions were characterized.

Fensch River showed higher microbial contribution, highlighting urbanized areas.

The Regime flow engendered OM content decrease and more degraded OM.

Seasonal variations were recorded with high autochthonous contribution during summer.


D Arsovski 1 ', Olivier

X Bonnet'

S Drilholle

L Tomovic

A Béchet

A Golubovic

& A Besnard

Keywords: Immature: 0, 72 / Clemmys guttata Immature: 0, 72 Catch-curve analysis Bjorndal, Bolten

Delayed maturity and high survival rates of immatures and adults characterize long-lived species' life histories. Understanding how these traits interact in stochastic environments is essential to understand early life in long-lived species and to assist conservation planning. Unfortunately, available informRödel of demographic traits in immatures are often fragmentary and the logistical difficulties of capturing and recapturing this cohort leave little room for improvement. Published immature chelonian survival estimates vary broadly, are often not age-specific, and lack precision. In an attempt to overcome this issue, we developed a novel modelling approach based on Capture-Recapture data to obtain robust age-specific survival probabilities in two chelonian species (the freshwater European pond turtle, Emys orbicularis, and the terrestrial Hermann's tortoise, Testudo hermanni). More specifically, we show that implementing a linear relationship between survival probabilities and age does not distort survival estimates and improves precision. Both species display a progressive increase in survival with age, reaching a plateau at the ages of four to five. As maturity occurs later in both species (8-12 years-old), the survival plateau might be governed by the final hardening of the carapace, which brings obvious survival benefits. Taking advantage of the flexibility of multievent models we encourage a systematic field approach even when very large samples from immatures cannot be amassed.

Only so can demographic traits in chelonians, and more generally in long-lived species, be properly explored.

Introduction

Delayed sexual maturity, high imature and adult survival, and variation in the correlative nature of these traits define the life histories of long-lived animals [START_REF] Dunham | Lifehistory patterns in squamate reptiles[END_REF][START_REF] Charnov | On evolution of age at maturity and adult lifespan[END_REF][START_REF] Congdon | Delayed sexual maturity and demographics of Blanding's turtles (Emydoidea blandingi): implications for conservation and management of long-lived organisms[END_REF][START_REF] Congdon | Demographics of common snapping turtles (Chelydra serpentina) implications for conservation and management of long-lived organisms[END_REF]. Understanding their ecology, evolution and finally conservation biology requires robust demographic analyses based on thorough insight on these traits [START_REF] Heppel | Application of life-history theory and population model analysis to turtle conservation[END_REF][START_REF] Gans | Using long-term data and a stage-classified matrix to assess conservation strategies for an endangered turtle (Clemmys guttata)[END_REF]. Nevertheless, developmental changes throughout a prolonged immature phase can induce huge variation in trait values; e.g. before (delayed) maturity mean annual survival increases linearly with age (see for instance [START_REF] Congdon | Demographics of common snapping turtles (Chelydra serpentina) implications for conservation and management of long-lived organisms[END_REF][START_REF] Jorgenson | Effects of age, sex, disease, and density on survival of bighorn sheep[END_REF]Fernàndez-Chacon et al., 2011;[START_REF] Hastings | Cohort effects and spatial variation in age-specific survival of Steller sea lions from southeastern Alaska[END_REF][START_REF] Delong | Age-and sex-specific survival of California sea lions (Zalophus californianus) at San Miguel Island, California[END_REF]. This complicates the understanding of trait covariation and mandates age-, or at least stage-specific estimates.

Longevity and delayed maturity are often expressed to the extreme in chelonians [START_REF] Wilbur | Life-history evolution in turtles[END_REF]. At present day, an ever-increasing set of threats [START_REF] Klemens | Turtle conservation[END_REF] have made that combination of life-history traits a chelonian Achilles' heel, with almost 70% of all 335 species threatened or recently extinct (almost 46% of which endangered or critically endangered); for many, conservation status has not been assessed [START_REF] Van Dijk | Turtles of the world, 7th edition: annotated checklist of taxonomy, synonymy, distribution with maps, and conservation status[END_REF]. In order to counter this trend robus t age-specific survival estimates are necessary to provide age-specific survival elasticities. Indeed, developing reliable projections of population growth using matrix models [START_REF] Caswell | Matrix population models. Construction, analysis, and interpretation[END_REF], evaluating conservation priorities [START_REF] Congdon | Delayed sexual maturity and demographics of Blanding's turtles (Emydoidea blandingi): implications for conservation and management of long-lived organisms[END_REF]Heppe l, 1998;[START_REF] Gans | Using long-term data and a stage-classified matrix to assess conservation strategies for an endangered turtle (Clemmys guttata)[END_REF] and estimating conservation efficiency (e.g. after reintroduction and/or headstarting in Heppel, Crowder & Crouse, 1996a;[START_REF] Mitrus | Headstarting in European pond turtles (Emys orbicularis): does it work?[END_REF][START_REF] Canessa | Challenges of monitoring reintroduction outcomes: insights from the conservation breeding program of an endangered turtle in Italy[END_REF] rely on such endeavours in long -lived animals.

Alas, the elusive nature of chelonian early life precludes the amassing of large and dense CR (Capture-Recapture) datasets of aged immature turtles, actively hindering this goal. Although chelonian adult survival is relatively well-studied [START_REF] Congdon | Demographics of common snapping turtles (Chelydra serpentina) implications for conservation and management of long-lived organisms[END_REF][START_REF] Henry | Survival rates and demography of the Hermann's tortoise Testudo hermanni in Corsica, France[END_REF][START_REF] Chaloupka | Estimates of sex-and ageclassspecific survival probabilities for a southern Great Barrier Reef green sea turtle population[END_REF][START_REF] Olivier | Assessing spatial and temporal population dynamics of cryptic species: an example with the European pond turtle[END_REF][START_REF] Bertolero | Testudo hermanni (Gmelin 1789) -Hermann's tortoise[END_REF][START_REF] Bertolero | The importance of including survival release costs when assessing viability in reptile translocations[END_REF], sporadic insight on early survival fails to paint a clear picture. Indeed, a rapid review of the sparse literature on agespecific early life chelonian survival revealed a wide spectrum of estimates ranging from 11% in neonate European pond turtles (Emys orbicularis) to 92% in juvenile Loggerhead turtles (Table 1). Much is likely a Alas, the elusive nature of chelonian early life precludes the amassing of large and dense CR (Capture-Recapture) datasets of aged immature turtles, actively hindering this goal. Although chelonian adult survival is relatively well-studied consequence of inherent interspecific differences; in juvenile tortoises survival estimates range from eight to 90%, whereas in juvenile sea turtles they almos t always exceed 70% (Table 1). Perhaps even more importantly, the plethora of methodological approaches (last column, Table 1) likely contributes largely to this variation (see also [START_REF] Pfaller | Phylogeny, biogeography and methodology: a metaanalytic perspective on heterogeneity in adult marine turtle survival rates[END_REF]. 3

Here, we present a general analytical framework that allows for age-specific survival estimates to be obtained from sparse CR data (i.e. small and varying numbers of individuals per age class). We applied this approach on two distantly related (within Testudinoidea in [START_REF] Crawford | A phylogenetic analysis of turtles[END_REF], Near Threatened (IUCN 2017) chelonians: the European pond turtle and the Hermann's tortoise (Testudo hermanni). In T. h. hermanni (the western subspecies) age -specific survival progressively increases with age from 39% in neonates to 97% in 6-year olds and older (Fernandez-Chacon et al., 2011); juvenile survival estimates of its eastern counterpart (T. h. boettgeri) are lacking. Immature European pond turtle survival exhibits a very wi de range (11% to 90% in Table 1), and robust age-specific analyses are lacking.

Materials and methods

Studied species and sites European pond turtle

The European pond turtle inhabits a broad range of habitats across Europe, northern Africa and westerns Asia. Nevertheless, it is often locally endangered and even extinct [START_REF] Fritz | Conservation actions for European pond turtlesa summary of current efforts in distinct European countries[END_REF]. The European pond turtle is largely impacted by diverse anthropogenic factors such as habitat loss, regulation of rivers and shoreline development that homogenize habitats and fragment populations [START_REF] Rogner | European pond turtle Emys orbicularis[END_REF].

During 16 years (1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012), a total sampling hand-capture effort of 598 h and 6158 trap-days in the Natural Reserve of Tour du Valat (Camargue, France, 250 ha 43°30 0 N, 4°40 0 E) resulted in 749 captured European pond turtles (for details on field methodology see [START_REF] Olivier | Assessing spatial and temporal population dynamics of cryptic species: an example with the European pond turtle[END_REF][START_REF] Ficheux | Rapid response of a long-lived species to improved water and grazing management: the case of European pond turtle (Emys orbicularis) in the Camargue, France[END_REF]. Among these, 298 could be aged using growth-rings [START_REF] Castanet | Les méthodes d'estimation de l'âge chez les chéloniens[END_REF][START_REF] Olivier | Ecologie, traits d'histoire de vie et conservation d'une population de cistude d'Europe Emys orbicularis en Camargue[END_REF] for a total of 809 captures. Sex was assigned for sexually dimorphic individuals followed by permanent notch marking on the carapace [START_REF] Cagle | A system for marking turtles for future identification[END_REF]).

Hermann's tortoise

Hermann's tortoises are widespread across the European Mediterranean region. Nevertheless, the westerns subspecies has suffered major losses in habitat and therefore distribution range [START_REF] Stubbs | Testudo hermanni in France[END_REF][START_REF] Bertolero | Testudo hermanni (Gmelin 1789) -Hermann's tortoise[END_REF]. With rapid uncontro lled urbanization in the Balkans, its eastern counterpart might be heading for a similar fate. Local illegal and legal tortoise ha rvest of huge proportions in the past decades (Cites trade report on Testudo hermanni, 2017) have massively contributed to this trend.

During 9 years (2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016), we studied a very dense (~60 adults per hectare, [START_REF] Bonnet | A prison effect in a wild population: a scarcity of females induces homosexual behaviors in males[END_REF] population on Golem Grad Island (Prespa Lake, Macedonia, ~18 ha, 40°52 0 09″N, 20°59 0 17″E). Each year the area was intensively patrolled (2-12 observers). Overall, 169 search days enabled us to capture 1830 individuals. Among them, 466 could be aged at 1225 capture occasions. Every captured individual was sexed when sexually dimorphic, aged, and permanently marked using a notche-code on the marginal-scutes [START_REF] Cagle | A system for marking turtles for future identification[END_REF]. In the course of recaptures, age was re-estimated blindly (for error rate and more details on ageing and general CR methodology see [START_REF] Bonnet | A prison effect in a wild population: a scarcity of females induces homosexual behaviors in males[END_REF][START_REF] Arsovski | When carapace governs size: variation among age classes and individuals in a free ranging ectotherm with delayed maturity[END_REF].

Multievent model design

We performed goodness-of-fit tests on the datasets based on the CJS model. Attributed to the detection of trap dependence and transience (see Results) that might be induced either by age-specific variations of recapture and survival probability but also by capture heterogeneity [START_REF] Olivier | Ecologie, traits d'histoire de vie et conservation d'une population de cistude d'Europe Emys orbicularis en Camargue[END_REF][START_REF] Ficheux | Rapid response of a long-lived species to improved water and grazing management: the case of European pond turtle (Emys orbicularis) in the Camargue, France[END_REF], we used multievent modelling [START_REF] Pradel | Multievent: an extension of multistate capture-recapture models to uncertain states[END_REF]. Events are field observations masking possibly multiple states; multievent models estimate the probability of observing a state given the event it is masked under using hidden Markov chains. Accordingly, sex was coded as a state in order to deal with sex uncertainty in young individuals. In order to deal with potential heterogeneity in recapture rates, we built mixture models [START_REF] Pledger | Open capturerecapture models with heterogeneity: I. Cormack-Jolly-Seber Model[END_REF] states following five successive transition matrices: survival from t to t + 1 (matrix Φ, Table 3a), dispersal between sites, given the individual survived (matrix Ψ, Table 3b), and change of recapture heterogeneity class given the individual survived and changed site (matrix F, Table 3c). The approach was simpler on the Hermann's tortoises, not parameterizing matrices Ψ and F since individuals did not change sites, nor were caught in traps. Note that for easier implementation, Ψ is a 9*17 matrix where eight departure states can transit to a "stay" or "moved" state. F is thus a 17*9 matrix, that allows "moved" individuals to change recapture probability class (since we suspect recapture heterogeneity to be related to trap location we assumed that "stay" individuals would not change recapture heterogeneity class). Finally, individuals can be recaptured or not. For easier implementation, this event matrix was split into two matrices, one allowing to model recapture probability given the states (matrix P, Table 3d), the other to estimate the probability of correctly sexing individuals (matrix S, Table 3e).

Building biological scenarios

Since young and small European pond turtles are less mobile and therefore less likely to be captured in a trap, two age classes were considered when modelling recapture probability (additional explorations would require a bigger sample): neonateto-2-year-olds and older than 2 years. Initially, survival-atage was a linear covariate with a plateau at age six. The more numerous tortoise dataset allowed multiple combinations of age-recapture probability to be explored, while maintaining an age effect on survival. Subsequently, in both species we explored multiple additive relationships with age, site, sex, time and capture heterogeneity.

Afterwards, maintaining the best parametrization of recapture probabilities we explored the effects of sex and site on survival. Lastly, we explored different parametrizations of the age effect on survival probabilities, namely linear, logarithmic and quadratic versus discrete, and different age at plateau. We superimpose our results from the best-fit discrete model against those aided by covariates and note the possible benefits and drawbacks. All models were implemented in E-SURGE [START_REF] Choquet | Program E-SURGE: a software application for fitting multievent models[END_REF] separately per species, ranking competitive models using Akaike information criteria (AIC, [START_REF] Burnham | Model selection and multimodel inference: a practical information-theoretic approach[END_REF].

Results

All 298 European pond turtles had hatched between 1990 and 2011, and all 466 Hermann's tortoises between 1999 and 2015. The chronological breakdown of captured animal numbers per hatching year can be found in Table 4. In both data-sets the goodnessof-fit tests were significant (chi 2 = 127.35 and 72.09, d.f. = 60 and 31, respectively, P < 0.001) with significant transience and trap-dependence (both P < 0.001), suggesting strong survival or recapture heterogeneity.

European pond turtle

The best-fitted model (Table 5) implied an additive time variation of both age classes ' recapture probabilities, the second (>2 years old) also exhibiting heterogeneity and sex differences. Removin g the effect of recapture heterogeneity increases AIC sharply (by 82.6), confirming its importance. The younger age-class displayed similar capture probabilities as the low heterogeneity class of the older age class (on average ranging between 0.23 and 0.3 4, between capture occasions). Higher heterogeneity class males were consistently higher than the respective females, with average respective estimates of 0.84 and 0.72. Survival retained the linear age -effect and did not vary between sites and sexes. Exploring a survival age-plateau resulted with two very close models (DAIC = 0.36), with survival summiting at 4 and 5 years of age. Nevertheless, neonate survival estimates are significantly different (0.10 f 0.05 vs. 0.20 f 0.07); the discrete model was unable to provide these estimates due to a lack of neonates. All three models demonstrate a steep increase in survival probability between zero and 4 years of age (Fig. 1). The differences in survival probabilities between the discrete and covariate models we re low, ranging between one and nine percent among age-classes. Nevertheless, the discrete model was much less precise (Fig. 1).

Hermann's tortoise

After model selection, regarding recapture probability 1-to 5-year-olds formed a separate cohort whereas all further age classes were considered separately. All are site dependent, while only those from the Plateau experience recapture heterogeneity (lowering AIC by 71). Among them, the higher heterogeneity class ranged between 0.56 and 0.71, the low heterogeneity class from 0.01 to 0.07 and Beach tortoises somewhere in the middle between 0.32 and 0.40. All exhibit additive time variation. Exploring different parameterizations of the age -effect on survival resulted with a logarithmic to linear relationship reaching a plateau at 4-6 years of age, or a discrete model summiting at the age of four [DAIC between these models ranged between 0.6 and 1.3, Table 5; models whose DAIC from the best fit model <2 are considered to also have substantial support [START_REF] Burnham | Model selection and multimodel inference: a practical information-theoretic approach[END_REF]]. Despite lowering AIC, adding site and/or sex effects on discrete model survival failed to provide precise estimates, thus the age-effect discrete model was used for comparison purposes. With covariates, best model fit was achieved when female survival from the Plateau was considered separately. Plateau female survival probabilities increased linearly with age from 0.08 when neonate to 0.86 when 5 -years-old. Conversely, male tortoises and females from the Beach expressed significantly higher survival at all ages starting at 0.32 with a plateau at 0.97 (Fig. 2). Superimposing the discrete model estimates (despite being slightly lower due to lack of separation of Plateau females) showed a broad overlap of confidence intervals but was unable to estimate neonate (N = 1) survival (Fig. 2).

Discussion

By means of advanced multievent CR modelling, this study demonstrates that even sparse datasets can be used to obtain agespecific estimates of survival in long-lived species. Consequently, it demonstrates that, as in other long-lived vertebrates, the substantially prolonged immature phase of both the Hermann's tortoise and the European pond turtle entail a steep and progressive increase in survival with age.

The age-linear covariate (continuous) models are streamlined versions of their discrete counterparts, suggesting no bias in the age-specific survival estimates. Moreover, the continuous models clearly outperformed the discrete models in terms of precision, especially in the sparser dataset of the European Table 5 Model 5), superimposed against the estimates and 95% confidence spread of mean age-specific survival probabilities of two separate cohorts (females from the Plateau, and both sexes from the Beach along with males from the Plateau) from the same population obtained from the best-fit covariate model (Model rank 1, Table 5).

pond turtle (Fig. 1). Most notably, the discrete models failed to estimate neonate survival due to the lack of marked neonates. The continuous models partially solve this issue by projecting the linear relationship of age and survival on neonate surviva l. Nevertheless, such predictions bear considerable uncertainty and should only be considered surrogates until the y can be supported by proper data collected on this age class. Attributed to the difficulty of capturing and recapturing elusive immature reptiles, researchers have often opted for indirect estimates of juvenile survival, such as catch -curve analyses (Table 1), or indirect mathematical approaches [START_REF] Pike | Estimating survival rates of uncatchable animals: the myth of high juvenile mortality in reptiles[END_REF]. The only robust study comes from a reintroduction program of western Hermann's tortoise adults allowing for all future recruits to be monitoredan exceptional case providing 19 years of data and precise discrete estimates (Fernàndez-Chacon et al., 2011); superimposing our eastern subspecies estimates reveals no differences between the taxa, and simply corroborates the idea that age -linear covariates helps streamline results without added bias. The following results thus seem encouraging; ultimately, substantial field effort followed by appropriate statistical approaches can pro-vide precise trait estimates, indispensable to matrix models underlying evolutionary ecology research, and efficient conservation policies.

Our results seemingly conform to existing literature (Table 1)mean survival is lowest in neonates, yet never below 20%, and it increases progressively with age (Fig. 1 and2). [START_REF] Mitrus | Headstarting in European pond turtles (Emys orbicularis): does it work?[END_REF] estimated surprisingly low survival values for neonate European pond turtles (0.11); whilst this might reflect the true situation, the author provides "minimal annual survivorships (numbers recaptured/number captured in previous year)", thus in practice actually describing a mix between survival and recapture rates. The age-survival relationships do not vary between our studied species despite their ecological differences and distant phylogenetic relatedness, possibly alluding a similar trend throughout this diverse clade (183 species in [START_REF] Crawford | A phylogenetic analysis of turtles[END_REF]. Furthermore, several other long-lived vertebrates also exhibit a linear increase in age-specific survival (see for instance [START_REF] Delong | Age-and sex-specific survival of California sea lions (Zalophus californianus) at San Miguel Island, California[END_REF]Forslund & P€art, 1995;[START_REF] Jorgenson | Effects of age, sex, disease, and density on survival of bighorn sheep[END_REF][START_REF] Schwarz | Estimation of juvenile survival, adult survival, and age-specific pupping probabilities for the female grey seal (Halichoerus gryprus) on Sable Island from capture-recapture data[END_REF][START_REF] Shine | Patterns of survival, growth and maturation in snakes and lizzards[END_REF]. Different species reach their respective survival plateaus at different ages, often corresponding with maturity [2-3 years in bighorn sheep [START_REF] Jorgenson | Effects of age, sex, disease, and density on survival of bighorn sheep[END_REF] note that first reproduction usually occurs later), 4 years in grey seals [START_REF] Schwarz | Estimation of juvenile survival, adult survival, and age-specific pupping probabilities for the female grey seal (Halichoerus gryprus) on Sable Island from capture-recapture data[END_REF], 7 years in Steller sea lions [START_REF] Hastings | Cohort effects and spatial variation in age-specific survival of Steller sea lions from southeastern Alaska[END_REF], 5 years in California sea lions [START_REF] Delong | Age-and sex-specific survival of California sea lions (Zalophus californianus) at San Miguel Island, California[END_REF]]. Both Hermann's tortoises and European pond turtles reach maturity between the ages of eight and 12 (six in [START_REF] Olivier | Ecologie, traits d'histoire de vie et conservation d'une population de cistude d'Europe Emys orbicularis en Camargue[END_REF][START_REF] Rogner | European pond turtle Emys orbicularis[END_REF][START_REF] Bertolero | Testudo hermanni (Gmelin 1789) -Hermann's tortoise[END_REF], yet arrive at a survival plateau earlier -at approximately four-to 5-yearsold (Fig. 1 and2). This is likely a consequence of the final hardening of the carapace, which can bring obvious survival benefits prior maturity [START_REF] Wilbur | Life-history evolution in turtles[END_REF]. After maturity, European pond turtles from Camargue experience sex differences in survival [START_REF] Olivier | Assessing spatial and temporal population dynamics of cryptic species: an example with the European pond turtle[END_REF]; nonetheless, our results overrule the possibility of it originating prior maturity. When immature neither species is obviously sexually dimorphic [START_REF] Rogner | European pond turtle Emys orbicularis[END_REF][START_REF] Bertolero | Testudo hermanni (Gmelin 1789) -Hermann's tortoise[END_REF], thus sex-specific habits or environmental pressures (e.g. sexual coercion in the Hermann's tortoise [START_REF] Hailey | Population density and adult sex ratio of the tortoise Testudo hermanni in Greece: evidence for intrinsic population regulation[END_REF]] that could influence survival prior maturity are either elusive, or likely not present. In adult Hermann's tortoises sex-specific survival is not common apart from few populations with a bias in sex-ratio [START_REF] Hailey | Population density and adult sex ratio of the tortoise Testudo hermanni in Greece: evidence for intrinsic population regulation[END_REF]. Interestingly, the Golem Grad tortoise population has the highest reported bias in sex-ratio of this species, reaching its maximum on the Plateau where only 5% of all adult individuals are female [START_REF] Bonnet | A prison effect in a wild population: a scarcity of females induces homosexual behaviors in males[END_REF]. Our best-fit model implies significantly lower survival of younger females from this locality from birth (Fig. 2). We hypothesize this result to be a consequence of male coercion on the oldest females in our dataset, later projected onto the youngest age classes by the covariate. As such, this example is also a cautionary message on the use of covariates! Nevertheless, adult survival insight is needed, and more data for the discrete model to confirm these claims. Pin-pointing the age at which Plateau females are initially affected should be explored, but is beyond the scope of the current study.

The high variation in juvenile survival estimates that fail to give age-or at least stage-specific estimates in Table 1 is likely owed to the age-class that was predominant in the respective datasets. This mandates prudence when interpreting them in an evolutionary-ecological framework or applying them to conservation policies. Our results corroborate that immature survival increases with age in long-lived species, additionally suggesting it occurs rather steeply in chelonians. This is likely a consequence of ontogenic shifts in morphology, physiology and behaviour and therefore alterations in interactions with the environment [START_REF] Golubovié | Ontogenetic shift of antipredator behaviour in Hermann's tortoises[END_REF]. It would come as a surprise if shifts in age-specific survival do not translate to age-specific shifts in other aspect of a species' life history. Indeed, after the age of five Golem Grad tortoises experience ever-increasing agespecific body size variation (Fig. 1, [START_REF] Arsovski | When carapace governs size: variation among age classes and individuals in a free ranging ectotherm with delayed maturity[END_REF]. Armed with a hard carapace they can reach a survival plateau and therefore enough confidence to start expressing individuality in their respective environments; interestingly, our models' preference to consider tortoise capture probabilities independently per age after the age of five corroborates this notion.

Remarkably, behavioural shifts might follow this trend as well: Hermann's tortoises alter their antipredator behaviours after maturity [START_REF] Golubovié | Ontogenetic shift of antipredator behaviour in Hermann's tortoises[END_REF]. We suggest that future early life studies on species with a long immature phase should focus on age-specific considerations of other aspects of animals' life histories.

Conclusion

Reliable projection matrices based on accurate early life agespecific survival estimates are essential for fundamental and conservation purposes. We suggest a simple way of facilitating the process of obtaining these estimates even with sparse datasets. Systematic approaches to survey young animals should be unified, and the resulting (even sparse) age-specific sets of data can usefully be modelled using advanced statistical techniques. Continuous annual surveys of small and elusive animals might seem as an inefficient allocation of labour considering the output, but hopefully the advancements in direct methodological approaches will gradually change this notion.

Tableau 2

 2 Abbreviations (left column) and descriptions (right column) of states that European pond turtles from Camargue (Esquineau and Faïsses), France and Hermann's tortoises from Golem Grad (the Plateau and the Beach), Macedonia can assume and transit among

Tableau 3

 3 Elementary matrices describing transitions among elementary states ('m/M' and 'f/F' refer to males and females, respectively, capitals denoting high recapture probability and vice versa; '1' and '2' refer to the two sites of both datasets: Esquineau/the Plateau and Faïsses/the Beach, respectively; Table 2): (a) survival matrix 0, / is the probability to survive from t to t + 1; (b) dispersal matrix Ψ, w is the probability to stay faithful to the site of first capture from t to t + 1, whereas after a dispersal event the elementary state is prefixed a 'd' (e.g. M1 -> dM1; (c) change of heterogeneity class matrix F, f is the probability of reaching the high recapture probability class; (d) first event matrix P, p is the probability of being recaptured after which the departure state is prefixed a 'c', whereas when failed to be recaptured the prefix is '0'; (e) second event matrix S, s is the probability to assess the sex of the individual, 0 -unobserved, 1 -observed as male in Esquineau/on the Plateau, 2observed as female in Esquineau/on the Plateau, 3 -observed as unsexable in Esquineau/on the Plateau, 4 -observed as male in Faïsses/on the Beach, 5 -observed as female in Faïsses/on the Beach and 6 -observed as unsexable in Faïsses/on the Beach

Figure 2

 2 Figure 2 Estimated age-specific mean survival probabilities and 95% CIs of young Hermann's tortoises from Golem Grad Island, Macedonia obtained from a discrete model (Model rank 9, Table5), superimposed against the estimates and 95% confidence spread of mean age-specific survival probabilities of two separate cohorts (females from the Plateau, and both sexes from the Beach along with males from the Plateau) from the same population obtained from the best-fit covariate model (Model rank 1, Table5).

Tableau 1 Review of published chelonian age-or stage-specific survival estimates prior to maturity. Some studies reported ambiguous age categories here presented as immature. When explicitly reported, materials and methods are also provided

		Immature (age-specific)
	Species	survival	Materials & methods	Reference
	Land tortoises
	T. h. hermanni	Neonate: 0.39 f 0.10	19 years of CR, Cormack-Jolly-Seber	Fernandez-Chacbn et al. (2011)
		1-year-old: 0.45 f 0.08	(CJS) model
		2-year-old: 0.77 f 0.08
		3-year-old: 0.68 f 0.07
		4-year-old: 0.86 f 0.06
		5-year-old: 0.88 f 0.05
	T. h. hermanni	Neonate: 0.54	14 years of CR, CJS model	Bertolero (2002)
		1-2 years-old: 0.64
		3-9 years-old: 0.91
	T. h. hermanni	0-2 years-old: 0.52	7 years of CR	Henry et
		3-9 years-old: 0.88
	Testudo graeca	Neonate: 0.39	Thread trailing	Keller, Diaz-Paniagua & Andreu (1998)

Table 3

 3 Continued.

Table 4 Breakdown of numbers of captured European pond turtles from Camargue, France and Hermann's tortoises from Golem Grad island, Macedonia per hatching-year

 4

	Hatching-year	European pond turtle	Hermann's tortoise
	1990	3	/
	1991	6	/
	1992	11	/
	1993	12	/
	1994	18	/
	1995	11	/
	1996	4	/
	1997	6	/
	1998	16	/
	1999	16	7
	2000	16	10
	2001	6	16
	2002	4	14
	2003	16	34
	2004	26	36
	2005	26	41
	2006	28	44
	2007	36	64
	2008	15	87
	2009	15	48
	2010	4	12
	2011	3	5
	2012	/	19
	2013	/	15
	2014	/	7
	2015	/	7

Acknowledgements

We are thankful for field help from numerous collaborators, particularly Camille Buyck, Mitko Tasevski, Sébastien Ficheux, Sonja Nikolié, Marc Cheylan, Jean-Marie Ballouard, Elisabeth Rosecchi, Bogoljub Sterijovski and Rastko Ajtié, and for financial support from the Ministry of Education, Sciences and Technological Development (grant No. 173043) of Serbia, the Rufford Small Grants Foundation (grant no. 20915-1) and the Tour du Valat Foundation. We thank the French Ministry of Environment and National Park 'Gali~cica' (permit no. 03-246) for granting us work permits.

 5), superimposed against the estimates and 95% confidence spread of mean age-specific survival probabilities of the same population obtained from the best-fit covariate model (Model rank 1, Table 5).


Thomas Maullin-Sapey

Camille Maumet

Thomas E Nichols

Detecting and Interpreting Heterogeneity and Publication Bias in Image-Based Meta-Analyses

Introduction

With the increase of data sharing, meta-analyses are becoming increasingly important in the neuroimaging community. They provide a quantitative summary of published results and heightened confidence due to higher statistical power. The gold standard approach to combine results from neuroimaging studies is an Image-Based Meta-Analysis (IBMA) [START_REF] Salimi-Khorshidi | Meta-Analysis of Neuroimaging Data: A Comparison of Image-Based and Coordinate-Based Pooling of Studies[END_REF] in which group-level maps from different studies are combined.

Recently, we have introduced the IBMA toolbox, an extension for SPM that provides methods for combining image maps from multiple studies [START_REF] Maumet | IBMA: An SPM toolbox for NeuroImaging Image-Based Meta-Analysis[END_REF]. However, the current toolbox lacks diagnostic tools used to assess critical assumptions of meta-analysis, in particular whether there is inter-study variation requiring random-effects IBMA, and whether publication bias is present. Here, we present two new tools added to the IBMA toolbox to detect heterogeneity and to assess evidence of publication bias.

Methods

The heterogeneity detection is performed with Cochran's Q statistic [START_REF] Hardy | A comparison of methods to detect publication bias in meta-analysis[END_REF]. Cochran's Q tests a null hypothesis of zero inter-study variance, and is large when study deviations are larger than expected by intra-study noise alone. If the user wishes to investigate the cause of heterogeneity at a specific voxel, they can generate a forest plot which displays a confidence interval for each study.

Publication bias detection is performed with a range of test statistics, including regression based methods such as Egger's regression and Macaskill's regression [4], ranking based methods such as Trim and Fill [START_REF] Duval | Trim and fill: A simple funnel-plot-based method of testing and adjusting for publication bias in meta-analysis[END_REF] and correlation based methods such as Begg's Correlation [START_REF] Begg | Operating Characteristics of a Rank Correlation Test for Publication Bias[END_REF]. All tests are based on detecting non-random arrangement of effect estimates relative to expected behavior based on sample size or intra-study variance. For example, if small studies all fall above a meta-analysis population mean, it suggests there may be missing small studies below the mean. If evidence of publication bias is found at some voxels, it can be explored with Galbraith plots and Funnel plots at individual voxels. The IBMA toolbox allows input of NIDM packs We used a set of 21 fMRI studies investigating the response to painful stimuli from one lab. To evaluate our tools a biased dataset was created: In a small, 3 × 3 × 3 voxel region the 9 studies with the largest mean activation were selected, discarding 12 studies, thus simulating publication bias in that region.

Maps of diagnostic test statistics were created to attempt to detect the simulated bias, and funnel plots were used to verify visually the bias.

Results

The IMBA toolbox's Q-Statistic found evidence for heterogeneity at nearly every voxel in the brain (Fig 1). While all results were from one lab and used the same software, the stimuli and designs are heterogeneous over studies.

The IBMA toolbox's publication bias test statistics successfully identified the region in which bias was induced (Fig 2). It can be seen that publication bias was detected both in the region bias was induced at (highlighted in red in Fig 2A ) and in many other regions of the brain. This is to be expected as selecting studies in a manner such that publication bias is induced in a specific region is likely to result in studies with larger variance being included in the selection. This in turn will results in more publication bias being induced across the brain purely by chance.

Figure 1: The thresholded Z map, from a meta-analysis of the 21 pain studies, reported in [START_REF] Maumet | IBMA: An SPM toolbox for NeuroImaging Image-Based Meta-Analysis[END_REF] (A) and the Q-statistic P-Value map thresholded at a corresponding FDR value of 0.05 (B). Forest plots for two voxels, both with extreme observed heterogeneity, one with no significant observed signal (an observed meta-analysis Z-stat of 0.5732) (1) and one with significant observed signal (an observed meta-analysis Z-stat of 3.5217) [START_REF] Maumet | IBMA: An SPM toolbox for NeuroImaging Image-Based Meta-Analysis[END_REF]. P-values are displayed with a -log10 scale with large values indicating strong evidence of heterogeneity. The most significant heterogeneity is found in the pain networks, such as the insular cortex and thalamus. However, heterogeneity is also found in areas with no Z-significance, such as cerebellar, visual and motor areas, suggesting one or more individual studies may have activated these areas.

Figure 2: Site where bias was induced, shown in red (A), the unweighted Egger's Regression -log 10 p-value map thresholded using an FDR-corrected p<0.05 (B) and the Trim and Fill L estimator of number of studies missing due to publication bias, thresholded using the common rule of thumb of 4 studies (C). Funnel Plots for the region bias in which was induced (1) and, for comparison, a region at which no bias was detected (2). The site where publication was induced is visible in Egger's results and the Trim and Fill L estimator, as are several other areas.

Conclusions

The IBMA toolbox is an open-source and freely available extension of SPM. This toolbox is as a first step towards making efficient, statistically valid synthesis of neuroimaging literature easily accessible to the research community.

As data sharing within the neuroimaging community increases and more statistic maps become readily available, we believe, processes such as the diagnostic tests provided by the IBMA toolbox will become a common component of any published IBMA.

  [7] or NIfTI images, facilitating the use of results from different software packages. It is open software and makes use of non-regression testing on GitHub [8].

-Maumet C., et al. (2016). Sharing brain mapping statistical results with the neuroimaging data model. Scientific Data. doi:10.1038/sdata.2016.102

-IBMA -The IBMA Toolbox [Internet]. [cited 18 Dec 2017]. Available: https://github.com/NeuroimagingMetaAnalysis/ibma

Acknowledgments

This work was supported by the Wellcome Trust. We gratefully acknowledge the use of the pain dataset from the Tracey pain group, FMRIB, Oxford. The majority of this work was conducted while TMS, TEN and CM were at the University of Warwick.