Montarges-Pelletier Emmanuelle
email: emmanuelle.montarges@univ-lorraine.fr.

Duriez Caroline

Ghanbaja Jaafar

Jeanneau Laurent

Falkenberg Gerard

Micro-scale investigations of the fate of heavy metals associated to iron-bearing particles in a highly polluted stream

Keywords: river particles, metallic contaminants, iron, micro-spectroscopy, microanalyses

As it flows through a dense steel making area, the Fensch River does transport iron-rich particles and colloids, displaying high contents in metallic contaminants (Zn, Cr, Pb, Cu, Ni, As). Chemical analysis using ICP-MS was carried out on three compartments, waters, suspended material and sediments, along the river linear. The variations of metallic trace elements concentrations along the river, were shown to being partially related to external inputs (industrial and domestic waste waters, urban surfaces leaching). However, some discrepancies of elements partitioning were evidenced. Pb, Cu, Mn tend to concentrate in suspended particulate and in dissolved fraction, while Cr and As follow Fe trend and concentrate within sediments of the most downstream station, just before the junction with Moselle waters. Zn appears strongly associated to iron rich-particles, resulting in a decrease of its concentration in waters for the last station. Along the Fensch linear, the variation of metal partitioning between water and particulate phases is accompanied with strong modifications of nature and mineralogy of iron-rich particles, as evidenced by microanalyses using electron and X-ray beams. The combination of bulk analyses using ICP-MS 2 and microanalyses, applied to the three compartments allowed us to propose a three step process "settling-weathering-resuspension" to explain the Zn partitioning.

Introduction

Metallic pollution in aquatic systems represents a major environmental concern due to the persistence and toxicity of heavy metals. Studies on metal-degraded aquatic systems should include the analysis of metal contents and speciation in at least three compartments, the dissolved fraction, the sediments and the suspended solids (particles and colloids) that have been shown to play a major role in the transport and speciation of toxic substances [START_REF] Mavrocordatos | Characterisation of colloids by electron microscopy[END_REF][START_REF] Mccarthy | Subsurface transport of contaminants[END_REF][START_REF] Stumm | Aquatic colloids as chemical reactants: surface structure and reactivity[END_REF][START_REF] Warren | Suspended particle grain size dynamics and their implications for trace metal sorption in the Don river[END_REF][START_REF] Buffle | A generalized description of aquatic colloidal interactions: the three-colloidal component approach[END_REF]Schemel et al. 2000). Indeed, natural colloids and particles display a much larger specific surface area, due to their small size. Also, because of their heterogeneous composition, of their low crystallinity, they display a high proportion of surface groups likely to bind metals, and as a consequence a higher surface reactivity. The study of such solid phases, besides global metals concentrations, requires specific methods, taking into account the high heterogeneity and the amorphous character of most of their constituents. Then, micro-scale and nano-scale information must be obtained, which calls for the use of spectro-microscopic techniques. In that context, electron microscopy (SEM, TEM, and STEM) has been extensively used (e.g., [START_REF] Mavrocordatos | Characterisation of colloids by electron microscopy[END_REF][START_REF] Filella | Particle size and mineralogical composition of inorganic colloids in waters draining the adit of an abandoned mine, Goesdorf, Luxembourg[END_REF]. Such techniques provide high spatial resolution and can be advantageously combined with Energy Dispersion of X-rays Spectroscopy (EDXS), which helps in the identification of poorly crystallized phases and metallic contaminants scavengers, that can not be identified by classical XRD.

However, these techniques present a few drawbacks (high vacuum conditions, beam damage, relatively high detection limits of EDXS). It is then worthwhile to combine them with X-ray based techniques such as X-ray microfluorescence (e.g., [START_REF] Isaure | Quantitative Zn speciation in a contaminated dredged sediment by µ-PIXE, µ-SXRF, EXAFS spectroscopy and principal component analysis[END_REF][START_REF] Mclean | Lead speciation in House Dust from Canadian Urban homes using EXAFS, microXRF and microXRD[END_REF]) and X-ray microscopy (TXM and STXM). This latter technique can be performed in wet conditions and provides spectroscopic information that can be spatially resolved at a sub-micronic scale (e.g. [START_REF] Bluhm | Soft X-ray microscopy and spectroscopy at the molecular environmental science beamline at the Advanced Light Source[END_REF][START_REF] Dynes | Speciation and quantitative mapping of metal species in microbial biofilms using scanning transmission X-ray microscopy[END_REF][START_REF] Miot | Iron biomineralization by anaerobic neutrophilic iron-oxidizing bacteria[END_REF].

The present work is a case study focusing on a highly polluted river located in the North East of France, the Fensch river. Sediments and suspended particulate material (SPM) from the Moselle-Fensch junction were previously studied and displayed high concentrations in heavy metals such as Zn, Sn, Cr and Pb as well as a high iron content [START_REF] Montarges-Pelletier | The junction of Fensch and Moselle rivers, France; mineralogy and composition of river materials[END_REF]. A strong correlation was evidenced from bulk analyses between iron and heavy metals contents in SPM and sediments (Zn, Cr, Pb). This correlation at macro-scale suggested that most of the heavy metals were concomitantly entering the river system. The sediments accumulated in the vicinity of the Fensch-Moselle junction were shown to integrate the contamination of the entire watershed, past and actual. In order to study the origin and fate of heavy metals in the Fensch river, sampling was extended from the source in Fontoy to the junction with Moselle in Illange. The molecular characterization of organic matter [START_REF] Jeanneau | Evolution of the source apportionment of the lipidic fraction from sediments along the Fensch river, France: A multimolecular approach[END_REF], and more specifically of the lipidic fraction had already evidenced the evolution of organic contaminants origin on the Fensch linear. The following paragraphs aim to show that iron particles and associated heavy metals are not simply transported and/or accumulated in sediments, but are also modified in terms of speciation and distribution between compartments. Thus, the presented results will focus on the distribution of heavy metals between the three compartments, i.e. dissolved fraction, SPM and sediments, as well as on the interactions between heavy metals and suspended materials, and specifically iron-rich suspended colloidal and particulate materials. This will be achieved by combining global elemental analysis and various microspectroscopic techniques i.e. TEM-EDXS, SEM-EDXS, micro-XRF and STXM. This latter technique allows obtaining spatially resolved NEXAFS spectra at Fe L 2,3 edges that are particularly sensitive to iron valency and coordination symmetry (e.g., [START_REF] Rivard | Combination Of Multi-Scales And Multi-Edges X-Ray Spectroscopies For The Investigation Of Kaolinite And Metallic Iron Interaction Products (Anoxic Conditions, 90°C)[END_REF][START_REF] Yoon | In Situ Characterization of Aluminum-Containing Mineral-Microorganism Aqueous Suspensions Using Scanning Transmission X-ray Microscopy[END_REF][START_REF] Bluhm | Soft X-ray microscopy and spectroscopy at the molecular environmental science beamline at the Advanced Light Source[END_REF][START_REF] Benzerara | Alteration of submarine basaltic glass from the Ontong Java Plateau: a STXM and TEM study[END_REF]). The analytical data collected at different scales, from bulk to nanometer, unraveled the influence of settling-resuspension cycle of sediments on the fate of metals within such highly contaminated surface waters.

Experimental section

Study area

The study area is located in the downstream zone of the Moselle basin, past the main cities of the region, Nancy and Metz, upstream the border with Luxembourg and Germany, where Moselle river finally discharges within the Rhine in Koblenz. Fensch river flows through a highly industrialized valley, well-known for steel-making activities. For more than a century, this small river has been considered as a natural sewer and was then the receiving end of numerous industrial and domestic inputs. Consequently, it was classified as the most polluted river in France in the 80's. As it is one of the tributaries of the Moselle river, its contamination represents a serious threat to the quality of Moselle waters that are used as drinking water resource. Despite recent improvement in industrial wastewaters treatment, past and present industrial and domestic inputs, as well as leaching of urban surfaces maintain a high anthropogenic pressure on this hydrosystem. Furthermore, as a significant portion (> 50%) of the river is covered by public and private facilities (Figure 1), dredging operations have been rather limited, and large amounts of highly polluted sediments remain in place, thus maintaining a significant environmental hazard. The complete geographic situation was already described in previous papers [START_REF] Jeanneau | Impact of a highly contaminated river on a more important hydrologic system: changes in organic markers[END_REF][START_REF] Montarges-Pelletier | The junction of Fensch and Moselle rivers, France; mineralogy and composition of river materials[END_REF]. An aliquot of 100 mL was immediately filtered at 0.22 µm on cellulose syringefilters, split into three distinct containers dedicated to DOC analysis, cations, and anions concentrations measurements, respectively. Samples for cations analysis were subsequently acidified with HNO 3 1 N. Samples were stored at 4°C until analysis. The resulting solution was analyzed by ICP-MS (trace elements) and by ICP-AES (major elements) (SARM, CRPG Vandoeuvre-lès-Nancy). The DOC was determined with a Dohrmann DC 190. Anions (F -, Cl -, NO 3 -, SO 4 2-et PO 4 3-)

Sampling methods and elemental analysis

were measured by ion chromatography (Dionex ICS-3000).

Suspended matter was retrieved from the centrifugation at 15 000 g during 20 min of 20 L of raw water and freeze-dried. SPM could also be recovered from water filtration through pre-weighed Nucleopore Membrane (47mm diameter, 0.22 µm pore size), this fraction includes particulate matter as well as colloids.

Such an additional filtration procedure provides a quantitative evaluation of suspended solids loads within Fensch waters. The resulting filter membranes were air-dried and kept under neutral atmosphere before SEM and TEM/STEM experiments.

Superficial sediments (0-10 cm) were sampled at the different stations. For the station of Illange, sediments were cored on about 50 cm to investigate the evolution with depth, core samples were divided in three parts from the top to the bottom: 0-10 cm, 10-30 and 30-50 cm. The different sediments samples were then frozen, freeze-dried and sieved at 2 mm, 250 µm and 50 µm. The different analyses were performed mainly on the coarse and fine fractions 0-2 mm and 0-50 µm. For SPM and sediments, elemental composition was determined through ICP-MS and ICP-AES (SARM, CRPG Vandoeuvre-lès-Nancy).

Microscopic and microspectroscopic methods for solids

Electronic microscopies

For TEM (Transmission Electronic Microscopy) analysis, filter fragments were suspended in ethanol, releasing fine particles and colloids, and ultrasonicated during twenty minutes to enhance particle dispersion. One drop of the resulting suspension was put down on a Cu grid coated by a carbon membrane (EuroMEDEX, Mesh200). TEM was carried out using a CM20 Philips TEM with 200 kV accelerating voltage. The electron microscope is coupled with an EDAX EDXS. Stoichiometric ratios can then be calculated from atomic percentages given by EDX spectra, and compared with known mineralogical compositions. A spot size of about 40 nm was used to record EDX spectra with a counting time of 40 s and dead time below 25%. EDX calibration standards were run to obtain quantitative analyses of major and trace elements with a detection limit of about 1000 ppm.

Scanning Transmission Electron Microscopy observations were performed by

focusing the electron beam into a narrow spot of 5-10 nm that is scanned over the sample. The rastering of the beam across the sample is used for mapping by EDX spectroscopy. Images were recorded in bright field BF. The scan raster is 256 x 256 points with a dwell time of 30 µs per pixel. The area to be scanned is selected from a low magnification image.

For SEM (Scanning Electron Microscopy), a Hitachi 54800 Scanning Electron Microscope equipped with a Kevex 4850-S EDX was used. One filter fragment was taped with carbon-tape onto 2 cm 2 plates and carbon coated. Backscattered electron imaging (BEI) was used to locate the particles of interest. In that mode, brightness is related to the average atomic number of materials, and the mineral phases bearing heavy elements appear as bright spots that can be easily detected.

In order to increase the emission of backscattered electrons, the SEM microscope was generally operated with a beam current of 3 pA and an accelerating voltage of 20 kV (analyzed micro-volume of about 6 µm 3 ). However, to improve the quantification of low atomic number elements, EDX analysis was sometimes carried out at an accelerating voltage of 10 kV (sampling volume of 3 µm 3 ).

Synchrotron based X-ray Fluorescence

Synchrotron-based X-ray fluorescence experiments were performed at beamline L in Hasylab light source facility, Hamburg, Germany. Aqueous suspensions of river material were filtered on cellulose filters that were directly analyzed by Xray fluorescence. The monochromatic X-ray beam (15 keV) was focused on the sample using borosilicate glass polycapillaries with a resulting beam size of 10 µm. Samples were placed on a motor x,y,z stage with a movement precision of 1 µm. Maps were acquired on various zones of the filters with 10 µm step size and dwell times of 5 sec. Fluorescence spectrum on each point was collected using a Vortex-EX (Radiant Detection Technologies) Silicon Drift Detector with 50 mm 2 active area. The XRF spectra were evaluated using the AXIL software package [START_REF] Falkenberg | Upgrade of the x-ray fluorescence beamline at HASYLAB/DESY[END_REF][START_REF] Osan | Characterization of anthropogenic sediment particles after a transboundary water pollution of river Tisza using synchrotron radiation[END_REF]).

X-ray microscopy

STXM experiments were carried out on the PolLux beamline located at the Swiss Light Source (SLS) Paul Scherrer Institut Villigen, Switzerland [START_REF] Raabe | PolLux: A new facility for soft xray spectromicroscopy at the Swiss Light Source[END_REF]. The SLS storage ring runs at 2.4 GeV in "top-up" operation mode, which guarantees a constant electron beam current of 400 (±1.5 mA). The PolLux STXM beamline uses polarized X-rays from a bending magnet in the photon energy range between 200 and 1200 eV. X-ray beam is focused by a 25 nm zone plate. The lateral resolution is about 40 nm in routine operation, with a spectral resolving power (E/ ΔE) better than 3000 at iron L 2,3 edges, and a detected flux in helium of more than 2 x 10 6 photons/s. Raw river water (10-20 µl) was deposited between two silicon nitride windows, with a surface of 1 mm 2 and a thickness of 100 nm. The experimental protocols used for STXM data acquisition and analysis can be found elsewhere [START_REF] Jacobsen | Soft X-ray spectroscopy from image sequences with sub-100nm spatial resolution[END_REF][START_REF] Hitchcock | Chemical mapping with soft X-ray spectromicroscopy[END_REF][START_REF] Lerotic | Cluster analysis of soft X-ray microspectroscopy data[END_REF]). XANES spectra were obtained by performing image stacks collected by scanning the sample in x-y direction on an area of a few square microns at energy increments of 0.2 eV over Fe L 2,3 -edges energy range (695-730 eV). The AXIS2000 software was used for data and image processing (http://unicorn.mcmaster.ca) [START_REF] Hitchcock | Chemical mapping with soft X-ray spectromicroscopy[END_REF]. Images of a given stack were first aligned with each other using a correlation method and converted into optical density (OD) according to the following equation: OD = -ln(I/I 0 ) where I represents the transmitted intensity at the point of interest and I 0 represents the incident intensity and was derived from empty regions in the images (no particle areas). I 0 includes the absorption by silicon nitride windows. In order to separate particles with different Fe L-edges spectrum, two procedures were tested for each stack. On one part, a manual procedure, using the sub-routine Zimbra, was used to select regions of interest. On the other part, in order to check that all the diversity of spectra and thus the diversity of iron phases was covered by the manual procedure, we applied principal component analysis (PCA) and cluster analysis [START_REF] Lerotic | Cluster analysis of soft X-ray microspectroscopy data[END_REF][START_REF] Lerotic | Cluster analysis in soft X-ray spectromicroscopy: Finding the patterns in complex specimens[END_REF]. The spectra extracted using either Zimbra routine were all corrected in energy position, and a linear background was subtracted. Resulting spectra were all normalized setting their area to 1. Reference spectrum of berthierine (a silicate containing mainly Fe 2+ ) was multiplied by 0.8 as a correction factor to take into account the difference in occupancy of the 3d orbitals in Fe 2+ and Fe 3+ ions (Van der Laan and Kirkman 1992). Experimental spectra were fitted with linear combination of normalized reference spectra of one Fe 2+ phase and one Fe 3+ phase, applying the CGO (Conjugate Gradient Optimization) curve fit routine in aXis2000. The coefficients thus obtained were used to calculate Fe 2+ :Fe 3+ ratio in the probed zones. Regarding the general evolution of water chemistry along the Fesnch river, there is a strong increase of conductivity value and major ions concentrations in Knutange, the second station, followed by a slow decrease from Knutange to Concerning the solids, there is a simultaneous increase of Zn SPM and sediment contents in Seremange, as the values go from 780 mg.kg -1 to 1892 mg.kg -1 and from 566 to 1058 mg.kg -1 respectively. However, while Zn concentrations rather suggest a dilution phenomenon, for the last station of Illange, Zn contents reach 2765 mg.kg -1 in sediments and 3720 mg.kg -1 in SPM. Similar trends were observed for Cu, Mn and Pb (Online Resource).

Results

Field parameters and major ions in solution

The major ions analysis in waters previously evidenced that the Knutange Mine exhaust (KN) acts on water composition through an increase of major ions concentration (Table 1). However, as the water flow from the mine is well above the Fensch flow, it dilutes some of the elements present in Fensch waters before the mixture with mine waters. Those surcharge waters appear enriched in Ni The third plot (Figure 2 plot C) displays the evolution of Cr concentrations and contents in waters, SPM and sediments respectively. Cr concentrations regularly increase from Fontoy to Illange with a maximum value of 6.6 µg.L -1 in Illange.

For Cr, the SPM and sediments contents display the same evolution as waters concentrations and Cr contents in Illange reach 267 and 693 mg.kg -1 in SPM and sediments respectively. A similar trend was observed for As (Online Resource).

In the case of iron (Figure 2 Despite the fact that iron was not detected in solution, it can be compared to Cr as for both elements, SPM and sediments contents increase, and Cr and Fe contents in sediments are higher than in SPM.

Iron, heavy metals and metalloids (Zn, Ni, Pb, Cu, Co As) contents in sediments are definitely higher in Illange. Actually, the last section of the Fensch river, meaning about one kilometer before the Moselle river, is a stagnant reach, with a over-calibrated and uniform river bed, surrounded by imposing banks. This last section is then an accumulation zone for solid materials coming from the entire watershed (Montargès-Pelletier et al. 2007[START_REF] Jeanneau | Impact of a highly contaminated river on a more important hydrologic system: changes in organic markers[END_REF], 2008). Although

Illange sediments appear as a metallic contamination sink, detected metals do not display similar evolutions along the Fensch linear and are heterogeneously distributed among the three investigated compartments. This heterogeneity can be explain not only by the origin variations of metallic contamination, but also by the variation of chemical speciation and association mode with suspended particles. Because XRD only evidenced the presence of crystalline phases, it was mandatory to carry out spatially resolved investigations on SPM samples, in order to get a more precise picture of the mineralogy of iron particles and, as a consequence, a better idea of their ability to trap and transport heavy metals within the Fensch waters. Such microanalyses also provide the nature of metal carriers. Actually, no more diatoms, or scarcely only, could be observed for the following stations. A closest view (Figure 3B) reveals mineral aggregates, which, on the basis of EDXS analysis, were shown to be constituted of numerous submicrometric phyllosilicates (that were identified to illites and interlayered illite/smectite), quartz, feldspars and calcite grains, covered by parts of silica diatoms skeletons. Scarce bright spots were assigned to iron oxihydroxide particles. ). EDXS analysis of individual iron particles evidence Fe/O atomic ratios ranging from 1 to 0.6, with a mean value at 0.75 suggesting the predominance of wuestite, magnetite and hematite particles. For such particle, electron beam was hardly transmitted and was set at the edge of the particle to favor the detection of oxygen fluorescence photons, that tend to be re-absorbed by iron rich spherical particles.

Status of

Fontoy

Figure 5 presents typical STXM images obtained on particles collected at Seremange. The presence of iron rich particles (black spots) is evidenced through the division of high resolution images respectively collected before iron edge and at iron edge (Figure 5A). A stack of images was then collected on a reduced zone (red square), from which spectroscopic data could be extracted at the pixel scale (50 nm). PCA routine was run for this Seremange stack, in order to rise up the different spectra. However, the results were influenced by the presence of thick iron particles. In order to reveal the diversity of iron phases, manual definition of regions of interests (ROIs) was preferred. The resulting ROIs are shown on Figure 5B (dashed lines), and associated Fe L 2,3 edge spectra on figure 5D. Reference spectra are displayed on Figure 5C, to compare sample spectra with those obtained on various iron-bearing reference minerals. For ferric and ferrous iron, Fe L 2,3 edges can be primary described as transitions from a ground state Fe 2p 6 3d 5 (2p 6 3d 6 ) to a final excited state Fe 2p 5 3d 6 (2p 5 3d 7 ) respectively. Calculated spectra based on the nearly atomic transition of these configurations have shown different fine structures for these two valence states (van der Laan and Kirkman 1994). In the case of the 4 standard minerals used here, the oxidation state of iron strongly affects the spectral features of the different samples. This is particularly marked by the presence of a signal at 708 eV that is clearly much more intense for Fe 2+ bearing minerals (Berthierine and Magnetite, bottom curves on Figure 5C).

L 2 peaks also display slight modifications in the relative intensities of major contributions at 722.5 and 724 eV. In the case of Berthierine (that can extrapolated to Fe 2+ oxide), the low energy component of L 2 edge increases in intensity while the high energy component decreases. A supplementary signal at 720 eV also indicates the presence of ferrous iron. This peak is absent in both maghemite and ferrihydrite (top curves).

Each ROI spectrum was fitted as a linear combination of ferrous and ferric iron mineral phases, providing Fe 3+ percentage. On the basis of these linear combination fittings, Seremange spectra could be classified in two main groups.

One first group evidences the presence of ferrihydrite-like phases, while the second group evidences the presence of mixed valence oxides with Fe 3+ percentage going from 49 to 70%. SPM collected at Seremange then clearly display significant amount of Fe 2+ bearing phases. This result goes well with Fe:O ratios obtained from TEM and STEM investigations, and supports the hypothesis of mixture of different oxides going from pure Fe 2+ wuestite or FeO to pure Fe 3+

Fe 2 O 3 or FeOOH.nH 2 O.

The occurrence of iron oxides with relative high contents in ferrous iron may be linked to the location of that sampling station, just downstream the first set of steel-making facilities (high furnaces) and just at the exit of the longest covered section of the Fensch river (3 km). The low oxygen content in water at this location confirms the reductive conditions. Still, it must be pointed out that according to literature data [START_REF] Todd | Surface oxidation of chalcopyrite (CuFeS 2 ) under ambient atmospheric and aqueous (pH 2-10) conditions: Cu, Fe L-and O K-edge X ray spectroscopy[END_REF]) none of the collected spectra corresponds to iron sulfides. Zn could be detected for 75% of studied points, with atomic percentage ranging between 0.2 and 27%.

Maisons-Neuves (MN)

A typical STXM image of SPM collected at Maisons-Neuves is presented in Figure 7. In that case, PCA routine was carried out to evidence the distinct spectra. In contrast with what was observed in Seremange (Figures 4 and5) Fe 2+ rich particles are very scarce (small blue particle in Figure 7 C, spectrum on Figure 7D). It must also be noticed that numerous bacteria are systematically observed on all the images. This can be directly related to the direct input of untreated domestic waste waters and/or to the presence just upstream of this collection point of a wastewater treatment facility using a biological activated sludge process. The role of these bacteria and of the associated Natural Organic Matter (NOM) in the increased mineralogical modifications of iron rich materials is likely, although no direct evidence for such processes can be provided.

Illange (ILL)

TEM

Status of metals (Zn) in suspended materials: synchrotron-based microfluorescence and TEM-EDXS

X-ray fluorescence maps were acquired for SPM collected in Illange and several zones of about 640 microns per 700 microns were investigated using a 10 µm micro-beam at 15 kV. In order to approach the links between iron-rich particles and trace metals, elemental maps were built on the basis of their emission lines intensity. Furthermore, resulting maps representing the spatial distribution of Ca, Fe, Zn, Pb and Cu were treated point by point to yield correlation graphs, to which linear regression was applied (Figure 10 and Table 2). Ca was used as a "witnesselement", its abundance in the sample was sufficiently high so that it could be readily detected. 2), showing that the presence of pure Fe-oxides modify the correlation.

For the other elements than Zn, i.e., Pb, Cu, the results were more difficult to analyze due to their lower abundance in the sample. Indeed, the distribution of those elements and the resulting correlation factors with iron-rich particles was strongly influenced by the presence of few Cu/Pb hotspots, those hot-spots being iron free or with a rather low iron content. These microfluorescence maps suggest then that Zn, Cu, Pb are present with at least two different status, the commonest one being related to iron-rich particles, the second one related to iron-free particles or iron-poor particles.

TEM and STEM analyses were also carried out to detect the occurrence of trace elements by EDXS. However, due to the relative high level of detection, i.e. 0.1%, Zn could be detected for a sufficient number of particles, and Cr and Pb were only occasionally detected.

For Fontoy, trace elements are almost all below the detection limit of EDXS except for Zn that is evidenced at relatively low concentrations for few particles (0.1-0.5%). For Seremange SPM sample, Zn was detected for about 65% of For the following stations, Maison-Neuve and Illange, Zn could be detected for 75% and 76% of studied points respectively, with atomic percentage ranging between 0.2 and 27% for Maison-Neuve and between 0.2 and 25% for Illange.

The nature of Zn carriers are qualitatively similar to those encountered in Seremange (Figure 9), but Zn was never more occurring as being associated to iron oxides. Zn-sulfides (Figure 9D) particles represent 4% or analyzed particles in Illange, and thus are far less encountered than Zn-bearing aggregates (Figure 9 A and B). Moreover, in Illange, SPM display Zn-bearing particles with no presence of iron or relatively low presence of iron (figure 9 micrograph C, 5% of investigated particles). Such particles, presenting a fibrous shape, are constituted of aluminum oxyhydroxides and calcium phosphates and Zn was detected with atomic ratio ranging between 3 and 6%.

Discussion

Along the Fensch river, the evolution of metals concentrations in solution is not identical for all metals (Figure 2 and Online resource). The elemental analyses on SPM and sediments tend to evidence an enrichment of SPM in Cu, Zn, Mn, Pb towards water and surface sediments for the last station on the Fensch linear, in the vicinity of the junction with the Moselle river. The variation of SPM content, of DOC which both tend to increase from upstream to downstream are not sufficient to explain the evolution of partitioning, and the modification of SPM composition might be retained as a possible explanation [START_REF] Shäfer | The influence of dissolved organic, suspended particulates, and hydrology on the concentration, partitioning and variability of trace metals in two contrasting Wisconsin watersheds (USA)[END_REF]. This metallic enrichment of SPM is apparently associated with the evolution of iron status in SPM, for which a global increase in oxidation state along the Fensch linear was revealed by SEM, TEM (Fe:O atomic ratio) and STXM. The evolution of iron oxidation state in SPM is combined to intense mineralogical modifications leading to significant morphological changes (Figures 6789). Various mechanisms can explain the observed evolution. First of all, due to their high density, anthropogenic iron oxide particles such as those observed at Seremange station (Figure 4), tend to settle as shown by XRD patterns from sediments (Online resource). Although it was not specifically studied in this work, it is now admitted that biotic and abiotic oxidation processes occur at the water-sediment interface (Santschi et al. 1990, Warren and[START_REF] Warren | Biogeochemical controls on metal behaviour in freshwater environments[END_REF]. Fensch waters and Fensch sediments are relatively rich in domestic organic matter [START_REF] Jeanneau | Impact of a highly contaminated river on a more important hydrologic system: changes in organic markers[END_REF][START_REF] Jeanneau | Evolution of the source apportionment of the lipidic fraction from sediments along the Fensch river, France: A multimolecular approach[END_REF], and it can be readily suggested that a relatively high biotic activity stands in the first layers of sediments. The abundance of microbes in the Fensch waters is evident and undoubtedly provokes the iron oxidation and mineralogical transformation. Indeed, as iron is used as electron donor for specific bacterial metabolisms [START_REF] Fortin | Formation and occurrence of biogenic iron-rich minerals[END_REF]Langley 2005, Mitsunobu et al. 2012), its cycle is commonly associated with microbial activity. In the case of the Fensch river, the oxidation processes of mineral particles should be prevented due to the relative high amount of organic matter which constitutes an O 2 "consumer" for its own decomposition. Furthermore, opulent organic matter may also act itself as a major factor of iron-particles modification. First, iron readily forms stable complexes with organic matter [START_REF] Stumm | Aquatic colloids as chemical reactants: surface structure and reactivity[END_REF]Morgan 1996, Rose et al. 1998), and the relatively high concentration in dissolved organic carbon (DOC, see table 1 andfigure 11) could influence the iron oxides dissolution. Secondly, organic matter certainly recovers the surface of iron particles when released in the aqueous medium, and participates then to the general processus of dissolutionreprecipitation. The organic coating of particles was already reported for sediments in a previous paper, with the use of FTIR in reflectance mode (Montarges-Pelletier et al 2007).

For a upstream-dowmstream trend, settling and biochemical modifications resulted not only in an increased occurrence in sediments of iron oxides such as magnetite, wuestite, and hematite but also in the apparition of hetereogeneous aggregates composed of hairy iron oxyhydroxides. The mineralogical modifications may occur at different locations : in the water column, or in the upper levels of sediments and due to the relatively weak depth of Fensch river (inferior to 1 m), the latter is certainly more efficient. However, the chronicity, the kinetics of weathering processes is difficult to precise and due to frequent remobilization phenomena of Fensch sediments, the cycle of Fensch SPM is not straightforward to describe. Furthermore, the fact that Illange sediments show relatively high contents in Fe 2+ bearing oxides (wuestite, magnetite) can also be a result of a selective remobilization of iron particles, enhancing the re-suspension of hairy and low-density oxyhydroxides. The succession of these three steps of settling, intense weathering and resuspension is the main explanation for the above described evolution of iron status in SPMs along the Fensch linear. This assumption was reported on a graphical summary (Figure 11).

Although organic matter was commonly reported as metal-scavenger in natural waters (e.g. [START_REF] Buffle | Measurement of complexation properties of humic and fulvic acids in natural waters with lead and copper ion-selective electrodes[END_REF][START_REF] Logan | Complexation of Cu2+ and Pb2+ by peat and humic acid[END_REF][START_REF] Murphy | The role of sorbed humic substances on the distribution of oranic and inorganic contaminants in groundwater[END_REF], Dupré et al 1999), the abundance of iron particles in the Fensch waters tends to minimize the role of organic matter in terms of metal sorbents. The iron cycle in Fensch waters certainly influences the fate of metals, and certainly in a predominant way.

The mineralogical modifications of iron particles may have direct consequences on the amount of heavy metals present in SPMs through two associated mechanisms. First, the formation of filamentous structures increases the specific surface area available for ion adsorption. These enhanced surface properties may explain also why such iron oxyhydroxides are associated with carbonates and phosphates phases. Second, newly formed iron oxy-hydroxides are efficient scavengers of heavy metals as shown in numerous studies (e.g. [START_REF] Tessier | Trace metals in oxic lake sediments: possible adsorption onto iron oxyhydroxides[END_REF][START_REF] Honeyman | Metals in aquatic systems[END_REF][START_REF] Schwertmann | RM Iron oxides in: Dixon JB and Weed SB[END_REF]Warren and Hack 2001;[START_REF] Waychunas | Nanoparticulate Iron Oxide Minerals in Soils and Sediments: Unique Properties and Contaminant Scavenging Mechanisms[END_REF].

Cr, Ni and Zn were selected to describe three distinct evolution trends in the Fensch waters. Chromium is supposed to be chemically bound to iron oxides as the trend of its contents (except in solution) follows iron content in SPM and sediments, but direct evidence could not be provided with the undertaken analytical procedure.

The Zn behaviour in Fensch waters could be precised. Microanalyses (micro-XRF and TEM-EDXS) evidence three different status for Zn in particulate fraction, including amorphous Zn-sulfides [START_REF] Priadi | X-ray Absorption Fine Structure Evidence for Amorphous Zinc Sulfide as a Major Zinc Species in Suspended Matter from the Seine River Downstream of Paris, Ile-de-France, France[END_REF], and Zn associated to iron-rich aggregates. While we could not directly evidence the chemical association mode between Zn and those complex particles, the resuspension of those iron-rich particles might be one key to explain the increase of Zn concentration in SPM (Figures 2 and 11). From the ICP-MS analyses on waters, SPM and sediments (Figure 2 and online resource), we had supposed that Mn, Cu and Pb were following the same trend as Zn. Those elements could be detected only occasionnally and the number of particles bearing Cu or Pb is not sufficient to adress some hypothesis on the nature of the bearing phases.

Thus, although SEM, TEM, STXM, micro-XRF and associated spectroscopies were shown to be provide helpful information, their use is strongly dependent on the heavy metals concentration in solid phases. While we could propose a schema for Zn fate and partitioning within the Fensch rivers, it was difficult to rise some conclusions for the other metals such as Pb, Cu or Cr.

The potential hazard that these heavy metals associated to iron-rich SPM will represent after the confluence with the Moselle river strongly depends on their mode of association. Various scenarios can be proposed: (i) outer sphere complex adsorption on iron oxy-hydroxides; (ii) inner-sphere complexes; (iii) coprecipitation of mixed mineral phases [START_REF] Stumm | Aquatic colloids as chemical reactants: surface structure and reactivity[END_REF]. The first case could represent a significant environmental threat as, with evolving physico-chemical conditions, weakly adsorbed heavy metals could be released in solution through dilution and/or ion exchange. Discriminating between these various cases could be achieved by following the speciation of heavy metals in SPMs before and after the confluence using (micro)spectroscopic techniques such as Extended X-ray Absorption Fine Structure. Such a study will be the object of a future publication. modifications, going from rather ferrous oxides to ferric oxyhydroxides. These modifications provoke a modification of heavy metals (Zn) partitioning between water, suspended solids and sediments. The decrease of Zn in solution is associated with pH values ranging between 7.74 and 7.94, relatively high concentrations in suspended solids (SS in mg.l -1 ) and dissolved organic carbon (DOC in mg.l -1 ). Other physical and chemical parameters were provided on Table 1. 112; 180; 66 196.6; 380; 85 197; 418; 73 179.3; 310;

1-Evolution of trace and major elements along the Fensch river, supplementary data

The following graphs present the evolution of Cu, Pb, Mn and As concentrations and contents in waters, SPM and sediments along the Fensch linear. Cu, Pb and Mn were considered to belong to Zn group on the basis of the evolution of their contents in SPM. Indeed, for Pb and Cu concentrations in waters do not display strong variations along the river (Pb : 0.05-0.2 µg.L -1 ; Cu 3-6 µg.L -1 ) but Cu content in SPM (24-266 mg.kg -1 ) present a relatively strong increase in Illange, reaching 266 mg.kg -1 for the last station. Pb was also considered as analogous to Zn and Cu, considering that in Illange, Pb content was close in both SPM and sediments (346 mg.kg -1 and 400 mg.kg -1 respectively).

2-XRD patterns of sediments and SPM

X-ray diffraction data were obtained using a D8 Bruker diffractometer equipped with a (θ, 2θ) goniometer using Co Kα 1 radiation (l = 1.7902 Å). X-ray diffractogramms were collected on powder samples in ambient conditions, within the 2Θ range [3, 65°], with 0.036° step and 3s collecting time. The XRD pattern obtained for Seremange SPM presents a very low signal:noise ratio due to the amorphous character of this material. Because the data were collected with few mg of material, the diffraction pattern presents a broad band at low angle values, due to the sample holder. Besides classical minerals such as quartz, calcite that predominates the pattern due to their relatively good cristallinity, hematite and magnetite diffraction bands could be identified (orange and red on the figure SI-3).

3-STEM-EDXS spectra from FONTOY particles

The figure below was obtained on Fensch SPM collected in Fontoy, at the most upstream station on the Fensch linear. For this station, SPM was mainly constituted of diatoms, clay minerals and carbonates. The following figure presents the elemental maps obtained for one particle using STEM-EDXS, as well as one spectrum collected for one point of interest on the particle. The elemental maps, acquired at a nanometer scale, show that the particle is mainly constituted of phyllosilicate layers, illite and smectite clays. The part of diatom skeleton (on the top of the micrograph) provides silicon signal only and a nanometric calcium phosphate could be evidenced and identified through the maps of Ca and P.

Figure 1

 1 Figure 1 displays the different sampling sites used in the present study, Fontoy

  Figure2presents the evolution of waters concentrations and SPM and sediments

(figure 2

 2 plot B) as this element display an increase in water concentrations at this station (4.8 µg.L -1 in Fontoy and 11.6 µg.L -1 in Knutange). For the following stations, Ni concentration tends to decrease, reaching 7.2 µg.L -1 in Illange. Ni contents in SPM do not present strong variations, ranging from 31 to 61 mg.kg -1 , and the maximum value occurs in Seremange and not in Illange as it was the case for Zn. As far as sediments are concerned, Ni contents increase regularly along the Fensch linear and the maximal value is measured in sediments from the last station, Illange. A similar trend was observed for cobalt (Online Resource).

  plot D), contents are given as percentage of iron oxide Fe 2 O 3 . Fe 2 O 3 in SPM increases downstream, with an edge in Seremange at 7.9 %. There is an increase of iron-rich particles inputs from several industrial facilities between Nilvange and the exit of this small watershed, and sediments show similar evolution of iron contents. The X-ray diffractograms of sediments along (Online Resource), confirm the increasing presence of iron-phases. Besides classic sedimentary mineral phases (quartz, calcite, dolomite and feldspars), magnetite Fe 2 O 3 , hematite Fe 3 O 4 , wuestite FeO are encountered, iron oxides being the predominant iron phases detected through XRD.

  Iron in suspended particles: microanalyses using SEM,TEM and STXMThe comparison of heavy metals and iron contents in the dissolved, particulate, sediment fractions strongly suggest the association of heavy metals with ironbearing colloids and particles. This could be further analyzed by focusing on four stations: Fontoy, Seremange, Maison-Neuve and Illange. Fontoy is close to the source of the Fensch and should be the least affected by industrial or domestic discharge although previous works evidenced the presence of human waste markers at this point[START_REF] Jeanneau | Impact of a highly contaminated river on a more important hydrologic system: changes in organic markers[END_REF]). The station of Seremange is the first easily accessible station located in a heavily industrialized area, downstream two steel manufacturing and making plants (Corus-Rail and Arcelor-Mittal). Maisons-Neuves and Illange are located downstream. Furthermore, the high iron contents in SPM and sediments call for a better knowledge of iron bearing phases in those matrices. It is then of prime interest to analyze if the modifications of heavy metals partitioning occurring along the Fensch river is linked to changes in the status and speciation of iron-bearing suspended materials.

Figure

  Figure 3A displays a general SEM view of SPM from Fontoy. The most obvious

Figure 3

 3 Figure 3 presents two STEM micrographs (C and D). Associated elemental maps

Figure 6

 6 Figure6presents some SEM and TEM observations of SPM collected at

  micrographs of SPM from Illange are shown in Figure 8. The amount of iron-rich filamentous structures is even higher than what was observed in Maisons-Neuves. EDXS analyses reveal that these structures are rich in iron and in heavy metals, essentially Zn, occasionnally Pb. The Fe:O ratio in these filaments is relatively low, around 0.5-0.4 which indicates not only a more oxidized status, but also the predominance of ferrihydrite-like materials or amorphous Fe-oxyhydroxide. Such filamentous or hairy particles are often combined with calcium phosphates and aluminosilicates (clay minerals). The EDX spectrum shown on Figure 8-B1 represents about 68 % of investigated particles with TEM-EDXS.

Figure 9

 9 Figure9presents one of the stacks acquired for SPM from Illange. Those STXM

Figure 10 presents

 10 Figure 10 presents Fe and Zn distribution zones obtained for two distinct zones on

  investigated particles, while Pb and Cr were rarely detected, and about 6% of particles display Pb in their elemental composition. Atomic percents of Zn range between 0.2% and 46%, and particles with Zn content below 1% represents 75% of the particles displaying Zn. Morevover, half of the particles for which Pb was detected are the particles with the highest Zn atomic percent. As far as Zn is concerned, its occurrence is particularly correlated to that of iron, as shown by STEM -EDXS elemental maps (Figure4D & E). Zn is occurring with different kinds of particles or aggregates of particles, from the most prevalent to the rarest, (i) Zn associated to iron oxides, (ii) Zn associated to heterogeneous aggregates essentially constituted of iron oxyhydroxides, aluminosilicates (clays), phosphates and carbonates, (iii) Zn sulfides. Those three kinds of Zn-bearing particles are sorted from the Zn-poorest to the Zn-richest.

Fig. 1 :

 1 Fig.1: Presentation of the study site. Fensch river takes its source in Fontoy and joins the Moselle river in Illange. Sampling stations are Fontoy FO, Knutange KN, Seremange SE, Maisons-Neuves MN, and Illange IL. The schematic map also presents the three tributaries Petite Fensch, Marspich and Kribsbach, the covered parts along the Fensch linear and the increase of industrial occupancy of surfaces from upstream to downstream. WWTP is located few dozens of meters before the sampling point MN.

Fig. 2 :Fig. 3 :Fig. 4 :

 234 Fig.2: Evolution of iron and heavy metal contents in waters (<0.22 µm), SPM and sediments from Fontoy (upstream) to Illange (downstream). A: Zn, B: Ni, C: Cr, D: Fe 2 O 3 . For A to C plots, left axis displays waters concentrations in µg.L -1 , and right axis displays solid contents for trace metals (in mg.kg -1 ) or for iron (Fe 2 O 3 %). White columns correspond to waters concentrations, grey columns to SPM contents and filled columns to sediments contents. Iron was not detected in all collected waters, the detection level being 0.03 mg.L -1 . Fig.3: SEM micrographs in backscattered electrons mode, from Fontoy SPM. A: general view (scale bar 300 µm). B: mineral aggregate (scale bar: 30 µm). TEM micrographs (C and D). A part of diatom skeleton can be observed on micrograph C, the red arrows points a calcium phosphate bullet.Fig.4: Seremange SPM, electron microscopies observations. A, B and C: SEM micrographs of iron-bearing particles collected in Fensch waters at the Seremange station, in backscattered electrons mode. From the left to the right: general view with a bright iron plate , a detailed view of star-like and spherical iron oxide. D and E: STEM micrographs with associated Zn and Fe elemental maps.

Fig. 5 :

 5 Fig.5: STXM data for Seremange SPM. A: Iron-rich particles map (scale bar 5 µm) obtained from the division of high resolution images collected respectively before and at Fe L 3 -edge (700 and 710 eV); B and C: regions of interest and corresponding Fe L 2,3 edges XANES spectra (solid lines). Each spectrum area is normalized to 1, linear combinations fits (dashed lines) of normalized spectra of Fe 2+ reference and Fe 3+ reference yield the ferric iron percentages. D: Fe L 2,3 spectra for standards Berthierine (Fe II -phyllosilicate); Magnetite, Fe 2+ Fe 3+ 2 O 4 ; Maghemite Fe 3+ 2 O 3 ; Ferrihydrite FeOOH.nH 2 O.

Fig. 6 :

 6 Fig.6: SEM and TEM micrographs of Maison-Neuve SPM. A: general SEM view displaying iron plate and heterogeneous aggregate (scale bar 30 µm), few diatoms skeletons could be noticed on this general view; B: Spherical aggregates of iron nanoparticles (scale bar 10 µm); C, D: TEM micrographs of iron-rich aggregates displaying spherical iron oxides as well as iron oxyhydroxides with hairy texture.

Fig. 7 :

 7 Fig.7: STXM treatment of Maison-Neuve SPM. A: High resolution Fe L 2,3 transmission image acquired at 710 eV. B: Iron-rich particles map (scale bar 2 µm ) obtained from the division of high resolution images collected respectively before and at Fe L 3 -edge (705 and 710 eV). Regions of Interest are marked with colored dashed lines. C : Fe L 2,3 edges XANES spectra (solid lines), each spectrum area is normalized to 1, linear combinations fits (dashed lines) of normalized spectra of Fe 2+ reference and Fe 3+ reference yield the ferric iron percentages.

Fig. 8 :

 8 Fig.8: TEM micrographs and EDX spectra for Illange SPM. A and B: Iron-rich particles, showing hairy texture, mainly constituted of iron oxyhydroxides, calcium phosphates and aluminosilicates (clays). B-1: Typical EDXS spectrum of iron-rich particles (Fe/O close to 0.5) displaying Zn at 2.5%. C: Aluminum oxyhydroxides, calcium phosphates and Zn (6%). D: Zn sulfides, with EDX spectrum (D-1).

Fig. 9 :

 9 Fig.9: STXM experiments for Illange SPM. A and B are high resolution images respectively collected below Fe-L 2,3 edges (700 eV) and near the maximum of Fe-L 3 edge (710 eV). Dashed lines on B show the regions of interest for spectrum extraction. C: All extracted spectra are identical and after normalization of its area to 1, a linear combinations fit (dashed lines) using normalized spectra of Fe 2+ reference and Fe 3+ reference suggests the predominance of a pure Fe 3+oxyhydroxide. Fig.10: X-ray Micro-Fluorescence on Illange SPM. SPM were prepared as deposit on cellulose filters. Fe and Zn distribution maps acquired at 15 keV, 640 µm per 700 µm, each pixel is 10 µm x 10 µm, counting time is 10 s. The intensities are plotted via a color scale, from the lowest to the highest intensities measured in a selected area, and normalized to a 0-100 scale. Correlation graphs on the left were built from the maps and plot about 4470 dots, except the aberrant and negative values. Correlation factors reported on table 2 were obtained from linear regressions.

Fig. 11 :

 11 Fig.11: Graphical summary. Due to successive steps of selective settling, weathering at the watersediment interface and remobilization, suspended iron rich particles undergo mineralogical

Figure SI- 1 :

 1 Figure SI-1 : Evolution heavy metal contents in waters (<0.22 µm), SPM and sediments from Fontoy to Illange. A:Cu, B: Mn, C: Pb, D: As, E: Co. Left axis displays waters concentrations in mg.L -1 , right axis displays solid contents in mg.kg -1 , except for Mn as this axis corresponds to MnO percent. White columns correspond to waters concentrations, grey columns to SPM contents and filled columns to sediments contents.

Figure SI- 2 :

 2 Figure SI-2: Evolution of the X-ray diffractograms obtained on Fensch sediments (0-2mm fraction), a: Fontoy; b: Knutange; c: Seremange; d: Maisons-Neuves; e: Illange. Identified crystalline phases quartz (Q), calcite (C), orthoclase (F K ), dolomite (D), hematite (H), magnetite (M), wuestite (W).The X-ray diffractograms of the sediments along the Fensch linear confirm the increasing presence of iron-phases. Although XRD analysis does not provide the whole mineralogy of iron phases, it shows that oxides are the predominant crystalline iron phases: magnetite Fe 2 O 3 , hematite Fe 3 O 4 and wuestite FeO.

FontoyIllangeFigure

  Figure SI-3: X-ray diffractogram obtained on Fensch SPM Seremange;. Identified crystalline phases quartz (Q), calcite (C), orthoclase (F), dolomite (D), hematite (H), magnetite (M), illite (I), kaolinite (K).

  Figure SI-3: Fontoy SPM, STEM micrograph, elemental maps and EDXS spectrum. (scale bar 2.5 µm)

Table 1 :

 1 Coordinates of sampling stations, physico-chemical parameters, major ions. The measured parameters are compared to mean, maxima and minima values provided by Regional Water Agency (mean value; MAX; MIN; SIERM database, 2001-2011 period, http://rhinmeuse.eaufrance.fr/); n.d. : not determined; S.S : suspended solid; DOC : dissolved organic carbon. *For the Illange station (IL), there is no SIERM data.

Table 2 :

 2 Correlation factors between Fe and Ca, and few metals Zn, Cu and Pb, calculated using linear regression on distribution maps. For Cr, data were not exploitable due to the low level of detection. For the table display, the zones were classified as a function of Zn(Fe) correlation factor, decreasing from Zone 1 to Zone 8. The mean and maximum intensity of Fe Kα line (number of counts, with dead time correction) for the zone is also indicated.

	Parameter /	Fontoy	Knutange	Seremange	Maisons Neuves	Illange*
	Station	FO	KN	SE	MN	IL
	Coordinates	49°21'05.0" N	49°20'42.2" N	49°19'18.6" N	49°19'41.4" N	49°19'52.4" N
								05°59'58.7" E	06°01'44.0" E	06°05'41.3" E	06°09'09.7" E	06°09'49.3" E
								229 m	197 m	188 m	162 m	156 m
	Temperature °C	8.7	10.7	5.2	6.5	9.4
	Conductivity µS.cm -1	707	2680	2320	1968	1917
	Conductivity SIERM	633; 725; 422	1561; 3193; 560	1605; 3205; 623	1663; 2658; 416	-
	pH							7.37	7.5	7.74	7.82	7.94
	pH SIERM					8 ± 0.3	7.7 ± 0.2	8 ± 0.2	7.9 ± 0.2	-
	S.S. mg.L	-1	49	20	101	94	115
	S.S. SIERM	4.4; 50; 1	5.6; 170; 1	10.3; 93; 2	22; 262; 3	-
	DOC mg.L	-1	2.78	2.66	4.15	5.3	5.42
	DOC SIERM	1.8; 6; 1	1.6; 6.5; 0.5	2.3; 4.4; 1	4; 14; 1	-
	HCO3 -SIERM	287.9; 362; 180	371.7; 508; 216	318; 478; 28	288; 453; 176	-
	SO4 mg.L	-1	72.4	1138	222	126	92.7
	SIERM							65.4; 160; 21	644; 1730;70	563.9; 1450; 97	427.1; 1070; 96	-
	Cl mg.L	-1					24.9	45	104	120	125
	SIERM							19.6; 69; 12	26.1; 81; 16	85.4; 535; 29	168.5; 326; 24	-
	NO3 mg.L	-1	10.9	2.4	5.0	6.1	7
	NO3 SIERM		11.8; 50; 1	5.2; 15; 0.5	6.8; 17; 1	7; 16; 1	-
	Ca mg.L	-1				101.4	287.3	234.0	209.7	211.7
	Ca SIERM

Acknowledgements

The authors greatly acknowledge the EU for funding their travel to HASYLAB and SLS facilities through the FP7 ELISA (Transnational Access Project). We also thank synchrotron facilities HASYLAB, SLS for providing beam time on L and PolLux beamlines respectively. PolLux beamline is funded by the BMBF. This work was financially supported by CNRS-INSU (ECODYN program) and Region Lorraine through the Research network Zone Atelier Moselle. A special thanks goes to Francis Borghesi from NANCIE who set up the laboratory van before each sampling campaign, and to Ali FALL from CAVF (Fensch Valley Cities Community), for selecting and providing access to the different stations. The authors also acknowledge Benjamin WATTS and Jorge RAABE from PolLux beamline for the technical support.

CONCLUSION

The previous paragraphs dealt with the study of a highly contaminated river flowing through steel making facilities and associated dense urban areas. Besides the measurement of metals and major elements in three compartments : suspended particulate, sediments and water (< 0.22 µm), a detailed description of suspended particulate was provided through a quite wide panel of microanalytical tools, including synchrotron based techniques. Microanalyses provided crucial information about SPM composition that could not be found elsewhere, through bulk techniques such as XRD. The mineralogical and textural modifications of iron-rich suspended particles along the Fensch linear were correlated with the variations of metals content, in particular Zn, within sediments, SPM and waters.

The comparison of SPM and sediments composition strongly suggest the modifications being due to a three step process (settling-weatheringresuspension). Such an assumption should be more precisely tested throught the study of iron and zinc speciation at a bulk scale.

Supplementary Information


Jocelyn Méré

Christophe Chopard

Anne Bonhoure #

Juliette Morlon-Guyot

Bruno Beaumelle
email: bruno.beaumelle@irim.cnrs.fr

Increasing stability and toxicity of Pseudomonas exotoxin by attaching an antiproteasic peptide *

Trypsin-like activities are present within the endocytic pathway and allow cells to inactivate a fraction of incoming toxins, such as Pseudomonas exotoxin (PE), that require endocytic uptake before reaching the cytosol to inactivate protein synthesis. PE is a favorite toxin for building immunotoxins. The latter are promising molecules to fight cancer or transplant rejection, and producing more active toxins is a key challenge. More broadly, increasing protein stability is a potentially useful approach to improve the efficiency of therapeutic proteins. We report here that fusing an antiproteasic peptide (bovine pancreatic trypsin inhibitor, BPTI) to PE increases its toxicity to human cancer cell lines by 20-40 fold.

Confocal microscopic examination of toxin endocytosis, digestion and immunoprecipitation

experiments showed that the fused antiproteasic peptide specifically protects PE from trypsinlike activities. Hence, the attached BPTI acts as a bodyguard for the toxin within the endocytic pathway. Moreover, it increased the PE elimination half-time in mice by 70%, indicating that the fused BPTI stabilizes the toxin in vivo. This BPTI-fusion approach may be useful for protecting other circulating or internalized proteins of therapeutic interest from premature degradation.

Immunotoxins are promising agents for treating cancer and transplant rejection [START_REF] Pastan | Immunotoxin therapy of cancer[END_REF][START_REF] Pastan | Immunotoxin treatment of cancer[END_REF][START_REF] Hetzel | Improved immunotoxins with novel functional elements[END_REF].

These hybrid molecules are usually prepared by linking a monoclonal antibody (sometimes restricted to its antigen binding portion) to a toxin devoid of its receptor-binding domain [START_REF] Pastan | Immunotoxin treatment of cancer[END_REF].

Most immunotoxins are prepared using toxins targeting the protein synthesis system, such as diphtheria toxin (DT), Pseudomonas exotoxin (PE) or ricin [START_REF] Pastan | Immunotoxin treatment of cancer[END_REF]. Production of more potent versions of these toxins to obtain more efficient immunotoxins would undoubtedly facilitate the clinical use of these therapeutic agents.

Ricin is an heterodimeric protein. The A-chain is an enzyme that is able to inactivate protein synthesis, resulting in cell death. The B-chain is a lectin that is responsible for cell binding [START_REF] Lord | Ricin: structure, mode of action, and some current applications[END_REF]. The bacterial toxins DT and PE are made of three structural and functional domains. They enable the toxin to bind to its receptor, translocate to the cytosol and inactivate protein synthesis, respectively [START_REF] Deng | Molecular mechanisms of the cytotoxicity of ADPribosylating toxins[END_REF] (see Fig. 1 for details). The catalytic activity of PE and DT catalyses the ADP-ribosylation of elongation factor 2 (EF2), thereby blocking protein synthesis.

Cells can process internalized proteins before they reach lysosomes. A number of proteolytic activities, including trypsin-and furin-like activities, have indeed been associated with various elements of the endocytic pathway, and toxins are attractive tools for studying this processing. While ricin, perhaps due to its packed structure [START_REF] Montfort | The three-dimensional structure of ricin at 2.8 A[END_REF] is intrinsically resistant to most animal-cell proteases [START_REF] Beaumelle | Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol[END_REF], toxicity of DT and PE is tightly associated with endosomal proteolysis [START_REF] Gordon | Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases[END_REF]. Cell-associated trypsin or furin can cleave DT within the Arg-rich interchain loop (Fig. 1). This processing results in toxin activation that is required for cytotoxicity [START_REF] Tsuneoka | Evidence for involvement of Furin in cleavage and activation of Diphtheria Toxin[END_REF] and activated, nicked DT is resistant to further trypsin digestion (up to 1µg/ml [START_REF] Drazin | Structure and activity of diphtheria toxin. II. Attack by trypsin at a specific site within the intact toxin molecule[END_REF]).

Upon endocytosis, PE can be similarly activated by trypsin-and furin-like activities that cut the toxin after Arg279, within a disulfide stabilized loop at the beginning of the translocation domain (Fig. 1). Cleavage at this site, then disulfide reduction, generates a 37 kDa carboxyl-terminal active fragment that translocates to the cytosol following retrograde transport to the ER and is involved in the intoxication process [START_REF] Chiron | Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin[END_REF][START_REF] Morlon-Guyot | Processing of Pseudomonas exotoxin A is dispensable for cell intoxication[END_REF]. Entire PE can also cross the endosome membrane to reach the cytosol [START_REF] Morlon-Guyot | Processing of Pseudomonas exotoxin A is dispensable for cell intoxication[END_REF][START_REF] Méré | Acidtriggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure[END_REF]. Nevertheless, PE processing by trypsin-like activities during endocytosis can result in toxin inactivation due, for instance, to a cleavage after Arg490, i.e. within its catalytic domain [START_REF] Chiron | Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin[END_REF]. An attempt was first made to protect PE from cell trypsin-like activity by inhibiting this processing after Arg490, using point mutations within this area of the molecule. Surprisingly, mutant PE that are resistant to cleavage after Arg490 by purified trypsin showed improved stability in mouse blood, but not enhanced toxicity as compared to PE [START_REF] Brinkmann | Alteration of a protease-sensitive region of Pseudomonas exotoxin prolongs its survival in the circulation of mice[END_REF]. Similar data were obtained when PE was conjugated to polyethylene glycol. This modification prolonged the PE elimination half-life in mice, while cytotoxicity remained unaffected [START_REF] Kuan | Pseudomonas exotoxin A mutants. Replacement of surface exposed residues in domain II with cysteine residues that can be modified with polyethylene glycol in a site-specific manner[END_REF].

Once delivered to the cytosol, incoming toxins will meet the proteasome that can also neutralize toxins. The identity of the first residue is a major determinant for recognition by this degradation system [START_REF] Varshavsky | The N-end rule: functions, mysteries, uses[END_REF]. This N-end rule also applies for toxins, and mutating the first residue of the DT A-chain revealed a linear relationship between intracellular stability and toxicity. Nevertheless, none of these mutants were more toxic than native DT [START_REF] Falnes | Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway[END_REF].

Because neither point mutations [START_REF] Brinkmann | Alteration of a protease-sensitive region of Pseudomonas exotoxin prolongs its survival in the circulation of mice[END_REF][START_REF] Falnes | Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway[END_REF], nor conjugation to polyethylene glycol [START_REF] Kuan | Pseudomonas exotoxin A mutants. Replacement of surface exposed residues in domain II with cysteine residues that can be modified with polyethylene glycol in a site-specific manner[END_REF] succeeded to produce more active toxins, it therefore remained to be determined whether improving toxin stability would enable generation of more potent molecules. In this study, we showed that fusing bovine pancreatic trypsin inhibitor (BPTI) to PE enhances its cytotoxicity, intracellular stability as well as its elimination half-life in mice. This approach could be applied to stabilize other clinically valuable circulating proteins such as hormones and growth factors that are susceptible to trypsin [START_REF] Duprez | Receptor-mediated endocytosis of interleukin 2 in a human tumor T cell line. Degradation of interleukin 2 and evidence for the absence of recycling of interleukin receptors[END_REF][START_REF] Renfrew | Sequential processing of epidermal growth factor in early and late endosomes of rat liver[END_REF].

Experimental Procedures

Reagents

Most chemicals were obtained from Sigma, cells from the American Tissue Culture Collection and pure RTB from Inland Laboratories (Austin, Texas). Purified furin and 6biotin-17-NAD were from R&D systems. Goat and rabbit anti-PE antibodies were from List Biological Laboratories and Sigma, respectively, and were affinity purified before use [START_REF] Alami | Involvement of ATP-dependent Pseudomonas exotoxin translocation from a late recycling compartment in lymphocyte intoxication procedure[END_REF].

The DT used in this study is an attenuated version (DT-E148S) [START_REF] Lemichez | Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery[END_REF] suitable for expression in E. coli.

DNA manipulations

A conventional PCR-based approach was used to prepare all constructions [START_REF] Beaumelle | Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol[END_REF], in pET-3d, using appropriate restriction sites. Target plasmids carrying DT-E148S [START_REF] Lemichez | Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery[END_REF], RTA [START_REF] Beaumelle | Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol[END_REF], PE [START_REF] Taupiac | A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly[END_REF] and BPTI [START_REF] Ostermeier | The folding of bovine pancreatic trypsin inhibitor in the Escherichia coli periplasm[END_REF] have been described. Chimeras in which BPTI was linked to the Nterminus of toxin X consisted of the OmpA signal sequence followed by the five residues from mature OmpA (Ala-Pro-Lys-Asp-Asn; to enable cleavage by the signal peptidase), Pro-Gln, BPTI, a spacer peptide (Ala-Ser-Ala-Ser-Thr-Pro-Glu-Pro-Asp-Pro-Glu-Lys-Leu (7)) and X. BPTI and DTB were connected by the same spacer when BPTI was attached to the DTB carboxyl terminal (see Fig. 1 for construction details). All PCR-amplified DNA was sequenced to check for the absence of mutations. The complete sequence of BPTI-PE is provided as supplemental Fig. 1.

Expression and purification of fusion proteins

Chimeras were expressed in (DE3) BL21-transfected E. coli, and purified from the periplasm using ion exchange chromatography. A CM-sepharose column was used for RTA [START_REF] Chaddock | Mutagenesis and kinetic analysis of the active site Glu 177 of ricin[END_REF], and a Q-Sepharose column for bacterial toxin (DT and PE [START_REF] Taupiac | A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly[END_REF]) purification. Western blots using anti-toxin and anti-BPTI antibodies were used to confirm the identity of the chimeras. PE, BPTI-RTA and recombinant RTA (rRTA) were > 95% pure after this single chromatographic step, while DT, BPTI-DT, DT-BPTI and BPTI-PE were further purified on a mono-P column, to reach > 95% homogeneity. To associate rRTA or BPTI-RTA to RTB, equimolecular amounts of A-and B-chains (30 µM) were treated with 8 mM reduced glutathione for 3 h at room temperature before overnight dialysis at 4°C [START_REF] Beaumelle | Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol[END_REF].

In vitro cleavage of PE and BPTI-PE by proteases

Toxins (5 µg) were incubated at 37°C for 30 min with trypsin concentrations ranging from 0.3 to 30 µg/ml, in110 mM NaCl, 20 mM Tris-HCl, pH 7.5, or in 110 mM NaCl, 20 mM sodium acetate, pH 5.5, in a final volume of 20 µl. To study furin processing, toxins (6 µg) were incubated at 25°C for 4 to 16 h with 0-25 U furin in 15 µl of 1 mM CaCl2, 100 mM sodium acetate, pH 5.5. Digestions were terminated by boiling for 3 min in reducing sample buffer before SDS/PAGE and Coomassie blue or Sypro ruby staining. Furin activity was assessed by monitoring the production of the 37 kDa C-terminal and the 28 kDa N-terminal fragments from PE [START_REF] Ogata | Processing of Pseudomonas Exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol[END_REF]. Furin processing of BPTI-PE generates a larger N-terminal fragment (~34 kDa), due to the presence of BPTI. The bands corresponding to the entire toxin and processed fragments were quantified using imageQuant (GE Healthcare).

Assay for PE catalytic activity

The ADP ribosylation activity of PE was tested as described (26) using partially purified EF2 [START_REF] Chung | The mechanism of ADP-ribosylation of elongation factor 2 catalysed by fragment A from diphtheria toxin[END_REF]. Toxins were first activated using 4 M urea and 40 mM dithiothreitol [START_REF] Brinkmann | Alteration of a protease-sensitive region of Pseudomonas exotoxin prolongs its survival in the circulation of mice[END_REF]. Each assay contained 6 µl of buffer (100 mM dithiothreitol, 0.5 mM EDTA, 100 mM Tris-HCl, pH 8), 3 µl of purified EF2, 3 µl of biotin-labeled NAD (150 µM), and 3 µl of toxin (0-5 pmoles).

Control tubes contained everything but enzyme. Incubation was performed at 25°C for 30 min and was stopped by adding reducing sample buffer and boiling. After SDS/PAGE, the gel was blotted onto nitrocellulose, blocked in 3% BSA and biotin was detected using extravidin-peroxidase and ECL + (GE Healthcare). Films were exposed within their linear range of detection, scanned and EF2 bands were quantified using ImageQuant. No other bands were present.

Assay for BPTI anti-trypsin activity

This FRET assay was performed as described before [START_REF] Twining | Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes[END_REF], with minor modifications. Briefly, 0-40 pmole of PE, BPTI-PE or BPTI were incubated with 20 pmoles of trypsin and 2 µg of casein-fluorescein in 0.2 ml of TBS (150 mM NaCl, 20 mM Tris, pH 8.0) in a white 96-well plate. Fluorescence was monitored every 30 min for 90 min using a polarstar omega multiplate reader equipped with 485/520 nm excitation/emission filters. Controls did not contain trypsin. Under these conditions, casein-fluorescein digestion by trypsin for 1 h resulted in a 3-fold increase in fluorescence.

Assay for BPTI trypsin-binding activity

Trypsin-agarose (5 µl of gel ~215 pmoles trypsin) was washed twice in TBS and incubated for 15 min at 25°C with 75, 150 or 260 pmoles of PE, BPTI-PE or BPTI in 20 µl of TBS.

After washing three times with TBS containing 0.1% Tween-20, gel-bound proteins were eluted by boiling in reducing sample buffer before SDS/PAGE. Gels were stained with Sypro ruby, imaged and bands were quantified using ImageQuant.

Immunoprecipitation

PE (5 nM) was added to 1.5 x 10 7 subconfluent L929 cells. After 4 h at 37°C, cells were washed with PBS and a chase of 30 min was performed to allow internalisation of plasma membrane-bound toxin. Cells were then lysed in 1 ml of RIPA buffer [START_REF] Taupiac | A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly[END_REF]. After 5 min on ice, insoluble material was removed by centrifugation at 4°C. The cleared lysate received 6 µg of goat anti-PE antibodies, was mixed for 1 h on a rotating wheel before adding 20 µl of protein G-sepharose. After 1 h on the wheel, immune complexes were recovered by centrifugation, washed, eluted by boiling in sample buffer and separated by SDS/PAGE. The gel was transferred to nitrocellulose for visualization using anti-PE antibodies then peroxidase-conjugated protein G and ECL + . Bands from five different experiments were quantified using ImageQuant.

Confocal microscopy

L929 cells grown on coverslips were labeled for 45 min at 37°C with 100 nM transferrinfluorescein and 15 nM of PE or BPTI-PE in DMEM supplemented with 0.2 mg/ml BSA.

Cells were washed, fixed in 3.7% paraformaldehyde that was then quenched using 50 mM ammonium chloride. After permeabilization in PBS containing 0.05% saponin and 1 mg/ml BSA, cells were labeled with goat anti-PE antibodies that were revealed using rhodaminelabeled donkey anti-goat IgG. Cells were finally mounted for examination under a Leica confocal microscope [START_REF] Morlon-Guyot | Processing of Pseudomonas exotoxin A is dispensable for cell intoxication[END_REF]. The ImageQuant software (GE Healthcare) was used to analyze cell images (30 < n< 40) in order to quantify signals from endocytosed material. Internalized transferrin-fluorescein signal was used to normalize PE data (see Results).

Toxicity measurements

Cells (5000 Jurkat, 6000 L929, 8000 K562, 10000 A431, 30000 MCF7 or HepG2) in RPMI /10% FCS were seeded in 96-well plates. Toxin solutions (and monensin or antibody as indicated) were added after 2 h at 37°C. One day after, 35 S-methionine/cysteine (trans-label, 0.25 µCi) was added for 24 h. Medium was then gently removed (after plate centrifugation for non-adherent cells) and cells were solubilized in 0.1N NaOH before protein precipitation with TCA. Proteins were collected onto fiberglass filters, and washed before radioactivity determination. Background incorporation was obtained from cells treated with 1 mM cycloheximide [START_REF] Beaumelle | Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol[END_REF].

Pharmacokinetics

Female Swiss OF1 mice (25-30 g, Iffa-Credo) were injected with PE or BPTI-PE (2 µg in 200 µl PBS/ 0.2% BSA) into the tail vein. Blood samples (30 µl) were collected 2 to 120 min after injection. Each mouse was bled no more than five times. Samples were allowed to clot on ice, then centrifuged to obtain the serum. Toxin concentrations were assayed by ELISA. To this end, 96-well plates were coated with goat anti-PE antibodies (1 µg/ml), then blocked with PBS/milk (8% w/v) for two hours. Sera and standards diluted in PBS/BSA (3%) were added before successive 1 h incubations with rabbit anti-PE antibodies (1:2000) and peroxidaseconjugated goat anti-rabbit antibodies in PBS/milk. After each incubation step, plates were washed with PBS/Tween (0.05%). Finally, a solution of 3,3',5'5-tetramethylbenzidine (Sigma) was added. After 15 min, the reaction was stopped with 0.5 M sulfuric acid, absorbance at 450 nm was read and the data were analyzed using the Pk-fit software package [START_REF] Farenc | Pk-fit: a pharmacokinetic/pharmacodynamic and statistical data analysis software[END_REF].

Results

A large fraction of internalized PE is inactivated by trypsin-like activity in L929 cells [START_REF] Brinkmann | Alteration of a protease-sensitive region of Pseudomonas exotoxin prolongs its survival in the circulation of mice[END_REF].

We reasoned that it should be possible to increase PE stability within the endocytic pathway by providing it with an antiproteasic moiety that would act as a bodyguard. A specific trypsin inhibitor was needed, since PE processing by furin is important for cytotoxicity [START_REF] Chiron | Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin[END_REF]. BPTI (aprotinin, a small 6.5 kDa polypeptide [START_REF] Ostermeier | The folding of bovine pancreatic trypsin inhibitor in the Escherichia coli periplasm[END_REF]) was a good candidate to fulfill this function since it inhibits trypsin but not furin [START_REF] Fryling | Characterization of a cellular protease that cleaves Pseudomonas Exotoxin[END_REF]. As control toxins, we used ricin and DT, which are not inactivated by proteolysis during initial uptake by most cell lines, and are only degraded when they reach lysosomes [START_REF] Van Deurs | Inhibition of the vacuolar H(+)-ATPase with bafilomycin reduces delivery of internalized molecules from mature multivesicular endosomes to lysosomes in HEp-2 cells[END_REF][START_REF] Umata | The cytotoxic action of Diphtheria Toxin and its degradation in intact cells are inhibited by Bafilomycin A1, a specific inhibitor of vacuolar-type H + -ATPase[END_REF].

Fig. 1 shows the chimeras used in this study. BPTI was fused to RTA, DTA, DTB, or PE via a linker peptide [START_REF] Beaumelle | Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol[END_REF]. BPTI has three disulfide bonds that stabilize the molecule [START_REF] Ostermeier | The folding of bovine pancreatic trypsin inhibitor in the Escherichia coli periplasm[END_REF].

Fusion proteins were therefore expressed and purified from E.coli periplasm to enable disulfide bridge formation.

Fused BPTI impairs DTA and RTA translocation

Fusing BPTI to DTB, which does not bear the toxic catalytic activity and is not transported to the cytosol [START_REF] Beaumelle | Selective translocation of the A chain of diphtheria toxin across the membrane of purified endosomes[END_REF], generated DT-BPTI that was as toxic as DT (Fig. 2A). Nevertheless, things were different when BPTI was fused to DTA-chain since the resulting BPTI-DT was virtually not toxic (Fig. 2A). This result is in agreement with data obtained using a dihydrofolate reductase (DHFR) -DT conjugate [START_REF] Klingenberg | Ability of methotrexate to inhibit translocation to the cytosol of dihydrofolate reductase fused to Diphtheria toxin[END_REF], which indicated that DHFR has to unfold during DHFR-DTA translocation to the cytosol. BPTI is a tightly folded protein that is difficult to transfer through membranes [START_REF] Vestweber | A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites[END_REF]. Hence, the fused BPTI likely hampered DTA translocation to the cytosol. Similar data were obtained using ricin. BPTI-RTA was associated with RTB to prepare BPTI-ricin. The latter was found to be as stable as control ricin within cells according to the results of both confocal microscopic examination and immunoprecipitation using anti-RTA antibodies (data not shown). Nevertheless, BPTI-ricin was 50-fold less toxic to L929 than control ricin (Fig. 2A). Just as for DT, these data confirm a previous study using DHFR, and showing that RTA passenger protein has to unfold for translocation to the cytosol [START_REF] Beaumelle | Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol[END_REF].

Altogether these data indicate that the fused BPTI did not interfere with cytotoxicity as long as the carrier toxin chain does not have to cross membranes. Conversely, fusing BPTI to A-chains inhibits toxicity, likely because BPTI unfolding for membrane translocation is difficult [START_REF] Vestweber | A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites[END_REF].

BPTI fusion enhances PE toxicity

The most striking result from this study was obtained when BPTI was fused to the PE Nterminus: toxicity to L929 mouse fibroblasts was enhanced by 40-fold, i.e. IC50 below 10 -13 M were reached (Fig. 2A). Since PE is one of the favorite toxins for building immunotoxins (2), we examined whether BPTI-PE was also superior to PE for killing human tumor cell lines.

We tested Jurkat (acute T-cell leukemia), K562 (chronic myelogenous leukemia), HepG2 (hepatocellular carcinoma), MCF7 (mammary gland adenocarcinoma) and A431 (epidermoid carcinoma) cell lines. In all cases, BPTI-PE was 20-42 fold more efficient than PE in killing these cells (Fig. 2B).

It was important to control whether fusion was actually required for toxicity enhancement. We therefore assessed the effect of a large excess (over a thousand-fold) of soluble BPTI on PE cytotoxicity. No improvement in toxicity can be achieved by adding BPTI to the cell culture medium (Fig. 2C). The fused BPTI was therefore directly responsible for the gain in PE toxicity. We then examined whether toxicity enhancement could be related to a toxic effect of the fused BPTI. To this end, we specifically neutralized the PE moiety of the BPTI-PE chimera using anti-PE antibodies or monensin. The latter protect cells from PE by impairing PE insertion into the endosomal membrane that is required before translocation [START_REF] Méré | Acidtriggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure[END_REF][START_REF] Alami | Involvement of ATP-dependent Pseudomonas exotoxin translocation from a late recycling compartment in lymphocyte intoxication procedure[END_REF]. PE and BPTI-PE toxicities were inhibited to the same extent by anti-PE antibodies or monensin (Fig. 2C). The PE moiety of the BPTI-PE chimera was therefore entirely responsible for cell-killing activity.

BPTI-PE displays both trypsin-inhibition and PE catalytic activity

To determine the basis of BPTI-PE cytotoxic activity we first checked whether PE catalytic activity was affected by the fusion. It can be seen in Fig. 3 that EF2-ADP-ribosylating activities of PE and BPTI-PE are virtually identical. Hence, BPTI fusion does not enhance PE toxicity by increasing its enzymatic activity. We also examined whether the BPTI portion of the BPTI-PE chimera was functional in trans. To this end we used a conventional FRET protease assay in which the substrate is casein-fluorescein whose fluorescence is quenched by fluorescence homotransfer, and increases upon proteolysis. A comparison of the anti-trypsin activities of BPTI and BPTI-PE in this assay showed that BPTI-PE preserved ~45 % of the BPTI capacity to protect casein against trypsin (Fig. 4A). Because BPTI-PE is 10-fold bigger than BPTI, we concluded that BPTI-PE is an effective trypsin inhibitor and that the BPTI portion of the chimera was functional. To confirm this point, we assessed whether BPTI-PE could bind trypsin. It is indeed well established that BPTI has a strong affinity for trypsin (Kd ~ 6 x 10 -14 M) and that the resulting BPTI-trypsin complex is extremely stable (half-life of ~17 weeks) [START_REF] Vincent | Trypsin-pancreatic trypsin inhibitor association. Dynamics of the interaction and role of disulfide bridges[END_REF]. Both BPTI and BPTI-PE, but not PE, efficiently bound to trypsin-agarose, confirming that BPTI is functional in BPTI-PE (Fig. 4B).

BPTI-PE is correctly processed by furin

Because PE processing by furin upon uptake is thought to be important for cytotoxicity [START_REF] Pastan | Recombinant toxins as novel therapeutic agents[END_REF], we examined whether BPTI-PE was processed by furin. PE cleavage by furin generates a characteristic C-terminal 37 kDa fragment, and a N-terminal 28 kDa fragment [START_REF] Ogata | Processing of Pseudomonas Exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol[END_REF]. The size of the latter increases to ~34 kDa upon BPTI fusion. We performed a furin concentration-and time-dependent study of PE and BPTI-PE processing by purified furin. Results showed that these proteins were processed by furin with a similar efficiency (Fig. 5). Collectively, these biochemical data indicated that the fusion of BPTI to PE affect neither the catalytic activity nor the furin-mediated processing of the toxin, while BPTI antiproteasic activity was moderately affected by the fusion.

Fused BPTI protects PE from endosomal trypsin-like activities

We then tested the extent of PE protection from trypsin due to BPTI fusion, i.e. protection in cis. We performed these assays both under neutral or acidic conditions, using pH 5.5 that is an average pH within early endosomes [START_REF] Gruenberg | Membrane transport in the endocytic pathway[END_REF]. Strikingly, even for high trypsin concentrations (up to 30 µg/ml) that entirely digest PE, BPTI-PE remains intact whatever the pH (Fig. 6). Hence, the fused BPTI fulfilled its role in vitro. We then examined whether this was also the case within cells.

Immunoprecipitation experiments (Fig. 7A and7B) showed that, for the same extracellularly applied toxin concentration, the amounts of intact toxin, furin-generated 37 kDa fragment and other proteolysis products generated by cells during uptake were greatly enhanced by the BPTI fusion. For cytotoxic fragments, i.e. entire toxin and the 37 kDa fragment, a two-to six-fold increase in the intracellular concentration (5-fold on average) was observed after attaching BPTI. These immunoprecipitation data also showed that fused BPTI prevents PE degradation by cell trypsin-like activities while preserving sensitivity to furin. Such PE processing is thought to take place within the endocytic pathway (8), and we performed immunofluorescence experiments to examine whether endosomal BPTI-PE was significantly protected from degradation as compared to endosomal PE. PE uptake kinetics was not affected by BPTI fusion (data not shown). As observed earlier for PE [START_REF] Morlon-Guyot | Processing of Pseudomonas exotoxin A is dispensable for cell intoxication[END_REF][START_REF] Ornatowski | Elevated furin levels in human cystic fibrosis cells result in hypersusceptibility to exotoxin A-induced cytotoxicity[END_REF], both PE and BPTI-PE were concentrated upon endocytosis within the pericentriolar recycling compartment of L929 cells, which is efficiently labeled with fluorescent transferrin. While the same amount of toxin was applied to cells, the resulting intracellular staining was much more intense when BPTI-PE was used (Fig. 8A).

Quantification of confocal images using fluorescent transferrin to normalize PE data showed that a 5-fold stronger signal was obtained when cells were labelled with BPTI-PE compared to when PE was used (Fig. 8B). This result confirms immunoprecipitation data and indicates that the BPTI fusion protects PE during endocytic uptake.

The BPTI fusion increases PE elimination half-time in mice

To finish, it was interesting to examine the effect of the BPTI fusion on PE elimination in vivo. The toxin levels in mice sera after intravenous injection of PE or BPTI-PE were assayed by ELISA. Toxicity assays that enabled quantification of biologically active molecules provided identical results (not shown). Toxin disappearance from the serum was biexponential (Fig. 9) indicating that the toxin distributes according to a two-compartment model [START_REF] Makoid | Basic Pharmacokinetics[END_REF]. The data were fitted using a pharmacokinetic data analysis software [START_REF] Farenc | Pk-fit: a pharmacokinetic/pharmacodynamic and statistical data analysis software[END_REF]. Results of this analysis are presented in Table 1. The initial distribution phase was rapid for both toxins (t ½ distribution 0.6-0.8 min), but the BPTI fusion increased ~3-fold the PE apparent distribution volume, thereby explaining the rapid initial drop in BPTI-PE concentration during the first 15 min. It can be seen from the second part of the curve (after 15 min) that BPTI-PE was more slowly eliminated than PE, with corresponding elimination half-times of 31 min and 18 min, respectively. These effects on the pharmacokinetics properties of PE directly resulted from BPTI fusion since a control conjugate between bovine RNAse A and PE behaved like PE (data not shown). Hence, BPTI fusion induces interesting modifications of PE pharmacokinetics parameters with a greater distribution volume and a longer elimination half-time.

Discussion

The main finding of this study was the strong increase in PE toxicity resulting from BPTI fusion to its N-terminus, i.e. to the PE receptor binding domain. This toxicity enhancement was due to the protection provided to PE by the fused BPTI toward trypsin-like activities present within the endocytic pathway. Nevertheless, when conjugated to an A-chain of a trypsin insensitive toxin such as DT or ricin, the stable folding of BPTI [START_REF] Vestweber | A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites[END_REF] severely hampered (RTA) or even blocked (DTA) toxin A-chain delivery to the cytosol which is required for toxicity. Different conclusions can therefore be drawn from the effect of BPTI fusion to these three toxins. First, RTA appears to be a more efficient vehicle than DTA for bringing folded proteins into the cytosol. Secondly, BPTI fusion seems to selectively increase the toxicity of trypsin vulnerable toxins such as PE. BPTI fusion to PE resulted in the stabilization of several intracellular fragments, such as the entire protein and the C-terminal 37 kDa fragment (Fig. 7). These results were therefore not informative regarding whether PE intoxication requires processing, and especially the production of the 37 kDa fragment [START_REF] Morlon-Guyot | Processing of Pseudomonas exotoxin A is dispensable for cell intoxication[END_REF].

The molecular mechanism underlying the superior antiproteasic properties of the BPTI fusion relative to unconjugated BPTI is unclear. Nevertheless, it seems reasonable to assume that the antitrypsin peptide should be kept as close as possible to the target protein to ensure optimum protection. Hence, the fused BPTI seems to mimic a high local concentration of BPTI and acts as a bodyguard for the toxin. BPTI is 10-fold smaller than PE, and the 59 basic residues (Arg+Lys) of PE, i.e. its potential trypsin processing sites are widespread between domains. It is thus unlikely that BPTI, which is fused to the large domain I of PE, could protect the entire toxin by steric hindrance. BPTI fusion induced interesting modifications in PE pharmacokinetic parameters in mice, enhancing the apparent distribution volume by 3-fold and the elimination half-life by 72%. The latter benefit probably results from protection provided by the fused BPTI with respect to circulating or cell-associated trypsin-like activities. Hence, BPTI tagging should allow more efficient PE delivery to tissues and a longer half-life in the bloodstream after injection. PE resistance to trypsin cleavage after Arg490 was already obtained through mutations around this trypsin processing site [START_REF] Brinkmann | Alteration of a protease-sensitive region of Pseudomonas exotoxin prolongs its survival in the circulation of mice[END_REF]. The weak toxicity of these mutants compared to BPTI-PE can look surprising. This difference in cytotoxicity is likely due to the existence of other trypsin-processing sites within PE in addition to the two reported loops, one containing Arg279 and the other Arg490 [START_REF] Chiron | Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin[END_REF]. Since fragments generated by cleavage within these loops are stable enough to be recovered by immunoprecipitation under appropriate conditions [START_REF] Chiron | Cleavage of Pseudomonas Exotoxin and Diphtheria toxin by a furin-like enzyme prepared from beef liver[END_REF], they are likely stable, dead-end products of proteolysis. Since PE owns more than 40 Arg, other trypsin sites likely exist and, according to the superiority of BPTI-PE over PE-Arg490 mutants, their cleavage likely triggers complete degradation of the molecule.

Several improved PE variants have been produced. It was first found that replacing PE C-terminal sequence with the ER retrieval motif KDEL generated a 2-to 3-fold more active toxin [START_REF] Seetharam | Increased cytotoxic activity of Pseudomonas and two chimeric toxins ending in KDEL[END_REF][START_REF] Smith | Internalized Pseudomonas exotoxin A can exploit multiple pathways to reach the endoplasmic reticulum[END_REF]. More recently, a 7-fold more toxic molecule was obtained when PE translocation domain was mutated to favor insertion into the endosome membrane [START_REF] Méré | Acidtriggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure[END_REF].

Nevertheless, with a 20-to 40-fold increase in toxicity BPTI-PE is by far the most efficient PE mutant ever obtained. Moreover, BPTI is weakly immunogenic, and already used in humans during surgery interventions on heart [START_REF] Carrel | Aprotinin in pediatric cardiac operations: a benefit in complex malformations and with high-dose regimen only[END_REF] or liver [START_REF] Himmelreich | Increased urokinase-type plasminogen activator (u-PA) levels in graft liver perfusate and decreased single chain u-PA activation with higher levels of aprotinin[END_REF]. Hence, BPTI-PE could have some therapeutic applications through the generation of more potent imunotoxins (1-3). More generally, the BPTI fusion approach could also be useful for stabilizing, and therefore maintaining the biological activity of circulating proteins of therapeutic interest such as cytokines or hormones.

TABLE 1

Pharmacokinetics parameters of PE and BPTI-PE in mice. Mice were injected in the tail vein with 66 µg/kg of PE or BPTI-PE. Three groups of three mice were used for each toxin (n = 9). Seric PE concentrations were monitored by ELISA over 120 min following injection. The log toxin concentration-time plot shows a biphasic response (Fig. 9), indicating that the toxin distributes according to a two-compartment model [START_REF] Makoid | Basic Pharmacokinetics[END_REF]. Data were analyzed using Pk-fit [START_REF] Farenc | Pk-fit: a pharmacokinetic/pharmacodynamic and statistical data analysis software[END_REF] and a two-compartment model to determine pharmacokinetics parameters of toxins. The significance of differences between data was assessed using an unpaired, two-sided Student's t-test (***, p < 0.001; **, p < 0.01). Results are mean + SD. [trypsin] (µg/ml) size. These data were used to calculate the pharmacokinetics parameters presented in Table 1.

Figure 1 .

 1 Figure 1

  Figure 2A

Figure 2 .

 2 Figure 2. Cytoxicity of BPTI fusion proteins. (A) Vero and L929 cells are highly sensitive to DT and PE, respectively, and were used to determine the IC50 of these toxins. DT used in this study is a ~500-fold attenuated mutant (E148S) (21). BPTI-DT was essentially not toxic. Data are mean of three independent experiments performed in quadruplicate ± S.E.M. The significance of differences between data was assessed using an unpaired, two-sided Student's t-test (***, p < 0.001). (B) BPTI-PE is more toxic than PE against human cancer cell lines. The relative toxicity, that represents the increase in toxicity due to BPTI fusion, is the IC50 (PE) / IC50 (BPTI-PE) ratio for the indicated cell line. IC50 (PE) were 80 nM (A431), 20 nM (MCF7), 20 pM (HepG2), 30 nM (K562) and 2 nM (Jurkats). (C) Analysis of BPTI-PE toxicity. The IC50 of PE and BPTI-PE were measured on L929 cells in the absence or presence of 10 µM BPTI, 10 nM anti-PE or 100 nM monensin, as indicated. Percentages of toxicity are IC50 (control)/IC50 (treated) (%).

Figure 3 .Figure 4 .

 34 Figure 3

Figure 5 .Figure 6 .

 56 Figure 5

Figure 7 .

 7 Figure 7. Intracellular stability of PE and BPTI-PE. L929 cells were incubated for 4 h at 37°C with 5 nM PE or BPTI-PE before washing and chasing for 30 min. Cells were lysed for PE immunoprecipitation before SDS-PAGE, transfer and anti-PE Western blot. (A) Immunoblot; IP, toxin immunoprecipitated from treated cells and Pure, purified toxins. (B) Quantificationof the main immunoprecipitated toxin fragments. Films from three independent experiments were scanned and bands corresponding to the entire toxin, active 37 kDa fragment and other fragments were quantified for both PE and BPTI-PE. The intensity ratio (BPTI-PE/PE) was calculated for each band to normalize data that are mean + SEM. The 28 kDa fragment of PE is poorly immunoprecipitated and is not visible on these films.

Figure 8 .Figure 9 .

 89 Figure 8A

  Méré et al. Supplemental Fig.1. Protein sequence of BPTI-PE.

	MKKTAIAIAVALAGFATVAQAAPKDNPQGRPDFCLEPPYTGPCKARI
	IRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGAASASTPE
	PDPEKLAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG
	VLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYS
	YTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTI
	EMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPR
	REKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLA
	GNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHR
	QPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGG
	DLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSL
	TCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNW
	TVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDA
	IWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGF
	YRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGW
	PLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPG
	KPPREDLK
	Signal
	From mature OmpA
	BPTI
	Spacer
	Mature PE

Acknowledgements

We thank Chritine Farenc for the pharmacokinetic analysis, Patrice Boquet (Nice) and Daniel Gillet (Paris) for providing us with the DT-E148S construct, as well as Prof. G. Georgiou and Dr. Zhan (Austin) for the BPTI clone and anti-BPTI antibodies.

* This study was supported by the Association de la Recherche sur le Cancer.

Supplemental Information Paragraph

Supplemental materials can be accessed free of charge online at http://pubs.acs.org