Patricia Garcia-Muñoz
email: garciamunoz@unistra.fr

Werner Dachtler

Bernd Altmayer

Ralf Schulz

Didier Robert

Frank Seitz

Ricki Rosenfeldt

Nicolas Keller
email: nkeller@unistra.fr

Reaction pathways, kinetics and toxicity assessment during the photocatalytic degradation of glyphosate and myclobutanil pesticides: Influence of the aqueous matrix

Keywords: photocatalysis, glyphosate, myclobutanil, oxidation pathway, wastewater matrix effect

Assessment of reaction pathways, kinetics and water toxicity during the photocatalytic degradation of glyphosate and myclobutanil pesticides has been performed in different aqueous matrices of increasing complexity, from the single pesticides to the mix of their commercial formulations. Using Aeroxide®-TiO2-P25 as reference UV-A (Ultraviolet A) light photocatalyst, the ability of photocatalysis to degrade glyphosate and myclobutanil pesticides in water was evidenced independently of the aqueous matrix complexity, complete mineralization into CO2, phosphate and chloride ions being achieved. Further, an unusual volcano-like TOC evolution profile resulting from the proposed glyphosate degradation pathway was observed whatever the aqueous matrix. Increasing the water matrix complexity from single pesticides to the commercial formulation mix reduced the degradation kinetics and consequently extended the time necessary for complete mineralization but, did not influence the overall pesticide fate profiles. This behavior was associated to the competitive adsorption of the organic matter onto the catalyst and to the presence of ions and inorganic matter.

The co-presence of glyphosate and to lesser extent of Roundup® formulation additives strongly impacted the myclobutanil fate profile, due to preferential adsorption/degradation of glyphosate. By contrast, despite their impact on the degradation pathway, the inorganic additives of the Systhane® formulation influenced to a lesser extent both myclobutanil removal duration and TOC removal compared to glyphosate/Roundup® products. The treatment allowed for most of the cases a strong reduction of acute toxicity to aquatic invertebrate test organisms (D.magna) whatever the water matrix complexity, while the ecotoxicity was reduced by half for the complex formulation mix.

GRAPHICAL ABSTRACT

Synthetic pesticides have been intensively used worldwide since half a century for protecting crops. In the center of Europe, particularly in the surroundings of the Upper Rhine river, viticulture remained one of the crops most subjected to pesticide treatments for protecting from local pests and consequently maintaining exploitation yields. Within this area, the main fungicide treatments applied generate 70% of the total grapevine growing costs [START_REF] M'bra | Heterogeneous photodegradation of Pyrimethanil and its commercial formulation with TiO2 immobilized on SiC foams[END_REF]. For instance, French vineyards represent 30% of the overall pesticide use, while they account for only 3% of all farmland.

Unfortunately, while their use allowed a strong increase in the crop yields and the development of intensive farming, their appearance in freshwater bodies and soils negatively affects the environment and the human health. One important sources of these compounds for the natural water bodies is the washing water arising from the cleaning of agricultural spraying machineries following application.

While wastewater effluents should therefore be treated prior to discharge, conventional treatments based on mechanical, physical, chemical or biological processes are facing with the increase in the volume and the load of the pollutants in these effluents, the reduction of the biodegradability of the pollutants, as well as with the heterogeneity of the water matrix [START_REF] Castro-Català | Invertebrate community responses to emerging water pollutants in Iberian river basins[END_REF][START_REF] Oller | Combination of Advanced Oxidation Processes and biological treatments for wastewater decontamination-A review[END_REF][START_REF] Münze | Pesticides from wastewater treatment plant effluents affect invertebrate communities[END_REF]. Among the Advanced Oxidation Processes (AOPs) that are emerging as feasible alternatives or complements to conventional wastewater treatment plants for treating waters contaminated by refractory compounds [START_REF] Malato | Decontamination and disinfection of water by solar photocatalysis: Recent overview and trends[END_REF], photocatalysis has gained important attention due to a high efficiency and relatively low costs, when working with low charges of pollutant, as well as to its ability to degrade the biorecalcitrant and emerging contaminants [START_REF] Esplugas | Comparison of different advanced oxidation processes for phenol degradation[END_REF][START_REF] Malato | CaÌ ceres, Treatment of 2,4-Dichlorophenol by Solar Photocatalysis: Comparison of Coupled Photocatalytic-Active Carbon vs. Active Carbon[END_REF]. It allows achieving a complete mineralization of refractory substrates thanks to its ability to oxidize short-chain acids and enables operating efficiently in an extended working pH range, while taking advantage of a versatile and environmentally-friendly implementation [START_REF] Malato | Decontamination and disinfection of water by solar photocatalysis: Recent overview and trends[END_REF].

Although intense efforts have been made to enhance the degradation rate of pesticides and more generally the mineralization yields of photocatalytic processes, TiO2 still remains the photocatalyst par excellence provided that a UV-A (Ultraviolet A) light irradiation is targeted, so that it has been employed in the photodegradation of a large variety of pesticides in the last decades [START_REF] M'bra | Heterogeneous photodegradation of Pyrimethanil and its commercial formulation with TiO2 immobilized on SiC foams[END_REF][START_REF] Kushniarou | Solar photocatalytic reclamation of agro-waste water polluted with twelve pesticides for agricultural reuse[END_REF][START_REF] Berberidou | Study of the decomposition and detoxification of the herbicide bentazon by heterogeneous photocatalysis: Kinetics, intermediates and transformation pathways[END_REF][START_REF] Carbajo | Effect of water composition on the photocatalytic removal of pesticides with different TiO2 catalysts[END_REF][START_REF] Vicente | Comparison of different TiO2 samples as photocatalyst for the degradation of a mixture of four commercial pesticides[END_REF]. The process time necessary for removing and mineralizing pesticides from water and consequently for reducing the associated toxicity is known to strongly differ depending on the active substance, since both composition and chemical structure of the target pesticide impact on the degradation pathways and the reaction intermediates [START_REF] Berberidou | Study of the decomposition and detoxification of the herbicide bentazon by heterogeneous photocatalysis: Kinetics, intermediates and transformation pathways[END_REF][START_REF] Tasca | Terbuthylazine and desethylterbuthylazine: Recent occurrence, mobility and removal techniques[END_REF]. Furthermore, pesticides often occur as mixtures in surface waters, and they are always applied as commercial formulations containing various kinds of organic and/or inorganic additives in addition to the active substance. Therefore, the ability of photocatalysis for treating water containing pesticide mixtures [START_REF] Carbajo | Effect of water composition on the photocatalytic removal of pesticides with different TiO2 catalysts[END_REF][START_REF] Vicente | Comparison of different TiO2 samples as photocatalyst for the degradation of a mixture of four commercial pesticides[END_REF] and commercial pesticide formulations [START_REF] M'bra | Heterogeneous photodegradation of Pyrimethanil and its commercial formulation with TiO2 immobilized on SiC foams[END_REF][START_REF] García-Ripoll | Treatment of aqueous solutions containing four commercial pesticides by means of TiO2 solar photocatalysis[END_REF], as well as the influence of the water matrix complexity, eg. the amount and composition of extra-load [START_REF] Seitz | Quantity and quality of natural organic matter influence the ecotoxicity of titanium dioxide nanoparticles[END_REF], on the pesticide degradation process and more generally on the efficiency of the water treatment, are highly worth being investigated.

The herbicide glyphosate and the fungicide myclobutanil are two fundamentally different compounds among the pesticides that are most frequently detected in the Upper Rhine river area [START_REF] Peschka | Trends in pesticide transport into the River Rhine[END_REF], glyphosate being a P-and N-containing aliphatic substrate, while myclobutanil is a N-and Clcontaining substrate with both C-C and N-C aromatic rings and aliphatic branch. Used as the commercial formulations Roundup® and Systhane®, respectively, they appeared at relatively high concentrations in wastewater because of their prolonged use. Glyphosate (C3H8NO5P) is a broad spectrum organophosphate systemic herbicide that has shown resistance to natural degradation pathways due to its inert C-P bond. Recent studies have demonstrated that glyphosate has negative effects on ecosystems and humans. It provokes severe effects to many non-targeted organisms, with for instance effects on reproduction in soil invertebrates, changes in the zooplankton behavior, reduction of fertility in some fishes [START_REF] Niemeyer | Do recommended doses of glyphosate-based herbicides affect soil invertebrates? Field and laboratory screening tests to risk assessment[END_REF][START_REF] Andrade | Shifts in Zooplankton Behavior Caused by a Mixture of Pesticides[END_REF]. Furthermore, anxiety and depression have been observed for mice following chronic exposure. Detected in surface and groundwater, myclobutanil (C15H17ClN4) is a triazole pesticide used to avoid fungi infection, the main environmental risk of which being related to the potential formation of bioactive (toxic) metabolites [START_REF] Chen | Occurrence, fate and ecological risk of five typical azole fungicides as therapeutic and personal care products in the environment: A review[END_REF]. Moreover, fungicides are generally known to be capable of causing endocrine activities [START_REF] Westlund | Investigation of the presence and endocrine activities of pesticides found in wastewater effluent using yeast-based bioassays[END_REF] and chronical effects [START_REF] Vu | Toxicological effects of fungicide mixtures on the amphipod Austrochiltonia subtenuis[END_REF] even at low concentrations of 3 µg/L. It has to be mentioned that no direct and straightforward relationship is established between the Total Organic Carbon (TOC) depletion and the toxicity reduction of an effluent [START_REF] Kã ¶hler | Organic Pollutant Removal versus Toxicity Reduction in Industrial Wastewater Treatment:  The Example of Wastewater from Fluorescent Whitening Agent Production[END_REF][START_REF] Liang | Elimination and ecotoxicity evaluation of phthalic acid esters from textile-dyeing wastewater[END_REF][START_REF] Supowit | Mass Balance of Fipronil and Total Toxicity of Fipronil-Related Compounds in Process Streams during Conventional Wastewater and Wetland Treatment[END_REF], mainly due to formation of potentially-harmful reaction intermediates, to the presence in the commercial formulations of non-inert inorganic additives, as well as to synergistic interaction effects between the aqueous substances along the treatment. For instance, Kohler et al. obtained 80% of TOC reduction with simultaneously an important increase in the toxicity [START_REF] Kã ¶hler | Organic Pollutant Removal versus Toxicity Reduction in Industrial Wastewater Treatment:  The Example of Wastewater from Fluorescent Whitening Agent Production[END_REF], while Supowit et al. treated fipronil-contaminated water and indicated that the generation of harmful intermediates provoked a rise in toxicity values [START_REF] Supowit | Mass Balance of Fipronil and Total Toxicity of Fipronil-Related Compounds in Process Streams during Conventional Wastewater and Wetland Treatment[END_REF]. Therefore, the toxicity assessment of the effluent has to be performed along the process simultaneously to the determination of the reaction kinetics and of the TOC removal efficiency.

The ability of photocatalysis to degrade both glyphosate [START_REF] Assalin | Studies on degradation of glyphosate by several oxidative chemical processes: Ozonation, photolysis and heterogeneous photocatalysis[END_REF][START_REF] Chen | Study on the photocatalytic degradation of glyphosate by TiO2 photocatalyst[END_REF][START_REF] Echavia | Photocatalytic degradation of organophosphate and phosphonoglycine pesticides using TiO2 immobilized on silica gel[END_REF][START_REF] Janin | Solar photocatalytic mineralization of 2,4dichlorophenol and mixtures of pesticides: Kinetic model of mineralization[END_REF] and myclobutanil [START_REF] Janin | Solar photocatalytic mineralization of 2,4dichlorophenol and mixtures of pesticides: Kinetic model of mineralization[END_REF][START_REF] Fenoll | Solar detoxification of water polluted with fungicide residues using ZnO-coated magnetic particles[END_REF] pesticides has already been demonstrated. Therefore, the novelty of this work is to study their photocatalytic degradation in different aqueous matrices of increasing complexity, from the single pesticides to the mix of their Roundup® and Systhane® commercially available formulations, and to assess the reaction pathways that take place in different matrices, the kinetics and the water ecotoxicity during the treatment. We highlighted that, in addition to its influence on the reaction kinetics, the aqueous matrix complexity strongly impacted on the reaction pathways followed for reaching the mineralization of the substrates. To this end, the study has been performed using the well-known commercial Aeroxide © TiO2-P25, considered de facto as the reference photocatalyst under UV-A light, as a first step necessary for shedding light on the influence of the aqueous matrix complexity on the water treatment efficiency during the degradation of glyphosate and myclobutanil pesticides.

2-MATERIALS AND METHODS

Materials and formulations

Analytical grade reagents were used without any further purification. Glyphosate (N-(phosphonomethyl)glycine, >99%) and Myclobutanil ((RS)-2-p-chlorophényl-2-(1H-1,2,4-triazole-1ylméthyl)hexanenitrile, >99%) were purchased from HPC High Purity Compounds (Cunnersdorf, Germany).

Roundup®-720 and Systhane®-200 products were commercially available formulations from Monsanto and Dow agrosciences, containing 720 g/L glyphosate and 200 g/L myclobutanil pesticides as active compounds, respectively. Table 1 and Table 2 show the composition expressed in mass percentage of the Roundup® and Systhane® products and the main physico-chemical parameters of the ready-to-use diluted formulations. Roundup® and Systhane® formulations displayed very distinct compositions. Beside the presence of their respective active compound, in both formulations with a ca. 40 wt.% fraction, Roundup® has been reported to contain additional carbon-containing molecules such as isopropylamine and surfactants, whereas by contrast Systhane® was characterized by the presence of several oxides and salts. Therefore, as a result of the diversity of the carbon sources within the Roundup® formulation, the glyphosate active compound amounted only for a fraction of the total TOC recorded for the commercial formulation, while in the case of the Systhane® formulation, the TOC only resulted from the myclobutanil itself. Both Roundup® and Systhane® solutions were prepared with a concentration of the active compound similar to that of the synthetic pesticide mixed solution, i.e. at 25 mg/L and 35 mg/L, respectively. We have to stress that the highly viscous nature of the commercial Systhane® paste somehow jeopardizes the initial myclobutanil concentration in the case of the degradation tests with the commercial products. This has been specified where appropriate.

Commercial Aeroxide © TiO2-P25 was obtained from Evonik. The isoelectrical point of the TiO2-P25 powder has been measured at 6.3 by zeta potential measurement.

Typical reaction procedure

Experiments were carried out in a batch beaker-type glass reactor at atmospheric pressure, room temperature (20 °C) and using O2 from the atmosphere as oxidant, air being pumped into the reactor at 150 mL/min for preventing from any oxygen deficiency into the reaction media. The reactor was placed in a thermostated chamber for maintaining a constant temperature during the tests. The photocatalytic tests have been performed under stirring at 600 rpm with a reaction volume of 250 mL at a 1 g/L Aeroxide© TiO2-P25 catalyst load. Independently of the aqueous matrix complexity, stirring the suspension for 0.5 h in the dark enabled the establishment of the dark adsorption/desorption equilibrium before turning on the irradiation (Figure S1). The suspension was further exposed to a 60 W/m 2 irradiance UV-A light centered at 365 nm, provided by Philips 24W/10/4P lamps. At each time interval, the solution has been sampled and further filtered through 0.20 μm porosity filter for removing the photocatalyst powder, before being analyzed. All glyphosate and myclobutanil degradation experiments have been triplicated, with standard deviation remaining within the ±3% relative range, so that no standard error bars have been reported on the graphs for sake of clarity.

Regardless of the water matrix, neither glyphosate or myclobutanil degradation, nor TOC reduction has been observed in the absence of the photocatalyst. This indicated that the photolysis of the pesticides or of the carboned additives in the commercial formulations can be neglected under UV-A in the experimental conditions.

Analysis techniques

The analysis of glyphosate and of both aminomethylphosphonic acid (AMPA) and primary amine (sarcosine) intermediates was performed by mass spectrometry with an Agilent 1260 Infinity HPLC Quadrupole Time of Flight Mass Spectrometer 6530A. Myclobutanil was analyzed by UV-Visible spectrophotometry (Cary-100 Scan Varian Inc.) by following the disappearance of the main absorption peak at λ = 220 nm as reaction samples were withdrawn from the reaction medium, while the analysis of the main high carbon number reaction intermediates was performed by mass spectrometry with an Agilent 1260 Infinity HPLC Quadrupole Time of Flight Mass Spectrometer 6530A. A typical chromatogram obtained during the photocatalytic treatment of a solution containing glyphosate and myclobutanil pesticides can be seen in Figure S2. Anions and cations as well as the short-chain acids were analyzed by ion chromatography (Metrohm 790IC) using a conductivity detector. A Metrosep A supp 5-250 column (25 cm length, 4 mm diameter) was employed as stationary phase, while an aqueous solution containing 3.2 mM Na2CO3 and 1 mM NaHCO3 was employed as mobile phase at a flow rate of 1 mL min -1 . Total Organic Carbon (TOC) measurements were performed using a TOC analyzer (Shimadzu, model TOC-L) for determining the organic carbon load.

Ecotoxicological tests

The ecotoxicity of the aqueous pesticide solutions (untreated and after photocatalytic treatments) was addressed after filtration by performing 48 h acute toxicity tests with Daphnia magna according to the OECD guideline 202 (2004) [START_REF]Daphnia sp. Acute Immobilisation Test[END_REF]. In the following, the aqueous pesticide solutions will be named test items. Prior each test, the test item was serially diluted in the test medium (daphnia culturing water (ASTM-medium [START_REF]Daphnia sp. Acute Immobilisation Test[END_REF]). The highest possible concentration was 50% test item and 50% test medium, since the daphnids do not survive in deionized water.

Briefly, juveniles (age < 24 h) were exposed in groups of five organisms (n=4) to the dilution series of the test item in the test medium. At the end of each test, immobile organisms were counted and if applicable the EC50 value (ie. the test item concentration (in %) at which 50% of the daphnids were immobilized) was calculated for each test item by applying a dose-response modeling. Thereby the EC50 values were expressed as percent of test item (% v/v) within the total test solution (= %TI).

So, the lower the %TI, the higher is the toxicity. This enables a direct comparison of the toxicity before and after photocatalytic treatment without having the information about i) the exact concentration of the active ingredient and ii) further implications such as eg. the impact of additional formulation ingredients.

However, the toxicity outcomes (= 48 h-EC50 values (%)) were statistically compared using confidence interval testing to unravel a potential change of toxicity during the photocatalytic treatment of the pesticide-containing solutions.

3-RESULTS AND DISCUSSION

Degradation of single pesticides

Degradation of glyphosate

Figure 1 shows the evolution with time under irradiation of both the glyphosate removal and the phosphate release in %, of the TOC and of the pH value. The evolution of the concentration of the short-chain acids, of both nitrate and ammonium ions, and of the aminomethylphosphonic acid (AMPA) intermediate has been also reported. A high adsorption of glyphosate onto the TiO2 surface has been observed during the adsorption/desorption equilibrium period in the dark (≈50%), that was accompanied with a notable rise in pH from 4.2 to 6. This resulted from the favoured interaction at the initial acidic pH of the negatively-charged glyphosate (pKa=0.8) with the positively-charged titania surface. After the adsorption/desorption equilibrium has been reached, a complete glyphosate depletion has been observed within the first 30 min of reaction. By contrast, an unusual volcano-like profile was observed for the TOC evolution during the course of the photocatalytic process, that exhibited a maximum at t60 before the complete mineralization of glyphosate was achieved at t240. It was worth noting that this initial increase of TOC was occurring together with the glyphosate disappearance, the simultaneous release of phosphates that closed the phosphorous balance and the detection of reaction intermediates such as AMPA and acetic acid. This suggested that the initially produced radicals were employed preferentially for cleaving bonds such as P-C or N-C rather than C-C bonds, since no reduction of the overall carbon content was observed at the beginning of the process. So, the increase in TOC observed for 60 min was proposed to be due to the desorption of the first reaction intermediates resulting from the P-C and N-C bond cleavage. Following the cleavage of these bonds to yield lower molecular weight compounds, typical oxidation steps took place, provoking the appearance of other short-chain acids (formic and oxalic acids) as a consequence of a classical oxidation route, and leading to the total mineralization of the initial compound after 240 min (Figure 1) [START_REF] Dang | Photodegradation mechanisms of phenol in the photocatalytic process[END_REF][START_REF] Rani | Photocatalytic degradation of toxic phenols from water using bimetallic metal oxide nanostructures[END_REF][START_REF] García-Muñoz | Treatment of hospital wastewater through the CWPO-Photoassisted process catalyzed by ilmenite[END_REF]. This was in agreement with the pH evolution profile during the test, with an initial drop at the beginning of the test, and a subsequent increase back to 6, as a result from the oxidation of the short-chain organic acid intermediates and potentially from the formation of ammonia.

The glyphosate degradation has been suggested to occur through two different pathways (Figure 2), both involving the breakdown of the glyphosate molecule into smaller compounds with no reduction of the overall carbon content. The first pathway may consist in the hydroxylation of the electrophilic N-C bond, that led to the formation of AMPA (aminomethylphosphonic acid, one of the most frequently detected degradation intermediates of glyphosate in natural water bodies) and to acetic acid (Figure 2A-right), in agreement with Assalin et al. [START_REF] Assalin | Studies on degradation of glyphosate by several oxidative chemical processes: Ozonation, photolysis and heterogeneous photocatalysis[END_REF]. Please note that the AMPA pathway may bypass acetic acid and form glyoxalic acid directly, as proposed by Jaisi et al. [START_REF] Jaisi | Mechanisms of Bond Cleavage during Manganese Oxide and UV Degradation of Glyphosate: Results from Phosphate Oxygen Isotopes and Molecular Simulations[END_REF]. In the second pathway, the attack of the P-C bond by the oxidizing radicals (HO• or O2•-) would result in the cleavage of the molecule with the direct release of phosphate to water and the formation of the sarcosine (2-(Methylamino)acetic acid) intermediate (Figure 2A-left). The formation of sarcosine was reported notably by Jaisi et al. [START_REF] Jaisi | Mechanisms of Bond Cleavage during Manganese Oxide and UV Degradation of Glyphosate: Results from Phosphate Oxygen Isotopes and Molecular Simulations[END_REF] and Echavia et al. [START_REF] Echavia | Photocatalytic degradation of organophosphate and phosphonoglycine pesticides using TiO2 immobilized on silica gel[END_REF]. The carbon-containing compounds suffered from further degradation yielding the corresponding aliphatic structures (Figure 2B), subsequently completely mineralized into their corresponding ions and CO2 (Figure 2C). The proposed degradation route was in agreement with previous studies [START_REF] Echavia | Photocatalytic degradation of organophosphate and phosphonoglycine pesticides using TiO2 immobilized on silica gel[END_REF].

Initially, the glyphosate solution showed no toxicity for daphnids (EC50 > 50 %TI; Table 3), since the initial glyphosate concentration of 25 mg/L was below the 48 h-EC50 for aquatic invertebrates (40 mg glyphosate/L) according to the toxicity information from the Pesticide Properties Data Base. However, a moderate increase in toxicity was observed for the sample treated for 120 min with an EC50 value of 41 %TI, before the solution exhibited again subsequently no toxicity after 240 min of treatment (no 48h-EC50 determinable), in good agreement with the complete oxidation of the different intermediate compounds (Figure 1). This toxicity profile could be ascribed to the formation of potentially toxic reaction intermediates.

Degradation of myclobutanil

Figure 3A shows the evolution with time under irradiation of the myclobutanil removal rate in %, of the TOC and of the pH value, while that of the concentration of short-chain acids as well as of both nitrate and ammonium ions is shown in Figure 3B, together with that of the chloride release in %. First, the establishment of the adsorption/desorption equilibrium in the dark evidenced a removal of 20% of myclobutanil (pKa=2.3). Myclobutanil was subsequently removed within 150 min of reaction while the TOC evolution displayed a usual decreasing profile since the beginning of the reaction for reaching a complete mineralization within 240 min of reaction. The complete mineralization was confirmed by the presence of short chain acids during the reaction, intermediates generated at the latest oxidation stages, as well as by the complete closure of the chloride balance by chloride ions at 240 min, that evidenced the absence of any residual organochloride species at the end of the reaction. No formation of chlorinated short-chain organic acids was observed throughout the test.

While other authors have reported that the chloride elimination was the first step in the oxidation of aromatic substrates [START_REF] Barreto-Rodrigues | Dechlorination and oxidative degradation of 4-chlorophenol with nanostructured iron-silver alginate beads[END_REF][START_REF] Komtchou | Degradation of atrazine in aqueous solution with electrophotocatalytic process using TiO2-x photoanode[END_REF], in the present study the chloride release was only observed after 60 min of reaction. A similar delayed chloride release was observed by Lai et al. [START_REF] Lai | TiO2 photocatalytic degradation and transformation of oxazaphosphorine drugs in an aqueous environment[END_REF] during the photodegradation of oxazaphosphorine with titania. Figure 4 shows the reaction intermediates and the final products detected during the photocatalytic degradation of myclobutanil, and Figure S3-A shows the intensity profile of the main aromatic intermediates detected by HPLC/MS, that displayed the typical profile for sequential reactions with intermediate compounds. First, the formation of C15-Cl and C10-Cl organochloride intermediates during the reaction corroborated the late release of chlorides. By considering that the attack modes of HO° on aromatic heterocyclic compounds can be summarized as hydroxylation, group substitution and H-abstraction, we suggested that myclobutanil was firstly hydroxylated -as in a typical mechanism -, providing an intermediate with a higher molecular weight than the parent compound (MW=304). This intermediate has been proposed to follow two degradation routes. In the route A, it would undergo a second hydroxylation producing a MW=221 Cl-containing C10 intermediate with the elimination of an aliphatic compound, while the route B would form a MW=192 Cl-free C9 compound with the release of a Cl-phenol molecule. In both cases, sequential hydroxylation steps would provoke the opening of the aromatic rings, with consequently the formation of short-chain organic acids, further mineralized to CO2. The detection in the reaction media of chloride, nitrate and ammonium ions coming from the pesticide heteroatoms corroborated this ring-opening and so the complete mineralization. Independently from the degradation routes proposed, this evidenced that the photocatalytic degradation of myclobutanil did not differ phenomenologically from the usual photo-oxidation of aromatic ring-pollutants via different AOPs [START_REF] Munoz | Application of CWPO to the treatment of pharmaceutical emerging pollutants in different water matrices with a ferromagnetic catalyst[END_REF].

The ecotoxicological investigations with myclobutanil showed a total detoxification of the solution single substance for daphnids already after 60 min of photocatalytic treatment. This is evidenced by a significant increase of the 48 h-EC50 value from 6 %TI to not measurable effects above 56 %TI. In the literature, 48 h-EC50 values for myclobutanil were found to be dependent on its individual enantiomers. However, for the rac-compound the value was calculated to be 9.24 mg/L, which is approximatively 7.5 times higher than the value in the present study (which is 1.2 mg myclobutanil/L). This, however, is not in a sharp contrast when also considering the 95% CI ranging from 0 to 7.2 mg myclobutanil/L of the EC50 values. In the study of Sun et al., there is unfortunately no error provided for the EC50 of myclobutanil [START_REF] Sun | Acute Toxicity, Bioactivity, and Enantioselective Behavior with Tissue Distribution in Rabbits of Myclobutanil Enantiomers[END_REF]. However, the comparably wide confidence interval (CI) in the present study let suggest that a statistically significant difference between both EC50 values may not be given.

Degradation of glyphosate and myclobutanil pesticide mixture

The influence of the co-presence of both glyphosate and myclobutanil in water on the treatment performances was first screened using pesticide concentrations identical to those used for the single pesticide degradation tests (ie. at a glyphosate and myclobutanil initial concentration of 25 mg/L and 20 mg/L, respectively, and the results are shown in Figure S3). The unusual TOC and myclobutanil concentration profiles obtained -with a slight increase in TOC and a delayed myclobutanil removal after an initial concentration plateau -, led us to further perform the study with a twice higher myclobutanil initial concentration while maintaining unchanged that of glyphosate, in order to highlight and to make this unusual behavior more visible, i.e with a glyphosate and myclobutanil initial concentration of 25 mg/L and 40 mg/L, respectively.

Figure 5 shows the evolution with time under irradiation of both the glyphosate and the myclobutanil substrate removal rates in %, of the TOC, of the AMPA intermediate concentration and of the chloride release in %. The evolution profile of the short chain acids, the N-compounds and of the myclobutanil aromatic intermediates is shown in Figure S6-A and Figure S4-B, respectively. First, it could be seen that glyphosate preferentially adsorbed on the TiO2 surface during the dark adsorption period, while no significant adsorption of myclobutanil was observed (Figure S1c). The glyphosate adsorption was higher than in the case of the pure glyphosate solution (80% vs. 50%), as a result of the less acidic pH of the solution (5.6 vs. 4.2), that increased the glyphosate dissociation and consequently the concentration of the anionic form of glyphosate interacting with the positivelycharge surface of TiO2. Glyphosate was first removed within 90 min of reaction, whereas the oxidation of myclobutanil only took place once the complete glyphosate removal was reached. It seems that the glyphosate adsorption onto the catalyst surface limits the oxidation of the co-present pesticide, due to the surface saturation by the glyphosate compound after the adsorption equilibrium, as confirmed by the pre-adsorption experiment in the dark. Furthermore, complete mineralization of myclobutanil and complete removal TOC were achieved within 420 min and 900 min of reaction.

The detection of AMPA as reaction intermediate and the release of phosphate with a full closure of the phosphorous mass balance, suggested that glyphosate is being degraded via a similar mechanism as for the single pesticide. However, the kinetic rate constant of glyphosate depletion was reduced from 0.096 to 0.027 min -1 (Table 4), with a delayed on both the AMPA and the phosphate evolution. By contrast, once the oxidation of myclobutanil has been initiated, no significant difference was observed in terms of kinetic rate constant for the myclobutanil degradation compared to that obtained with the pure substrate (k' = 0.027 min -1 vs. 0.021 min -1 for myclobutanil alone), indicating that its depletion was not affected in terms of kinetics by the presence of the phosphates and of the glyphosate degradation intermediates. The chloride balance was closed by the release of chloride ions, indicating the absence of any residual organochloride species at the end of the reaction. Also, it is worth noting that the TOC evolution displayed a volcano-profile in TOC values at the beginning of the test, as in the case of the treatment of the single herbicide. The phosphorous mass balance was closed in two steps: first, with an initial release of the 70% of stoichiometric phosphates accompanied by the degradation of AMPA, followed by the subsequent release of the remaining 30% of phosphates for longer reaction times (≈ 600 min). Also, a second maximum was observed at ≈ 650 min in the TOC profile after the closure of the phosphorous balance, so that this might also be attributed to the desorption of intermediates resulting from a N-C bond cleavage (the experiment has been triplicated for validating the presence of this maximum, and the obtained standard errors are reported on the TOC profile). Indeed, the residual presence of N-C bond-containing sarcosine after 480 min and 540 min of reaction was evidenced by LC MS/MS. The presence of intermediates from the myclobutanil degradation might hinder the adsorption of this N-C bond-containing intermediate issued from the glyphosate degradation, so that this volcano-profile was observed for longer treatment times, and was not observed when glyphosate was treated as single pollutant. As far as myclobutanil intermediates were concerned, the same metabolites were identified, indicating that the copresence of pesticides did not interfere in the myclobutanil oxidation routes (Figure S4-B).

For the mixture of myclobutanil and glyphosate (25:40 weight ratio), an interim but nonstatistically significant increase of toxicity for daphnia was observed after 180 min. In detail, the initial 48 h-EC50 value of 24 %TI decreased by approximately 20% down to 20 %TI (Table 3). This toxicity increase may be related to the presence of reaction intermediates issued from the glyphosate degradation, since an interim increase of toxicity was also observed during the case of the single glyphosate treatment. We also cannot rule out the existence of a synergistic interaction effect between the substrates and/or the reaction intermediates issued from the degradation of both pesticides [START_REF] Loureiro | Toxicity of three binary mixtures to Daphnia magna: Comparing chemical modes of action and deviations from conceptual models[END_REF]. Hence, a treatment of 900 min was needed to decrease the toxicity until values above calculable EC50 values occurred. This is in good agreement with the complete mineralization of both substrates at 900 min.

Degradation of active substances within commercial formulations

The degradation of the Roundup® solution was performed with an initial glyphosate concentration of 25 mg/L and an initial TOC value of 20 mg/L, within which the glyphosate active compound amounted only for 5.3 mg/L (Figure 1, Table 2). This extra-load of organic carbon coming from the components contained in Roundup will be also oxidized unselectively. However, this extraload did not alter the glyphosate concentration profile, and only the duration necessary for achieving the complete disappearance of glyphosate was extended to 100 min. Like in the case of glyphosate alone, the volcano-like TOC profile observed at the beginning of the test could be attributed to the C-N and C-P bond cleavage. It was therefore proposed that the mechanism followed the same pathway and that the components contained in the Roundup did not interfere into the glyphosate mineralization routes. However, the competitive adsorption on the catalyst surface of glyphosate and its degradation intermediates with isopropylamine, the surfactants and their degradation byproducts impacted on the kinetic rate constant of glyphosate removal (reduced to k' = 0,011 min -1 ) and on the intermediate evolution (AMPA, phosphate and aliphatic acids) that appeared for longer reaction times. As a result, the reaction time necessary for achieving the complete TOC removal was extended to 420 min, while the closure of the phosphorus balance confirmed that the total mineralization was reached. Further, the presence of cations like Na + , K + or NH4 + (corroborated by the conductivity measurements) and of anions like CO3 2-in the matrix was detrimental for photocatalysis due to the scavenging effect [START_REF] Malato | Decontamination and disinfection of water by solar photocatalysis: Recent overview and trends[END_REF][START_REF] Carbajo | Effect of water composition on the photocatalytic removal of pesticides with different TiO2 catalysts[END_REF].

For the untreated Roundup formulation, no toxicity for daphnia was observable, likely due to a too low initial concentration of 12.5 mg/L (equal to 50 %TI, Table 3). This is in line with the information from the product safety data sheet of Roundup®, which indicates a 48-h LC50 for Daphnia as high as 930 mg/L [START_REF] Schuette | Environmental fate of Glyphosate[END_REF].

A Systhane® solution with a myclobutanil concentration of 35 mg/L and a TOC value of 22 mg/L was treated (Figure 6B). Although the TOC measured was only related to the active compound, the reaction time needed for reaching the complete myclobutanil conversion and mineralization was slightly increased up to 240 min and 420 min, compared to 150 min and 240 min, respectively, in the case of myclobutanil alone, with a decrease by half of the apparent kinetic rate constant for myclobutanil removal down to k'=0,013 min -1 . Compared to the oxidation of myclobutanil as single substrate, a delay in the intermediate evolution was observed too (Figure S4-C), and the inorganic chloride was only released after 120 min of irradiation (Figure 6B), 60 min delayed compared to the single oxidation. The total depletion of the main organochloride compounds after 180min of reaction (Figure S4C) was corroborated by the release of chloride ions that closed the chloride mass balance after 420 min of treatment. This demonstrated that, although Kaolin was recently employed as adsorbent for removing pollutants from water [START_REF] Vardikar | Sonochemical synthesis, characterization and sorption study of Kaolin-Chitosan-TiO2 ternary nanocomposite: Advantage over conventional method[END_REF][START_REF] Amer | Removal of As(V) from aqueous solution by adsorption onto nanocrystalline kaolinite: Equilibrium and thermodynamic aspects of adsorption[END_REF][START_REF] Xu | Preparation of Magnetic Kaolinite Nanotubes for the Removal of Methylene Blue from Aqueous Solution[END_REF], it did not act as sorbent for storing chloride ions issued from the myclobutanil degradation.

The delay of the intermediates evolution has been attributed to the presence of cations and anions present in the commercial formulation (corroborated by the detection with ionic chromatograph), which partially inhibited the process, but also to a screening effect resulting from the presence of inorganic particles in suspension, which reduced the light penetration into the solution and consequently was lowering the treatment efficiency. This hypothesis was supported by the substantial increase in the rate of myclobutanil disappearance obtained when using a filtered Systhane® solution (Figure S5). Surprisingly, only C8 and C9 metabolites were identified as reaction intermediates among those detected when the reaction was performed with the synthetic ultrapure water, whether myclobutanil was as single substrate or mixed with glyphosate (Figure S4). This indicated that the degradation pathway was forced to go via the route B (C8) (Figure 4 route B) rather than via the route A (C10) (Figure 4 route A). This suggested that the co-existence of the cations and anions and of the inorganic oxide particles within the Systhane® formulation matrix could affect the oxidation pathway and so drive selectively the mineralization process towards the oxidation route B. Recently, Meephon et al. [START_REF] Meephon | Surface-Dependence of Adsorption and Its Influence on Heterogeneous Photocatalytic Reaction: A Case of Photocatalytic Degradation of Linuron on Zinc Oxide[END_REF] observed during the study of the linuron degradation with ZnO photocatalyst that changes in the adsorption alignment of linuron onto the catalyst surface led to different degradation intermediates. In this work, the authors concluded that the exposed surface of the photocatalyst was the key-factor driving the degradation pathway. In our case, the co-presence of inorganic and ionic substances could alter the way myclobutanil adsorbs at the surface and consequently provoke the formation of different intermediates during the process.

The ecotoxicity screening for the Systhane® formulation showed a successful reduction of the toxicity of the solution already after 80 min of photocatalytic treatment (Table 3). Indeed, the 48-h EC50 value for the untreated product (40 %TI; 13.9 mg myclobutanil/L) increased to > 50 %TI after 80 min and 360 min of treatment.

Finally, the mixture of both commercially available items was treated and the results are shown in Figure 6C and in Figures S6 andS7. The initial myclobutanil concentration was approximatively half that of the single Systhane® tests, due to the highly viscous nature of the commercial Systhane® paste that somehow slightly jeopardizes the initial concentration. This explained the more visible initial adsorption of myclobutanil when compared to the single Systhane® solution test. While the results showed that glyphosate was the first pesticide to be degraded within 120 min of test, a delay in both the myclobutanil and TOC reduction was observed, together with a delay in the intermediate appearance, similarly to the above-reported cases. While both phosphorous and chloride balances were fully closed by the release of phosphate and chloride ions, respectively, it is worth noting that only the intermediates corresponding to the proposed degradation route B have been observed, similarly to the case of the pesticide mix. Also, probably due to the lower kinetic rates in the case of the complex matrix, the presence of sarcosine was evidenced during the test (Figure S7). Interestingly, full mineralization was achieved faster than in the case of the glyphosate and myclobutanil mixture, ie. 420 min vs. 900 min. This might possibly result from a positive role of adsorbent played by the inorganic Kaolin additive from Systhane® that might reduce the strong competitive adsorption occurring between the different carboned molecules observed in the case of the pesticide mix. The apparent kinetic rate constant for glyphosate degradation was not affected by the co-presence of Systhane®, and although an additional drop of the kinetic rate constant for the myclobutanil degradation was observed compared to Systhane® (with k' = 0,002 min -1 , Table 4), both the complete removal of the target pesticides and the reduction in the acute toxicity with the treatment time were observed. This confirmed the viability of the photocatalytic process to treat complex commercial formulation-containing wastewaters in the case of glyphosate and myclobutanil pesticides.

Evaluation of the matrix effect

Figure 7 summarizes the influence of the water matrix on the evolution of both the glyphosate and the myclobutanil pesticide concentration upon photocatalysis. The increase in the water matrix complexity did not alter the overall glyphosate evolution profile, with the time required for removing glyphosate being extended from 30 min to 120 min (Figure 7A). By contrast, in addition to the increased time necessary for the elimination of myclobutanil from 150 min to 360 min, the copresence of glyphosate and of its additives in the Roundup® formulation strongly influenced the overall myclobutanil evolution profile, as a result of the preferential adsorption of glyphosate on the TiO2 surface. Indeed, the co-presence of glyphosate -and to a lesser extent of the Roundup® additives -caused a delay in the myclobutanil disappearance before a usual disappearance profile was observed. It was worth noting that the additives from Systhane® had a less pronounced influence on the myclobutanil removal duration than the glyphosate and the additives from Roundup® had, probably due to their inorganic nature.

A duration of 240 min has been selected for evidencing the influence of the water matrix complexity on the efficiency of the photocatalytic treatment in terms of TOC removal (X240), since it allowed the complete removal and mineralization of single pesticides (Figure 8). In the case of the mixtures of pesticides, the X240 TOC removal efficiency strongly dropped down to 55% and 25% for the 25:20 and 25:40 glyphosate:myclobutanil weight ratios, respectively, all the more pronounced as the myclobutanil concentration increased. By contrast, a high TOC removal efficiency in the 65-85% range was maintained in the case of the commercial formulations. Although the mineralization efficiency was influenced by the presence of the additional organic components in the Roundup® formulation -and in a lesser extent by that of the additional inorganic matter in the Systhane® product, this suggested that the mineralization efficiency was more affected by the co-presence of myclobutanil than by the presence of the inorganic additives or by that of the organic additives that are more easily oxidized than myclobutanil.

Both pesticides and their corresponding commercial formulations differ strongly in terms of chemical structure and composition, glyphosate being an aliphatic-type structure while myclobutanil is mainly constituted by aromatic rings, and either organic or inorganic additives being used in the commercial formulations. Table 4 shows that the apparent kinetic rate constants for the pesticide degradation were highly impacted by the presence of the matrix components, -although in a very different way according to the substrate, the degradation mechanism for glyphosate being notably mediated by the adsorption onto the surface catalyst -; an overall drastic reduction was observed from 0.096 min -1 and 0.021 min -1 for the single glyphosate and myclobutanil degradation, respectively, to 0.011 min -1 and 0.002 min -1 for the commercial formulation mixture. However, for both pesticides, the degradation kinetic rate constant maintained in the commercial Roundup® and Systhane® formulation mix about 10% of the corresponding value obtained in the case of the single pesticide.

Interestingly, the ecotoxicity of the untreated Systhane® x Roundup® mixture was higher than that of the single substances, being eg. approximately 3-fold more toxic for daphnids than the untreated Systhane® solution (Table 3). As the Roundup® solution solely did not show any effects on daphnids, this indicated the occurrence of a synergistic effect. The photocatalytic treatment of the commercial formulation mixture statistically significantly reduced the water toxicity already after 60 min of process, and by approximatively 50% after 360 min of treatment (25 %TI vs. 54 %TI), although a total degradation and mineralization of the myclobutanil and glyphosate active substances in the Systhane® and Roundup® products was analytically detected (Figure 7). This might result from the existence of a synergistic interaction effect involving species remaining in the treated water as trace level.

CONCLUSION

Independently of the water matrix complexity, both glyphosate and myclobutanil pesticides were degraded using Aeroxide TiO2-P25 as reference photocatalyst under UV-A irradiation, with a complete mineralization yield into CO2, chloride and phosphate ions. The glyphosate degradation has been proposed to occur via both AMPA and sarcosine pathways with an unusual volcano-like profile for the TOC evolution resulting from the degradation pathway, while the photocatalytic degradation of myclobutanil did not phenomenologically differ from the usual photo-oxidation of aromatic ringpollutants by AOPs. The overall pesticide evolution profiles were not affected by the water matrix complexity, except that the time necessary for the complete removal of both pesticides increased with increasing the matrix complexity. We showed that the myclobutanil evolution profile was strongly influenced by the co-presence of glyphosate -and to lesser extent of its additives in the Roundup® formulation -, as a result of the preferential glyphosate adsorption on the TiO2 surface that delayed the myclobutanil disappearance. By contrast, we provided further evidence that the inorganic additives from Systhane® had a lower influence on the myclobutanil removal duration and on the TOC removal efficiency when compared to that of the glyphosate/Roundup® products, although they impacted on its degradation route.

However, although the apparent kinetic rate constants for glyphosate and myclobutanil degradation and the mineralization efficiency were globally reduced with the increase in the water matrix complexity, complete mineralization was achieved via photocatalysis, with a full closure of the phosphorus and chlorine heteroatom balance as phosphate and chloride ions even in the case of the commercial formulation mix, for which a significant decrease in the acute toxicity of the water for daphnids was consequently obtained.

Works are ongoing for studying the reaction pathway and the influence of the aqueous matrix complexity in the degradation of both pesticides under simulated solar light using visible-light responsive photocatalysts, and for treating real wastewater in field experiments using immobilized photocatalysts for demonstrating further the viability of the photocatalysis approach and increasing the practical significance of the work.

C

Table 1. Main components of the Roundup®-720 and Systhane®-200 commercial formulations as identified from the manufacturer's datasheets and the literature [45,[START_REF] Giesy | Ecotoxicological risk assessment for Roundup® herbicide[END_REF] . NA: mathematically not assessable *: in square brackets, the EC50 values in mg/L based on the nominal concentration are given. This is only possible for single substances and initial solutions.

Roundup®

Figure 1 Figure 1 .Figure 2 Figure 2 .Figure 3 Figure 3 .

 112233 Figure 1

Figure 4 Figure 4 .

 44 Figure 4

Figure 5 Figure 5 .Figure 6 .

 556 Figure 5

Table 2 .

 2 Physico-chemical parameters of the ready-to-use diluted commercial formulations. of the active ingredients b obtained by TOC analysis c TOC value corresponding to the measured concentration of the active compounds

Table 3 :

 3 48-h EC50 values and respective lower and upper 95% Confidence Intervals (LCI, UCI) of the test items for Daphnia magna at different times of the photocatalytic treatment, expressed in percentage of test item volume within the test solution (= %TI).

	Samples	t (min)	48 h-EC50 expressed in %TI [expressed in mg/L]*	95% LCI	95% UCI
		Initial	> 50 [> 25.0]	NA	NA
	Glyphosate	60 120	55 41	38 NA	72 NA
		240	>50	NA	NA
		Initial	6 [1.2]	0	36
	Myclobutanil	60 150	>56 >56	NA NA	NA NA
		240	>56	NA	NA
		Initial	24	12	36
	Glyphosate x Myclobutanil	180	20	16	23
		900	NA	NA	NA
		Initial	>50 [> 12.5]	NA	NA
	Roundup	80	>50	NA	NA
		300	>50	NA	NA
		Initial	40 [13.9]	25	55
	Systhane	80	>50	NA	NA
		360	>50	NA	NA
		Initial	25	22	27
	Roundup x Systhane	60 180	38 42	34 34	42 50
		360	54	47	61

Acknowledgements.

The authors want to thank the European Fund for regional development (EFRE/FEDER) for the financial support of the PHOTOPUR project which is performed within the framework of Interreg V and the Sciences Offensive.


Christelle Kevin Nay

Koechlin Ramonatxo

Rochdi Sarraj

Marie-Laure Island

Luz Orfila

Loïc Treffel

Marie-Pierre Bareille

Alexandre Beck

Guillemette Gauquelin-Koch

Martine Ropert

Christelle Koechlin-Ramonatxo

Sarah Rochdi

Arnaud Beck

MD Olivier Loréal
email: olivier.loreal@univ-rennes1.fr

Frédéric Derbré

PhD Frederic Derbré
email: frederic.derbre@univ-rennes2.fr

Simulated microgravity disturbs iron metabolism and distribution in humans: Lessons from dry immersion, an innovative ground-based human model

Keywords: erythropoiesis, iron misdistribution, bedrest, spaceflight, muscle atrophy erythropoiesis, iron misdistribution, bedrest, spaceflight, muscle atrophy

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

INTRODUCTION

Physical function decreases in astronauts and patients in hospital, especially due to exposure to extreme physical inactivity. In both cases, microgravity and bedrest reduce work capacity and complicate recovery [START_REF] De Santo | Anemia and erythropoietin in space flights[END_REF][START_REF] Vico | Effects of long-term microgravity exposure on cancellous and cortical weight-bearing bones of cosmonauts[END_REF]. Iron is required due to its role in many biological functions including protein, lipid, carbohydrates and nucleic acid metabolisms. However, abnormally accumulated in organs, iron promotes oxidative stress through the Fenton reaction, and thus may play a role in the development of osteoporosis and muscle atrophy [START_REF] Reardon | Iron injections in mice increase skeletal muscle iron content, induce oxidative stress and reduce exercise performance[END_REF][START_REF] Tsay | Bone loss caused by iron overload in a murine model: importance of oxidative stress[END_REF]. In addition, when abnormally distributed in the body, iron could also affect erythropoiesis, and so contribute to anemia [START_REF] De Santo | Anemia and erythropoietin in space flights[END_REF][START_REF] Smith | Red blood cell and iron metabolism during space flight[END_REF].

Previous studies reported that serum ferritin levels are higher in astronauts and bedridden subjects, suggesting increased body iron stores [START_REF] Smith | The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station[END_REF][START_REF] Morgan | Bone metabolism and nutritional status during 30-day head-down-tilt bed rest[END_REF]. Using the hind limb unloading (HU) model to simulate microgravity in rats, we recently showed that short-term exposure to HU causes iron misdistribution, characterized by increased splenic iron store [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF]. Interestingly, hepatic hepcidin expression and blood hepcidin levels increase in rodents exposed to simulated microgravity [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF][START_REF] Xu | The regulation of iron metabolism by hepcidin contributes to unloading-induced bone loss[END_REF][START_REF] Nay | Intermittent reloading does not prevent iron availability decrease and hepcidin upregulation caused by hindlimb unloading[END_REF]. As hepcidin controls iron release from cells, especially spleen macrophages, it could play a key role in microgravity-induced iron misdistribution by limiting the expression and activity of ferroportin, the cell iron exporter that allows iron recycling from macrophages in plasma [START_REF] Zhang | Control of Systemic Iron Homeostasis by the Hemojuvelin-Hepcidin Axis12[END_REF]. Notably, it has been suggested that iron metabolism has strong interactions with other essential metals, especially due to the fact that some iron proteins could also participate in the metabolism of other metals [START_REF] Loréal | Iron, hepcidin, and the metal connection[END_REF][START_REF] Cavey | Spleen iron, molybdenum, and manganese concentrations are coregulated in hepcidin-deficient and secondary iron overload models in mice[END_REF][START_REF] Cavey | Mouse genetic background impacts both on iron and non-iron metals parameters and on their relationships[END_REF][START_REF] Valberg | Alteration in cobalt absorption in patients with disorders of iron metabolism[END_REF][START_REF] Hansen | Proteins involved in iron metabolism in beef cattle are affected by copper A c c e p t e d m a n u s c r i p t 15 deficiency in combination with high dietary manganese, but not by copper deficiency alone[END_REF]. Except few data collected in astronauts during spaceflight [START_REF] Zwart | Iron status and its relations with oxidative damage and bone loss during long-duration space flight on the International Space Station[END_REF], the effects of microgravity on hepcidin regulation in humans remains poorly understood despite its major role as an integrative signal in the control of systemic iron metabolism. Therefore, our objective was to characterize the effects of simulated microgravity and extreme physical inactivity on iron metabolism and distribution in relation with hepcidin modulation.

For this purpose, we used the dry immersion (DI) model that accurately reproduces the effects of microgravity [START_REF] Navasiolava | Long-term dry immersion: review and prospects[END_REF], and exposed healthy young volunteers for 5 days to this innovative ground-based human model. In addition, a group of participants underwent venoconstrictive thigh cuffs protocol, a countermeasure to microgravity used to sequester fluids in the lower limbs (Figure 1). 1. There was no significant difference between groups at baseline (Table 1).

Overall study design

The study was conducted at the MEDES space clinic, Toulouse, France from 19/11/2018 to 23/03/2019. Subjects arrived in the evening of BDC-5 and left in the morning of R+2. The experimental protocol included four days of ambulatory baseline measurements before immersion (BDC-4 to BDC-1), five days (120 hours) of dry immersion (DI1 to DI5) and two days of ambulatory recovery (R0, R+1). Besides, a week prior to beginning the protocol the subjects went to MEDES for pre-immersion muscle biopsy and resting metabolic rate measurement. General protocol of strict DI was conducted according to methodology detailed in De Abreu et al. 2017 [START_REF] De Abreu | Multi-System Deconditioning in 3-Day Dry Immersion without Daily Raise[END_REF]. Two subjects, one from Control group and one from Cuff group, underwent dry immersion simultaneously in the same room, in two separate baths (except for two subjects, one Cuffs and one Control, C and M, who had no mate).

Thermoneutral water temperature was continuously maintained. Light-off period was set at 23:00-07:00. Daily hygiene, weighing and some specific measurements required extraction from the bath. During these out-of-bath periods, subjects maintained the -6° head-down position, a reliable position to maintain physiological effects of microgravity used in bedrest studies [START_REF] Pavy-Le Traon | From space to Earth: advances in human physiology from 20 years of bed rest studies (1986-2006)[END_REF]. Total out-of-bath supine time for the 120 h of immersion was 9.7±1.3h. On DI1-DI4 out-of-bath time was 1.1±0.6 h/day. On DI5 out-of-bath time was 5.3±1.1 h, because of muscle biopsy and Magnetic resonance imaging (MRI). Otherwise, during DI, subjects remained immersed in a supine position for all activities and were continuously observed by video monitoring. Body weight, blood pressure, heart rate and tympanic body temperature A c c e p t e d m a n u s c r i p t were measured daily. The range of adequate water intake was fixed at 35-60 ml/kg/day; within this range water intake throughout the protocol was ad libitum (measured). The menu composition of each experiment day was identical for all participants and dietary intake was individually tailored and controlled during the study. The individual energy intake was calculated by multiplying resting metabolic rate with physical activity levels of respectively 1.6 and 1.3 before and during DI. Daily caloric intake was around 2625 kcal for baseline and 2160 kcal for the dry immersion period. Daily intake for iron was approximately 10 and 15 mg. Subject N was finally excluded from all statistical analysis for the present study due to higher abnormal values before and after DI for transferrin saturation (48.7 and 65.3%), serum ferritin levels (449.8 and 615 µg/l) and iron liver content (70.0 and 71.1 µmol/g).

Cuff

Subjects randomized to Cuff group wore the thigh cuffs during the 5 days of DI, from 10h to 18h at DI1 and from 8h to 18h at DI2-DI5. Thigh cuffs are elastic strips, adapted to each subject to have the same effects on lower limb distensibility as at counterpressure of about 30 mmHg. Individual adjustment was determined for each subject with calf plethysmography, performed in the supine position at BDC-2. At DI1, thigh cuffs were put on immediately prior to the onset of immersion at 10h.

Body composition

Body mass (kg) was measured on a weighing trolley at baseline and every morning during dry immersion. Fat and fat free mass were measured by DEXA (Hologic, QDR 4500 C, MA, USA) 4 days before DI (BDC-4), and after 5 days of DI (DI5).

Hematology and iron status

Venous blood was sampled at 7 a.m. in fasting state from a forearm vein 4 days before dry immersion (BDC-4), 2 hours before subject reambulation (R+0) and 2 days after reambulation (R+2). White blood cells (WBC) and red blood cells (RBC) counts, hemoglobin, hematocrit, reticulocytes by the Advia 2120 (Siemens Healthcare Diagnostics, Deerfield, Illinois, USA), an automated hematology analyzer. Serum iron, transferrin, bilirubin, myoglobin, haptoglobin concentrations were measured in the biochemistry laboratory (Rennes Pontchaillou Hospital) on Cobas 8000 analyser Roche® (Roche Diagnostics, Meylan, France). Serum iron and bilirubin concentrations were measured by standard colorimetric methods; serum transferrin and haptoglobin levels were determined by immunoturbidimetry; serum ferritin and myoglobin concentrations were measured by ECLIA (Electro-Chemiluminescence ImmunoAssay). Total iron binding capacity (TIBC) was calculated using the formula:

A c c e p t e d m a n u s c r i p t (transferrin in g/l x 25) and transferrin saturation (%) was calculated as follows: (iron in µmol/l / TIBC) × 100. Reference interval of local laboratory values of serum iron, transferrin saturation, ferritin, haptoglobin, myoglobin were 12.5-25 μmol/L, 23-46%, 55-345 μg/L, 0,3-2g/l, 10-92ng/ml respectively.

MRI protocol

MRI is a sensitive technique to quantify iron variations in tissues. Indeed, iron is a paramagnetic substance that becomes superparamagnetic (with a strong decrease of the T2*), when several atoms are grouped. Using this concept, Gandon et al. developed algorithms to quantify iron content in spleen and liver [START_REF] Gandon | Non-invasive assessment of hepatic iron stores by MRI[END_REF]. For this study, a 1.5-T Siemens-Avanto magnetic resonance imaging system was used in Toulouse Hospital (CHU Rangueil). MRI sessions were performed before DI at BDC-3 and at DI-5 evening. Subjects were transferred from DI bath to MRI apparatus in head-down bed rest -6° in order to maintain the effects of simulated microgravity. Subjects were in supine rest position during all the MRI acquisition.

Five gradient echo sequences were performed, following the protocol proposed by Gandon et al. [START_REF] Gandon | Non-invasive assessment of hepatic iron stores by MRI[END_REF]. A typical T1-weighted sequence with an in-phase echo time of 4 ms and a 90° pulse angle was followed by four sequences with a 20° pulse angle and in-phase echo times ranging from 4 to 21 ms, to progressively increase T2-weighting. Every sequence had an acquisition time of 15 s. Magnetic resonance gradient echo sequences used are summarized in Table 2.

For each sequence three circular regions of interest (ROIs) were drawn in the right lobe of the liver, one in the spleen and two in the left and right paraspinal muscles. The liver-to-muscle and spleen-to-liver SI ratio (SIR) were then calculated for each sequence using the algorithm provided by

RESULTS

Participant Flow

The participant flow is represented in Figure 2. This clinical trial was originally designed to assess the effects of thigh cuffs on the hematological, cardiovascular and musculoskeletal system responses induced by 5 days of DI. As cuffs did not have any significant effects on iron metabolism parameters (Figure 3) during simulated microgravity, the results and discussion section are only focused on the effects of DI on iron metabolism.

Dry immersion affects both body weight and composition

After 5 days of DI, body lean mass decreased significantly in all participants (-2.5%, p<0.001;

Table . 3 and Supplemental Fig. 2B), as well as the leg lean mass (-2.9%, p<0.001; Table . 3).

Total body mass and total fat mass concomitantly decreased (-2.5% and -0.1% respectively, p<0.001 for both; Table 3 and Supplemental Fig. 2A and2C), so the fat mass proportion of the participants remained unchanged (Table . 3 and Supplemental Fig. 2D). Thigh cuffs had no significant impact on body weight and body composition (Table . 3).

Dry immersion alters iron homeostasis and distribution in the body

Serum ferritin levels significantly increased in response to DI (+25%, p=0.003; Figure 3I).

MRI analysis of body iron stores showed that 5 days of DI induced iron accumulation in the spleen (+29%; p=0.009; Figure 3A and3C), but not in the liver (Figure 3B and 3C). We did not observe any correlation between spleen iron and serum ferritin levels (r=0.04, p=0.81). DI also increased serum iron concentration, and transferrin saturation level (+22% and +25% respectively, p<0.001 for both; Figure 3D and3E), whereas serum transferrin concentration remained unchanged (Figure 3F). The serum hepcidin level significantly increased after DI A c c e p t e d m a n u s c r i p t (+66%, p<0.001, Figure 3G). We did not observe any significant effect of thigh cuffs on iron homeostasis and body iron distribution (Figure 3).

Effects of dry immersion on erythropoiesis, hemolysis and myolysis parameters

Dry immersion increased red blood cells (RBC) and white blood cells (WBC) counts (+13% and +25% respectively, p<0.001 for both; Table 4), as well as hemoglobin and hematocrit levels (+12% and +13% respectively, p<0.001 for both;

DISCUSSION

The present study was designed to explore in humans the consequences of simulated microgravity and extreme physical inactivity on iron metabolism and distribution in relation with the modulation of hepcidin, the central regulator of systemic iron homeostasis. Using an innovative ground-based model of DI and MRI, we demonstrated that short-term exposure to simulated microgravity causes iron sequestration in the spleen. Such spleen iron accumulation could be related to the increase of serum hepcidin levels we observed, since this peptide limits iron egress from macrophages. Our data also support that hemolysis and skeletal muscle atrophy could contribute to this iron misdistribution.

The dry immersion model allows scientists to accurately and rapidly mimic most of physiological effects of microgravity and extreme physical inactivity [START_REF] De Abreu | Multi-System Deconditioning in 3-Day Dry Immersion without Daily Raise[END_REF][START_REF] Demangel | Early structural and functional signature of 3-day human skeletal muscle disuse using the dry immersion model[END_REF][START_REF] Treffel | Craniomandibular System and Postural Balance after 3-Day Dry Immersion[END_REF]. Short-term DI studies are sufficient to induce significant changes in fluid volume redistribution [START_REF] De Abreu | Multi-System Deconditioning in 3-Day Dry Immersion without Daily Raise[END_REF][START_REF] Robin | DI-5-CUFFS: Venoconstrictive Thigh Cuffs Limit Body Fluid Changes but Not Orthostatic Intolerance Induced by a 5-Day Dry Immersion[END_REF], cardiorespiratory responses [START_REF] Navasiolava | Long-term dry immersion: review and prospects[END_REF][START_REF] Tomilovskaya | Dry Immersion as a Ground-Based Model of Microgravity Physiological Effects[END_REF] and muscle plasticity [START_REF] Demangel | Early structural and functional signature of 3-day human skeletal muscle disuse using the dry immersion model[END_REF][START_REF] Shenkman | Human muscle atrophy in supportlessness: effects of short-term exposure to dry immersion[END_REF] [START_REF] Demangel | Early structural and functional signature of 3-day human skeletal muscle disuse using the dry immersion model[END_REF][START_REF] Fitts | Functional and structural adaptations of skeletal muscle to microgravity[END_REF][START_REF] Dirks | Dietary feeding pattern does not modulate the loss of muscle mass or the decline in metabolic health during short-term bed rest[END_REF][START_REF] Risso | Neocytolysis: none, one or many? A reappraisal and future perspectives[END_REF]. These results suggest that the iron metabolism parameters we studied should truly reflect conditions related to microgravity and physical inactivity.

The increase of serum ferritin levels in response to DI agrees fully with previous reports in real and simulated microgravity studies [START_REF] Smith | The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station[END_REF][START_REF] Morgan | Bone metabolism and nutritional status during 30-day head-down-tilt bed rest[END_REF]. Such a rise in ferritin levels could indicate that iron overload develops during DI. However, a ferritin increase can also be related to other etiologies including acute and chronic inflammation [START_REF] Kohgo | Body iron metabolism and pathophysiology of iron overload[END_REF]. To clarify the situation, we used MRI that allows a non-invasive quantification of iron concentration variations before and after DI, both in the liver and the spleen, two key organs involved in iron metabolism and storage [START_REF] Gandon | Non-invasive assessment of hepatic iron stores by MRI[END_REF]. Here, for the first time according to our knowledge, we found in humans that short-term exposure to simulated microgravity causes an increase in iron concentration increase in the spleen, whereas the liver remains spared from iron excess. Importantly, these data are in agreement with our previous findings obtained in rats, in which 7 days of hindlimb unloading increased spleen iron concentrations, but did not affect liver iron store [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF]. These results suggest that spleen iron sequestration occurring during the first days of microgravity could be a potential mechanism to protect other organs from iron excess. However, even if we were not able to determine spleen volume variations, we cannot exclude that microgravity could affect this parameter, and so spleen iron concentrations. It would be thus interesting to explore in humans the effects of long-duration exposure to microgravity on iron content in other organs known to be functionally affected by this condition (e.g. liver, bone, heart). In this way, preclinical studies performed on mice support that iron starts to accumulate in the liver and bone when hindlimb unloading is prolonged from 3 to 4 weeks [START_REF] Xu | The regulation of iron metabolism by hepcidin contributes to unloading-induced bone loss[END_REF][START_REF] Yang | Iron overload involved in the enhancement of unloading-induced bone loss by hypomagnetic field[END_REF] suggesting that the risks of iron-related disorders need to be considered for long-duration space travel.

Hepcidin, the gatekeeper of systemic iron metabolism, is known to promote ferroportin degradation, thus inhibiting iron export from enterocytes and macrophages, and leading to iron sequestration in cells, especially in the spleen which is a macrophage-rich organ [START_REF] Zhang | Control of Systemic Iron Homeostasis by the Hemojuvelin-Hepcidin Axis12[END_REF].

Our data showing that DI increased serum hepcidin levels support that increased spleen iron concentrations would be due to iron sequestration in this organ more than to a variation in spleen volume. The main inducers of hepcidin expression are the increase of transferrin saturation and iron stores levels, especially in the liver, but also the inflammatory process [START_REF] Fung | Manipulation of the hepcidin pathway for therapeutic purposes[END_REF].

A c c e p t e d m a n u s c r i p t

Our data supports that the hepatic iron accumulation is likely not involved in the hepcidin concentration increase, as MRI did not reveal any significant change in hepatic iron concentrations after DI period. Conversely, both the increase of serum iron and transferrin saturation levels could have played a role in promoting hepcidin synthesis as suggested by reports linking serum transferrin saturation level and hepcidin expression [START_REF] Hentze | Two to tango: regulation of Mammalian iron metabolism[END_REF]. However, microgravity could rapidly induce a low-grade pro-inflammatory state that could also participate in the hepcidin level increase [START_REF] Bosutti | Calorie restriction modulates inactivity-induced changes in the inflammatory markers C-reactive protein and pentraxin-3[END_REF]. Indeed, despite the normal wr-CRP levels, we found higher levels of serum haptoglobin and ferritin concentrations, both proteins known to be induced during inflammation. The increase of serum iron and transferrin saturation levels were not expected, as an increase in hepcidin concentration is reported to decrease both parameters by limiting iron egress. Therefore, it must be considered whether the rise of transferrin saturation is the initial element leading to iron metabolism disturbance in our conditions. The mechanisms leading to such an increase are likely multifactorial. The first one could be the hemoconcentration due to the decrease of plasma volume. In the present study, a decrease of 15 to 20% of plasma volume has been reported between the first and the fifth day of DI [START_REF] Robin | DI-5-CUFFS: Venoconstrictive Thigh Cuffs Limit Body Fluid Changes but Not Orthostatic Intolerance Induced by a 5-Day Dry Immersion[END_REF]. This reduction of plasma volume remain quite close to those observed during bed rest studies and spaceflight for the same duration [START_REF] Convertino | Clinical aspects of the control of plasma volume at microgravity and during return to one gravity[END_REF][START_REF] Alfrey | Control of red blood cell mass in spaceflight[END_REF][START_REF] Leach | Regulation of body fluid compartments during short-term spaceflight[END_REF]. Even if this decrease in plasma volume could slightly contribute to increase of some blood analytes, it cannot explain the rise of transferrin saturation calculated from a ratio of serum iron and transferrin concentrations.

Furthermore, our data suggest that hemolysis and myolysis could also be involved by promoting iron release in blood compartment.

Regarding hemolysis, it is well known that 65-70% of the total body iron is contained in hemoglobin of RBC [START_REF] Andrews | Disorders of iron metabolism[END_REF][START_REF] Muñoz | Disorders of iron metabolism. Part 1: molecular basis of iron homoeostasis[END_REF]. Noteworthy, senescent RBC are engulfed by macrophages in the reticuloendothelial system, especially in spleen macrophages during the erythrophagocytosis process. Specifically, the hemoglobin they contained is catabolized by the cytosolic hemeoxygenase-1 to release the heme-bound iron, whereas biliverdin is converted in unconjugated bilirubin [START_REF] Zhang | Molecular mechanisms of normal iron homeostasis[END_REF][START_REF] Nairz | Pumping iron"-how macrophages handle iron at the systemic, microenvironmental, and cellular levels[END_REF]. Hemolysis phenomenon is biologically characterized by increased serum unconjugated bilirubin levels, combined in case of intravascular hemolysis, to a reduction of serum haptoglobin level [START_REF] Barcellini | Clinical Applications of Hemolytic Markers in the Differential Diagnosis and Management of Hemolytic Anemia[END_REF]. Previous studies evaluating these markers observed that serum bilirubin levels remained unaffected in humans exposed to real or simulated microgravity, thus suggesting that significant hemolysis did occur under microgravity [START_REF] Alfrey | Control of red blood cell mass in spaceflight[END_REF][START_REF] Ryan | Haemoglobin mass alterations in healthy humans following four-day headdown tilt bed rest[END_REF][START_REF] Trudel | Hemolysis during and after 21 days of head-down-tilt bed rest[END_REF]. On contrary, we observed that 5 days of DI increased both serum unconjugated bilirubin and haptoglobin levels, suggesting that if hemolysis occurs A c c e p t e d m a n u s c r i p t under DI, this hemolysis is extravascular [START_REF] Mebius | Structure and function of the spleen[END_REF]. The slight hemolysis we observed could ultimately be due to the rapid destruction of young red blood cells (i.e. neocytolysis), a phenomenon previously described in astronauts during the first days of spaceflight [START_REF] De Santo | Anemia and erythropoietin in space flights[END_REF][START_REF] Alfrey | Neocytolysis: physiological down-regulator of red-cell mass[END_REF].

Interestingly, neocytolysis is triggered when a decrease in EPO production occurs, splenic endothelial cells responding to this withdrawal by increasing permeability and promoting phagocytosis of young red cells by macrophages [START_REF] Trial | Erythropoietin Withdrawal Alters Interactions Between Young Red Blood Cells, Splenic Endothelial Cells, and Macrophages: An In Vitro Model of Neocytolysis[END_REF]. Thus, the decrease of serum EPO levels we observed after 5 days of DI supports that this EPO withdrawal could favor iron uptake by splenic macrophages. Combined with the increase of serum hepcidin levels limiting iron export form macrophages, this decrease in EPO levels probably contribute to splenic iron sequestration observed in the present study.

Myolysis may also play a role in the increases of serum iron and transferrin saturation levels.

Indeed, skeletal muscle contains 10 to 15% of body iron, mainly as heme-bound iron of myoglobin [START_REF] Andrews | Disorders of iron metabolism[END_REF][START_REF] Muñoz | Disorders of iron metabolism. Part 1: molecular basis of iron homoeostasis[END_REF]. In agreement with previous short-term bedrest and DI studies [START_REF] Demangel | Early structural and functional signature of 3-day human skeletal muscle disuse using the dry immersion model[END_REF][START_REF] Fitts | Functional and structural adaptations of skeletal muscle to microgravity[END_REF][START_REF] Dirks | Dietary feeding pattern does not modulate the loss of muscle mass or the decline in metabolic health during short-term bed rest[END_REF], our data showed that total and leg lean mass are significantly reduced after 5 days of DI compared with baseline. These data strongly support the development of skeletal muscle atrophy during DI period. Importantly, at the same time, the serum myoglobin levels significantly increased after 5 days of DI. Similarly to hemoglobin, macrophages are able to phagocyte myoglobin to degrade myoglobin-associated heme, and to recycle the heme-bound iron [START_REF] Corna | The Repair of Skeletal Muscle Requires Iron Recycling through Macrophage Ferroportin[END_REF]. Thus, we cannot exclude that phagocytosis of myoglobin by splenic macrophages could also promote both spleen iron sequestration and the increase of unconjugated bilirubin release to bloodstream.

In summary, our study demonstrates for the first time that in young healthy men, dry immersion rapidly induces iron misdistribution characterized by increased spleen iron level.

Our data suggest that hemolysis and myolysis combined with the increase the hepcidin level are involved in iron sequestration in spleen. Consequently, these results question the longterm effects of microgravity and extreme physical inactivity on iron metabolism, and their potential deleterious consequences on the health of astronauts, especially as iron-induced oxidative stress could be facilitated by radiation exposure in space [START_REF] Yang | Effects of Iron Overload and Oxidative Damage on the Musculoskeletal System in the Space Environment: Data from Spaceflights and Ground-Based Simulation Models[END_REF]. Whereas the current Dietary Reference Intake for iron is 8 mg/day for men [START_REF] Smith | Assessment of Nutritional Intake During Space Flight and Space Flight Analogs[END_REF] A c c e p t e d m a n u s c r i p t

  ANSM : French National Agency for Medicines and Health Products Safety BDC: baseline data collection DI : dry immersion EPO: erythropoietin HIC : hepatic iron store HU : hindlimb unloading SIC : splenic iron store

  were recruited to be exposed to 5 days of dry immersion. Two subjects were excluded before BDC-5 for reasons unrelated to protocol. A total of eighteen subjects were included in the study and randomly divided at BDC-2 into Control or Cuffs group (9/9 split). Participants were anonymized and designated in data sets by single letters. Subjects B, E, F, I, K, M, O, Q and S were included in control group while A, C, D, G, H, J, N, P and R were included in Cuffs group. All subjects were informed about the experimental procedures and gave their written consent. The experimental protocol conformed to the standards set by the Declaration of Helsinki and was approved by the local Ethic Committee (CPP Est III: October 2, 2018, n° ID RCB 2018-A01470-55) and French Health Authorities (ANSM: August 13, 2018, ClinicalTrials.gov Identifier: NCT03915457). Baseline group characteristics are detailed in Table

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Dry immersion method: the subject is immersed in thermoneutral water covered with an elastic waterproof fabric (Photo MEDES)

A c c e p t e d m a n u s c r i p t

  Table. 4). To explore both hematologic and iron homeostasis responses to dry immersion, erythropoiesis, hemolysis and myolysis biomarkers were assessed in blood compartment. Serum EPO levels and blood reticulocyte fraction were significantly decreased after 5 days of DI (-22% and -17% respectively, p=0.009 and p=0.003 respectively; Table. 4). Unconjugated bilirubin and haptoglobin levels were significantly increased in the bloodstream after 5 days of DI (+24% and +21% respectively, p=0.037 and p<0.001, Table.4 and Figure3H, respectively). Serum myoglobin levels were also higher after 5 days of DI compared to baseline (+15%, p=0.011;

Table.

[START_REF] Tsay | Bone loss caused by iron overload in a murine model: importance of oxidative stress[END_REF]

). Wide range C-reactive protein (wr-CRP) level remained unchanged by DI (Table

.

4). Thigh cuffs participants exhibited lower RBC count compared to CTL participants, both before and after exposure to DI (-7% before DI and -10% after DI, Cuff effect: p=0.009;

Table. 4)

Table 1 .

 1 , the mean iron content of the standard International Space Station menu is around 20 ± 6 mg/day[START_REF] Smith | Assessment of Nutritional Intake During Space Flight and Space Flight Analogs[END_REF]. These values can even reach 35 mg/day for some crewmembers due to consumption of commercial food items Baseline group characteristics at BDC-2. Data are the mean ± SD. Statistical significance was checked using an unpaired T-test.

	m a n u s c r i p t
	A c c e p t e d

fortified with iron (51). Our results finally indicate that high iron content in the astronaut's food, or iron supplementation during periods of extreme physical inactivity, are questionable

Table 2 . Magnetic resonance gradient echo sequences used in MRI protocol

 2

		CTL (n=9)	Cuff (n=9)	Source of Variation
		BDC-4	DI5	BDC-4	DI5	Time	Cuff Interaction
	Total body mass (kg)	73.79 ± 7.24	71.37 ± 7.09	73.41 ± 9.12	71.48 ± 8.68	p<0.001 NS	NS
	Body lean mass (kg)	55.50 ± 4.74	54.16± 4.54	55.78 ± 6.73	54.33 ± 6.47	p<0.001 NS	NS
	Leg lean mass (kg)	18.42 ± 1.93	17.74 ± 1.71	18.29 ±2.53	17.62 ± 2.16	p<0.001 NS	NS
	Body fat mass (kg)	17.67 ± 3.34	17.22 ± 3.31	17.62 ± 3.69	17.14 ± 3.63	p<0.001 NS	NS
	Body fat mass (%)	24.0 ± 2.8	24.0 ± 2.8	23.9 ± 3.2	23.9 ± 3.4	NS	NS	NS

. TR, repetition time; TE, echo time; FA, flip angle; PD, proton density; TA, acquisition time.

Table 3 .

 3 Impact of 5 days of DI on body weight and composition. Data are the mean ± SD.

	m a n u s c r i p t
	Statistical significance was checked using a two-way ANOVA, sources of variation (time, Cuff and interaction) are reported, and the significance level was set at 0.05 (CTL, n=9; Cuff, n=8). DI, dry immersion; CTL, controls (no thigh cuffs). A c c e p t e d

Table 4 . Laboratory work-up before and after 5 days of dry immersion.

 4 Data are the mean ± SD. Statistical significance was checked using a two-way ANOVA, sources of variation (time, Cuff and interaction) are reported, and the significance level was set at 0.05

(CTL, n=9; Cuff, n=8). DI, dry immersion; CTL, controls (no thigh cuffs); WBC, white blood cells; RBC, red blood cells; EPO, erythropoietin; wr-CRP, wide range C-reactive protein.

ACKNOWLEDGEMENTS

The authors thank the volunteers and the staff of MEDES for participation in this protocol at the MEDES space clinic in 2018 and in 2019. This work was supported by the Centre National d'Etudes Spatiales. The work was also supported by INRAE and Brittany Council and partially supported by an AlimH department grant. The authors thank Stephanie Zindilis and Elisabetta Andermarcher for expert manuscript editing. ACKNOWLEDGEMENTS The authors thank the volunteers and the staff of MEDES for participation in this protocol at the MEDES space clinic in 2018 and in 2019. This work was supported by the Centre National d'Etudes Spatiales. The work was also supported by INRAE and Brittany Council and partially supported by an AlimH department grant. The authors thank Stephanie Zindilis and Elisabetta Andermarcher for expert manuscript editing.

CONFLICT OF INTEREST STATEMENT

No conflict of interest, financial or otherwise related to this work, is declared by the author(s).

AUTHOR CONTRIBUTIONS

Exclusion criteria

 Past record of orthostatic intolerance,  Cardiac rhythm disorders,  Chronic back pains,  Vertebral fracture, scoliosis or herniated disc,  Glaucoma,  HTA,  History of migraines,  History of hiatus hernia or gastro-esophageal reflux,  History of thyroid dysfunction, renal stones, diabetes,  History of head trauma,  Past records of thrombophlebitis, family history of thrombosis or positive response in thrombosis screening procedure (anti thrombin III, S-protein, C-protein, factor V Leiden mutation and the mutation 20210 of the prothrombin gene),  Individuals exhibiting mutations involved in hereditary hemochromatosis (HAMP, HFE, HFE2, SCL40A1 and TRF2),  Abnormal result for lower limbs echo-doppler,  History or active claustrophobia,  History of genetic muscle and bone diseases of any kind,  Bone mineral density: T-score ≤ -1.5 on the hip,  Osteosynthesis material, presence of metallic implants or any other contra-indication for MRI,  Poor tolerance to blood sampling,  Having given blood (more than 8ml/kg) in a period of 8 weeks or less before the start of the experiment,  Antibiotic treatment in the 2 previous months before the beginning of the study,  Vegetarian or vegan,  History of food allergy,  Positive reaction to any of the following tests: HVA IgM (hepatitis A), HBs antigen (hepatitis B), anti-HVC antibodies (hepatitis C), anti-HIV1+2 antibodies,  Subject already participating or in the exclusion period of a clinical research,  Refusal to give permission to contact his general practitioner,  Incarcerated persons,  Subject who, in the judgment of the investigator, is likely to be non-compliant during the study, or unable to cooperate because of a language problem or poor mental development,  Subject who has received more than 4500 Euros within 12 months for being a research subject.  Subject under guardianship or trusteeship.