Steven Roger

Alexandra Ducancelle

Hélène Le Guillou-Guillemette

Catherine Gaudy

Françoise Lunel

HCV virology and diagnosis

Keywords: hepatitis C virus, virology, diagnosis

INTRODUCTION

Hepatitis C virus (HCV) infection is a major cause of severe liver disease including chronic hepatitis, cirrhosis and hepatocellular carcinoma. The HCV burden in public health is estimated at about 71 million people worldwide by World Health Organization (WHO) with at least 400,000 people that died every year from HCV disease [1]. New hepatitis C treatments with oral direct-acting antivirals (DAAs) showing high rates of response, with short treatment duration [START_REF] Pawlotsky | New Hepatitis C Therapies: The Toolbox[END_REF] have been available. HCV can now be eradicated with minimal side effects. Unfortunately, there is no vaccine yet available, but the development of a safe prophylactic vaccine remains a medical priority [START_REF] Roingeard | Hepatitis C Vaccine: 10 Good Reasons for Continuing[END_REF]. For this purpose, Hepatitis B-C subviral envelope particles can be produced by industrialized procedure. It seems to be very promising as this HBV-HCV vaccine candidate has been shown to elicit a broadly cross neutralizing activity against HCV [START_REF] Beaumont | Mixing particles from various HCV genotypes increases the HBV-HCV vaccine ability to elicit broadly cross-neutralizing antibodies[END_REF]. Despite this revolution in the HCV-treatment, one of major challenge to achieve a global eradication of HCV remains to reduce the under diagnosis. The low rate of diagnosis is a major obstacle in resources limited countries and is mainly due to the cost of molecular tools, that are essential to diagnose and follow chronic HCV infection. In another hand, the mild clinical symptoms observed in HCV chronic disease, may explain that the majority of HCV infected individuals are unaware of their infection, because HCV testing is not generalized, like it is for HIV.

HCV was discovered in 1989 after many years of work, by several researchers, who recently obtained the Nobel price [START_REF] Feinstone | Transfusion-Associated Hepatitis Not Due to Viral Hepatitis Type A or B[END_REF][START_REF] Kolykhalov | Transmission of Hepatitis C by Intrahepatic Inoculation with Transcribed RNA[END_REF][START_REF] Choo | Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome[END_REF]. This major discovery allowed the description of the HCV genome and later on of the virus replication and cell cycle, and also, importantly, the development of diagnostic tests for the detection of HCV antibodies (Ab) and RNA who were a priority in transfusion.

In this review, we will try to get into the virology and cell biology of HCV. Thereafter, we will discuss the different categories of laboratory tests to diagnose/explore HCV infected subjects.

HCV VIROLOGY a. HCV genome

HCV is a positive sense, single-stranded RNA virus, composed of approximately 9600 nucleotides that belongs to the Flaviviridae family and is part of the Hepacivirus genus [START_REF] Kapoor | Characterization of a canine homolog of hepatitis C virus[END_REF][START_REF] Drexler | Evidence for Novel Hepaciviruses in Rodents[END_REF]. Figure 1 shows the HCV genome organization. The viral genome encodes a single open reading frame (ORF) of 3010 amino acids flanked by 5′ and 3′ non-translated regions (NTRs) which are critical for viral replication [START_REF] Tanaka | A Novel Sequence Found at the 3′-Terminus of Hepatitis C Virus Genome[END_REF] and translation [START_REF] Tsukiyama-Kohara | Internal ribosome entry site within hepatitis C virus RNA[END_REF]. The 5′ NTR contains an internal ribosome entry site (IRES).

Translation of the HCV protein starts from the 5′-UTR IRES [START_REF] Tsukiyama-Kohara | Internal ribosome entry site within hepatitis C virus RNA[END_REF] and generates a single polyprotein, which is processed by cellular proteases to produce the structural proteins that form the viral particles (core and envelope glycoproteins E1 and E2), or is processed by viral proteases to generate nonstructural (NS) proteins (p7, NS2, NS3,NS4A, NS4B, NS5A, and NS5B) (Figure 1).

b. Variability of HCV

HCV displays a high genetic diversity which results from defects in the repair activity of the RNAdependent RNA polymerase (resulting in nucleotide substitutions) and from the absence of 5' to 3' exonuclease activity (lack of error correction) [START_REF] Steinhauer | Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase[END_REF]. The mean frequency of nucleotide mutations varies from 1.4 x 10 3 to 1.9 x 10 3 substitutions per nucleotide and per year. This estimate is based on comparisons of the major sequences of complete genomes obtained after eight years of evolution in a chimpanzee and 13 years in a human [START_REF] Ogata | Nucleotide sequence and mutation rate of the H strain of hepatitis C virus[END_REF], [START_REF] Okamoto | Genetic drift of hepatitis C virus during an 8.2-year infection in a chimpanzee: Variability and stability[END_REF]. Some of the mutations accumulating during replication are silent or synonymous. These mutations have no impact on the amino-acid sequence of the viral protein, but may affect the secondary structure of the genomic RNA. Other so-called non-synonymous mutations lead to changes in protein sequence and the emergence of variants. Other mutations lead to the production of defective viral particles and are therefore lethal. The regions of the genome corresponding to essential viral functions (domains involved in translation or replication), or displaying major structural constraints (non-coding 5' and 3' ends) are the best conserved; indeed, the non-coding 5' region is the most highly conserved region of the genome, with more than 90% identity between the sequences of distantly related strains (4). The region encoding the capsid is also well conserved, with around 80% sequence identity between isolates [START_REF] Feinstone | Transfusion-Associated Hepatitis Not Due to Viral Hepatitis Type A or B[END_REF]. The most variable region of the genome is that encoding the envelope glycoproteins, E1 and E2. The sequences of hypervariable regions (HVR1 and HVR2) of E2 in strains isolated from different patients may differ by more than 50%. The polypeptides encoded by these hypervariable regions are therefore very tolerant to amino-acid substitutions [START_REF] Smith | Evolution of the hypervariable region of hepatitis C virus[END_REF][START_REF] Labonté | Modulation of Hepatitis C Virus RNA-dependent RNA Polymerase Activity by Structure-based Site-directed Mutagenesis[END_REF]. HVR1 contains a central highly variable domain (residues 8 to 18), flanked by six conserved positions (positions 2, 6, 7, 20, 23, and 26) that are assumed to be important for the structure of the whole HVR1 domain [START_REF] Gaudy | Significance of Pretreatment Analysis of Hepatitis C Virus Genotype 1b Hypervariable Region 1 Sequences To Predict Antiviral Outcome[END_REF][START_REF] Puntoriero | Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1can induce antibodies cross-reacting with a large number of viral variants[END_REF]. HVR1 was shown to be a decoy antigen that diverts the host immune response away from more conserved neutralizing epitopes [START_REF] Ray | Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy[END_REF].The classification of HCV, is based on the topology of the trees obtained by phylogenetic relationships between viral variants. HCV variants can be classified into seven clades, and then into subtypes corresponding to subgroups of the most closely related viruses within a clade [START_REF] Smith | Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: Updated criteria and genotype assignment web resource[END_REF]. The virus genotype is indicated by an Arabic number (from 1 to 7), associated with a lower-case letter to indicate the subtype. Genotypes differ from each other by around 25-35% and subtypes differ from each other by around 15-25% at the nucleotide level, according to the rules defined by the International Committee on Taxonomy of Viruses) [START_REF] Simmonds | ICTV Virus Taxonomy Profile: Flaviviridae[END_REF]. In a single-host, HCV exists as pools of related genetic variants, referred to as quasispecies [START_REF] Farci | The quasispecies of hepatitis C virus and the host immune response[END_REF][START_REF] Tsukiyama-Kohara | Hepatitis C Virus: Viral Quasispecies and Genotypes[END_REF]. The genome instability of HCV was reported as viral quasispecies [START_REF] Farci | The quasispecies of hepatitis C virus and the host immune response[END_REF]. The RNA-dependent RNA polymerase of HCV does not have proofreading activity, and HCV has a high error rate, [START_REF] Powdrill | Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance[END_REF], leading to genetic heterogeneity and quasispecies which were first identified via the hypervariable region (HVR) in the envelope E2 protein. HVR-1 and HVR-2 are located in E2 region (Figure 1). HVR-1 mutations can facilitate escape from host immune responses. HVR-1 and HVR-2 mutations are generated very rapidly and confer adaptive advantages for the virus i.e. tropism, virulence and drug resistance [START_REF] Ootsuyama | Humoral Immune Response to Hypervariable Region 1 of the Putative Envelope Glycoprotein (gp7O) of Hepatitis C Virus[END_REF][START_REF] Cerny | Immunological Aspects of HCV Infection[END_REF]. A high rate of amino acid substitutions per site per year have been described in acute and chronic cases of HCV infections [START_REF] Higashi | Characterization of hypervariable region in hepatitis C virus envelope protein during acute and chronic infection[END_REF]. The sequence variations in HVR-1 may reflect the existence of various quasispecies in the acute phase, the persistence of which could explain chronic infection [START_REF] Gaudy | Significance of Pretreatment Analysis of Hepatitis C Virus Genotype 1b Hypervariable Region 1 Sequences To Predict Antiviral Outcome[END_REF].

c. HCV Genotypes

The HCVDB (http://hepatitis.ibcp.fr), DDJB (http://www.ddbj.nig.ac.jp) and Los Alamos (http://hcv.lanl.gov) databases currently include a large number of HCV sequences, making it possible to compare an isolate with a large number of reference sequences [START_REF] Tulio De Oliveira | BioAfrica -Oxf HCV Subtyping Tool n[END_REF]. Although currently restricted to specialist laboratories, the gold standard method for HCV genotyping is sequencing of the NS5B region followed by phylogenetic analysis including comparison to reference sequences [START_REF] Laperche | Unique NS5b Hepatitis C Virus Gene Sequence Consensus Database Is Essential for Standardization of Genotype Determinations in Multicenter Epidemiological Studies[END_REF][START_REF] Guillou-Guillemette | Genetic diversity of the hepatitis C virus: Impact and issues in the antiviral therapy[END_REF].

Phylogenetic analysis permitted to classify HCV into 7 major genotypes and 67 subtypes [START_REF] Smith | Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: Updated criteria and genotype assignment web resource[END_REF] (Figure 2). Genotypes 1 and 3 are the most prevalent, accounting for more than 30% of all infections [START_REF] Messina | Global distribution and prevalence of hepatitis C virus genotypes[END_REF].

Genotypes 2, 4, 5, and 6 account for less than 10% of infections, respectively. Genotype 7 has been found in few people from Central Africa [START_REF] Murphy | Hepatitis C Virus Genotype 7, a New Genotype Originating from Central Africa[END_REF][START_REF] Smith | Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: Updated criteria and genotype assignment web resource[END_REF].

Figure 2 : Phylogenetic analysis of complete HCV genome [START_REF] Cuypers | Genetic Diversity and Selective Pressure in Hepatitis C Virus Genotypes 1-6: Significance for Direct-Acting Antiviral Treatment and Drug Resistance[END_REF].

As shown in figure 3, in Europe and North America, genotype 1 is the most prevalent in most countries, followed by genotypes 2 and 3. Genotype 2 shows the highest prevalence in Central Africa, and genotype 3 accounts for most infections in India, Pakistan, Thailand, and other Asian countries and in Northern European countries. Genotypes 4 and 5 have probably recently increased in prevalence due to emigration from the Middle East and Africa, and transmission by intravenous drug users. Genotype 4 is mostly found in Central Africa and the Middle East, and genotype 5 frequency is higher in Southern Africa. Prevalence of genotype 6 is high in East and Southeast Asia, and is mostly found in Laos and Vietnam (Figure 3). to 150 years ago. Many subtypes have been identified (2c to 2l) among genotype 2 strains, mainly found in West Africa [START_REF] Pybus | The Epidemic Behavior of the Hepatitis C Virus[END_REF]. The spread of genotype 3 began about 40 years ago. Infection of HCV genotype 3a was increased in India from 1940s to 1990s, followed a decrease after 21th century [START_REF] Bressanelli | Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus[END_REF][START_REF] Akkarathamrongsin | Molecular Epidemiology and Genetic History of Hepatitis C Virus Subtype 3a Infection in Thailand[END_REF]. Genotypes 4 and 6 emerged much earlier: 350 and 700 years ago, respectively [START_REF] Simmonds | Fleming Lecture. The origin and evolution of hepatitis viruses in humans[END_REF]. Genotype 4 may be originated in central Africa and multiple strains have been exported to north Africa since ~mid 19th century, explaining the high prevalence in Egypt [START_REF] Iles | Phylogeography and epidemic history of hepatitis C virus genotype 4 in Africa[END_REF]. Spread of genotype 4 was probably due to emigration during the 2 nd World War. Multiple HCV genotype 6 (6a, 6e, 6l etc.) and 1(1a,1b) subtypes are found in Vietnam and probably increased following Vietnam War [START_REF] Li | The genetic diversity and evolutionary history of hepatitis C virus in Vietnam[END_REF].

Conversely, the limited number of subtypes in Europe (where genotype 1, 2, 3 are the more frequently found), North America and Japan is consistent with the more recent introduction of HCV into these populations.

d. Clinical Significance of Quasispecies and Genotypes

The main difference between HCV genotypes 1 and 2 were in their responsiveness to interferon (IFN) treatment [START_REF] Orito | Hepatitis C virus serotype II responds more favorably to interferon-a therapy n[END_REF]. That's why, at the past area of IFN based regimens, genotype determination played a crucial role for prediction of treatment responsiveness. Compared with patients infected with HCV genotype 1b , a higher proportion of patients with genotype 2a showed sustained responses to IFN-α [START_REF] Messina | Global distribution and prevalence of hepatitis C virus genotypes[END_REF][START_REF] Murphy | Hepatitis C Virus Genotype 7, a New Genotype Originating from Central Africa[END_REF]. These results confirm the previous recommendation of HCV genotyping before starting IFN-α treatment [START_REF] Pawlotsky | EASL Recommendations on Treatment of Hepatitis C[END_REF]. However, in the context of pangenotypic DAAs genotype determination is not essential to determine the type of treatment.

The quasispecies diversity of HCV was also suspected to be involved in the responsiveness to IFN. Nonstructural (NS) NS5A is a phosphoprotein found in a basally phosphorylated form. The amino acid sequence is also varying depending on the genotype. Mutations in NS5A HCV were found to confer sensitivity to IFN [START_REF] Gaudy | Significance of Pretreatment Analysis of Hepatitis C Virus Genotype 1b Hypervariable Region 1 Sequences To Predict Antiviral Outcome[END_REF][START_REF] Tulio De Oliveira | BioAfrica -Oxf HCV Subtyping Tool n[END_REF][START_REF] Laperche | Unique NS5b Hepatitis C Virus Gene Sequence Consensus Database Is Essential for Standardization of Genotype Determinations in Multicenter Epidemiological Studies[END_REF].

A region, called the IFN-sensitivity-determining region (ISDR) located in the C-terminal portion of NS5A) (Figure 1) was initially described by japonese authors [START_REF] Enomoto | Mutations in the Nonstructural Protein 5a Gene and Response to Interferon in Patients with Chronic Hepatitis C Virus 1b Infection[END_REF]. Contradictory data about ISDR were reported from other countries than Japan, especially from Europe and USA [START_REF] Laperche | Unique NS5b Hepatitis C Virus Gene Sequence Consensus Database Is Essential for Standardization of Genotype Determinations in Multicenter Epidemiological Studies[END_REF][START_REF] Guillou-Guillemette | Genetic diversity of the hepatitis C virus: Impact and issues in the antiviral therapy[END_REF][START_REF] Messina | Global distribution and prevalence of hepatitis C virus genotypes[END_REF].

In HCV-1b infected patients, IFN therapy response seems to be mostly influenced by the mutation within the ISDR region in NS5A gene [START_REF] Shen | Mutations in ISDR of NS5A gene influence interferon efficacy in Chinese patients with chronic hepatitis C virus genotype 1b infection[END_REF]. Inshauspe et al [START_REF] Inchauspe | Genomic structure of the human prototype strain H of hepatitis C virus: Comparison with American and Japanese isolates[END_REF] identified another domain localised in the C-terminal region of NS5A and termed the V3 domain. It is length of 27 amino-acid and harbours a high variability level. Duverlie et al [START_REF] Duverlie | Sequence analysis of the NS5A protein of European hepatitis C virus 1b isolates and relation to interferon sensitivity n[END_REF] demonstrated a relationship between the mutation level in V3 and the response to interferon therapy. Our lab and others confirmed this correlation [START_REF] Guillou-Guillemette | Genetic diversity of the hepatitis C virus: Impact and issues in the antiviral therapy[END_REF]. HCV quasispecies diversity at baseline and under interferon-ribavirin combined treatment was studied in the entire NS5A gene and in each region of interest (PKR-bd, ISDR and V3). The V3 domain showed a higher quasispecies diversity in responder patients samples; confirming the potential role of the genetic diversity in the response to the interferon-based therapy [START_REF] Guillou-Guillemette | Genetic diversity of the hepatitis C virus: Impact and issues in the antiviral therapy[END_REF][START_REF] Puig-Basagoiti | Dynamics of hepatitis C virus NS5A quasispecies during interferon and ribavirin therapy in responder and non-responder patients with genotype 1b chronic hepatitis C[END_REF][START_REF] Veillon | Analyse des mutations des domaines ISDR et V3 de la protéine NS5A du virus de l'hépatite C avant le traitement par l'interféron avec ou sans ribavirine[END_REF] Liver steatosis is frequently observed in Chronic HCV disease [START_REF] Lonardo | Pathogenesis and significance of hepatitis C virus steatosis: An update on survival strategy of a successful pathogen[END_REF], and genotype 3 seems to be associated with non-alcoholic fatty liver disease (NAFLD) [START_REF] Bechmann | The interaction of hepatic lipid and glucose metabolism in liver diseases[END_REF]. Insulin resistance (IR) was also reported in patients infected with genotype 1 and 4 [START_REF] Moucari | Insulin Resistance in Chronic Hepatitis C: Association With Genotypes 1 and 4, Serum HCV RNA Level, and Liver Fibrosis[END_REF][START_REF] Thompson | Viral clearance is associated with improved insulin resistance in genotype 1 chronic hepatitis C but not genotype 2/3[END_REF].

Studies have also suggested a potential involvement of the HCV proteins and Viral quasispecies heterogeneity in the establishment of a chronic hepatitis and in the occurrence and outcome of Hepatocellular carcinoma (HCC) [START_REF] El-Shamy | Impact of HCV core gene quasispecies on hepatocellular carcinoma risk among HALT-C trial patients[END_REF][START_REF] Amougou-Atsama | The role of hepatitis C virus genotypes and core mutations in hepatocellular carcinoma in Cameroon[END_REF].

HCV infects and replicates in human hepatocytes. Animal model is the chimpanzee, which played a major role in the discovery of HCV [START_REF] Bukh | Animal Models for the Study of Hepatitis C Virus Infection and Related Liver Disease[END_REF].Other animal models are HCV-permissive mouse models [START_REF] Billerbeck | Animal Models for Hepatitis C[END_REF]. Important advances have been achieved with the replicon system [START_REF] Manns | Treating viral hepatitis C: efficacy, side effects, and complications[END_REF][START_REF] Mchutchison | Interferon Alfa-2b Alone or in Combination with Ribavirin as Initial Treatment for Chronic Hepatitis C[END_REF] and the identification of a full-length isolate of HCV (JFH1) that could recapitulate the full viral life cycle in the human hepatoma cell line Huh7 (cell culture HCV, HCVcc) [START_REF] Lohmann | Hepatitis C Virus RNA Replication[END_REF].The viral life cycle was studied using such viral systems [START_REF] Lindenbach | Complete Replication of Hepatitis C Virus in[END_REF][START_REF] Wakita | Production of infectious hepatitis C virus in tissue culture from a cloned viral genome[END_REF] and more recently primary human hepatocytes [START_REF] Lagaye | Efficient replication of primary or culture hepatitis C virus isolates in human liver slices: A relevant ex vivo model of liver infection[END_REF].

e. HCV particle

HCV virion structure is still poorly known. Its association with lipoproteins is one of its major characteristic [START_REF] Thomssen | Association of hepatitis C virus in human sera with Ilipoprotein n[END_REF]. HCV particles are 50-80 nm in diameter [START_REF] Catanese | Ultrastructural analysis of hepatitis C virus particles[END_REF] and contain the single-stranded RNA genome associated with the core and the envelope glycoproteins, E1 and E2 [START_REF] Vieyres | Incorporation of Hepatitis C Virus E1 and E2 Glycoproteins: The Keystones on a Peculiar Virion[END_REF]. The HCV genome interacts with the core protein to form the nucleocapsid , and is surrounded by a lipid membrane, in which are anchored the envelope glycoproteins. Apolipoproteins can also be found in association with HCV particles [START_REF] André | Characterization of Low-and Very-Low-Density Hepatitis C Virus RNA-Containing Particles[END_REF][START_REF] Chang | Human Apolipoprotein E Is Required for Infectivity and Production of Hepatitis C Virus in Cell Culture[END_REF]. The lipid composition of cell culture-HCV produced particles looks like very-low density lipoproteins (VLDL) and low-density lipoproteins (LDL) with cholesteryl esters [START_REF] Merz | Biochemical and Morphological Properties of Hepatitis C Virus Particles and Determination of Their Lipidome[END_REF].

HCV envelope glycoproteins play a role in receptor binding and mediate the fusion process between the viral envelope and an endosomal host cell membrane [START_REF] Krey | The Disulfide Bonds in Glycoprotein E2 of Hepatitis C Virus Reveal the Tertiary Organization of the Molecule[END_REF]. Crystal structures of the E2 glycoprotein was recently published [START_REF] Kong | Hepatitis C Virus E2 Envelope Glycoprotein Core Structure[END_REF][START_REF] Khan | Structure of the core ectodomain of the hepatitis C virus envelope glycoprotein 2[END_REF]. Papers have suggested that E1 should be the fusion protein or a fusion partner of an E1E2 fusion complex [START_REF] Lavillette | Characterization of Fusion Determinants Points to the Involvement of Three Discrete Regions of Both E1 and E2 Glycoproteins in the Membrane Fusion Process of Hepatitis C Virus[END_REF][START_REF] Douam | Critical interaction between E1 and E2 glycoproteins determines binding and fusion properties of hepatitis C virus during cell entry: Hepatology[END_REF].

f. HCV life cycle HCV entry

The HCV virion is associated with lipoproteins to form a complex particle called lipoviroparticle [START_REF] Dubuisson | Virology and cell biology of the hepatitis C virus life cycle -An update[END_REF].

The life cycle began by binding to glycosaminoglycans (GAGs) and SRB1. Then, the virus follows a multi-steps process. A series of specific cellular entry factors are inolved, including SRB1, CD81, tight-junction proteins, CLDN1 and OCLN, EGFR, transferrin receptor (TfR), and NPC1L1 and signaling proteins. Entry of HCV is probably restricted to hepatocytes. After binding to several components of the host cell, HCV particle is internalized by clathrin-mediated endocytosis and fusion takes place in endosomes [START_REF] Douam | Critical interaction between E1 and E2 glycoproteins determines binding and fusion properties of hepatitis C virus during cell entry: Hepatology[END_REF][START_REF] Dubuisson | Virology and cell biology of the hepatitis C virus life cycle -An update[END_REF].

HCV RNA translation and replication

IRES initiates translation of the HCV genome into a single polyprotein (Figure 1). Viral and host encoded proteases process the viral polyprotein into the mature proteins, core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B [START_REF] Moradpour | Hepatitis C Virus Proteins: From Structure to Function[END_REF]. Peptidases mediate cleavage of the structural proteins, core, E1, E2, and the p7/NS2 junction. NS3 mediates cleavage of NS4A and NS4B, and then NS4A associates with the N-terminus of NS3. The resulting NS3/4A protease complex can then cleave at the NS4B/5A and NS5A/5B junctions. The cleavage between NS2 and NS3 is mediated by the NS2 cysteine protease.

After translation, the HCV proteins are associated with membranes derived from the endoplasmic reticulum (ER). NS3/4A, NS4B, NS5A, and NS5B constitute the viral proteins of the replication process, which replicates the positive sense RNA genome through a negative strand intermediate [START_REF] Lohmann | Hepatitis C Virus RNA Replication[END_REF].

The viral RNA-dependent RNA polymerase NS5B is the key enzyme of RNA synthesis. RNA genomes are translated to produce new HCV proteins, and are RNA templates for further RNA replication and they are assembled to form infectious virions.

HCV, to replicate its genome, induces massive rearrangements of intracellular membranes to create in the cytoplasm a micro-environment, called the "membranous web" [START_REF] Lohmann | Hepatitis C Virus RNA Replication[END_REF]. Analyses with electron microscopy and 3D reconstructions show double-membrane vesicles accumulating during the RNA replication peak. NS5A is the only protein capable to induce double-membrane vesicles [START_REF] Dubuisson | Virology and cell biology of the hepatitis C virus life cycle -An update[END_REF].

A large number of host factors influence translation and replication of HCV which were identified using different approaches [START_REF] Shulla | Hepatitis C virus-host interactions, replication, and viral assembly[END_REF].

Lipid droplets (LDs) are also found in ultrastructural studies of the membranous web. It is suggested that they play a role in the coordination of viral RNA synthesis and virion morphogenesis [START_REF] Dubuisson | Virology and cell biology of the hepatitis C virus life cycle -An update[END_REF]. By interacting with different cellular partners, NS5A plays a central role in the formation of the membranous web and the recruitment of the replicase around LDs.

HCV assembly and release

The implication of the viral non-structural proteins, like in other members of Flaviviridae family, is important in the HCV particle assembly. The core protein interacts with the genomic RNA to form the nucleocapsid. After cleavage of its C-terminus, core protein is loaded onto LDs. The junction between core-loaded LDs and the replication-complex-rich ER membranes is the site of virion assembly.

HCV glycoproteins E1 and E2 form a complex retained in the ER which migrate close to LDs where assembly takes place [START_REF] Miyanari | The lipid droplet is an important organelle for hepatitis C virus production[END_REF]. Interactions between NS2 and E1, E2, and p7 and are essential for the migration of E1E2 heterodimer at the virion assembly site [START_REF] Dubuisson | Virology and cell biology of the hepatitis C virus life cycle -An update[END_REF]. P7 is also necessary for the final steps of capsid assembly and envelopment [START_REF] Gentzsch | Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment[END_REF].

The other non-structural proteins are also involved in the assembly process. The C-terminal domain of NS5A is essential for NS5A interaction with the LD-bound core protein, and HCV assembly. Within the replicase, the NS3/4A And NS4B and NS5B, have been implicated in virus assembly. Thus, newly replicated viral genomes are transferred to the assembly sites via NS3/4A or NS5A, and NS2, and p7 connect replication complexes and core proteins to the glycoproteins [START_REF] Dubuisson | Virology and cell biology of the hepatitis C virus life cycle -An update[END_REF].

The next steps in HCV virion morphogenesis are linked to the metabolism of VLDL assembly but is poorly understood. In patients, HCV particles circulate as hybrid particles, known as lipoviroparticles (LVPs), combining components of both HCV and very low-density lipoprotein (VLDL). The importance of ApoE for the life cycle of HCV has been confirmed by studies showing ApoE to be required for LVP production [START_REF] Boyer | The Association of Hepatitis C Virus Glycoproteins with Apolipoproteins E and B Early in Assembly Is Conserved in Lipoviral Particles[END_REF]. LVPs circulate in the serum of patients as part of a mixed population, including lipoprotein-like particles and complete viral particles [START_REF] Piver | Ultrastructural organisation of HCV from the bloodstream of infected patients revealed by electron microscopy after specific immunocapture[END_REF].

DIAGNOSIS OF HCV INFECTION

As already mentioned in the introduction, the development of oral very effective, direct acting antivirals agents (DAAS), with very low toxicity has completely changed the management of HCV infection, with the opportunity to eradicate HCV and cure the estimated 71 million people living with HCV around the world. However, while DAAS are now available, the access to treatment is still difficult.

Moreover, very few people living with HCV are aware of their infection. The challenge is to offer access to affordable and simple diagnostic tools, especially in low and middle-income countries (LMICs) where HCV is frequent and the diagnostic step essential to eradicate HCV worldwide.

From a public health perspective, HCV diagnosis should be as more simple as possible and should be linked to the HCV cure process. It also has been underlined that liver assessment is very important for choosing treatment duration and particularly for monitoring patients with cirrhosis [START_REF] Calès | Evaluation and improvement of a reliable diagnosis of cirrhosis by blood tests Évaluation et amélioration du diagnostic fiable de cirrhose par les tests sanguins[END_REF][START_REF] Cale | Improved fibrosis staging by elastometry and blood test in chronic hepatitis C[END_REF].

In this chapter, we will discuss the different categories of laboratory tests: classical serologic tests able to detect anti HCV antibodies (Ab), named indirect tests, and assays that can detect and quantify HCV particle components such as HCV RNA or HCV Core Antigen (cAg) named direct tests. We will also discuss new virological tools for screening and monitoring HCV infection. All this tests are complementary for the diagnosis of infection, therapeutics decisions and to assess treatment efficacy (cure or eradication on infection) after the end of treatment.

Indirect Tests

Viral antigens used to develop serological assays were obtained by cloning HCV genome soon after the discovery of HCV. Serological tests were first developed for blood donors screening and prevent blood transmission.

Anti HCV antibodies (Ab) are generally identified using ELISA (enzyme linked immunosorbent assay) or chemiluminescence immuno-assay (CLIA) tests. We are now using third generation assays which incorporate several HCV epitopes from the different structural and nonstructural regions of HCV (C100-3, NS4 region in the first generation assays and C22 (Capsid region), C33 (NS3 region) and NS5 antigen in the third generation assay).

During acute infection, as in many other viral infections, there is a window period where specific anti-HCV Ab are not detectable. With the third generation assays, the window period can vary from patient to patient and from test to test: the seroconversion generally occurs after 4 to 8 weeks after the onset of infection [START_REF] Blach | Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study[END_REF][START_REF] Razavi | Hepatitis C virus prevalence and level of intervention required to achieve the WHO targets for elimination in the European Union by 2030: a modelling study[END_REF][START_REF] Chevaliez | Diagnosis and management of chronic viral hepatitis: Antigens, antibodies and viral genomes[END_REF][START_REF] Kamili | Laboratory Diagnostics for Hepatitis C Virus Infection[END_REF]. Fourth-generation assays are also known as "antigen-antibody combo" tests, the principle of these assays being the detection of both anti-HCV Ab and HCV Ag at the same time [START_REF] Larrat | Performance of an antigen-antibody combined assay for hepatitis C virus testing without venipuncture[END_REF].

These assays are positive earlier in acute infections and thus reduce the window period [START_REF] Marwaha | Current testing strategies for hepatitis C virus infection in blood donors and the way forward[END_REF].

Sensitivity and specificity of third generation assays are high in our countries, when tests are approved by European or American authorities (FDA) (nearby 99%). Some false positive results are described in various pathological conditions and in blood donors. It has been shown that high titles of anti HCV antibodies (high ratio) are more often true positive than low titles which are more often false positive [START_REF] Dubuisson | Virology and cell biology of the hepatitis C virus life cycle -An update[END_REF][START_REF] Gentzsch | Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment[END_REF].The sensitivity was sometimes in default with first generation assays especially in immunocompromised patients, however the third generation tests generally are able to detect anti-HCV anti-Ab in these patients except if immunosuppression is serious. In this case, serological tests may be negative despite active infection [START_REF] Marwaha | Current testing strategies for hepatitis C virus infection in blood donors and the way forward[END_REF].

Supplemental tests

Recombinant immunoblot assays (RIBA) were used in the past to confirm the ELISA tests. They were also useful to determinate false positivity in case of a weakly EIA signal in the absence of viremia [START_REF] Lunel-Fabiani | Virological tools for the diagnosis and follow up of hepatitis C: use and role of new tests[END_REF]. The results indicated the presence of specific anti-HCV antibodies, meaning that tested individuals have currently an active infection or have come into contact with HCV at some point. But they are now no more used.

Direct classical tests

HCV core Antigen detection was developed in the early 2000 [START_REF] Lunel | Antigène et charge virale[END_REF]. At the beginning, they were commercialized as shaper alternatives to nucleic acid test (NAT) i.e. to screen blood donors or to be used in low-and middle-income countries (LMICS), where laboratories are not well equipped. These assays are either ELISA or CLIA and require less expertise and are less expensive compared to NAT.

A lot of teams have evaluated HCV core Ag in transfusion or for diagnosis of infection and for monitoring treatments like interferon [START_REF] Gaudy | Usefulness of the Hepatitis C Virus Core Antigen Assay for Screening of a Population Undergoing Routine Medical Checkup[END_REF][START_REF] Veillon | Comparative evaluation of the total hepatitis C virus core antigen, branched-DNA, and amplicor monitor assays in determining viremia for patients with chronic hepatitis C during interferon plus ribavirin combination therapy[END_REF][START_REF] Pivert | Comparaison des cinétiques de l'ARN et de l'antigène de capside du virus de l'hépatite C dans le suivi thérapeutique des patients co-infectés par le virus de l'hépatite C et le virus de l'immunodéficience humaine, traités par bithérapie interféronribavirine, dans le cadre du protocole RIBAVIC[END_REF][START_REF] Chevaliez | Clinical utility of hepatitis C virus core antigen quantification in patients with chronic hepatitis C[END_REF][START_REF] Rockstroh | HCV core antigen as an alternate test to HCV RNA for assessment of virologic responses to all-oral, interferonfree treatment in HCV genotype 1 infected patients[END_REF] and more recently DAAS [START_REF] Lamoury | Hepatitis C virus core antigen: A simplified treatment monitoring tool, including for posttreatment relapse[END_REF]. New generation CLIA quantitative test (Architect, Abbott laboratories) has good sensitivity but less than that of real time PCR (limit of detection equivalent to 500-3000 HCV RNA IU/ml) [START_REF] Chevaliez | Clinical utility of HCV core antigen detection and quantification in the diagnosis and management of patients with chronic hepatitis C receiving an all-oral, interferon-free regimen[END_REF][START_REF] Chevaliez | New virological tools for screening, diagnosis and monitoring of hepatitis B and C in resource-limited settings[END_REF].

Qualitative or quantitative detection of HCV RNA Molecular tests are used to confirm HCV infection. They used either RT PCR or transcription mediated amplification (TMA). These tests are indicated to confirm replication in subjects found anti HCV Ab positive or to detect viremia in acute infection, during the window period (when Ab assays are still negative). Commercialized HCV RNA tests are very sensitive and able to detect and quantify less than 30 copies/ml (reference) or <15 IU/ml of HCV RNA or less [START_REF] Guillou-Guillemette | Detection and Quantification of Serum or Plasma HCV RNA: Mini Review of Commercially Available Assays[END_REF]. Quantification of HCV RNA is more often used.

The following table 1 summarizes the available, approved tests for HCV RNA detection and/or 298 quantification [START_REF] Guillou-Guillemette | Detection and Quantification of Serum or Plasma HCV RNA: Mini Review of Commercially Available Assays[END_REF][START_REF] Chevaliez | New Virologic Tools for Management of Chronic Hepatitis B and C[END_REF]. 299 These tests are so called POC tests because they can be used close to the patient, locally or can be sent by post without needing cold chain.

These tests use plasma [START_REF] Gupta | Point -of -care testing (POCT) in molecular diagnostics: Performance evaluation of GeneXpert HCV RNA test in diagnosing and monitoring of HCV infection[END_REF][START_REF] Mchugh | Multicenter Evaluation of the Cepheid Xpert Hepatitis C Virus Viral Load Assay[END_REF], or whole blood drowns by veno-punction. Other matrices, like capillary blood, using manual or automated device (lancette) which can be used in children (puncture of the finger or the heel). Capillary blood can be collected in a tube, a pipette or paper (DBS, dried blood spot). Oral fluid similarly can be also collected [START_REF] Larrat | Performance of an antigen-antibody combined assay for hepatitis C virus testing without venipuncture[END_REF]. DBS can be used to collect whole blood specimens for EIA detection of anti-HCV antibodies in a central laboratory. A second spot on the same paper can be used to test for HCV RNA (to be performed in anti-HCV antibody-positive samples) [START_REF] Tuaillon | Dried blood spot for hepatitis C virus serology and molecular testing[END_REF].

Rapid diagnostic tests (RDT)

Their principle is to capture antibodies on a solid surface where complementary antigens are attached.

Different technologies are used, like immune-chromatography, lateral flow tests, cassettes. There is a control line which can confirm the correct liquid migration.

RDT have a lot of advantages but also disadvantages: they are easy to use, do not need special equipment, have generally good sensitivity and specificity, when approved by international authorities like WHO or FDA. The result can be given very fast, permitting early management /cure of patients [START_REF] Chevaliez | Prospective assessment of rapid diagnostic tests for the detection of antibodies to hepatitis C virus, a tool for improving access to care[END_REF].

However, the interpretation of the result is subjective and the quality of the results may depend on the quality of matrices (i.e patients with low Ab quantity can be falsely negative) [START_REF] Chevaliez | New virological tools for screening, diagnosis and monitoring of hepatitis B and C in resource-limited settings[END_REF].

HCV diagnosis

HCV can be diagnosed by the detection of anti-HCV antibodies in the serum or plasma samples, where a non-reactive result is considered as absence of HCV infection. To assess that HCV antibodies implies active infection, it needs to be confirmed by a HCV RNA test.

In figure 4, we propose a simple algorithm for HCV testing applicable theoritically worldwide.

However, screening strategies for HCV infection should be defined according to the local epidemiology of HCV infection, ideally within the framework of national plans [START_REF] Pawlotsky | EASL Recommendations on Treatment of Hepatitis C[END_REF]. If acute infection is suspected, as previously mentioned, initial testing for HCV RNA or HCV core antigen may be required in immunocompromised patients, patients previously tested for HCV infection and those at risk of recent infection during the window period.

HCV infection can sometimes be cleared after the acute phase in a low percentage of cases [START_REF] Kumar | Genotyping & diagnostic methods for hepatitis C virus: A need of low-resource countries[END_REF][START_REF] Lingala | Natural History of Hepatitis C[END_REF] and individuals may remain anti-HCV Ab positive without any detectable viremia. Thus, if anti-HCV antibodies are detected, the presence of HCV RNA, or alternatively HCV core antigen (if HCV RNA assays are not available and/or not affordable) should be determined to identify patients with active infection [START_REF] Larrat | Performance of an antigen-antibody combined assay for hepatitis C virus testing without venipuncture[END_REF][START_REF] Pawlotsky | EASL Recommendations on Treatment of Hepatitis C[END_REF][START_REF] Panel | Hepatitis C guidance: AASLD-IDSA recommendations for testing, managing, and treating adults infected with hepatitis C virus: HEPATITIS C VIRUS GUIDANCE PANEL[END_REF]. Of course, clinical and biological evaluation is mandatory if acute or chronic infection is confirmed. : Nucleic Acid Amplification Test. 3 : HCV genotyping is only necessary in few cases to decide the treatment [START_REF] Pawlotsky | EASL Recommendations on Treatment of Hepatitis C[END_REF].

HCV genotyping methods

Generally, the most recommended methods to determine the HCV genotype are sequencing the highly conserved regions of the HCV genome such as NS5, core, E1 and 5' UTR. There are also kit-based assays using probes corresponding to genotype specific oligonucleotides and amplified products are then hybridized with these probes [START_REF] Kumar | Genotyping & diagnostic methods for hepatitis C virus: A need of low-resource countries[END_REF]. The most widely used method is based on reverse hybridization with the line probe assay [START_REF] Stuyver | Second-generation line probe assay for hepatitis C virus genotyping[END_REF]. Deep sequencing will soon be also useful for genotyping.

Together with prior treatment failure and the presence of severe fibrosis, the HCV genotype, including genotype 1 subtype, may still be useful in some cases to decide the treatment regimen and duration, particularly in previous non responders or in reinfections [START_REF] Pawlotsky | EASL Recommendations on Treatment of Hepatitis C[END_REF].

However, it is possible to treat individuals with pan-genotypic HCV drugs without HCV genotype/subtype, in particular in LMICs where molecular tests are not available or too costly, or to simplify therapy to improve access to care in some populations i.e, IV drug addicts [START_REF] Pawlotsky | EASL Recommendations on Treatment of Hepatitis C[END_REF].

CONCLUSION

The identification of HCV in 1989, a few years after the blood scandal, was one of the major event in the medical field, as recently recognized by the Medicine Nobel price. The main consequences of this discovery were the development of screening tests first used for blood donors, to avoid post transfusion hepatitis (which was observed in 5% of blood recipients, before the implementation of anti HCV Ab assays). Almost thirty years later, the increasing knowledge of HCV biology was also at the origin of the development of DAA able to achieve HCV cure. The last challenge will be to increase access to diagnostic and care of HCV infection, especially in low income countries.

Figure 1 :

 1 Figure 1 : HCV genome and viral particle structure. The HCV genome is composed of an open reading

Figure 3 :

 3 Figure 3 : Global distribution and prevalence of hepatitis C virus genotypes [38]. *Genotype 7 has

Figure 4 :

 4 Figure 4: Algorithm for hepatitis virus C testing. *Initial testing for HCV RNA or HCV core antigen is

  Before DAAS availability, the follow up of patients treated with Pegylated interferon and ribavirin was based on HCV RNA decline which was a good predictor of response. With DAAs, HCV RNA measurements are no longer required/necessary during treatment but they are recommended 3 months after the end of therapy, to confirm HCV eradication, or in case of reinfection suspicion[START_REF] Pawlotsky | EASL Recommendations on Treatment of Hepatitis C[END_REF].

	CAP/CTM HCV 2.0 (Roche) Xpert® HCV (Cepheid) VERIS HCV (Beckman) Aptima® HCV Quant Dx (Hologic)	0.650 mL plasma/serum 1 mL plasma/serum 1 mL plasma 0.5 mL plasma/serum Results are expressed in IU/ml. New virological tools for indirect/direct diagnosis 15-10 8 10-10 8 12-10 9 TMA 10-10 8 Point of care tests (POC):	2 IU/mL 9.3/8.8 IU/mL 4.0/6.1 IU/mL 12 IU/mL 4.3/3.9 IU/mL	12 15 10 12 10	4.6 5.7 1.75 1.75 2.5	No No Yes Yes Yes

Table 2

 2 summarizes the different available HCV rapid diagnostic tests[START_REF] Chevaliez | New virological tools for screening, diagnosis and monitoring of hepatitis B and C in resource-limited settings[END_REF]. 326

					Sensitivity -	Specificity -
				Volume	Reading
		Manufacturer	Sample Type		Whole Blood	Whole blood
				needed (µL)	time (min)
					(95%CI)	(95%CI)
			Oral fluid,		99.4% (97.7-	100% (97.9-
	Oraquick ®	Orasure	whole blood,	5 or 40 (oral	99.9)	100.0)
	HCV Rapid	Technologies,			20-40
	Antibody Test	Bethlehem, PA	serum,	fluid)	97.6% (oral	100% (oral
			plasma		fluid)	fluid)
				30 (serum,
			Whole blood,
	Toyo® anti-	Turklab, Izmir,		plasma) or	96.2% (93.3-	98.2% (94.8-
			serum,		5-15
	HCV test	Turkey		60 (whole	98.0)	99.4)
			plasma
				blood)
			Whole blood,
	Labmen®	Turklab, Izmir,			62.7 % (54.8-	100% (94.4-
			serum,	10	15
	HCV test	Turkey			69.5)	100.0)
			plasma
	Signal HCV	Span Divergent,	Serum,
				100	10
	v2.0	Udhna, India	plasma
		MP	Whole blood,
	MultiSure
		Biomedicals,	serum,	25	15
	HCV
		Singapore	plasma
	Assure HCV	MP	Whole blood,	5 (serum,
	Rapid test	Biomedicals,	serum,	plasma) or
		Singapore	plasma	50 (whole


Fabienne Battaglia-Brunet

Marina Le Guédard

Olivier Faure

Mickael Charron

Daniel Hube

Nicolas Devau

Catherine Joulian

Hugues Thouin

Jennifer Hellal

Influence of agricultural amendments on arsenic biogeochemistry and phytotoxicity in a soil polluted by the destruction of arsenic-containing shells

Keywords: arsenic, soil, water, fertilizers, microorganisms, omega 3 index

Agricultural soils can contain high arsenic (As) concentrations due to specific geological contexts or pollution. Fertilizer amendments could influence As speciation and mobility thus increasing its transfer to crops and its toxicity. In the present study, field-relevant amounts of fertilizers were applied to soils from a cultivated field that was a former ammunition-burning site. Potassium phosphate (KP), ammonium sulfate and organic matter (OM) were applied to these soils in laboratory experiments to assess their impact on As leaching, bioavailability to Lactuca sativa and microbial parameters. None of the fertilizers markedly influenced As

Introduction

High concentrations of the toxic element arsenic (As) in soils generally originate from mining and industrial activities, long-term applications of As-containing pesticides or the geochemical background. Among industrial activities, storage or destruction of As-bearing molecules used in chemical weapons during the wars has locally resulted in high As concentrations in soils [START_REF] Bausinger | Exposure assessment of a burning ground for chemical ammunition on the Great War battlefields of Verdun[END_REF][START_REF] Thouin | Characterization and mobility of arsenic and heavy metals in soils polluted by the destruction of arsenic-containing shells from the Great War[END_REF][START_REF] Hube | Industrial-scale destruction of old chemical ammunition near Verdun: a forgotten chapter of the Great War[END_REF].

When soils affected by As pollution are submitted to agricultural practices, arsenic speciation, bio-availability for plants and mobility towards the water phase may be changed. Major phenomena influencing As mobility [START_REF] Smith | Arsenic in the soil environment: a review[END_REF] include; [START_REF] Lemonte | Sea Level Rise Induced Arsenic Release from Historically Contaminated Coastal Soils[END_REF] pH which influences AsIII and AsV oxy-anions charge, (2) redox conditions, which influences As speciation and the stability of iron oxides that are essential As-bearing phases, and (3) competing substances, that may favour As desorption from solid phases. In particular, phosphate, a structural analogue of AsV can compete with As for sorption on iron oxides [START_REF] Smith | Chemistry of inorganic arsenic in soils: kinetics of arsenic adsorption-desorption[END_REF]. Thus, in agricultural soils, fertilizing practices involving phosphate amendments may affect As speciation and mobility. [START_REF] Brackhage | Readily available phosphorous and nitrogen counteract for arsenic uptake and distribution in wheat (Triticum aestivum L.)[END_REF] observed an increase of As mobility and uptake by wheat associated to P-fertilization in soil flooding conditions. Conversely, Nfertilization seemed to attenuate As mobility and plant uptake [START_REF] Brackhage | Readily available phosphorous and nitrogen counteract for arsenic uptake and distribution in wheat (Triticum aestivum L.)[END_REF][START_REF] Van Oort | Arsenic et vieilles parcelles[END_REF]. In addition, agricultural soils are often fertilized with organic matter (OM).

Many studies have described geochemical interactions between As and organic matter: modification of As speciation [START_REF] Redman | Natural organic matter affects arsenic speciation and sorption onto hematite[END_REF], formation of soluble complexes [START_REF] Saada | Adsorption of arsenic(V) on kaolinite and on kaolinite-humic acid complexes. Role of humic acid nitrogen groups[END_REF][START_REF] Redman | Natural organic matter affects arsenic speciation and sorption onto hematite[END_REF], competition for sorption sites [START_REF] Bauer | Mobilization of arsenic by dissolved organic matter from iron oxides, soils and sediments[END_REF], and influence of OM on microbial AsIII-oxidizing activity [START_REF] Lescure | Influence of organic matters on AsIII oxidation by the microflora of polluted soils[END_REF].

Finally, all types of amendments may impact the structure of soil microbial communities which exert a major influence on As speciation [START_REF] Yamamura | Potential for microbially mediated redox transformations and mobilization of arsenic in uncontaminated soils[END_REF]. Bacteria isolated from soils have been shown to oxidize AsIII and/or reduce AsV [START_REF] Macur | Bacterial populations associated with the oxidation and reduction of arsenic in an unsaturated soil[END_REF][START_REF] Inskeep | Detection, diversity and expression of aerobic bacterial arsenite oxidase genes[END_REF][START_REF] Bachate | Oxidation of arsenite by two βproteobacteria isolated from soils[END_REF], or to methylate this toxic metalloid [START_REF] Huang | Arsenic speciation and volatilization from flooded paddy soils amended with different organic matters[END_REF].

Filamentous fungi isolated from contaminated soils are able to reduce AsV and methylate As [START_REF] Su | Arsenic biotransformation by arsenicresistant fungi Trichoderma asperellum SM-12F1, Penicillium janthinellum SM-12F4, and Fusarium oxyporum CZ-8F1[END_REF]. Microbial transformations of As in soil have important implications because mobility, toxicity and bioavailability of this metalloid are closely related to its speciation (Smedley and Kinniburgh, 2002). The global AsIII-oxidizing activity of the microflora should tend to reduce the risk of As transfer from soil to surface water or groundwater. This global activity is the result of AsIII-oxidation and simultaneous AsV-reduction, that can occur in aerobic conditions through the activity of As resistance genes. All the modifications due to the amendments can lead to changes in the bioavailability and the toxicity of As for crops.

This study aimed to measure the impact of fertilizing practices on As mobility, speciation and transfer to crops in soils from a former chemical-ammunition-destruction facility dating from Revised manuscript with accepted corrections the interwar period and subsequently converted into agricultural land near Verdun, France [START_REF] Hube | Industrial-scale destruction of old chemical ammunition near Verdun: a forgotten chapter of the Great War[END_REF]. It is one of the most important historical areas of chemical ammunition destruction of WW I, containing arsenical chemical warfare agents, located in a sensitive zone for agriculture and groundwater. Fertilizers frequently applied for common crops such as barley, corn and wheat (potassium phosphate (KP) fertilizer, ammonium sulfate and OM) were applied to soils in a laboratory-scale experiment to assess their impact on the speciation, mobility and phytotoxicity of As as well as on As transforming microbial communities.

2.

Material and methods

Origin and characterization of soil samples

Soils were sampled from a field near Verdun, France, where there was previously a chemical ammunition destruction facility [START_REF] Hube | Industrial-scale destruction of old chemical ammunition near Verdun: a forgotten chapter of the Great War[END_REF]. The field was used as pasture in 2012 and was cultivated from 2012 to 2015 with wheat, barley and corn. Since 2015, it is fallow ground as farming was forbidden when the pollution was detected.

Surface soils (0-20 cm) were sampled with a spade in a highly polluted zone, and in a reference zone 25 m away. Each sample was taken as a composite of 5 points from 3 m × 3 m squares according to the GEMAS protocol (EGS 2008) but adapted to the small surfaces available. The soils were characterized (Table 1) according to the following methods: pH in water (NF ISO 10390), organic carbon (NF ISO 10694), total nitrogen (NF ISO 13878), Phosphorus (NF ISO 11263), total elements, major and trace elements (extraction with HF + HClO4, NF ISO 14689-1).

As species in soils were analysed by HPLC-ICP-MS after extraction with 10 mL H3PO4 1 M added to 0.4 g of freeze-dried sample, ground and homogenized by sieving (2 mm) and microwave heating [START_REF] Vergara Gallardo | Speciation analysis of arsenic in environmental solids reference materials by high-performance liquidchromatography-hydride generation-atomic fluorescence spectrometry following orthophosphoric acid extraction[END_REF]) in a closed system at 120°C during 20 min

Revised manuscript with accepted corrections (analyse performed by UT2A laboratory, Pau, France). The remaining solution was diluted to 50 mL with ultrapure water and then As species were analysed with HPLC-ICP-MS using quantification by standard additions to avoid matrix effects. As species separation was performed using an anion exchange column (Hamilton PRPX-100) and a mobile phase made of ammonium hydrogen phosphate 15 mM at pH 8.5. These analytical conditions enable the determination of AsIII, AsV and methylated As species (MMA and DMA). Details of the analytical methods are provided in SM1.

Table 1. Main characteristics of the two studied soils. (*) total element. Diphenylarsinic acid (DPAA) was analysed by HPLC-DAD and clark I, clark II, Clark oxide, triphenylarsine (TPA), and 9-phenylarsafluorene were analysed by GC-MS (Envilytix, Wiesbaden, Germany, details in SM1). They were detected only in the polluted soil that contained 0.2% DPAA and 0.1% TPA. These two last species were not quantified in the next steps of the study that focused on total As and bio-transformations of AsIII and AsV.

Microcosm experiments

Preparation

Microcosms were setup in 200 mL polystyrene pots, 50 mm diameter, whose bottoms were perforated with a 0.9 mm needle, to make 13 holes in each pot. In order to retain soil particles Revised manuscript with accepted corrections in the pot, a fine layer of glass wool was placed at the bottom of each pot and covered with 10 cm 3 of clean Fontainebleau sand. Both glass wool and sand were previously cleaned in 10 % HNO3, rinsed with demineralized water and dried before use.

Each microcosm was filled with 150 g dry soil, with or without amendments. Each condition was tested in triplicate. The quantities of amendments added to the soils in the microcosms were calculated based on the real quantities applied on site (SM2) for KP fertilizer and for ammonium sulfate. A third amendment, organic manure, was chosen as the site was used as a pasture for many years (Table 2). The amount of manure added to the soils in the microcosms was based on real quantities usually applied in cultivated fields, i.e. 10 tons.ha -1 . 4 % K and 0.6 % P. The two solid amendments (KP and manure) were added as powders to the dry soil and mixed for 24 h by rotation to homogenize. Ammonium sulfate was added as a 2.4 g.L -1 concentrated solution, 1.5 mL in each microcosm during the first watering.

Watering and incubation

Watering was always performed with Mont Roucous mineral water (pH 5.85; 3.1 mg.L -1 Na 2+ ; 2.4 mg.L -1 Ca 2+ ; 0.5 mg.L -1 Mg 2+ ; 2.0 mg.L -1 SO4 2-; 6.3 mg.L -1 HCO3 -; 3 mg.L -1 NO3 -), used to simulate rain water.

For the first watering, 53 mL of water was carefully poured on the soil surface. Then, 24 h after the first watering, the microcosms were watered again, sufficiently to recover 20 mL of percolated water in the underlying pot. The quantity of inlet and outlet water was recorded by weighing the recipients. Global aerobic non-saturated conditions were maintained.

Microcosms were incubated at 25°C in the dark, with 80 % atmospheric humidity. Soils never dried out during the incubation. Percolated water was filtered at 0.45 µm. Determination of As speciation was performed using an ion exchange method [START_REF] Kim | Separation of inorganic arsenic species in groundwater using ion exchange method[END_REF], the separated As species and the total As in water were determined by oven-AAS (details in SM1). Soils were watered at the beginning of experiment then after 1 week, 1 month and 3 months.

Final determination of biological parameters

At the end of the experiment (3 months), soils were sampled to determine biological parameters. All measurements were performed in triplicate.

Most Probable Number (MPN) determinations

Active As-transforming microorganisms present in microcosms were enumerated by the following MPN methods. To enumerate the active AsIII-oxidizing microorganisms (Thouin et Revised manuscript with accepted corrections al., 2016), wet soil (equivalent to 2.5 g dry soil) was placed in a sterile glass erlenmeyer flask with 10 ml of sterile physiological water (9 g.l -1 NaCl in demineralized water), agitated for 30 min at 25°C, then sonicated 2 x 20 s at 45 kHz. Triplicate suspensions were prepared for each soil. Soil suspensions were serially diluted in sterile physiological water up to dilution 10 -7 .

CAsO1 mineral medium [START_REF] Battaglia-Brunet | An arsenic(III)-oxidizing bacterial population: selection, characterization, and performance in reactors[END_REF] were added. A white precipitate appeared when AsIII was present, i.e. when AsIII-oxidizing bacteria were absent (negative well). Un-inoculated wells served as negative blanks, and wells containing CAsO1 medium with 100 mg.L -1 AsV served as a positive reference. The number of positive wells for each dilution was determined, and the most probable number of bacteria in dilutions was given by the Mc Grady table for 5 tubes.

To enumerate Active AsV-reducing microorganisms [START_REF] Thouin | Influence of environmental changes on the biogeochemistry of arsenic in a soil polluted by the destruction of chemical weapons: a mesocosm study[END_REF], soil suspensions were prepared as described for AsIII-oxidizing microorganisms then diluted in sterile physiological saline solution to a dilution of 10 -6 . CAsO1 basal mineral medium [START_REF] Battaglia-Brunet | An arsenic(III)-oxidizing bacterial population: selection, characterization, and performance in reactors[END_REF] was complemented with 20 mM lactic acid and AsV (100 mg.L -1 ). The medium was distributed in Microtest TM Tissue culture plates (96 wells), 250 µL per well.

Each well was inoculated with 25 µL of diluted soil suspension. Five wells were inoculated with for each dilution. Culture plates were incubated at 25°C for 10 days in anaerobic jars with Anaerocult packs (Merck). Presence of AsIII formed in the wells during incubation was revealed as described above. Positive well numbers were determined for each dilution, and the Brunet et al., 2002, details in SM1), As in the AsV-containing aqueous phase was quantified by flame AAS (Varian, Palo Alto, CA, USA). AsIII-oxidation and AsV-reduction rates were calculated between each point of analyse (evolution of AsV concentration divided by the time between sampling events).

Toxicity and transfer to plants

Impact of fertilizers on As bioavailability and phytoxicity was evaluated with the ecotoxicological test AFNOR XP X 31-233 (2012), using the Omega-3 Index based on the analysis of leaf fatty acid composition of Lactuca sativa grown under controlled conditions and by measuring the As leaf content in the same plants. Fifteen Lactuca sativa seeds were sown in plastic pots filled with 430 g of dry soil, with or without amendment, and control pots containing a standard soil (loam). Each condition was tested in triplicate using the same

Revised manuscript with accepted corrections quantities of amendments as for the microcosm experiment. Amendments were added to the soil at the beginning of the experiment and seedlings were grown for 17 days in a growth chamber under a 16 h/8 h photoperiod (8000 lx or 10000 lx white light intensity) and a 22°C/16°C day/night temperature. Pot location inside the growth chamber was randomly changed every 2 to 3 days to homogenize light exposure and watering. One week after germination, germination rates were determined and the number of young seedlings per pot was reduced. 14 days after germination, the aerial parts of seedlings were weighed, and the first leaf of some plants (or a section of it: 20 to 200 mg of fresh tissue) used to determine the leaf fatty acid composition, was placed in a glass tube containing 1 mL of a methanol/H2SO4 AsIII oxidation and AsV reduction rates were tested for homogeneity of variance and normal distribution. One-way analysis of variance (ANOVA) and Tukey HSD (Honestly Significantly Different) tests were carried out to test for any significant differences between the means. Differences between means at the 5% level (P < 0.05) were considered significant.

Results

Initial soil analyses showed that phosphate concentration was ten times higher in the polluted soil than in the reference soil, and the potassium concentration was two times higher in the polluted soil than in the reference soil (Table 1). Average concentrations of total As were close to 1000 ppm in the polluted soil and to 20 ppm in the reference soil. Concentrations of total As in the percolated water were significantly lower for the reference soil microcosms (1 to 6 µg.L -1 , Figure 1A) compared to polluted soil microcosms (2000 to 5000 µg.L -1 , Figure 1B). Although the ratio polluted/reference for total As was close to 50X in the solids, it was in the range of 1000X in the percolated water. This indicated that As was much more mobile in the polluted soil than in the reference soil. An increase of leached As was linked to the addition of KP fertilizer and a decrease of this leached As was linked to the addition of ammonium sulfate amendment in the polluted soil, at the first watering event (day 0, Figure 1B).

Cumulated amounts of total As leached from the soils (Figure 2) indicated that total As leached from the polluted soil was 1000X higher than the cumulated As leached from the reference soil. None of the amendments significantly increased or reduced As leaching compared with the blank experiment. A slight increase could be linked to the addition of KP fertilizer in the polluted soil and the lowest values of leached As were obtained with the ammonium sulfate amendment (Figure 2A and2B).

Revised manuscript with accepted corrections Speciation (quantification of AsIII and AsV) was monitored in water from microcosms containing the polluted soils only, as As concentrations were too low in the reference soil percolation water. AsIII concentrations of AsIII varied between 5 and 15 µg.L -1 in these leachates, representing 0.1 to 0.5 % of total As. Total AsIII leached during the experiment (Figure 2C) ranged from 700 to 1000 ng. These amounts of AsIII represent less than 1 % of the leached As, however they are higher than the total amounts of As leached from the reference soil. The amount of total leached AsIII was slightly higher with the KP amendment.

However, the difference with other conditions is not statistically significant. Globally, the amendments had no significant influence on As speciation in the leached water during this experiment.

Final pH values (SM3) were close to the initial values, the general tendency being a small decrease during the experiment. None of the amendments induced an increase in pH sufficient to mobilize As.

Numbers of AsIII-oxidizing microorganisms were in the range of 10 4 cells.g -1 for the reference soil, and 10 6 cells.g -1 for the polluted soil (Figure 3A), thus 100 times higher in the polluted soil. Numbers of AsV-reducing microorganisms were in the range of 10 6 cells.g -1 whatever the soil type (Figure 3B). Globally cell numbers were slightly lower in the reference soil compared to the polluted soil. However, this difference was less marked than it was for AsIII-oxidizing microorganisms. Non-parametric statistical tests (XLSTAT 2018.2.50583 -Kruskal-Wallis test Two-sample t-test and z-test), performed as the comparison of all values with reference soil and all values with polluted soil indicates that the two groups (a and b on

Figure 3) are significantly different for both AsIII-oxidizing and AsV-reducing bacteria.

However, no significant influence of the different amendments on the MPN of Astransforming microorganisms could be detected.

Revised manuscript with accepted corrections

Results of the AsIII-oxidizing activity tests (Figure 4A) clearly showed two trends: one observed with the group of polluted soil conditions, whose microbial communities oxidized AsIII very rapidly, and a second trend observed with the reference soils where kinetics were slower and clearly affected by the amendments. The maximum AsIII-oxidation rate was significantly higher with the polluted than with the reference soil. Considering exclusively the reference soil, the ammonium-amended soil gave a value of maximum AsIII-oxidation rate significantly lower than the non-amended soil, whereas the KP and organic amendments tended to decrease the AsIII-oxidizing rate but not significantly.

Results of the AsV-reducing activity tests (Figure 4B) indicated a rapid complete reduction of AsV after two days, for all soils. Thus, the AsV-reduction rate was similar for all conditions.

As contents in the lettuce leaves are shown in Figures 5A and5B. As contents in leaves are significantly higher in lettuces grown on polluted soils (7000 to 9000 µg.kg -1 DW) than in those grown on reference soils (60 to 120 µg.kg -1 DW). As transfer factor from soil to shoot (TFshoot/soil) for polluted soil is 2.7-fold higher than for the TFshoot/soil from the reference soil.

Thus, as observed with microcosm experiments, As from the polluted soil appeared more mobile and more phytoavailable than As from the reference soil.

While the phosphate concentration in the soil is 10X higher in the polluted soil compared with the reference soil, the phosphorus content in the lettuce leaves was similar for both soils (SM4). Concerning the amendment intake, results showed a significant (except for As in the leaves of plants grown on the reference soil) increase of As and P contents in lettuce leaves in both soils amended with KP (Figure 5 and SM4). The other amendments did not show any differences with the un-amended soil, regardless of the soil type (reference or polluted).

The Omega-3 Index is a standardized biomarker to evaluate the possible toxic effects of contaminants on plants (AFNOR XP X31-233, 2012). This biomarker measures the degradation of polyunsaturated fatty acids and decreases when lipid peroxidation, caused by an excess of reactive oxygen species (ROS) in the presence of contaminants, increases.

Results showed that the Omega-3 Index was significantly lower for lettuces grown on the polluted soil compared to the reference soil (Figure 6). This indicated that lipid peroxidation was higher in lettuces grown on the polluted soil, which was therefore more phytotoxic than the reference soil. This phytotoxicity of As in the polluted soil caused a decrease of the lettuce seedling growth, as shown in SM5 (69.02 mg of FW.plant -1 ±13.73 and 30.34 mg of FW.plant - 1 ±4.97 for reference soil and the polluted soil, respectively). Moreover, while there was no difference between the un-amended and amended reference soils, the Omega-3 Index increased significantly for lettuces grown on polluted soil amended with ammonium sulfate.

Thus, addition of ammonium sulfate in the polluted soil seemed to reduce As induced lipid peroxidation.

Discussion

Among the parameters controlling the behaviour of As in soils, redox conditions play a major role. Experiments performed with microcosm systems allowing control of redox conditions showed that low Eh values induced mobilization of As from a flood plain soil [START_REF] Frohne | Controlled variation of redox conditions in a floodplain soil: Impact on metal mobilization and biomethylation of arsenic and antimony[END_REF], a freshwater marsh delta soil [START_REF] Shaheen | Redox effects on release kinetics of arsenic, cadmium, cobalt, and vanadium in Wax Lake Deltaic freshwater marsh soils[END_REF] and an historically contaminated coastal soil [START_REF] Lemonte | Sea Level Rise Induced Arsenic Release from Historically Contaminated Coastal Soils[END_REF]. Here, non-saturated conditions were maintained in microcosms, so the mobility of As should mainly be controlled by other factors. Although

AsV adsorption on iron oxides is known to decrease with pH [START_REF] Dixit | Comparison of Arsenic(V) and Arsenic(III) sorption onto iron oxide minerals: implications for arsenic mobility[END_REF], As mobility in the polluted soil compared to the reference soil was probably not exclusively linked to soil pH (8.2 and 8.3 in the reference and the polluted soil, respectively, Table 1). As mobility could also be related to the higher concentration of phosphate in the polluted soil.

Indeed, phosphate concentration was ten times higher (and the potassium concentration two times higher) in the polluted soil than in the reference soil. One hypothesis to explain this is that the farmer might have provided higher quantities of KP fertilizer in this precise area in an attempt to improve crop yields. Indeed plant growth was strongly affected by the undetected pollution in this area of the field. Phosphate is an analogue of arsenate AsV [START_REF] Smith | Chemistry of inorganic arsenic in soils: kinetics of arsenic adsorption-desorption[END_REF] which competes with As for adsorption sites on iron oxides. In the percolation water, the highest total As concentrations were always observed with the KP fertilizer and could be linked to the addition of phosphate. Consequently increased As mobility may have increased the toxic impact on plant growth. In addition, as the polluted soil contained both more phosphate and more As than the reference soil, the ratio between the density of adsorption sites and concentrations of both elements was lower in the polluted than in the reference soil.

This has previously been observed in soils polluted with As, but with higher doses of phosphate fertilizers [START_REF] Davenport | Phosphate fertilizers influence leaching of lead and arsenic in a soil contaminated with lead arsenate[END_REF][START_REF] Peryea | Phosphate-Enhanced Movement of Arsenic Out of Lead Arsenate-Contaminated Topsoil and Through Uncontaminated Subsoil[END_REF]. In terms of concentration, As in the microcosm' percolation water was in the range 2-5 mg.L -1 , in the same range as concentrations already reported in water phases in contact with soils polluted by different sources of As. Thus, [START_REF] Cao | Effects of compost and phosphate amendments on arsenic mobility in soils and arsenic uptake by the hyperaccumulator, Pteris vittata L[END_REF] found 5-6 mg.L -1 of As in water leached from soil polluted with a chromium-copper-arsenic (CCA) pesticide, presenting a total As content of 135 mg.kg -1 , and Qi and Danahoe (2008) found 1 mg.L -1 of As when they used acid rainwater to leach a soil polluted by historical herbicide application and containing 300 mg.kg -1 As.

MPN of As-transforming microbes suggested that the pollution induced an increase of the abundance of active microorganisms able to modify As speciation in the soil (Figure 3). The difference observed between the reference soil and the polluted soil was more marked for AsIII-oxidizing than for AsV-reducing microbes, and that was true both for MPN and activity tests: AsIII-oxidizing microbes were roughly 100 times more abundant in the polluted soil (Figure 3A), and this soil presented a higher AsIII-oxidizing activity than the reference soil. [START_REF] Thouin | Characterization and mobility of arsenic and heavy metals in soils polluted by the destruction of arsenic-containing shells from the Great War[END_REF] showed that the AsIII-oxidizing rate increased with the As concentration

Revised manuscript with accepted corrections in samples of polluted technosoils, and suggested that the level of As concentration exerted a selective pressure on the microbial community, increasing its global efficiency to oxidize AsIII. Here, whereas amendments did not influence the AsIII-oxidizing activity of the polluted soil (Figure 4A), the fertilizers exerted a clear influence on this activity in the reference soil. AsIII-oxidizing activity was diminished by the ammonium sulfate amendment (Figure 4A). Yet, this fertilizer tended to decrease As mobility from soils towards percolation water, in particular at the beginning of the experiment with the polluted soil (Figure 1B). Our results are in agreement with those of a long-term (1929 to 2018) experiment studying the effects of agricultural amendments on the behaviour of trace elements. In that experiment, authors showed that ammonium-based fertilization induces an increase of immobilized As in the soil, compared with other types of amendments, due to reduced As leaching [START_REF] Van Oort | Arsenic et vieilles parcelles[END_REF]. This phenomenon might be linked to H + production during ammonium oxidation (nitrification). Even if no macroscopic pH decrease was observed here, protonation of the surface hydroxyl groups of iron oxides could increase AsV adsorption on these minerals in the soil [START_REF] Dixit | Comparison of Arsenic(V) and Arsenic(III) sorption onto iron oxide minerals: implications for arsenic mobility[END_REF]. Reduced As mobility from solids to the water phase might decrease As bioavailability, consequently reducing the selective pressure on the global microbial community and decreasing its efficiency to oxidize AsIII.

The organic fertilizer did not significantly influence As behaviour nor the activity and abundance of As-transforming microbes (Figures 1 to 4). In previous studies, OMwas reported to induce higher As mobility in soils [START_REF] Beesley | Assessing the influence of compost and biochar amendments on the mobility and toxicity of metals and arsenic in a naturally contaminated mine soil[END_REF]. However, contradictory results were reported about the impact of OM on As mobility [START_REF] Kumpiene | Stabilization of As, Cu, Cr, Pb and Zn in soils using amendments -a review[END_REF]. In terms of effects on microbial transformation of As, [START_REF] Lescure | Influence of organic matters on AsIII oxidation by the microflora of polluted soils[END_REF] showed that OM exerted a positive effect on AsIII-oxidation rates from 0 to 0.08 g.L -1 of organic carbon, then tended to decrease the AsIII-oxidation activity at higher concentrations. Here, the dose of organic fertilizer (0.26 %) was probably not sufficient to modify microbial activities.

Revised manuscript with accepted corrections

The solid and liquid phases of the soils contained AsV as the main As form, whereas the MPN and activity of AsV-reducing microbes was important according to Figures 3 and4.

Even when As is mainly in the form of AsV, it is not a static distribution of AsV between solids and water phases, but rather a dynamic equilibrium, involving both microorganisms oxidizing AsIII and microorganisms reducing AsV. In this system, bioreduction of AsV might play a role in As mobilisation. [START_REF] Turpeinen | Influence of microbes on the mobilization, toxicity and biomethylation of arsenic in soil[END_REF] have already observed that microbes increased the As mobility, mainly as AsV, from soils incubated in aerated conditions. We found AsV as the main As form leached from the polluted soil, with no significant effect of the amendments on this speciation, in soils that were not saturated with water, i.e. with no limitation of oxygen availability. Our results must be confirmed with further experiments performed with diverse polluted sites, and comparing biotic to abiotic conditions, and nonsaturated to saturated conditions. However, they suggest that microbial parameters would be very sensitive bio-indicators of the dynamics of As concentrations and speciation in the water phase.

Considering the pot experiments with plants, As transfer was significantly more important in lettuces grown on the polluted soil. These results showed that As was highly mobile and as a consequence highly phytoavailable in the polluted soil. This correlated with results from the microcosms where As was shown to be more mobile from soil to water within the polluted soil compared to the reference soil. In lettuces, amendment with KP induced an increase of both As and P contents in lettuce leaves, as already observed by [START_REF] Cao | Effects of compost and phosphate on plant arsenic accumulation from soils near pressure-treated wood[END_REF].

Phosphate-induced plant As uptake may have been related to the slight increase of As concentration in the leachate. The lettuces' Omega-3 Index showed that the high As content in the polluted soil increased lipid peroxidation in lettuce leaves compared to the reference soil (Figure 6). [START_REF] Bustingorri | Protective effect exerted by soil phosphorus on soybean subjected to arsenic and fluoride[END_REF] observed an increase of lipid peroxidation in soybean plants linked to the presence of As. Here, the increase of lipid peroxidation induced by the Revised manuscript with accepted corrections polluted soil, corresponding to nearly 50 % decrease of the Omega3 index (Figure 6) was coupled with a decrease of the lettuce seedling growth. Thus, in agreement with findings obtained from previous studies, the excessive production of ROS induced by As exposure promotes lipid peroxidation and causes damage in thylakoid membranes that may lower photosynthetic efficiency [START_REF] Abbas | Arsenic Uptake, Toxicity, Detoxification, and Speciation in Plants: Physiological, Biochemical, and Molecular Aspects[END_REF]. In agreement with previous results of As transfer from soil to lettuces (Figure 5), these results confirm that As is highly phytoavailable and as a consequence highly phytotoxic in the polluted soil.

The amounts of agricultural amendments applied to fields can influence the mobility, bioavailability and toxicity of As. Addition of ammonium sulfate to polluted soil was associated with an increase of the Omega-3 Index (Figure 6) and as a consequence a reduction of lipid peroxidation in lettuce leaves while no decrease was observed concerning the As uptake. This was not observed in the reference un-polluted soil, suggesting that the decrease of lipid peroxidation is possibly influenced by the contribution of ammonium sulfate to As detoxification. These results are in agreement with several studies showing that, under heavy metal stress, an application of sulfur stimulates plants [START_REF] Mishra | Thiol metabolism and antioxidant systems complement each other during arsenate detoxification in Ceratophyllum demersum[END_REF][START_REF] Duan | EuroGeoSurveys geochemical mapping of agricultural and grazing land in Europe (GEMAS) -field manual[END_REF].

In fact, sulfur (S) in higher plants is a vital component for the synthesis of some amino acids (Cysteine and Methionine) and metabolites such as gluthatione (GSH) and phytochelatins (PCs) involved in the detoxification of heavy metals. Some studies relate that As induces ROS production in plants leading to lipid peroxidation [START_REF] Shukla | Differential transcriptome modulation leads to variation in arsenic stress response in Arabidopsis thaliana accessions[END_REF] and the synthesis of PCs enzymatically synthesized from GSH. Then, PCs form complexes with As before its sequestration into the vacuoles through ABCC transporters [START_REF] Song | Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters[END_REF]. This induction of the biochemical pathways of plant S metabolism increases the S requirement under As stress (Leao et al., 2014;[START_REF] Khare | Arabidopsis ABCG34 contributes to defense against necrotrophic pathogens by mediating the secretion of camalexin[END_REF]. Therefore, the decrease of lipid peroxidation in lettuces grown on the polluted soil amended with ammonium sulfate may be explained by an improvement of the intracellular As detoxification processes due to an increase of the Revised manuscript with accepted corrections synthesis of GSH and PCs linked to an increase of S assimilation by plants. Similarly, nitrogen (N) could also play an important role in the detoxification of heavy metals in plants.

Indeed, many studies related that, under abiotic stress, nitrate supply stimulates root nitric oxide production (Sun et al, 2010;[START_REF] Simontacchi | Plant survival in a changing environment: the role of nitric oxide in plant responses to abiotic stress[END_REF] which is a bioactive signalling molecule involved in plants' response to heavy metal stress by detoxifying ROS [START_REF] Hassan | Toxic effect of Cd on rice as affected by nitrogen fertilizer form[END_REF][START_REF] Kaur | Exogenous nitric oxide (NO) interferes with lead (Pb)-induced toxicity by detoxifying reactive oxygen species in hydroponically grown wheat (Triticum aestivum) roots[END_REF]. Thus, according to these results, it seems that a better N and S nutrition status may protect the plants from As and as a consequence lead to a decrease of oxidative stress. However, more research is needed to fully understand the mechanisms of interactions between ammonium sulfate and As in plants and the importance of ammonium sulfate in detoxification.

Conclusions

The addition of fertilizing amendments, at the real average dose applied on the sites, did not strongly influence the speciation and the quantity of mobile As. Observed trends were an increase of As mobility with KP fertilizer, a decrease of mobility with ammonium sulfate amendment, and no effect of organic amendments. The quantity of As mobilised in the percolation water was bioavailable for plants and soil microorganisms. Results of an original combination of active As-transforming bacteria enumeration and As-related microbial activity tests showed that microbial parameters were strongly linked to As levels in water and in soils.

In particular, microbial AsIII-oxidizing activity proved to be very sensitive to low doses of ammonium sulfate. Although the impacts of KP and ammonium sulfate on As speciation were insignificant, the small effects observed on plant As-uptake and bacterial activities may have an impact in the long term. Consistent results were observed with microbial and plant parameters, in particular concerning the effect of ammonium sulfate fertilization. These tests

Revised manuscript with accepted corrections could be used as sensitive indicators of As bioavailability and toxicity in soils. Whereas microbial parameters can be quicker to obtain than plant indicators, As bioavailability for plants will always be very important to determine, particularly when these plants are cultivated and consumed. Max R mg.L -1 .h

  containing 100 mg.L -1 AsIII was distributed in Microtest TM Tissue culture plates (96 wells), 250 µL by well. Each well was inoculated with 25 µL of each soil suspension dilution. Five wells were inoculated for each dilution. Culture plates were incubated at 25°C for 10 days. Presence of AsIII in the wells was revealed by the formation of insoluble white complex AsIII-PyrrolidineDithioCarbamate (PDC): in each well 150 µL 0.1 M acetate buffer (pH 5) and 100 µL PDC solution (5 g.L -1 )

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Cumulated leached total As and AsIII. A: reference soil total As; B: polluted soil

Figure 3 .

 3 Figure 3. Most probable numbers of As-transforming microorganisms. A: AsIII-oxidizing

Figure 4 .

 4 Figure 4. Activity tests. A: AsIII-oxidizing activities; B: AsV-reducing activities. Error bars

Figure 5 .Figure 6 .

 56 Figure 5. As content in the lettuce leaves grown on A: the reference soil; B: the polluted soil

  Figure 1

  Figure 5

Table 2 .

 2 Amendments

		for microcosms. Hypothesis: Depth of amended soil 0.3 m, soil density
	1.3.
	Amendments	Real average	kg.ton -1 of soil Mass of amendment in the
		quantity applied per		microcosm, for 150 g of
		hectare (kg)		soil (mg)
	KP	273	0.07	10.5
	Ammonium	95	0.024	3.6
	sulfate
	Organic	10,000	2.56	384
	amendment
	KP fertilizer (00 18 18) was provided by Soufflet Agri Service (Neuville-aux-Bois, France). It
	contained 0.32 % organic C, 22.4 % K2O, 18.9 % P2O5, and less than 0.1 g. kg -1 of total N.
	Fertilizer granules were crushed to a powder before application (< 100 µm). Biomarine
	organic amendment (NF U 44-051 provided by Truffaut, France) was used as an organic
	amendment. It is composed a mixture of vegetal and animal wastes (horse manure, sheep
	manure, poultry manure, algae, grape marc), containing 33.9 % organic C, 31.8 g.kg -1 total N,

  Revised manuscript with accepted corrections most probable number of AsV-reducing microorganisms was given by the Mc Grady tablefor

	five tubes.
	2.2.3.2. Activity tests
	AsIII-oxidizing tests were performed in 250 mL Erlenmeyer flasks filled with 100 mL of
	CAsO1 medium (Battaglia-Brunet et al., 2002) supplemented with 100 mg.L -1 / AsIII and
	inoculated with soil (equivalent to 0.2 g dry weight). Flasks were incubated at 25°C in
	oxidizing conditions under agitation (100 rpm). AsV-reducing tests were performed in 250
	mL serum flasks filled with 100 mL of CAsO1 medium supplemented with 20 mM lactic
	acid, 0.2 g.L

-1

yeast extract and AsV (100 mg.L -1 ). Flasks were inoculated with soil (equivalent to 0.2 g of dry weight). Flasks were hermetically closed, flushed with N2, and incubated at 25°C in static conditions. Flasks were sampled every day in order to monitor the evolution of AsV concentrations: 5 mL of culture were filtered at 45 μm with cellulose acetate filters and frozen at -20°C until AsIII/AsV separation with the PDC/MIBK method (Battaglia-

Acknowledgements

This study was part of the AgriAs project, co-funded by the EU, the Academy of Finland, L'Agence Nationale de la Recherche (contract 16-WTW5-0003-04), Bundesministerium für Ernährung und Land-wirtschaft and Forskningsrådet FORMAS under the ERA-NET Cofund WaterWorks2015 Call. This ERA-NET is an integral part of the 2016 Joint Activities developed by the Water Challenges for a Changing World Joint Programme Initiative (Water JPI).