Jean-Guillaume Letarouilly

Odile Broux

Aline Clabaut
email: clabaut@univ-littoral.fr.

New insights into the epigenetics of osteoporosis

Keywords: Human osteoporosis, Epigenetics, Osteoblast, Osteoclast, Adipocyte, Differentiation

Osteoporosis is characterized by reduced bone formation and accumulation of adipocytes in the bone marrow compartment. The decrease in bone mass results from an imbalance between osteoclast-mediated bone resorption and osteoblastmediated bone formation. The deficiency of bone cells to replace the resorpted bone can be due to a preferential differentiation of bone marrow stromal cells into adipocytes at the expense of osteoblasts. Consequently, the processes that control the differentiation of osteoclasts, osteoblasts and adipocytes play a crucial role in bone metabolism. It is known that epigenetic mechanisms are critical regulator of the differentiation programs for cell fate and moreover are subject to changes during aging. Here, we summarize recent findings on the role of epigenetics in the modulation of mechanisms that may be associated with osteoporosis. In particular, we focus on disturbances in the bone remodeling process described in human studies that address the epigenetic regulation of the osteoblast/adipocyte balance.

Introduction

Bone is under constant turnover throughout life in order to maintain a healthy bone mass. Bone homeostasis depends on the balance between bone resorption by osteoclast and bone formation by osteoblast. Osteoporosis (OP) results from the impairment of this balance leading to a reduction in bone mass associated with a high risk of fragility fractures. It is most often caused by an increase in osteoclastic activity that is not sufficiently compensated by an increase in bone formation.

Recently another mechanism for OP involving the bone marrow adipocyte has been proposed. Indeed, evidence from numerous studies supports a link between bone loss and accumulation of medullary adipocytes [START_REF] Meunier | Osteoporosis and the replacement of cell populations of the marrow by adipose tissue. A quantitative study of 84 iliac bone biopsies[END_REF][START_REF] Justesen | Adipocyte tissue volume in bone marrow is increased with aging and in patients with osteoporosis[END_REF]. Osteoblasts and adipocytes are derived from the same multipotent precursor, the bone marrow mesenchymal stem cell (BMSC) [START_REF] Owen | Marrow stromal stem cells[END_REF][START_REF] Pittenger | Multilineage potential of adult human mesenchymal stem cells[END_REF], and it has been suggested that the excessive accumulation of marrow adipocytes is caused by preferential differentiation of BMSCs into adipocytes at the expense of osteoblasts [START_REF] Beresford | Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures[END_REF][START_REF] Gimble | Playing with bone and fat[END_REF][START_REF] Duque | Bone and fat connection in aging bone[END_REF]. The processes which maintain in balance the differentiation and the activity of osteoclasts, osteoblasts and adipocytes are controlled by specific key genes and are tightly regulated by endogenous and exogenous factors. The phenotypic modifications during cellular differentiation are also driven by epigenetic changes which represent another fundamental mechanism of cell fate determination and commitment [START_REF] Teven | Epigenetic regulation of mesenchymal stem cells: a focus on osteogenic and adipogenic differentiation[END_REF][START_REF] Perez-Campo | Epigenetic mechanisms regulating Mesenchymal Stem Cell differentiation[END_REF]. The first use of the term 'epigenetic' was made by Conrad Waddington in 1942 to describe interactions between genotype and the environment, which together shape the expressed characteristic traits of an organism, defined as the phenotype [START_REF] Waddington | The Epigenotype[END_REF]. It refers to potentially heritable changes in gene expression without modifications of the DNA sequence. At least, three mechanisms including DNA methylation, histone modifications and miRNA based pathways initiate epigenetic changes [START_REF] Han | Integrating epigenomics into the understanding of biomedical insight[END_REF] (Figure 1). As these changes are influenced by factors such as age, gender and environment, it is not surprising that there is more and more interest in the study of epigenetics in the pathogenesis of OP.

In this review, we summarize the impact of epigenetic modifications on the modulation of the bone remodeling process described in human clinical studies. In addition, we provide an overview of the recent in vitro findings on the epigenetic mechanisms that govern the osteoblast/adipocyte balance in human cells.

Epigenetic modifications

2.a. DNA methylation

DNA methylation is one of the most studied epigenetic mechanisms [START_REF] Moore | DNA methylation and its basic function[END_REF]. It consists of a methyl group being covalently bound to the 5' position of cytosine in palindromic cytosine-phosphate-guanine (CpG) dinucleotides, abbreviated to 5mC. CpG dinucleotides occur infrequently, only 2% of the mammalian genome contains short high-frequency stretches of CpGs called CpG islands that are mainly associated with gene promoters and regulatory regions [START_REF] Moore | DNA methylation and its basic function[END_REF]. The addition of methyl groups to cytosine is catalyzed by DNA methyltransferases (DNMTs). The predominant consequence of methylation is transcriptional repression, which can be mediated by directly or indirectly blocking the binding of essential transcription factors to their targets. However, DNA methylation pattern established early in vivo is often influenced by the external environment and small differences may have a big impact on the phenotype diversity that is linked to the onset of non-malignant diseases [START_REF] Moore | DNA methylation and its basic function[END_REF].

2.b. Histone modifications

The major protein complex around with DNA coiled and thereby condensed is called a nucleosome. Each nucleosome consists of eight proteins called histones (H): an H3-H4 tetramer and two H2A-H2B dimers. Histone methylation is one of the most common epigenetic modifications on histones. Lysine (K) and arginine (R) residues are the predominant sites of methylation, with the potential addition by the histone methyltransferases (KMTs) of one, two, or three methyl groups. These methyl groups regulate transcription mainly by blocking access to transcription factors and inhibiting gene transcription. The removal of methyl groups from histone lysine residues is catalyzed by histone lysine demethylases (KDMs) [START_REF] Kouzarides | Chromatin modifications and their function[END_REF]. Acetylation is another wellknown post translational modification of histones. In general, histone acetylation is associated with active gene transcription, whereas histone deacetylation is correlated with gene repression. Acetylation neutralizes the positive charge of histones, and thereby prevents the compaction of chromatin. On the contrary, histone deacetylation contributes to a more compacted chromatin, thus impending transcription. The dynamic balance between acetylation and deacetylation of histones is performed by two classes of enzymes: lysine/histone acetyltransferases (HATs) and lysine/histone deacetylases (HDACs) [START_REF] Kouzarides | Chromatin modifications and their function[END_REF].

2.c. MicroRNAs

MicroRNAs (miRNAs) are a class of small (18-25 nucleotides) non-coding singlestrand RNAs [START_REF] Sato | MicroRNAs and epigenetics[END_REF]. 28,645 miRNAs are referenced in the miRBase (http://www.mirbase.org/release 17; December 2017). Their role in gene regulation is essential as they are involved in a number of biological processes including cell proliferation, differentiation, developmental timing control, apoptosis and stress responses, as well as pathological states such as cancer [START_REF] Macfarlane | MicroRNA: Biogenesis, function and role in cancer[END_REF]. miRNAs can regulate up to 60% of protein-coding genes at the post-transcriptional level by blocking messenger RNA (mRNA) translation or by inducing mRNA degradation, preventing protein production [START_REF] Sato | MicroRNAs and epigenetics[END_REF].

Epigenetic mechanisms regulating bone remodeling in OP: Focus on clinical studies.

To maintain bone mass homeostasis, the bone remodeling cycle starts with the dissolution of bone mineral and enzymatic matrix degradation by osteoclasts.

Subsequent bone formation is performed by osteoblasts, producing osteoid matrix which will mineralize. In order to keep both processes in balance, controlled differentiation of osteoblasts and osteoclasts from their precursors is essential. The transcriptional factors Runx2 (Runt-related transcription factor 2) and Osx (Osterix) are key regulators of all stages of osteoblast differentiation also controlled by critical signaling pathway including the Wnt pathway [START_REF] Soltanoff | Signaling networks that control the lineage commitment and differentiation of bone cells[END_REF]. Once mature, osteoblasts that become entombed within the bone matrix, called osteocytes, also modulate the differentiation and activity of both cell types [START_REF] Soltanoff | Signaling networks that control the lineage commitment and differentiation of bone cells[END_REF]. Osteoclast originates from the hematopoietic lineage and its bone-resorptive activity is mainly regulated by the RANK/RANKL/OPG (receptor activator of nuclear factor kβ /RANK ligand /osteoprotegerin) system. RANKL is produced by osteoblasts and its binding to RANK, located on the surface of osteoclasts, activates the expression of osteoclastogenic genes. In contrast, OPG, also secreted by osteoblasts, acts as a decoy receptor by binding to RANKL and inhibits bone resorption. All these aforementioned factors interact with many transcriptional activators or repressors, increasing the complexity of these regulations [START_REF] Soltanoff | Signaling networks that control the lineage commitment and differentiation of bone cells[END_REF]. Furthermore, it has been shown that epigenetic mechanisms take part in the control of osteoclastogenesis and osteoblastogenesis [START_REF] Vrtačnik | Epigenetic mechanisms in bone[END_REF]. Thus it is expected that epigenetic changes may contribute to the altered bone remodeling associated with osteoporosis. Below are summarized the recent epigenetic findings reported in human studies with the exception of histone modifications for which no data were found. However, this epigenetic mechanism, explored in vitro and in animal experiments, are presented in recently published reviews [START_REF] Gayor | Epigenetic regulation of bone remodeling and its impact on osteoporosis[END_REF][START_REF] Marini | Epigenetic regulation of bone remodeling and its impact on osteoporosis[END_REF][START_REF] Husein | Epigenetics and bone remodeling[END_REF].

3.a. DNA methylation

Due to its relevant roles in age-related diseases, some teams showed interest in the involvement of DNA methylation in the pathogenesis of OP. In 2013, Delgado-Calle and his colleagues compared the DNA methylation patterns of bone from patients with OP and osteoarthritis (OA). Thanks to an Illumina methylation arrays analysis, the authors found differentially methylated regions in whole genome between patients with OP and OA [START_REF] Delgado-Calle | Genome-wide profiling of bone reveals differentially methylated regions in osteoporosis and osteoarthritis[END_REF]. In another study by the same group, methylation of Wnt pathway genes was analyzed in postmenopausal bone tissue samples from patients with OP and OA. Several genes in the Wnt pathway presented differences in methylation between both groups, which could contribute to the higher Wnt pathway activity observed in osteoarthritis [START_REF] García-Ibarbia | Contribution of genetic and epigenetic mechanisms to Wnt pathway activity in prevalent skeletal disorders[END_REF]. Another important actor of the Wnt pathway is sclerostin, the product of the SOST gene mainly secreted by osteocytes.

A higher number of CpG methylations was found in the SOST promoter in bone biopsies of postmenopausal OP compared to normal controls, correlated with decreased SOST mRNA expression and serum level [START_REF] Reppe | Methylation of bone SOST, its mRNA, and serum sclerostin levels correlate strongly with fracture risk in postmenopausal women[END_REF]. As SOST is described as an inhibitor of bone formation, the increase in SOST promoter methylation could be a counteracting mechanism to reduce the inhibition of wnt signaling in an attempt to promote bone. To go a step further, by combining transcript profiling with DNA methylation analyses in bone and blood of osteoporotic and healthy patients, they found other transcripts, inhibitors of bone formation, with methylation levels significantly different between osteoporotic and control patients [START_REF] Reppe | Distinct DNA methylation profiles in bone and blood of osteoporotic and healthy postmenopausal women[END_REF]. This is in contrast with another genome-wide analysis which failed to detect associations between DNA methylation changes in blood and bone mineral density [START_REF] Morris | Epigenome-wide association of DNA methylation in whole blood with bone mineral density[END_REF]. Also paradoxically, increased SOST protein expression was observed at the femoral neck in women with osteoporotic fractures, concomitantly with reduced expression of the deacetylase Sirtuin 1, a negative regulator of SOST [START_REF] El-Haj | Reduced Sirtuin1 expression at the femoral neck in women who sustained an osteoporotic hip fracture[END_REF]. However, in this study bone samples from OA patients were used as surrogate controls instead of healthy controls. These observations highlight a critical need to take into account data heterogeneity according to experimental design (sample sizes, controls, tissue types and technologies) and analytical tools. Besides the involvement of DNA methylation in osteoblastogenesis, much less is known in the regulation of osteoclast differentiation. To our knowledge, only one study investigated the influence of methylation in the increase of resorption associated with osteoporosis, with a focus on the RANKL/OPG system in human bones [START_REF] Delgado-Calle | Role of DNA methylation in the regulation of the RANKL-OPG system in human bone[END_REF]. Although the RANKL mRNA level and RANKL:OPG transcript ratio were significantly higher in bone from osteoporotic patients, there was no evidence for differential methylation of RANKL at the bone tissue level compared to control group. In this study, the experiments were conducted using bone samples from OA patients as controls and the biological interpretation of the results could be questioned. However, given the practical and ethical difficulties to obtain bone samples from healthy subjects, this limitation applies to most of the described studies. Notwithstanding these concerns, all the studies reported to date strongly suggest that perturbations in DNA methylation are present in osteoporosis and linked to altered bone remodeling.

3.b. miRNAs

Because of ethical and technical limitations, the number of studies investigating miRNAs expression in osteoporotic bone tissue is still limited. Li et al. first reported the association between the downregulation of miR-2861 and primary OP in 2 adolescents [START_REF] Li | A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans[END_REF]. Two more recent studies compared bone samples from patients with hip fractures and non-fractured controls with osteoarthritis. Using micro-array technologies, Garmilla-Ezquerra et al. found significant down-regulation of miR-187-3p and up-regulation of miR-518f in osteoporotic bone while zhang et al. focusing on the characterization of miR-221, showed its involvement in OP through regulating Runx2 [START_REF] Garmilla-Ezquerra | Analysis of the bone microRNome in osteoporotic fractures[END_REF][START_REF] Zhang | MicroRNA-221 is involved in the regulation of osteoporosis through regulates RUNX2 protein expression and osteoblast differentiation[END_REF]. Besides these few cell-specific studies, a higher number of reports investigated the differences in miRNAs signatures in human whole blood, serum, plasma or peripheral mononuclear cells between osteoporotic and non-osteoporotic subjects [START_REF] Seeliger | Five freely circulating miRNAs and bone tissue miRNAs are associated with osteoporotic fractures[END_REF][START_REF] Panach | Serum circulating MicroRNAs as biomarkers of osteoporotic fracture[END_REF][START_REF] Weilner | Differentially circulating miRNAs after recent osteoporotic fractures can influence osteogenic differentiation[END_REF][START_REF] Kocijan | Circulating microRNA signatures in patients with idiopathic and postmenopausal osteoporosis and fragility fractures[END_REF][START_REF] Heilmeier | Serum miRNA signatures are indicative of skeletal fractures in postmenopausal women with and without type 2 diabetes and influence osteogenic and adipogenic differentiation of adipose tissue-derived Mesenchymal Stem Cells in vitro[END_REF][START_REF] De-Ugarte | SNPs in bone-related miRNAs are associated with the osteoporotic phenotype[END_REF][START_REF] Wang | MiR-133a in human circulating monocytes: a potential biomarker associated with postmenopausal osteoporosis[END_REF][START_REF] Cao | MiR-422a as a potential cellular microRNA biomarker for postmenopausal osteoporosis[END_REF][START_REF] Chen | MiR-503 regulates osteoclastogenesis via targeting RANK[END_REF][START_REF] Li | Plasma miRNA levels correlate with sensitivity to bone mineral density in postmenopausal osteoporosis patients[END_REF][START_REF] Meng | Identification of miR-194-5p as a potential biomarker for postmenopausal osteoporosis[END_REF][START_REF] Chen | Evaluation of MicroRNA 125b as a potential biomarker for postmenopausal osteoporosis[END_REF][START_REF] Wang | Reduced miR-144-3p expression in serum and bone mediates osteoporosis pathogenesis by targeting RANK[END_REF][START_REF] Yavropoulou | Expression of microRNAs that regulate bone turnover in the serum of postmenopausal women with low bone mass and vertebral fractures[END_REF]. In this way, the up or down regulation of circulating miRNAs linked to changes in bone metabolism was detected (Table 1). These miRNAs are potentially involved in the regulation of osteoblastogenesis, osteoclastogenesis or both but not all miRNA targets are biologically validated. Generally, the identity of regulated miRNAs varies significantly across studies, and for some of them there is even conflicting results. For example, Seelinger et al. demonstrated an upregulation of miR-21-5p in the serum of osteoporotic patients with bone fractures compared with patients with non-osteoporotic fractures [START_REF] Seeliger | Five freely circulating miRNAs and bone tissue miRNAs are associated with osteoporotic fractures[END_REF]. This result was further confirmed by Panach et al. who found a strong correlation between the levels of mi-21-5p and CTx, a marker of bone resorption, in the serum of osteoporotic fractured patients compared with OA patients [START_REF] Panach | Serum circulating MicroRNAs as biomarkers of osteoporotic fracture[END_REF]. In contrast, two studies analyzing the plasma of osteoporotic patients and healthy controls demonstrated that the miR-21-5p levels were higher in the control groups [START_REF] Li | Plasma miRNA levels correlate with sensitivity to bone mineral density in postmenopausal osteoporosis patients[END_REF][START_REF] Yavropoulou | Expression of microRNAs that regulate bone turnover in the serum of postmenopausal women with low bone mass and vertebral fractures[END_REF]. These discrepancies among studies are likely due to the different nature of patients' population (osteoporotic with or without fractures) and selected controls (healthy, OA or with non osteoporotic fractures).

Moreover, given that fracture significantly affects bone homeostasis, one can assume that this has an impact on miRNA expression and can bias the results. Interestingly, a link between bone tissue and serum/blood expression patterns was found for some miRNAs, such as miR-125b upregulated in both serum and bone tissue of osteoporotic patients with fracture and in blood and bone tissue of osteoporotic women [START_REF] Panach | Serum circulating MicroRNAs as biomarkers of osteoporotic fracture[END_REF][START_REF] Chen | Evaluation of MicroRNA 125b as a potential biomarker for postmenopausal osteoporosis[END_REF]. This provides evidence that circulating miRNA levels may be directly correlated to cell-specific miRNA levels and thus to altered bone metabolism. Another evidence was provided by a recent study reporting substantial changes of several miRs that regulate osteoblast and osteoclast differentiation and function following treatment with anti-osteoporotic agents [START_REF] Anastasilakis | Changes of Circulating MicroRNAs in Response to Treatment With Teriparatide or Denosumab in Postmenopausal Osteoporosis[END_REF]. In particular, a decrease expression of miR-133a was found in the serum of osteoporotic postmenopausal women at 12 months of treatment with teriparatide, a bone anabolic drug .This finding is in line with two previous reports demonstrating an increase in miR-133a expression in the serum of osteoporotic patients [START_REF] Wang | MiR-133a in human circulating monocytes: a potential biomarker associated with postmenopausal osteoporosis[END_REF][START_REF] Li | Plasma miRNA levels correlate with sensitivity to bone mineral density in postmenopausal osteoporosis patients[END_REF]. miR-133 directly targets Runx2 and therefore affects osteoblastogenesis, it is thus coherent that its expression was decreased following a treatment that stimulates bone formation. Despite some differences in the published data, taken all together, these studies suggest that miRNAs could be useful to investigate alterations of bone metabolism and follow the response to antiosteoporotic treatment.

Can epigenetic modifications also explain the increase in medullary adiposity observed in OP?

In bone loss, the deficiency of bone cells to replace the resorpted bone can be related to an insufficient differentiation of BMSCs into osteoblasts. In particular, it could be the reflection of the imbalance between adipogenesis and osteogenesis, lately proposed as another mechanism for OP [START_REF] Beresford | Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures[END_REF][START_REF] Gimble | Playing with bone and fat[END_REF][START_REF] Duque | Bone and fat connection in aging bone[END_REF]. Recent works, mostly performed through in vitro investigations on human cells, suggested that this perturbed BMSC fate decision and commitment could be directly driven by epigenetic mechanisms (Figure 2).

4.a. Histone modifications

Growing evidence suggests that histone modifications play a critical role in the balance between osteoblast and adipocyte and could explain the increase of bone marrow adiposity observed in OP [START_REF] Deng | Histone methyltransferases and demethylases: regulators in balancing osteogenic and adipogenic differentiation of mesenchymal stem cells[END_REF][START_REF] Huang | Fate determination in mesenchymal stem cells: a perspective from histone-modifying enzymes[END_REF]. First, the negative impact of histone deacetylation on osteogenesis was suggested by in vitro investigations using chemical inhibitors of histone deacetylases [START_REF] De Boer | Inhibition of histone acetylation as a tool in bone tissue engineering[END_REF][START_REF] Cho | Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors[END_REF]. More recently, using an epigenetic library screen, another group investigated the effects of small-molecule inhibitors of histone deacetylase on BMSC differentiation [START_REF] Ali | Epigenetic library screen identifies Abexinostat as novel regulator of adipocytic and osteoblastic differentiation of human skeletal (mesenchymal) stem cells[END_REF][START_REF] Ali | CUCDC-907 promotes bone marrow adipocytic differentiation through inhibition of histone deacetylase and regulation of cell cycle[END_REF]. They identified several novel compounds that promoted adipocyte differentiation through an increase in acetylation (particularly at lysine H3K9) in the promoter region of adipocyte-related genes, including the master adipogenic transcription factor PPARgamma (peroxisome proliferator-activated receptor). Moreover, several studies highlighted the importance of histone methylation and demethylation in the BMSC fate. For instance, Hemming and colleagues revealed that the combination of methylation and demethylation might function as an epigenetic switch for BMSC lineage determination [START_REF] Hemming | EZH2 and KDM6A act as an epigenetic switch to regulate mesenchymal stem cell lineage specification[END_REF]. Indeed, they showed that EZH2 (the Enhancer of Zeste Homolog 2), a histone methyltransferase that catalyzes the trimethylation of histone H3 on lysine 27 (H3K27me3) on master regulatory genes, promoted adipogenic differentiation and inhibited osteogenesis. In contrast, by removing these repressive marks, lysine demethylase 6A (KDM6A) inhibited adipogenesis and promoted osteogenesis.

Several EZH2 targets, involved in the regulation of BMSC fate have been identified [START_REF] Chen | Myocyte Enhancer Factor-2 Interacting Transcriptional Repressor (MITR) is a switch that promotes osteogenesis and inhibits adipogenesis of Mesenchymal Stem Cells by inactivating Peroxisome Proliferator-activated Receptor 2[END_REF]. Among these targets, HDAC9c, a non-catalytic HDAC protein, have the capacity to negatively regulate the transcriptional activity of PPARgamma. Interestingly, Chen et al. observed that, in aged BMSCs, adipogenesis is enhanced as a result of increased EZH2 and decreased HDAC9c expression [START_REF] Chen | Enhancer of Zeste Homolog 2 and Histone Deacetylase 9c regulate age-dependent mesenchymal stem cell differentiation into osteoblasts and adipocytes[END_REF]. Additional studies support the notion that EZH2 is a central key of complex epigenetic regulation of bone marrow adipogenesis. Phosphorylation of EZH2 inhibited its methyltransferase activity and resulted in promoted osteogenesis [START_REF] Wei | CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells[END_REF]. Furthermore, Zhu et al. showed that HoxA-AS3, a long non-coding RNA, interacted with EZH2 to modulate the status of H3K27me3 in the promoter region of the osteoblastic transcriptional factor Runx2 and repressed its transcription [START_REF] Zhu | Long non-coding RNA HoxA-AS3 interacts with EZH2 to regulate lineage commitment of mesenchymal stem cells[END_REF].

4.b. miRNAs

Studies addressing the role of miRNAs as mediators of the balance between osteogenesis and adipogenesis begin to emerge. By targeting different downstream targets, these miRNAs can affect both lineage commitment and terminal differentiation (Table 2). Indeed, it has been shown that miR-204, miR-211 and miR-320 inhibit osteogenesis via Runx2 repression while miR-637 down-regulates differentiation through repression of Osterix [START_REF] Huang | MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation[END_REF][START_REF] Hamam | microRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal (skeletal) stem cells[END_REF][START_REF] Zhang | MiR-637 maintains the balance between adipocytes and osteoblasts by directly targeting Osterix[END_REF]. Qadir et al. demonstrated that miR-124 increases adipogenesis and negatively regulates osteogenesis by targeting several members of the Dlx (distal-less) family factors required for osteoblast differentiation and Runx2 expression [START_REF] Qadir | miR-124 negatively regulates osteogenic differentiation and in vivo bone formation of mesenchymal stem cells[END_REF]. Recently, Elsafadi et al. found that downregulation of LRP3 (Low-density lipoprotein receptor-related protein 3) expression by miR-4739 suppresses osteogenic differentiation [START_REF] Elsafadi | MicroRNA-4739 regulates osteogenic and adipocytic differentiation of immortalized human bone marrow stromal cells via targeting LRP3[END_REF]. miRNAs can also promote a switch between osteogenesis and adipogenesis by targeting inhibitors of adipocyte differentiation. For instance, in BMSCs of aging mice and human, miR-188 activates PPARgamma by suppressing two of its inhibitors, HDAC9 and RICTOR (RPTORindependent companion of MTOR complex), resulting in an increase number of adipocytes at the expense of osteoblasts [START_REF] Li | MicroRNA-188 regulates age-related switch between osteoblast and adipocyte differentiation[END_REF]. Lastly and very interestingly, perturbations in circulating miRNA levels in OP have been shown to be directly linked to in vitro altered adipocyte/osteoblast balance. Indeed, a microarray investigation of serum from osteoporotic patients and healthy controls revealed a significant reduction of miR-27a expression in postmenopausal osteoporotic patients compared to controls. Subsequent analysis on BMSCs from patients indicated that osteoblastogenesis was reduced, whereas adipogenesis was increased [START_REF] You | Table 2. miRNAs involved in the osteoblast/adipocyte balance miRNA Cellular model Target References miR-204/211 Human BMSCs Runx2 57 miR-320 Human BMSCs Runx2 58 miR-637 miR-637 Osx 59 miR-124 Human maxillary BMSCs Dlx5, Dlx3, Dlx2 60 miR-4739 Immortalized human BMSCs LRP3 61 miR-188 Human BMSCs HDAC9, RICTOR 62 miR-27a Human BMSCs Mef2c 63[END_REF].

Conclusions

Although being actively studied, advances in understanding the role of epigenetic factors in the pathophysiology of skeletal diseases, such as OP, are still preliminary.

Overall, experimental data presented in this review show that epigenetic events may have profound effect on commitment, differentiation and activity of the cells of the bone marrow environment and consequently may contribute to the pathophysiology of age-related bone loss. Studies that address the epigenetic regulation in the osteoblast/adipocyte balance are somewhat limited in mainly being performed in vitro. However, new high throughput technologies will likely contribute to increase the number of investigations, including those conducted on human samples. Promisingly, a link between bone and serum/blood expression patterns was found for some miRNAs, providing evidence that circulating miRNA levels may be directly correlated to altered bone metabolism. This finding together with the fact that miRNAs are stable in body fluids make them potential tools to detect early manifestations of OP.

Given that epigenetics is recognized as a significant contributor to pathological conditions, efforts to investigate the epigenetic marks in OP could pave the way for new therapeutic advances.

Legends

Figure 1 :

 1 Figure 1: Mechanisms of epigenetic regulations. (a) DNA methylation is a process catalyzed by DNA methyltransferases (DNMTs), preventing the binding of regulatory transcription factors to the promoter regions thereby repressing gene expression. (b) Histone methylation by histone methyltransferases (KTMs) and the removal of acetyl groups by histone deacetylases (HDACs) induce the closing of chromatin conformation and consequently transcriptional repression. In contrast, acetylation of lysine residues on histone tails, regulated by histone acetyltransferases (HATs), prevents the compaction of chromatin and promote transcriptional activation. (c) miRNAs negatively regulate gene expression by blocking mRNA translation or by inducing mRNA degradation.

Figure 2 :

 2 Figure 2: Epigenetic mechanisms regulating preferential human BMSC differentiation into adipocytes.Here are schematized the epigenetic modifications involved in the perturbations of cell fate commitment that favor adipogenesis at the expense of osteogenesis. The histone methyltransferase EZH2 facilitates adipogenesis (1) by trimethylation of histone H3 on Lysine 27 at the promoter of HDAC9c, resulting in suppression of its expression and reactivation of PPARgamma expression and (2) by downregulating, in association with the long non-coding RNA HoxA-AS3, the expression of the osteoblastic transcriptional factor Runx2. In addition, by targeting genes required for osteoblast differentiation, such as Runx2, miRNAs and histone deacetylases (HDACs) negatively regulate osteogenesis and increase adipogenesis. There is no available data concerning the role of DNA methylation in the increase of bone marrow adiposity observed in human OP.

Table 1 . Human clinical studies identifying miRNAs involved in the pathogenesis of osteoporosis a. miRNAs expression upregulated in patients with osteoporosis miRNA Study design References

 1

	miR-518f-3p	Bone samples from women with hip fractures (n=18) and non-fractured control group with osteoarthritis (n=18)	29
	miR-21-5p, miR-23-3p, miR-24-3p, miR-25-3p, miR-100-5p, miR-125b-5p	Serum and bone samples from osteoporotic fractured patients and non-osteoporotic fractured controls (n=30 per group/serum and n=20 per group/bone tissue)	31
	miR-122-5p, miR-125b-5p, miR-21-5p	Serum samples from osteoporotic fractured patients (n=15) and
		osteoarthritic controls (n=12)	32
	miR-3679, miR-4274	Bone samples from postmenopausal women with hip fracture (n=10) and osteoarthritis (n=10)	36
	miR-133a	Circulating monocytes from postmenopausal women with high (n=10) and low (n=10) BMD	37
	miR-422	Circulating monocytes from postmenopausal women with high (n=10) and low (n=10) BMD	38
	miR-133a-3p	Plasma samples from postmenopausal normal (n=40) , osteopenic (n=40) and osteoporotic women (n=40)	40
	miR-130b-3p, miR-151a-3p, miR-151b,	Blood samples from osteopenic (n=23) and osteoporotic women (n=25)
	miR194-5p, miR-590-5p		41
	miR-125b	Serum and bone samples from postmenopausal normal (n=30) and osteoporotic patients (n=30)	42
	miR-124, miR-2861	Serum from postmenopausal women with low BMD with (n=35) and without (n=35) fractures and healthy control group (n=30)

Acknowledgements

JG Letarouilly was supported by a grant from the "Société Française de Rhumatologie" (SFR).

The authors declare that they have no competing interest.

Disclosure of interest


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

Introduction

The recent increase of microplastics (MPs) occurrence into the aquatic environment is raising concern about their potential harm on diverse organisms [START_REF] Gall | The impact of debris on marine life[END_REF]. These plastics of a size below 5 mm [START_REF] Gesamp ; | Retention of microplastics in a major secondary wastewater treatment plant in Vancouver, Canada[END_REF] can be divided into either primary microplastics (directly produced MPs) or secondary microplastics (resulting from macroplastics fragmentation) [START_REF] Gesamp ; | Retention of microplastics in a major secondary wastewater treatment plant in Vancouver, Canada[END_REF][START_REF] Koelmans | Plastics in the marine environment[END_REF]. Their entry routes into the marine environment mainly come from land-based origin: accidental loss or mishandling plastics, dumping plastic wastes and mismanaged landfills i.e an absence of a fence surrounding the landfill with no appropriate synthetic material for covering wastes (reviewed by [START_REF] Duis | Microplastics in the aquatic and terrestrial environment: sources (with a specific focus on personal care products), fate and effects[END_REF]. Wastewater treatment plants (WWTP) add up to these entry routes and is considered as an important MPs pathway into the aquatic environments [START_REF] Talvitie | Do wastewater treatment plants act as a potential point source of microplastics? Preliminary study in the coastal Gulf of Finland, Baltic Sea[END_REF]. These WWTPs may collect both industrial and domestic water containing MPs used in various products [START_REF] Zbyszewski | Comparison of the distribution and degradation of plastic debris along shorelines of the Great Lakes, North America[END_REF][START_REF] Eerkes-Medrano | Microplastics in freshwater systems: A review of the emerging threats, identification of knowledge gaps and prioritisation of research needs[END_REF]. Several factors influence the MPs quantities being discharged by the WWTPs such as the volume of treated water, the flow rate, the filtration processes and sewer systems [START_REF] Mason | Microplastic pollution is widely detected in US municipal wastewater treatment plant effluent[END_REF][START_REF] Ziajahromi | Wastewater treatment plants as a pathway for microplastics: Development of a new approach to sample wastewater-based microplastics[END_REF]. As indicated in previous studies, most of the MPs can be effectively removed by secondary and tertiary treatment [START_REF] Lares | Occurrence, identification and removal of microplastic particles and fibers in conventional activated sludge process and advanced MBR technology[END_REF][START_REF] Murphy | Wastewater Treatment Works (WwTW) as a Source of Microplastics in the Aquatic Environment[END_REF], but it doesn't exclude that a large number of MPs is still being released in the aquatic systems [START_REF] Browne | Accumulation of Microplastic on Shorelines Woldwide: Sources and Sinks[END_REF][START_REF] Mathalon | Microplastic fibers in the intertidal ecosystem surrounding Halifax Harbor, Nova Scotia[END_REF][START_REF] Murphy | Wastewater Treatment Works (WwTW) as a Source of Microplastics in the Aquatic Environment[END_REF] and most specifically into river catchments [START_REF] Roex | Potential risk of microplastics in the fresh water environment 9[END_REF]. Removal efficiencies of microplastics in WWTPs range generally between 72-99.4% [START_REF] Gatidou | Review on the occurrence and fate of microplastics in Sewage Treatment Plants[END_REF][START_REF] Sun | Microplastics in wastewater treatment plants: Detection, occurrence and removal[END_REF] suggesting that most particles are either accumulated during the sedimentation process or the grease removal [START_REF] Magni | The fate of microplastics in an Italian Wastewater Treatment Plant[END_REF][START_REF] Murphy | Wastewater Treatment Works (WwTW) as a Source of Microplastics in the Aquatic Environment[END_REF]. These rates remain limited by current detection techniques.

Both fibers and fragments are observed in the WWTPs water [START_REF] Magnusson | Screening of microplastic particles in and down-stream a wastewater treatment plant[END_REF].

Their dispersion into the aquatic environment leads to their ingestion by aquatic species like bivalves [START_REF] Browne | Ingested microscopic plastic translocates to the circulatory system of the mussel Mytilus edulis (L.)[END_REF][START_REF] Railo | Application of an enzyme digestion method reveals microlitter in Mytilus trossulus at a wastewater discharge area[END_REF]. Mussels, Mytilus spp. are considered as suitable animals for monitoring chemical pollutants in aquatic environments [START_REF] Green | Contaminants in Coastal Waters of Norway[END_REF].

They are sessile animals capable of giving precise spatial information, easily collected where they form mussel's beds in shallow waters and contain enough tissue amounts for laboratory analysis. They are filter-feeders (feed mainly on phytoplankton) which increase their probability to ingest MPs and integrating them in the food chain [START_REF] Wright | The physical impacts of microplastics on marine organisms: a review[END_REF]. MPs ingestion might lead to several physiological issues for bivalves such as strong inflammatory responses in Mytilus sp. (Avio et al., 2015) or adverse consequences in Mytilus edulis digestive glands [START_REF] Von Moos | Uptake and effects of microplastics on cells and tissue of the blue mussel Mytilus edulis L. after an experimental exposure[END_REF]. Mussels are important prey source for many fish and they are highly consumed by humans making it critical to evaluate the amount of MPs ingestion.

Even though several studies evaluated WWTPs discharge into the aquatic systems, most of them focused on treatment plants discharging solely in the freshwater systems (Gies et al., 2018;[START_REF] Lares | Occurrence, identification and removal of microplastic particles and fibers in conventional activated sludge process and advanced MBR technology[END_REF][START_REF] Mintenig | Identification of microplastic in effluents of waste water treatment plants using focal plane array-based micro-Fourier-transform infrared imaging[END_REF][START_REF] Dris | Microplastic contamination in an urban area: a case study in Greater Paris[END_REF][START_REF] Murphy | Wastewater Treatment Works (WwTW) as a Source of Microplastics in the Aquatic Environment[END_REF][START_REF] Wolff | Determination of the microplastics emission in the effluent of a municipal waste water treatment plant using Raman microspectroscopy[END_REF]. A few assessed WWTPs discharges in the marine environment [START_REF] Talvitie | Do wastewater treatment plants act as a potential point source of microplastics? Preliminary study in the coastal Gulf of Finland, Baltic Sea[END_REF][START_REF] Talvitie | How well is microlitter purified from wastewater? -A detailed study on the stepwise removal of microlitter in a tertiary level wastewater treatment plant[END_REF][START_REF] Ziajahromi | Wastewater treatment plants as a pathway for microplastics: Development of a new approach to sample wastewater-based microplastics[END_REF][START_REF] Leslie | Microplastics en route: Field measurements in the Dutch river delta and Amsterdam canals, wastewater treatment plants, North Sea sediments and biota[END_REF]. To our knowledge, no study has evaluated microplastics input, dispersion and accumulation in coastal marine environment via wastewater treatment plant outlet in three different matrices. Also, no study has evaluated the role of coastal landfills in MPs entry into the coastal marine environment making its relevance not well-known.

. The aims of this research were (1) to assess the contribution of a major tertiary WWTP located in Le Havre (France) in MPs discharge into the marine coastal environment, (2) to follow the released MPs in the marine environment along a distance gradient from the discharge pipe in three matrices (surface water, bottom sediments and mussels), and (3) to evaluate the role of an abandoned coastal landfill as a source of MPs entry into the marine coastal environment.

Material and Methods

Study zone and sampling sites:

The sampling area was located in Le Havre, Northern France. The study zone investigated was divided into three areas: The Edelweiss Wastewater Treatment Plant (WWTP), Le Havre harbor and Sainte-Adresse coastal zone. The WWTP is located inside Le Havre harbor and receives the effluent of 20 municipalities (serving a population of 415 000 habitants and a volume of 80 000 m 3 per day). This tertiary WWTP was constructed in 2011 and is capable of treating collected water in five different steps before discharging it into the harbor's basin. Le Havre harbor is located at the mouth of the Seine estuary, in the Eastern English Channel. It is the second biggest French harbor in tonnage and receives 59% of containers traffic (Report GPMH, 2015). The Sainte-Adresse's site is located in the vicinity of the Dollemard coastal landfill. This landfill was constructed in 1960 and received tons of household, industrial and special wastes. This landfill was closed in 2000, yet the huge amount of dumped garbage led to its collapse on the bottom of the cliff resulting a 750 m waste distribution along the coast. During high tide, the waves collect the wastes sitting on the coast promoting their entry into nearby coastal waters (Report SCE, 2011).

In the WWTP, water samples from raw influent and effluent were collected as well as a sample of sewage sludge. In the marine environment, samples of water, sediments and mussels were collected in four sites (1 to 4) in the harbor, and one site (site 5) in the vicinity of the coastal landfill (Fig. 1). The sites were located from the WWTP outlet with respective distances of 50 m, 1.1 km, 1.6 km, 5.3 km and 10.3 km. These sampling sites were selected in order to follow MPs dispersion and concentration in the three analyzed matrices.

Sampling Methods:

The sampling was done between the 3 rd and the 4 th April 2018. Inside the WWTP, two samples of 500 mL raw influent and a sample of 100 g of sewage sludge were taken for analysis on the 3 rd of April at 3:00 pm. Since we wanted to sample all sites the same day to have the same environmental conditions, we chose to sample a large volume of water (one to two m 3 ) representative of the analyzed water body rather than making replicates. The first ten centimeters of the water column was pumped and filtered on stainless steel sieves of different mesh sizes of 500 μm, 200 μm, 80 μm and 20 μm. The filtering system consisted of a water pump (Bilge pump, SEAFLO © ) connected to a flowmeter (Turbine Digital Pro K24) that calculate the exact volume being filtered throughout the sieves. Before collecting the water samples, the pump and the pipe were rinsed with the seawater then with Milli-Q water. The 20 µm sieve was the fastest to get clogged so it was analyzed separately. Each sieve was rinsed with Milli-Q water inside clean glass bottles (previously washed with Milli-Q water) covered in aluminum foil and conserved at 4°C until analysis.

One kilogram (kg) of sediment samples was taken with a stainless-steel sediment core sampler from all sites except in site 4 where it was a rocky substrate. They were conserved inside aluminum foil then stored at -10°C until analysis.

Wild mussels (Mytilus sp.) specimens were collected in all sites (1 to 5). Around 20 individuals were sampled near the water filtration location. Each individual was immediately embedded in aluminum foil then frozen at -10°C until analysis.

Water temperature, salinity, dissolved oxygen and turbidity were measured at each site with a multiparameter probe (Mutiparameter HI 982, HANNA instruments).

Microplastics analysis

Contamination prevention

In order to prevent contamination, several precautions were followed. Cotton lab coats were worn throughout the experiments, and all steps (filtration, measurements and digestion) were realized under a laminar flow hood. All laboratory materials used were made of glass: bottles, petri dishes, Erlenmeyers, filtration system; and they were covered immediately after manipulation. All surfaces and equipment were rinsed before usage with filtered ethanol 70% and MilliQ water. Whereas for the solutions: Ethanol 70%, KOH 10%, ZnCl2, H2O2 30%; they were filtered 3 times on GF/A filters (Whatman, France) to eliminate any unwanted particles or fibers. Controls were made with every step in order to track the contamination during the work.

Water Samples

All water samples have undergone density separation (DS) using a solution of zinc chloride (ZnCl2) of a proper density of 1.8 g/cm 3 . This solution was chosen due to its high efficacy in retrieving microplastics [START_REF] Rodrigues | Effectiveness of a methodology of microplastics isolation for environmental monitoring in freshwater systems[END_REF]. Before adding ZnCl2 to our samples, a series of density separation trials were tested: different volumes of filtered ZnCl2 (50 mL, 100 mL, and 6 150 mL) were added to different separating funnels containing a solution of 600 mL of water and 5 g of sediments with seventeen different polymers of different densities (in order to replicate a solution similar to our samples). Samples were left 24 hours; they were shaken twice: once every 4 hours then left to rest for 16 hours to ensure the formation of two separate phases where the supernatant consisted of particles with a density < 1.8 g/cm 3 . The density separation efficacy was calculated following the formula (Phuong et al. 2017):

% = × 100
The percentage obtained was 82.7 ± 0.5% (above 80 %) and the volume of 50 mL was chosen.

Sludge samples

Sludge samples were put in aluminum foil and dried at 60°C. After testing several solutions (hydrogen peroxide (H2O2 30%), hydroxide potassium (KOH 10%) and sodium hypochlorite NaClO) and encountering filtration difficulties or a small sample analysis (<1 g), visual observation was chosen. Three dried subsamples of 3 g were put inside clean Petri dishes and observed under a stereomicroscope [START_REF] Murphy | Wastewater Treatment Works (WwTW) as a Source of Microplastics in the Aquatic Environment[END_REF]. Microplastics (including fibers, fragments and particles) were put on a microscopic slide for further counting and analysis (color, length and polymers type).

Sediment samples

Wet sediments collected were defrosted at room temperature. For each site, 3 subsamples of 100 g were taken and oven dried at 60°C for 24 hrs. From each subsample, 10 g of dried sediments [START_REF] Mathalon | Microplastic fibers in the intertidal ecosystem surrounding Halifax Harbor, Nova Scotia[END_REF][START_REF] Liebezeit | Microplastics in beaches of the East Frisian Islands Spiekeroog and Kachelotplate Bull[END_REF]Harrison et al., 2012) was weighed and placed inside an Erlenmeyer pre-rinsed with Milli-Q water. Firstly, 40 mL of 30% hydrogen peroxide H2O2 [START_REF] Nuelle | A new analytical approach for monitoring microplastics in marine sediments[END_REF]Avio et al., 2015) was added to the Erlenmeyer that was placed on a heating stirrer plate for 24 hour at 40°C with a rotation per minute of 100 rpm in order to remove organic debris. Afterwards, a density separation solution was used. Briefly, the solution containing the sediments were put inside a separating funnel and a volume of 40 mL of ZnCl2 (1.8 g/cm 3 ) was added [START_REF] Liebezeit | Microplastics in beaches of the East Frisian Islands Spiekeroog and Kachelotplate Bull[END_REF][START_REF] Imhof | Contamination of beach sediments of a subalpine lake with microplastic particles[END_REF][START_REF] Nuelle | A new analytical approach for monitoring microplastics in marine sediments[END_REF]; the mix was then agitated for 5 minutes and let sit for 24 hours. The supernatant containing the MPs was filtered on GF/A filters and stored inside clean glass Petri dishes.

Mussels samples

Mussels were thawed 4 hours prior to digestion. Ten mussels were taken from each site: the total length, width (cm), total and net weight (g) was taken for each individual. Their condition index (CI) was calculated as follow [START_REF] Afnor | Norme Francaise. Huitres creuses[END_REF]:

= ℎ ( ) $ % & ℎ ( ) ' × 100
This index is a good indicator for the physiological condition (tissue growth, energy reserves) of mussels [START_REF] Orban | Seasonal changes in meat content, condition index and chemical composition of mussels (Mytilus galloprovincialis) cultured in two different Italian sites[END_REF].

Mussels were digested using a solution of potassium hydroxide (KOH) 10% (m/v, ChimiePlus, France) was used in order to eliminate any organic material [START_REF] Dehaut | Microplastics in seafood: Benchmark protocol for their extraction and characterization[END_REF][START_REF] Kühn | The use of potassium hydroxide (KOH) solution as a suitable approach to isolate plastics ingested by marine organisms[END_REF]. The correct KOH 10% volume was chosen after testing different volumes (150 mL, 200 mL, and 250 mL) added inside Erlenmeyers containing mussels. Then, these Erlenmeyers were transferred to a heating magnetic stirrer at 60°C for 24 hours. After digestion, the digestat of each individual was filtered on GF/A filter and the digestion efficiency was calculated following the equation: clear and homogeneously colored (red, blue, green and black) [START_REF] Song | A comparison of microscopic and spectroscopic identification methods for analysis of microplastics in environmental samples[END_REF]. Measurements were done on the suspected items at their longest dimension using "Piximètre" software version 5.9 and they were divided into five different size classes: <20 µm; 20 -80 µm; 80 -200 µm; 200 -500 µm; >500 µm.

% = 100

Raman spectroscopy

Micro-Raman Xplora Plus (HORIBA Scientific®, France) was used in order to identify the polymer nature of suspected plastics. The machine is equipped with an automated plate protected by a door that can be shut during analysis so the filter placed inside would not affected by any airborne contamination.

Due to the time consumption that takes when analyzing filters under micro-Raman, five filters of mussels containing the highest number of potential MPs were chosen and analyzed. All sludge and sediments items, and subsamples of water items were also taken for Raman analysis. For identification, two lasers with a wavelength of 532 nm and 785 nm and a range of 200-3400 cm -1

were used with x10 and x100 objectives (Olympus). Filters were either analyzed manually or using automated ParticleFinder module for LabSpec 6. Each particle spectrum is compared to a polymer database identification software (KnowItAll, BioRad®) and a personal library made with standard polymers obtained from Goodfellow (France). Pigments spectra (Copper phthalocyanine, Hostasol Green, etc…) obtained were identified using an option called "Mixture

Analysis" existing in "KnowItAll software" that is capable to decompose a mixed spectrum into two spectra consisting of the dye and its associated polymer.

Statistical analysis

All statistical comparison tests were done using SPSS software (IBM SPSS STATISTICS 20).

Microplastics found in sediments and mussels were reported per gram of sample and in unit volume (per L) for the MPs in water samples. After a Shapiro-Wilk test, mussel's and water data did not follow a normal distribution and non-parametric tests (Kruskal-Wallis and Mann-Whitney U tests) were used to test for difference of ingested MPs between all sites (significance level: 0.05). The mussels' condition index and microplastics found in sediments followed a normal distribution and one way analysis of variance (ANOVA) test with a post-hoc Tukey test was used to determine if there was significant difference between sites (p < 0.05 significance level). Spearman's rank correlation was used to test any correlation between the number of ingested MPs and the mussels' condition index.

Non-metric Multidimensional Scaling (n-MDS) was performed on a Bray-Curtis distance matrix created from transformed data (square root). This test was used as an ordination method alongside ANOSIM and SIMPER tests to compare the contribution of each observed polymer in the similarity and dissimilarity between water, sediments and mussels of all sites. These tests were done using PRIMER v7.0 (Primer-E Ltd., Plymouth Marine Laboratory, UK).

Results

Contamination control

Blank samples only showed the presence of fibers with an average of 1.71 ± 1.5 during the dissection and digestion of mussels (representing the average found for a batch of 9 mussels representing a total one hour constant lab work under the laminar flow hood), 0.5 ± 1 for water samples density separation and 0.25 ± 0.43 for sediment samples, H2O2 digestion and density separation (also representing an hour of laboratory work). Fibers on background blanks were dominated by black (66.7% of total) and clear (33.7%). The absence of fragments, films, foams and beads indicates that the contamination was only limited to airborne fibers. The average numbers of fibers found in the control blanks were similar to other studies [START_REF] Horton | Large microplastic particles in sediments of tributaries of the River Thames, UK -Abundance, sources and methods for effective quantification[END_REF][START_REF] Bråte | Mytilus spp. as sentinels for monitoring microplastic pollution in Norwegian coastal waters: A qualitative and quantitative study[END_REF]. These blanks are important in any microplastics study in order to evaluate the number of procedural contamination every batch of samples is undergoing and, hence, correcting the results.

Wastewater Treatment Plant and microplastics dispersion

The amount of suspected MPs items in the raw sewage influent was 244 items/L; dominated by fragments with 183 fragments/L. This number decreased to 2.84 items/L in the effluent leading to an estimated MPs retention of 98.83% by the WWTP. Synthetic fibers constituted 25% and 40% of suspected MPs found in the influent and the effluent water sample, respectively. Blue fragments and black fibers dominated both the influent and the effluent. The collected sewage sludge had an average of 16.13 ± 1.2 suspected MPs/g with a dominance of fibers (12.46 ± 1.08 fibers/g). Red fibers, and blue and green fragments dominated the sludge samples.

Supplementary Table 1 shows the environmental parameters measured during sampling.

Temperature ranged between 8°C in site 3 and 9.1°C in site 1. Turbidity ranged between 4.23 N.T.U in site 2 and 17.6 N.T.U in site 1; it reached a higher value in site 5 (121 N.T.U). The number of suspected MPs in the surface water decreased with the increasing distance from the outlet flow of the WWTP (Fig. 2). The number of suspected MPs decreased by 96% at site 4 (5.3 km from effluent) when comparing to the amount of MPs initially emitted from the plant (2.844 items/L to 0.099 items/L). Yet in site 5, near the landfill, the amount of MPs was alarmingly high reaching 6 items/L (double amounts of MPs discharged from the WWTP). Particles (fragments, films or beads) dominated sites 2 and 5 whereas fibers dominated in sites 1, 3 and 4 (1; 0.27 and 0.073 fibers/L, respectively). Site 5 had the presence of secondary microplastics (fragmented shapes) but also primary microplastics (polystyrene raw microbeads represented 22% of identified particles). Blue fragments (45%) and black fibers (51%) dominated the water samples.

Microplastics in sediments

Microplastics analyzed were expressed in number of plastics found in 1 g of dried sediments.

Although there was no significant difference in MPs concentration among the analyzed sites (p > 0.05), the highest concentration was recorded in site 2 (1.04 ± 0.07 MPs/g) and the lowest in site 5 (0.41 ± 0.36 MPs/g) (Fig. 3). Fragments dominated the type of MPs found in all sampling sites representing 70.5%, 84%, 86.9, and 77.7% at sites 1, 2, 3 and 5, respectively. Site 1 had the highest number of fibers with an average of 0.2 fibers/g. Films were not observed in all sediment samples. Blue color dominated all fragments (69%) whereas blue and green colors constituted 71.4% of observed fibers.

Microplastics in mussels

For all sites, mussels collected had a shell length ranging between 1.4 and 7.72 cm with an average of 5.6 ± 1.6 cm. The net weight ranged between 0.5 and 20.4 g with an average of 6.52 ± 4.2 g. There were significant differences in mussels' wet weight between sites (p < 0.05): mussels from site 5 were significantly smaller than those from the others sites. To avoid disparity between different sized mussels, the number of ingested MPs was converted per gram of soft tissue (wet weight) and not per individual [START_REF] Bråte | Mytilus spp. as sentinels for monitoring microplastic pollution in Norwegian coastal waters: A qualitative and quantitative study[END_REF]. 93% of collected mussels had ingested suspected MPs. Mussels from site 5 ingested a significantly higher number of suspected MPs (2.75 ± 3.08 items/g) compared to the others sites (p < 0.05) except for site 4 (Fig. 4).

Mussels from site 2 had the lowest number of ingested MPs (0.41 ± 0.33 items/g). Fragments dominated mussels in all sites (̴ 52% in sites 1, 3 and 4; 72% in site 5) except in site 2 where fragments and fibers were equally distributed (̴ 40%). Among the investigated fragments, blue, red and transparent fragments (̴ 20% for each color) dominated. Whereas for the fibers, both blue and black colors represented 80% of the observed fibers. There was significant difference between the mussels' condition index (CI) (p < 0. 05) with site 1 being significantly different from site 5. Yet, there was no significant correlation (Spearman's correlation, r = -0.084; p > 0.05) between the CI index and the ingested items. The size class 20 -80 µm was the dominant size class in all water samples (Fig. 5 A). The size class 200 -500 µm had a higher dominance in site 2 (37.7%) in comparison to other sites. All sites had the presence of < 20 µm size class with an abundance less than or equal to 5%.

For the sediments, the dominant MPs size class in all sites was the 20 -80 µm representing between 40% at site 3 and 56% at sites 3 and 5 (Fig. 5 B). Plastics with a size class > 500 µm consisted mainly of fibers with an abundance ranging between 12 % (sites 2 and 3) and 22%

(sites 1 and 5).

Ingested items of a size < 200 µm were the most abundant in mussels representing 78.5%, 84.4%, 72.2%, 63.1%, 61.9% at sites 1, 2, 3, 4 and 5, respectively (Fig. 5 C). Among them, suspected MPs of size class 20 -80 µm were the most abundant in all sites with an average of 41% of the ingested items.

Polymers identification

In our study, approximately 19% of all items were identified with the Raman spectroscopy: half of mussels' filters and all items found in the sludge and sediments samples were analyzed. For the water, an average of 27 MPs was identified per site. Of the 398 items analyzed, 344 (86.4%)

were positively identified as a known polymer. Among the non-polymers items, only 2% were identified as cellulose-based fibers. The rest were non identifiable (for example if a sample is too thin, Raman tends to detect the underlying substrate instead of the sample Käppler et al., 2015).

In the WWTP, 10 types of polymers were identified with a dominance of polystyrene (PS) and polyethylene (PE) in the influent, sludge and effluent samples (Fig. 6 A). PS was dominant in the influent (37.5%) and the sludge (25%) whereas PE (39.7%) dominated the effluent sample. The other polymers only represented a limited proportion (̴ 3 -17%) of total MPs in the wastewater.

In the surface water samples, 11 types of polymers were identified with polystyrene (PS = 40%), polyamide (PA =18%) and polyethylene terephthalate (PET =14%) the most detected polymers.

Polypropylene (PP) and PET were mainly detected in site 5 and PA in sites 2, 3 and 5.

Sediments samples had eight different polymer types (Fig. 6 B). A copolymer ethylenepropylene was observed in site 1; it was the first copolymer identified in this study. PS was the most abundant polymer (38%) and observed in all sites.

Nine types of microplastics polymers were ingested by mussels (Fig. 6 C) with PS (33%), acrylonitrile butadiene styrene (ABS = 19%) and PP (16%) the most abundant polymers.

Polystyrene (PS) and acrynotrile butadiene styrene (ABS) tend to be found in blue or green colored MPs with a size below 200 µm. Polypropylene (PP) and polyethylene (PE) were found either in transparent or in multicolor shades (green, blue and red colors) with a size class greater than 200 µm. All PET found were of fibers whereas those in PA were both fibers and films.

There was no specific relationship between colors and size for the other polymers.

Comparison of the polymers composition identified in the different matrices (water, sediments and mussels) indicated a high dissimilarity between groups (ANOSIM, R=0.857, significance level = 0.1%). Non-metric multidimensional scaling (n-MDS) showed the dimensional distance separating these groups (a, b, c, d and e) (Fig. 7). Mussels and sediments of all sites (except sediments from site 5) were regrouped in the same category, indicating a similarity in polymers composition between mussels and sediments. Water samples (except site 5) formed two more or less similar groups (SIMPER test indicated a similarity of 57%). At site 5, polymer composition of water and sediment samples were very different from the other sampling sites (65% of dissimilarity). They are characterized by the high abundance of PET and PS. (2.84 items/L) were in the range of those reported in other WWTPs in Europe, USA and Australia : between 1-3160 particles/L and 0.0007-125 particles/L for raw and treated wastewater, respectively (reviewed by [START_REF] Sun | Microplastics in wastewater treatment plants: Detection, occurrence and removal[END_REF]. According to the results of the present study, Le Havre WWTP had a MPs removal efficiency of 98.83%. Removal efficiencies of microplastics in WWTPs range generally between 72-99.4% [START_REF] Gatidou | Review on the occurrence and fate of microplastics in Sewage Treatment Plants[END_REF][START_REF] Sun | Microplastics in wastewater treatment plants: Detection, occurrence and removal[END_REF]. Variations between studies are mainly caused by different types of wastewaters and treatments used in studied WWTPs with different sampling size limitations, preparation and identification methods [START_REF] Lares | Occurrence, identification and removal of microplastic particles and fibers in conventional activated sludge process and advanced MBR technology[END_REF][START_REF] Gatidou | Review on the occurrence and fate of microplastics in Sewage Treatment Plants[END_REF]. The dominance of fibers and fragments in WWTP effluent is consistent with other studies [START_REF] Mason | Microplastic pollution is widely detected in US municipal wastewater treatment plant effluent[END_REF][START_REF] Michielssen | Fate of microplastics and other small anthropogenic litter (SAL) in wastewater treatment plants depends on unit processes employed[END_REF][START_REF] Sutton | Microplastic contamination in the San Francisco Bay, California, USA[END_REF]), yet we found a higher proportion of fragments. Generally, fibers were the most abundant type of microplastics in the wastewater [START_REF] Leslie | Microplastics en route: Field measurements in the Dutch river delta and Amsterdam canals, wastewater treatment plants, North Sea sediments and biota[END_REF][START_REF] Lares | Occurrence, identification and removal of microplastic particles and fibers in conventional activated sludge process and advanced MBR technology[END_REF]. Le Havre harbor has a strong presence of industrial activities (petrochemicals, refineries)

Discussion

and is also the leading harbor in containers traffic. Several accredited industries in Le Havre collect both solid wastes (plastics, groceries, metal, glass, ashes for example) and liquid wastes (bilges water, hydrocarbon mud, wastewater, slops) resulting from cargo and ships' selfgenerated wastes. These collected wastes by Le Havre industries could end up in the treated water of the WWTP and potentially be an important fragment source in the influent.

The concentration of suspected MPs items in the sludge samples (16.13 ± 1.25 MPs/g of dried weight) is in the average of what has been observed in other WWTPs such as in Glasgow (19.67 ±4.51 MPs /g; [START_REF] Murphy | Wastewater Treatment Works (WwTW) as a Source of Microplastics in the Aquatic Environment[END_REF]. Microplastics concentration in sludge generally varies between 1 to 170 MPs/g [START_REF] Gatidou | Review on the occurrence and fate of microplastics in Sewage Treatment Plants[END_REF]. Normally, the detected amounts of MPs found in the sludge are in orders of magnitude higher than those found in treated wastewaters [START_REF] Talvitie | How well is microlitter purified from wastewater? -A detailed study on the stepwise removal of microlitter in a tertiary level wastewater treatment plant[END_REF][START_REF] Gatidou | Review on the occurrence and fate of microplastics in Sewage Treatment Plants[END_REF] suggesting the high potential of WWTPs in retaining microplastics from raw wastewater. Even though the number of MPs retained in the sludge might seem high, but when converting the number of sludge found per g to per m 3 (the studied WWTP generates about 800 g of sludge per m 3 of treated raw influent) a concentration of about 13 000

MPs per m 3 of raw wastewater treated are retained in the sludge. Compared to the number of MPs found in influent (244 000 MPs per m 3 ), a large number of MPs seems to have been either removed during the pretreatment processes, during grease and grit removal steps or during the secondary/tertiary treatments. For example, [START_REF] Murphy | Wastewater Treatment Works (WwTW) as a Source of Microplastics in the Aquatic Environment[END_REF] observed a high proportion of microplastics' removal during grit and grease removal (44.6%). The contribution of the different processes on MPs removal is still limited. However, recent experiments using a lab-scale sequencing batch biological WWTP showed that 52% of microbeads were accumulated in the activated sludge [START_REF] Kalčíková | Wastewater treatment plant effluents as source of cosmetic polyethylene microbeads to freshwater[END_REF]. Inside the Edelweiss WWTP, activated sludge is collected, dehydrated, incinerated and transformed to a non-harmful valuable mineral product. In case of agricultural usage, this obtained product will be free of plastic (MPs will be incinerated and won't cause any potential harm to the soil). Yet, WWTP incinerate their sludge without taking into consideration the plastics existing in their samples; plastics combustion can lead to the formation of two by-products: airborne particulate emission (soot) and solid residue ash (black carbonaceous color) (Reviewed by [START_REF] Verma | Toxic pollutants from plastic waste-a review[END_REF]. Soot has a high potential of health and environmental risks (particulate bound heavy metals and dioxins (Valavanidid et al., 2008)).

Incinerating sludge should be reconsidered due to the plastics presence and its toxicity when burned. We found differences in suspected MPs size classes in the raw influent, effluent and sludge. Large sized MPs (> 200 µm) were observed in the sludge indicating their retention during treatment and explaining their low presence in the effluent. This is generally the case where larger and heavier particles are more easily retained (e.g. during sedimentation), whereas smaller particles remain in suspension [START_REF] Murphy | Wastewater Treatment Works (WwTW) as a Source of Microplastics in the Aquatic Environment[END_REF][START_REF] Magni | The fate of microplastics in an Italian Wastewater Treatment Plant[END_REF]. The dominance of smaller particles (20 -80 µm) in the effluent may also have resulted from particles degradation during treatment. As reviewed by [START_REF] Sun | Microplastics in wastewater treatment plants: Detection, occurrence and removal[END_REF], polystyrene (PS = 27%), polyethylene (PE = 24%), polyamide (PA =12%), polyethylene terephthalate (PET) and polypropylene (PP) (11% each) were the most detected polymers in WWTPs. Polystyrene (PS) are used in packaging [START_REF] Marsh | Food packaging -roles, materials, and environmental issues[END_REF] such as polystyrene foam used for coffee cups, trays, takeaway food containers, eating utensils [START_REF] Thompsett | Design and construction of expanded polystyrene embankments. Practical design methods as used in the United Kingdom[END_REF][START_REF] Issam | Adhesion properties of adhesive prepared from waste polystyrene[END_REF]. The PET and PA are all widely used in synthetic clothes; while PE are used in food packaging, films, plastics bottles and beauty care products [START_REF] Lares | Occurrence, identification and removal of microplastic particles and fibers in conventional activated sludge process and advanced MBR technology[END_REF][START_REF] Ziajahromi | Wastewater treatment plants as a pathway for microplastics: Development of a new approach to sample wastewater-based microplastics[END_REF]. Some of the polymers found in our samples can be traced to maritime activities: for example, PUR is mainly used as rigid and flexible foams, coatings, adhesives, and binders (Zhang et al. 2015).

Release, dispersion and concentration of microplastics in the marine coastal environment

Low concentrations of MPs (e.g. < 0.1 MP/L) in the effluent may contribute significantly to MPs pollution in the receiving environment due to the large volumes that are continuously discharged by WWTPs [START_REF] Mason | Microplastic pollution is widely detected in US municipal wastewater treatment plant effluent[END_REF][START_REF] Mathalon | Microplastic fibers in the intertidal ecosystem surrounding Halifax Harbor, Nova Scotia[END_REF][START_REF] Talvitie | Do wastewater treatment plants act as a potential point source of microplastics? Preliminary study in the coastal Gulf of Finland, Baltic Sea[END_REF][START_REF] Talvitie | How well is microlitter purified from wastewater? -A detailed study on the stepwise removal of microlitter in a tertiary level wastewater treatment plant[END_REF][START_REF] Michielssen | Fate of microplastics and other small anthropogenic litter (SAL) in wastewater treatment plants depends on unit processes employed[END_REF][START_REF] Carr | Transport and fate of microplastic particles in wastewater treatment plants[END_REF]Ziajahromiet al., 2017). With an average discharge volume of 80 000 m 3 /day, the daily amount of MPs released in the marine environment by Le Havre WWTP would be approximately 227 x 10 6 MPs/day. Correcting this number with our Raman identification data results in an estimated daily discharge of 210 x 10 6 MPs/day. The daily discharge of microplastics by WWTPs varies according to the number of serving habitants, the volume of treated raw influent and also depending on the seasons or environmental conditions. For example, [START_REF] Talvitie | How well is microlitter purified from wastewater? -A detailed study on the stepwise removal of microlitter in a tertiary level wastewater treatment plant[END_REF] estimated that a Finnish WWTP treating a volume of 270 000 m 3 /day may discharge MPs with values as high as 1.7×10 6 and 1.4×10 8 particles/day in different days of the week.

The results of this study reveal the presence of MPs in all examined matrices (water, sediments and mussels) and in all sites, indicating their extensive dispersion in the coastal marine environment. To our knowledge, this is the first study that assessed MPs dispersion in the coastal marine environment along a gradient from the WWTP effluent in three different matrices. Our results showed that the number of MPs in the surface water decreased with the increasing distance from the WWTP outflow. The number of MPs decreased by 96% at site 4 (5.3 km from the outflow) when comparing to the amount of MPs in the effluent. Such spatial gradient of decrease in MPs was not observed for sediments and mussels samples. There was no relationship between MPs concentration found in the water and concentrations in sediments or mussels samples. The reason for this discrepancy between surrounding water and sediments or mussels samples may be related to several factors including wheatear conditions, water circulation, dispersion velocity and MP polymers density that may affect MPs sink to the water column and seabed. In addition, the concentration of MPs in surface water is directly affected by the amount of MPs found in the effluent which varies temporally (diurnal or daily variations) [START_REF] Talvitie | How well is microlitter purified from wastewater? -A detailed study on the stepwise removal of microlitter in a tertiary level wastewater treatment plant[END_REF]. When comparing different matrices or compartments, a more adapted sampling strategy should take into account the time lag necessary for MPs to sink in the water column, to be ingested by mussels and to be sedimented at the bottom. So, the concentration of MPs measured in the surface water near a source of MPs does not necessarily reflect their bioavailability to organisms living in the water column or in the bottom. [START_REF] Railo | Application of an enzyme digestion method reveals microlitter in Mytilus trossulus at a wastewater discharge area[END_REF] showed no significant difference in MPs concentration between mussels located 30 meters or 700 meters from a WWTP discharge pipe. Another study demonstrated that bivalves located downstream and upstream the WWTP accumulated more particles than those located directly near the effluent (Domogalla-Urbansky et al., 2018). The heterogenicity of the MPs distribution and of its concentration levels in different matrices are likely reflecting a complex interplay between the distance from the pollution sources, hydrodynamic features such as currents, gyres, fronts and shape of the coast line (Schmidt et al., 2018). In open aquatic environment, far from direct sources of MPs, a correlation was found between microplastic levels in the water and in the mussels. This is the case in open coastal waters along the China coasts where [START_REF] Qu | Assessing the relationship between the abundance and properties of microplastics in water and in mussels[END_REF] found a positive and quantitative correlation of MPs in mussels and in their surrounding waters.

In comparison with the MPs concentrations reported for other water bodies nearby the discharge of WWTP effluents, the concentrations of the present study were considerably higher. For example, MPs concentrations of 0.001 -0.002 MPs/L were reported for seawater collected 20 -200 meters from the WWTP effluent outlet in Sweden [START_REF] Magnusson | Screening of microplastic particles in and down-stream a wastewater treatment plant[END_REF]. In the Seine River (near Paris) MPs concentrations of 0.013 -0.1 MPs/L were recorded downstream the Seine-Centre wastewater treatment plant [START_REF] Dris | Microplastic contamination in an urban area: a case study in Greater Paris[END_REF]. In the bay of Brest (France), near an important military harbor, the mean MPs concentration in surface water was 0.00024 ± 0.00035 MPs/L [START_REF] Frère | Influence of environmental and anthropogenic factors on the composition, concentration and spatial distribution of microplastics: A case study of the Bay of Brest (Brittany, France)[END_REF]. In sediments, as for water, the MPs concentrations were also higher than those recorded in the Southern North Sea in the harbor of Nieuwpoort 166.7 ± 92.1 MP/kg DW [START_REF] Claessens | Occurrence and distribution of microplastics in marine sediments along the Belgian coast[END_REF] or in the bay of Brest 0.97 ± 2.08 MPs/kg DW [START_REF] Frère | Influence of environmental and anthropogenic factors on the composition, concentration and spatial distribution of microplastics: A case study of the Bay of Brest (Brittany, France)[END_REF], but in the range of those recorded in Mediterranean coastal sediments [START_REF] Alomar | Microplastics in the Mediterranean Sea: Deposition in coastal shallow sediments, spatial variation and preferential grain size[END_REF] or in the Venetian Lagoon [START_REF] Vianello | Microplastic particles in sediments of Lagoon of Venice, Italy: First observations on occurrence, spatial patterns and identification[END_REF]. Concerning mussels, the number of MPs ingested ranged between 0.4 -1 MPs/g w.w and was similar to those found by [START_REF] Li | Microplastics in mussels sampled from coastal waters and supermarkets in the United Kingdom[END_REF] in the UK coastal water (0.7-2.9 items/g w.w) but higher than those recorded by Van Cauwenbergh et al. The high number of MPs in surface water, sediments and mussels found in Le Havre harbor can be linked to the continuous discharge of the WWTP in this semi-enclosed basin. Harbor's geometry might contribute to a low flushing rate [START_REF] Claessens | Occurrence and distribution of microplastics in marine sediments along the Belgian coast[END_REF]; its morphology (the basin) along with a great amount of daily discharged MPs will result to a high accumulation of MPs and raises their chances of ending at the bottom. Small water bodies have higher MPs concentration than that in open sea [START_REF] Luo | Comparison of microplastic pollution in different water bodies from urban creeks to coastal waters[END_REF] explaining the great abundance of MPs found inside the harbor.

Several studies showed that fibers were the most dominant in sediments [START_REF] Alomar | Microplastics in the Mediterranean Sea: Deposition in coastal shallow sediments, spatial variation and preferential grain size[END_REF][START_REF] Cannas | Plastic litter in sediments from the coasts of south Tuscany (Tyrrhenian Sea)[END_REF][START_REF] Lots | A large-scale investigation of microplastic contamination: Abundance and characteristics of microplastics in European beach sediment[END_REF][START_REF] Blair | Secondary microplastics were prevalent in sediment in a freshwater UK urban river[END_REF] or mussels [START_REF] Catarino | Development and optimization of a standard method for extraction of microplastics in mussels by enzyme digestion of soft tissues[END_REF][START_REF] Bråte | Mytilus spp. as sentinels for monitoring microplastic pollution in Norwegian coastal waters: A qualitative and quantitative study[END_REF][START_REF] De Witte | Quality assessment of the blue mussel (Mytilus edulis): Comparison between commercial and wild types[END_REF] whereas in our study, fragments were the most dominant as observed in the sediments of the Venetian Lagoon [START_REF] Vianello | Microplastic particles in sediments of Lagoon of Venice, Italy: First observations on occurrence, spatial patterns and identification[END_REF]. The dominance of fragments over other types of MPs in sediments and mussels samples suggests an additional source of their entry other than the WWTP where textile-derived fibers are dominant [START_REF] Frère | Influence of environmental and anthropogenic factors on the composition, concentration and spatial distribution of microplastics: A case study of the Bay of Brest (Brittany, France)[END_REF][START_REF] Gatidou | Review on the occurrence and fate of microplastics in Sewage Treatment Plants[END_REF]. The main source of MPs fragments in the harbor may be related to anthropogenic activities and to the breakdown of larger plastic debris [START_REF] Cole | Microplastics as contaminants in the marine environment: A review[END_REF][START_REF] Vianello | Microplastic particles in sediments of Lagoon of Venice, Italy: First observations on occurrence, spatial patterns and identification[END_REF]. The shape and color characteristics of fragments found indicate that they are mainly secondary microplastics resulting from the decomposition of larger plastics. The MPs polymers composition identified in the sediments and mussels' samples were different from those identified from the effluent or surface water samples (Fig. 6) suggesting that there are other sources of MPs inputs into the harbor. Le Havre is a highly industrialized city and a leading chemical industrial platform in Europe. It is the 1 st French petrochemical complex, with 24.3 tons of crude oil processed per year. Many refineries and plastic companies (about 200 companies) are located in and around the harbor.

Coastal landfill as important source and route for MPs in the coastal environment

This study revealed strong MPs abundances in the coastal environment near the Sainte Adresse's abandoned coastal landfill (site 5). To our knowledge, no study to date has evaluated MPs concentrations in coastal areas near landfills. The concentrations found in the water and mussels samples are 2 to 4 times higher than those found in Le Havre Harbor. However lower MPs concentration in sediments was observed in site 5. This can be explained by the difference in sediments composition between sites. Site 5 consists of sandy sediments whereas for the other sites (inside the harbor), the sediments are sandy-muddy to muddy. [START_REF] Strand | Microplastic particles in sediments from Danish waters[END_REF] demonstrated that there is a strong relationship between MPs abundance and both organic (%TOC) and fine fraction (< 63 μm) content in sediments, supporting the hypothesis that MPs will accumulate in depositional areas. In the Lagoon of Venice, [START_REF] Vianello | Microplastic particles in sediments of Lagoon of Venice, Italy: First observations on occurrence, spatial patterns and identification[END_REF] detected the lowest microplastic concentrations in the outer Lagoon, where water currents are higher (> 1 m/s). Consequently, the highest concentrations were encountered in the inner Lagoon which is characterized by lower hydrodynamics and a higher fine particle (< 63 μm) fraction in the sediment.

Our results highlight the significant role that coastal landfills play in MPs entry into the marine coastal environment. Many environmental factors and microbial degradation [START_REF] Zettler | Life in the "plastisphere": microbial communities on plastic marine debris[END_REF][START_REF] He | Municipal solid waste (MSW) landfill: A source of microplastics?-Evidence of microplastics in landfill leachate[END_REF] may lead to the fragmentation of plastics to microplastics, and small plastic debris would be carried out by the discharge of leachate. Microplastics were identified in the leachate from both active and closed landfills [START_REF] He | Municipal solid waste (MSW) landfill: A source of microplastics?-Evidence of microplastics in landfill leachate[END_REF]. Microplastics polymer composition found at site 5 near the landfill was different from other sampling sites although this cannot provide conclusive evidence of these microplastics origins. This site had also a dominance of polystyrene (PS) (̴ 25 -42%) with a high abundance of PS microbeads (22%) in the water sample. PS is generally more prone to weathering by UV radiation than other plastics [START_REF] Alimi | Microplastics and nanoplastics in aquatic environments: aggregation, deposition, and enhanced contaminant transport[END_REF]. The landfill could potentially carry more PS plastic into the environment than the WWTP discharge. Although fragmentation and release of microplastics is a long-term process, our results confirm that landfill isn't the final sink of plastics, but a potential source of microplastics [START_REF] He | Municipal solid waste (MSW) landfill: A source of microplastics?-Evidence of microplastics in landfill leachate[END_REF]. This can pose serious environmental problems since microplastic is not listed as pollutant in the any country's regulations of landfill.

Mussels as sentinel species for MPs pollution

Mussels from all sites have mainly ingested small sized MPs (< 200 μm). This was similar to other studies that demonstrated that MPs below 300 μm were the most found in bivalves [START_REF] Leslie | Microplastics en route: Field measurements in the Dutch river delta and Amsterdam canals, wastewater treatment plants, North Sea sediments and biota[END_REF][START_REF] Naji | Microplastics contamination in molluscs from the northern part of the Persian Gulf[END_REF]Phuong et al., 2018). Although we found that small sized MPs dominated the water and sediments samples, it is currently unknown whether their abundance in mussels are due to mussels size preferences in regards to prey, or because the majority of MPs in the environment were typically in this size range. In laboratory exposure experiments, [START_REF] Browne | Ingested microscopic plastic translocates to the circulatory system of the mussel Mytilus edulis (L.)[END_REF] previously demonstrated that mussels are capable of ingesting plastic particles between 3 and 10 μm in size that they filter from the water phase. Several studies showed that bivalves are capable to ingest different type of polymers and the most common plastic inside their soft tissues differed from one study to another [START_REF] Avio | Microplastics pollution after the removal of the Costa Concordia wreck: First evidences from a biomonitoring case study[END_REF]Phuong et al., 2018;[START_REF] Digka | Microplastics in mussels and fish from the Northern Ionian Sea[END_REF][START_REF] Bråte | Mytilus spp. as sentinels for monitoring microplastic pollution in Norwegian coastal waters: A qualitative and quantitative study[END_REF]. Polystyrene (PS) was the most common polymer found in our mussels, followed by acrylonitrile butadiene styrene (ABS) and polypropylene (PP). They are the most common plastics used worldwide whether in packaging or other household materials. The comparison of ingested polymers by mussels indicated a high similarity with sediments (69% of average similarity -SIMPER test). Mussels, such as the blue mussel Mytilus edulis is often selected as model species as they inhabit a wide geographic range, are sedentary, and filter large volumes of water. Our study supports the idea that the blue mussels are a promising sentinel species for small MPs (< 1 mm) in the marine environment [START_REF] Bråte | Mytilus spp. as sentinels for monitoring microplastic pollution in Norwegian coastal waters: A qualitative and quantitative study[END_REF]Li et al., 2019). However, if they are suggested to be used as a standard bioindicator for MPs contamination in the marine environment [START_REF] Ospar | OSPAR Request on Development of a Common Monitoring Protocol for Plastic Particles in Fish Stomachs and Selected Shellfish on the Basis of Existing Fish Disease Surveys ICES Advice[END_REF], it is necessary to resolve a standardized method for mussel sampling and MPs extraction and identification in order to ameliorate the comparability between MPs data studies.

Conclusion

The investigated WWTP had microplastics retention efficiency of 98.83% yielding to daily discharges of 227 million MPs predominantly composed of fibers and fragments. These MPs were found in all examined matrices (water, sediments and mussels) and in all sites, indicating their extensive dispersion in the coastal marine environment. Polystyrene (PS), polyethylene (PE), polypropylene (PP) and acrylonitrile butadiene styrene (ABS) were the most common polymers identified by Raman spectroscopy. The number, size and type of MPs in the water, sediments and mussels differed but a similarity in polymer composition was found between mussels and sediments samples. Our study supports the idea that the blue mussels are a promising sentinel species for small MPs (< 200 μm) in the marine environment.

To our knowledge, this study is the first that assessed MPs dispersion in the coastal marine environment along a distance gradient from the WWTP outflow in three matrices. Our results

showed that the number of MPs in the surface water decreased with the increasing distance from

  filter weight after digestion (g) IFW: Initial filter weight (g) TW: Mussel total weight (g)The volume with the highest digestion efficiency was chosen for all wild mussels individuals. A volume of 250 mL of KOH% was chosen for the samples (%DE= 99.82 ± 0.3%) 2.4. Microplastics observation and identification: 2.4.1. Visual observation After digestion, filters were observed under 120x magnification using Leica M165 C Stereomicroscope and images of suspected MP particles were taken with a Leica M170 HD camera and LAS (Leica application suite) software. To prevent double-counting of MPs, filters were methodically examined from the highest edge to the lowest along the first column, lowest to highest edge along the second column and so on. All suspected MPs particles were counted, categorized by type (films, fragments, microbeads or fibers) and color (dark, red, yellow, blue, white or green). The following criteria were taken into consideration while counting as mentioned by[START_REF] Hidalgo-Ruz | Microplastics in the marine environment: a review of the methods used for identification and quantification[END_REF] (1) absence of cellular or organic structures; (2) a homogenous thickness across the particles; and, (3) homogenous colors. Specific criteria were taken for counting synthetic fibers: consistent in thickness with no taper toward the ends and are

3. 4 .

 4 Items size distribution in the three different matrices Suspected MPs were categorized into five different size classes as shown in Fig. 5. Inside the WWTP (Fig. 5 A), two MPs size classes (20 -80 µm and 80 -200 µm) dominated in the raw sewage influent samples and were equally distributed (30%) whereas the other size classes: < 20 µm and 200 -500 µm represented a respective 14% and 17.5% of the sample. The effluent water sample was dominated by small MPs from the size class (20 -80 µm) representing 42% of the sample. Two size classes: 80 -200 µm and 200-500 µm were equally distributed in the effluent; each one represented a percentage of 20.8%. Whereas in the sludge, the size class 200-500 µm dominated with 56.25% of the suspected MPs followed by size classes 80-200 µm and > 500 µm (18.75% each). Smaller items were less abundant in the sewage sludge with only 6.25% items in the size class of 20-80 µm with no observed items in the size class <20 µm.

4. 1 .

 1 Wastewater treatment plant as a MPs source in the marine environment Many recent studies have highlighted the crucial role of municipal wastewater treatment plants as potential pathways of microplastics entry into the aquatic environment. The detected amounts of suspected MPs items in the raw sewage influent (244 items/L) and treated wastewater effluent

(

  2015) in Belgian, Dutch and French North Sea coasts (0.2 ± 0.3 items/g w.w) or in the French Atlantic coast by Phuong et al. (2018) (0.23 ± 0.20 items/g d.w).

  the WWTP outflow. It revealed strong MPs abundances (2 to 4 times higher than those found near the WWTP outlet) in the vicinity of the abandoned coastal landfill suggesting that it contributes more than the WWTP effluent as routes of MPs entry into the marine coastal environment. This study was limited by the number of water samples taken even though high volumes of water were filtered. Yet, it gives an idea about the MPs being released by the WWTP and coastal landfill. However, more researches should concentrate on microplastic sources and their temporal variations in relation with different factors such as environmental conditions (seasons, precipitation, wind, water current, tide,..) and factors associated with the operation of the WWTP (the volume of treated raw influent, timing of effluent discharge,..).Such knowledge could help controlling different MPs sources in order to prevent related MPs pollution and, should be, therefore, more comprehensively investigated in the future.FundingMaria Kazour is financially supported by a PhD fellowship from the National Council for Scientific Research (Lebanon) and Université du Littoral Côte d'Opale (France). This work has been financially supported by the European Union (ERDF), the French Government, the French Region Hauts-de-France and Ifremer, in the framework of the project CPER MARCO 2015-2020.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Localization of the WWTP (Le Havre wastewater treatment plant) and the sampling sites: 1 (near the effluent of the WWTP), (2) upstream and (3) & (4) downstream the effluent, (5) Sainte-Adresse's site (near the coastal landfill).

Figure 4 :

 4 Figure4: Average (±SD) number of suspected microplastics (items/g of mussels' wet weight) by shape categories in mussels collected in all the investigated sites (statistics: letters indicate significant differences between sites; p < 0.05).

Figure 5 :Figure 6 :Figure 7 :

 567 Figure 5: Proportion of size class composition of suspected microplastics in the three analyzed matrices A) WWTP and the surface water, B) sediments and (C) mussels from the different sampling sites (site 1 being the closest to the effluent to site 5 being the farthest near the landfill; see figure 1).

Acknowledgments

The authors would like to thank the administration of Le Havre harbor (especially Miss. Magali HAUTOT who helped obtaining permissions to enter the harbor and was our companion during sampling), the CODAH and the wastewater treatment plant "EDELWEISS" board and crew for allowing us to collect our samples.

Declaration of interests: None