Angelica Miglioli

Teresa Balbi

Lydia Besnardeau

Remi Dumollard

Laura Canesi
email: laura.canesi@unige.it

Laura Canesi Distav

Bisphenol A interferes with first shell formation and development of the serotoninergic system in early larval stages of Mytilus galloprovincialis

Keywords: Bisphenol A, mussel, early development, shell formation, tyrosinase, hox1, serotonin

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

Introduction

Bisphenol A (BPA), a monomer used in the production of epoxy resins and polycarbonate plastics is continuously produced and released into the environment in large quantities globally through sewage discharges, landfill leachate, surface runoff, and release from plastic debris. Although it degrades quickly, due to its continual inputs, it is pseudo-persistent and ubiquitous in different aquatic compartments, with reported concentrations between ng and µg/L (Flint et and Seebacher, 2020). For freshwater, measurements are on average in the range between 10 and some 100 ng/l with mean maximum concentrations of 2000 ng/L for freshwater and above 8000 ng/L in marine waters (ECHA, 2017 and refs. therein). In Europe, concentrations of BPA were above detection limits in 70% and 61% of freshwater and marine water samples [START_REF] Staples | Distributions of concentrations of bisphenol A in North American and European surface waters and sediments determined from 19 years of monitoring data[END_REF]. BPA is weakly estrogenic and a potential endocrine disrupting chemical-EDC in mammals, and it also represents a potential concern for aquatic organisms, as shown by its reproductive and developmental effects on aquatic vertebrates and invertebrates, in particular on early life stages of molluscs and amphibians [START_REF] Canesi | Environmental Effects of BPA[END_REF]; ECHA, 2017; Cuvillier-Hot and Lenoir, 2020; [START_REF] Wu | Effect of the plastic pollutant bisphenol A on the biology of aquatic organisms: A meta-analysis[END_REF]. In July 2019, the General Court of the European Union confirmed that BPA must be listed as a substance of 'very high concern' (https://www.ehn.org/bisphenol-a-2639174350.html).

Marine invertebrates are sensitive to environmental stressors, especially during early larval stages.

In addition, taxa characterized by calcified structures (e.g. molluscs, echinoderms) are markedly vulnerable to global changes, including ocean acidification and pollution [START_REF] Hamdoun | Embryo stability and vulnerability in an always changing world[END_REF][START_REF] Przeslawski | A review and meta-analysis of the effects of multiple abiotic stressors on marine embryos and larvae[END_REF]. In this context, bivalve molluscs represent an important ecological group that, by living in complex environments such as coastal ecosystems, experience both natural environmental fluctuations (pH, temperature, salinity) and exposure to anthropogenic chemicals, and also show rapid adaptation capabilities to environmental fluctuations [START_REF] Bitter | Standing genetic variation fuels rapid adaptation to ocean acidification[END_REF]. In the mussel Mytilus galloprovincialis, BPA has been shown to affect early development in the 48h embryotoxicity assay in a wide concentration range [START_REF] Fabbri | Adaptation of the bivalve embryotoxicity assay for the high throughput screening of emerging contaminants in Mytilus galloprovincialis[END_REF]. Further studies demonstrated that BPA, from nanoMolar concentrations, significantly affected transcription of several genes, including those involved in biomineralization and the serotonin receptor 5-HTR, both at the trocophora and D-veliger stage (24 and 48h pf post fertilization-pf, respectively ) [START_REF] Balbi | Impact of bisphenol A (BPA) on early embryo development in the marine mussel Mytilus galloprovincialis: Effects on gene transcription[END_REF]. However, it is not known whether the observed changes in gene transcription induced by BPA are associated with impaired shell growth and formation of serotoninergic neurons, two key processes occurring during the first stages of larval development.

In M. galloprovincialis, the processes leading to the formation of the first larval shell have been recently investigated. The time course of both organic matrix and CaCO3 deposition was evaluated, from the initial shell only made of organic matrix and characterized by a saddle shape (the shell field), to development of the first calcified larva, the D-veliger [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF]. These processes were associated with upregulation of genes involved in biomineralization (Tyrosinase, Chitin synthase, Carbonic anhydrase, Extrapallial Protein). In particular, the results underlined the pivotal role of tyrosinase in matrix deposition and remodelling and in driving first shell calcification [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF].

In the present work, the effects of BPA (0.05, 0.5 and 5 µM) on the mechanisms involved in shell formation, and on the development of the serotoninergic system, were investigated in mussel larvae at close times pf (24, 28, 32, and 48h). Deposition of the organic matrix and of the calcified shell were evaluated by confocal imaging of Calcofluor/Calcein staining. The effect of BPA on larval phenotypes at 48hpf was also evaluated. Transcription of tyrosinase (TYR) was quantified by qPCR and its spatial pattern of expression by In Situ Hybridization-ISH. The effects of BPA on localization of transcripts for M. galloprovincialis Homeobox 1 (HOX1), a conserved transcription factor involved in ectodermal patterning and differentiation [START_REF] Barucca | Hox and paraHox genes in bivalve molluscs[END_REF]Perez-Parallè et al, 2005) and in molluscan larval shell formation and neurogenesis (Huan, et al. 2020) was also evaluated by ISH. Development of the serotoninergic system was investigated by evaluating transcription of the serotonin receptors 5-HTR by qPCR and the presence of serotoninergic neurons by 5HT immunoreactivity.

Materials and Methods

Animal handling, gamete collection and fertilization

All procedures were carried out as previously described [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF]. and details are reported in SI. Fertilized eggs were exposed to different concentrations of BPA, depending on the endpoint measured (final concentrations 0.05, 0.5 and 5 µM, corresponding to 11.4, 114 and 1140 µg/L). These concentrations fall within the range previously utilized for evaluating the effects of BPA on mussel larval development at 48 hpf [START_REF] Fabbri | Adaptation of the bivalve embryotoxicity assay for the high throughput screening of emerging contaminants in Mytilus galloprovincialis[END_REF].

Effect of Bisphenol A on formation of the first larval shell: morphometric analysis

Fertilized eggs were exposed to BPA in 24-well plates at 30 min pf. Stock solutions of BPA (0.5 M in Dimethyl-sulfoxide/DMSO) were diluted in MFSW at 100 µM and tenfold serially diluted to obtain the desired concentrations (100 nM, 1 µM and 10 µM -2x with respect to final concentration). The time-course of shell formation was investigated at different times pf, between the trochophore and the D-veliger stage (24, 28, 32 and 48 hpf). For details on early larval development see SI. Control samples in MFSW added with the vehicle at the final concentration utilized (0.01% DMSO) were run in parallel. Fluorescent dyes were employed to visualize the components of the larval shell as previously described [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF]: Calcofluor White Fluorescent Brightener 28 (Sigma Aldrich, Lyon, France) for organic matrix and Calcein (Sigma Aldrich, Lyon, France) for CaCO3 deposition, respectively, as previously described [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF] and details are reported in SI .

Larvae were imaged at each experimental time point with a Leica SP8 Confocal Laser Scanning Microscope (CLSM -Leica, France) scanning sequentially Calcofluor, Calcein and Brightfield signals with a 0.5 µm Z-stack interval. Channels were merged, 3D rendered and rotated to measure the area (in µm 2 ) of one organic/calcified valve in each larva by manual drawing using IMAGEJ software (Kapsenberg et al., 2018). Area measurements were performed on larvae obtained from 5 independent parental pairs (N=5) and on least 15 individuals for each experimental condition. Data were normalized with respect to controls for each parental pair in different experimental conditions (times pf and exposure).

At 48 hpf, the effect of BPA was also evaluated by scoring shell malformations and measuring shell lengths in larvae from 5 independent parental pairs and in at least 50 larvae for each parental pair (see SI).

In situ hybridization -ISH

The ISH protocol previously described for M. galloprovincialis larvae [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF] was used to investigate the expression patterns of Mg-tyrosinase (Mg-Tyr, GenBank: KV583276.1) and Mg-Homeobox1A (Mg-HOX1A, GenBank: FN597580.1). Primer pairs were designed to amplify approximately 1-1.5 Kb cDNA fragment of Mg-Tyr (5'-ATGCGATTCTTTATACATGAAA-3';3'-TTGGTGGTTTTGGTACATGT-5') and Mg-HOX1A (5'-ATGAATACGGAGAGTGATTACACA-3'; 3'-TCGACTGTCCTGACATAAACCCAT-5'). The amplicons were then used to synthesize sense and antisense digoxigenin-labelled RNA probes as previously described [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF]. Larvae (control and BPA-exposed) were grown in 50 mL culture flasks as described above. At different times pf [START_REF] Przeslawski | A review and meta-analysis of the effects of multiple abiotic stressors on marine embryos and larvae[END_REF][START_REF] Schmittgen | Analyzing real-time PCR data by the comparative CT method[END_REF][START_REF] Voronezhskaya | Neuronal development in larval mussel Mytilus trossulus (Mollusca: Bivalvia)[END_REF]

qPCR

Eggs were fertilized in polystyrene 6-well plates and treated with 2x BPA solution in order to obtain the final nominal concentration of 0.05 μM in a 8 mL volume. Control samples were run in parallel.

Larvae were collected at 24, 28, 32 and 48 hpf by a nylon mesh (20 μm pore-filter) and washed with artificial seawater (ASW) [START_REF] Balbi | Impact of bisphenol A (BPA) on early embryo development in the marine mussel Mytilus galloprovincialis: Effects on gene transcription[END_REF]. Three wells for each stage were pooled in order to obtain approximately 7000 embryos/replicate. RNA extraction, retro-transcription and qPCR were carried out as previously described (Balbi et al., 206;[START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF] and are described in SI.

5-HT immunocytochemistry

Antibodies against the neurotransmitter serotonin (5-HT) were used to visualize the development of the serotonergic system in control, 0.05 and 0. Embryos were imaged with a Leica Sp8 CLSM; Hoechst, 5-HT (ir) and Brightfield signals were sequentially scanned with a 0.3 µm Z-stack interval. Using ImageJ software, channels were merged and subsequent Z-stacks were assembled in order to assess the number of immunoreactive (ir) cells by counting Hoechst stained nuclei in the 5-HT ir region as shown in Fig. S1.

Statistical analysis

All data, obtained from 5 or 4 independent parental pairs, depending on the experiment (mean ± SD) were analysed by one-way nonparametric ANOVA (Kruskal-Wallis test) followed by the Tukey's test (p<0.05). All statistical calculations were performed using GraphPad Prism 5 software (GraphPad Inc.).

Results

Effect of Bisphenol-A on shell biogenesis and larval development

Fertilized eggs were exposed to different concentrations of BPA (0.05, 0.5 and 5 µM) and the growth of the organic matrix and calcified shell was evaluated by morphometric analysis of stained larvae at different times pf, as previously described [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF]. In Fig. 1 representative images are reported of Calcofluor/Calcein staining in control larvae and larvae exposed to different concentrations of BPA (from left to right) at 24, 28, and 32hpf, showing the organic matrix (blue)

and calcified component (green) of the growing shell. As previously described, in control samples at 24hpf, the initial larval shell was characterized by a saddle shape (the shell field) completely made of organic matrix, with no signs of calcification [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF]. BPA exposure induced alterations of the organic matrix, that was reduced to a thin line at the highest concentration tested (5 µM). At 28hpf, when first calcification was visible in control larvae, increasing concentrations of BPA induced changes in both structures. At 32hpf, control samples showed increased calcification, with evident accretion rings, while the hinge region was still not calcified. At this time point, all BPA concentrations affected the organic matrix as well as the calcified shell. In particular, larvae exposed to both 0.5 and 5 µM BPA had the typical appearance of immature stages, and no accretion rings were visible in the calcifying shell. At 48hpf, control larvae reached the normal D-veliger stage, characterized by extensive calcification and a straight hinge. In BPA-exposed larvae, Dveligers were also observed; however, lower concentrations of BPA induced shell malformations, in particular in the hinge region, and also altered the pattern of calcification. At the highest concentration tested (5 µM), BPA led to disruption of both shell matrix and CaCO3 deposition.

The effects of BPA on the growth of organic matrix and calcified shell were quantified by measuring the areas of each shell component (µm 2 ) as previously described [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF].

Data are reported in Fig. 2, as % values recorded in BPA-exposed larvae with respect to controls at 24 (A), 28 (B) and 32hpf (C). At 24hpf, BPA induced a dose-dependent decrease of the area occupied by the organic matrix (blue), that was significant at 0.5 and 5 µM BPA (-28% and 64%, respectively; p<0.05 (Fig. 2A). A similar effect on the organic matrix was observed at 28 hpf (Fig. 2B). Moreover, at this stage BPA induced a concentration dependent decrease in the calcified areas (green) that paralleled those observed for the organic matrix; such a decrease was significant at all the concentrations tested (from -20% to -50% at 0.05 and 5 µM, respectively; p<0.05) (Fig. 2B).

Similar decreases in both shell components were observed at 32hpf at all the concentrations tested (Fig. 2C), and they were associated with evident phenotypic changes (Fig. 1). The results are summarized in Fig. 2D, where data are reported as organic matrix:calcified shell ratios in different experimental conditions. In control larvae this ratio showed a significant decrease from 28 to 32 hpf, reflecting progressive calcification. BPA induced a concentration-dependent increase in the area occupied by the organic matrix with respect to that of the calcified shell, indicating a delay in CaCO3 deposition.

In Fig. 3A main phenotypical alterations induced by BPA at 48 hpf are reported (malformed hinge, protruding mantle, arrested trocophora). BPA induced a concentration dependent decrease in the percentage of normal D-larvae (from 94% in controls to 18% at the highest BPA concentration) (Fig. 3B). A small increase in the percentage of malformed shells (convex or indented hinge) was observed with respect to the number of total larvae at all the concentrations tested (from a minimum of 2.7% to a maximum of 9.4% in control and larvae exposed to 5 µM BPA. Moreover, a progressive delay in development was observed, indicated by the increase in the proportion of immature D-veligers, characterized by protruding mantle, at all the concentrations tested (from 3.4% in controls to 25.7% in larvae exposed to 5 µM BPA). In addition, a clear concentration dependent increase in the proportion of larvae withheld at the trocophora stage was observed, reaching 50% of total larvae at the highest concentration tested. When the average shell length of all D-veligers (normal, malformed, immature) was measured, both 0.5 and 5 µM BPA also induced a significant decrease in size with respect to controls (Fig. 3C) (p<0.05).

Effect of BPA on gene expression in the shell field: tyrosinase and HOX1

After screening different genes involved in first shell formation (such as CS, CA, EP) (not shown), was first quantified by qPCR in control larvae and larvae exposed to the lowest concentration of BPA (0.05 µM). In Fig. 4, data on the time course of the level of TYR mRNA transcripts in control and BPA-exposed larvae at different times pf are reported with respect to eggs. In control larvae, a large upregulation was observed from 24hpf, that peaked at 32hpf (with an increase of about 8000folds with respect to eggs), followed by a decrease at 48 hpf. Such trend was affected by exposure to BPA, that significantly increased TYR transcription at 24 and 32 hpf (+67% and +34 % compared to controls respectively, p<0.05). The effects of BPA on the TYR expression pattern were then evaluated by ISH (Fig. 5). As previously described [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF] in larvae grown in physiological conditions, TYR was highly expressed in the shell field area from 24 hpf, and its localization subsequently paralleled the growth of the organic matrix as the trochophore developed into the first D-Veliger at 48 hpf, when the signal was weaker and restricted to the margins of the growing shell (Fig. 5, first column). At 24 and 28 hpf, lower concentrations of BPA (0.05 and 0.5 µM) did not significantly affect TYR expression pattern. However, from 32hpf, an altered spatial pattern was observed at all the concentrations tested, that was more evident at 48 hpf, when the TYR signal was detected in the whole larval body. At the highest concentration (5 µM) BPA affected localization of TYR transcripts at all times pf.

ISH was also utilized to investigate the expression pattern of HOX1 mRNA and the results are reported in Fig. 6. In control larvae, HOX1 expression was highest at 24 hpf, when it was localized at the external margins of the shell field, but not in its centre, as clearly observed in the dorsal view of the larva. From 28 hpf, the signal was concentrated at two distinct, small areas along the hinge region (lateral view, black arrows), and it progressively weakened until becoming hardly detectable at 48hpf. BPA clearly affected HOX1 expression pattern at all times pf and at increasing concentrations. At 24 hpf, HOX1 transcripts progressively filled the centre of the shell field; at 28hpf the two distinct sites of expression (black arrows) were closer to each other until almost in contact in correspondence to the indented hinge at the highest concentration tested. At 32 hpf, BPAexposed larvae showed a stronger signal (similar to that observed in control larvae at 28 hpf)

indicating developmental delay, and the two sites of expression were closer with respect to controls.

Finally, at 48 hpf, BPA apparently increased the HOX1 signal, but it strongly altered its pattern of expression in the whole larvae at all concentrations tested.

Effect of BPA on development of the serotonergic system

Levels of mRNA transcripts for the serotonin receptor 5-HTR were quantified by qPCR in control larvae and larvae exposed to BPA (0.05 µM) and data on the time course of 5-HTR transcription at different times pf are reported with respect to eggs in Fig. 7. In control samples, after a first upregulation at 24 hpf (about 150-folds with respect to eggs), a decrease in mRNA abundance for 5-HTR was observed at both 28 and 32 hpf, followed by strong upregulation at 48 hpf (about 700 folds vs eggs). BPA exposure did not affect transcription of 5-HTR between 24 and 32 hpf, whereas a significant decrease was observed at 48 hpf (-64% with respect to controls; p<0.05).

5-HT immunofluorescence labelling was utilized to detect serotonin positive cells by confocal microscopy, together with Hoechst staining to identify cell nuclei (Fig. S1), in control larvae and larvae exposed to 0.05 µM BPA at (24, 28, 32 and 48hpf). The results are reported in Fig. 8 and Fig. 9, respectively. In control larvae 5-HT immunoreactivity (ir) was detectable from 24 hpf and it was concentrated in few cells (Fig. 8A, columns a and b) that were located in the pre-trochal area of the larva, close to the apical cilia (Fig. S2). As shown more detail in Fig 8A (column c), two 5-HTir cells closely adjacent to each other could be identified in trochophora at 24hpf. As the larva developed, the number of 5-HT-ir cells progressively increased to three at 28 hpf, and to four in the late trocophora (32 hpf), where the emergence of tiny neurites could be also observed. In BPAexposed larvae 5-HT immunoreactivity, was clearly affected with respect to control larvae; in particular, no more than two 5-HT-ir cells could be observed from the early to the late trocophora stage (from 24 to 32 hpf) (Fig. 8B). At 48 hpf (Fig. 9), control D-veligers showed up to seven serotonin positive neurons, organized into a compact pyramidal structure (columns a and b), and emerging axons could be clearly identified expanding towards the hinge in the dorsal side of the larva (Fig. 9A, column c). At this stage, in BPA-exposed larvae, different effects were observed depending on the BPA-induced phenotype. In D-veligers showing protruding mantles, only five, loosely connected 5-HT-ir cells were observed (column c). Moreover, ectopic 5-HT-ir cells were occasionally observed in the protruding mantle (not shown). In arrested trocophorae, no more than three 5-HT-ir neurons could be observed. Higher concentrations of BPA (0.5 µM) more severely affected 5-HT immunoreactivity (Fig. S3 and Fig. S4). From 24 to 32 hpf, only one and two weakly 5-HT-ir cells could be observed (Fig. S3). At 48hf, a stronger reduction in 5-HT-ir cell number was observed in malformed and immature D-veligers and in arrested trocophorae, where three, four and two serotonin positive cells could be observed, respectively (Fig. S4).

Discussion

The results of this work obtained in the mussel M. galloprovincialis represent the first data on the effects of BPA, a contaminant of emerging concern, on first shell formation and development of serotoninergic system, two key processes of early larval development in bivalves. Different effects of BPA could be observed from the lowest concentration tested (0.05 µM, corresponding to 11.4 µg/L), that falls within the environmental concentrations of BPA detected in some coastal areas [START_REF] Corrales | Global Assessment of Bisphenol A in the Environment[END_REF].

Increasing concentrations of BPA affected the growth of both components of the shell (organic matrix and CaCO3 deposition), this mainly resulting in developmental delay. When the overall impact of BPA on the growth of the two shell components was quantified by the organic matrix:calcified shell ratio, a concentration and time dependent increase was observed at increasing times pf, indicating an unbalance in the processes of shell formation. In particular, BPA induced alterations in the pattern of deposition of both the organic matrix, that were progressively detectable from the early trochophora stage, and of CaCO3, with barely detectable shell accretion rings and nonhomogeneous calcification at later stages. These effects were reflected at 48 hpf by a concentration dependent increase in the frequency and severity of altered shell phenotypes, resulting in develop- Here we show that in M. galloprovincialis HOX1 was strongly expressed at 24 hpf in the dorsal side of the trocophora around the shell field, but not in its centre, and expression progressively declined at longer times pf. Exposure to BPA, from environmental concentrations clearly induced dose-dependent alterations in the transcription pattern of HOX1 at all times pf. Although other Mytilus HOX genes will participate in larval development, these results clearly show that expression of HOX1, a transcription factor involved in ectodermal patterning, is concentrated in the shell field, thus supporting its role in initial shell formation, and that it represents a significant target for BPA during larval development. These results support the hypothesis that BPA can affect shell biogenesis from the very beginning of the morphogenetic process.

Previous data showed that both BPA and the natural estrogen 17β-estradiol (at 10 µg/L, a concentration close to the lowest utilized in the present work) could also affect neuroendocrine signalling in early larvae, including transcription of the serotonin receptor 5-HTR [START_REF] Balbi | Impact of bisphenol A (BPA) on early embryo development in the marine mussel Mytilus galloprovincialis: Effects on gene transcription[END_REF]. When transcription of 5-HTR was evaluated in control larvae, upregulation was observed at all times pf with respect to eggs, with highest levels of mRNA transcripts in early trocophorae and D-veligers (at 24 and 48 hpf, respectively). BPA induced significant downregulation of 5-HTR only at 48 hpf, confirming previous data [START_REF] Balbi | Impact of bisphenol A (BPA) on early embryo development in the marine mussel Mytilus galloprovincialis: Effects on gene transcription[END_REF]. In order to evaluate if BPA could affect development of the serotoninergic system, we looked for the presence of 5-HT immunoreactive neurons. In bivalves, neurogenesis begins with the emergence of the apical sensory cells Exposure to, environmental concentrations of BPA affected the development of 5-HT-ir neurons.

A decrease in the number of 5-HT-ir cells was detected from 28hpf. At 48 hpf, the extent of reduction in cell number was associated with different degrees of developmental delay (from seven in normal D-veligers, to five in early veligers and three in larvae withheld at the trocophora stage, respectively). Higher concentrations resulted in even stronger reductions in the number of serotonin positive neurons, that also in this case were associated with altered phenotypes at 48hpf (malformed, immature, arrested).

The results demonstrate that in mussels exposure to BPA interferes with the development of another ectodermal structure, i.e. neurogenesis of the serotoninergic system. Although the reduction in number 5-HT-ir cells was detectable from the trocophora stages, the most evident effects were detected at 48 hpf, when both the number of 5-HT-ir cells and the amount of 5-HTR transcripts were reduced. Overall, the results indicate that exposure to BPA can induce neurodevelopmental toxicity in M. galloprovincialis.

Current knowledge about the correlation between developmental exposure to BPA and levels of 5-HT is relatively scarce. In mammalian models, it has been proposed that early life exposure to BPA may alter the metabolism of 5-HT and other neurotransmitters, causing neurodevelopmental disorders and behavioural defects [START_REF] Castro | Identification of dopamine-and serotonin-related genes modulated by bisphenol A in the prefrontal cortex of male rats[END_REF][START_REF] Xin | Mice exposed to bisphenol A exhibit depressive-like behavior with neurotransmitter and neuroactive steroid dysfunction[END_REF]. In male zebrafish, BPAinduced behavioural changes were associated with reduced levels of 5-HT in the hypothalamus and down-regulation of tyrosine hydroxylase in brain tissues [START_REF] Li | Impact of low-dose chronic exposure to Bisphenol A (BPA) on adult male zebrafish adaption to the environmental complexity: Disturbing the color preference patterns and reliving the anxiety behavior[END_REF]. In molluscs, 5-HT signalling is involved in a number of key physiological processes from reproduction, to galloprovincialis evaluated by qPCR at different times pf. Data, reported as fold changes with respect to eggs, represent the mean ± SD (N=4). Data were analysed by one way ANOVA Kruskall-Wallis followed by Tukey's test and statistical differences between control and BPA-exposed samples at each developmental stage are reported (* p < 0.01). Expression of all transcripts was significantly different at all times pf with respect to eggs (p < 0.01, not shown). A) In control larvae, 5-HT immunoreactivity was concentrated in compact developing neurons from 24 hpf. In column c) the presence of two, three, and four-five 5-HT-ir cells can be identified, respectively, from 24 to 32 hpf (*).

B) In larvae exposed to BPA (0.05 µM), 5-HT immunoreactivity was apparently more scattered.

Moreover, only two 5-HT-ir cells could be observed from 24 to 32 hpf.

Figure 9

Confocal images of serotonin immunoreactive cells (5-HT-ir) in control and BPA-exposed early larval stages of M. galloprovincialis at 48 hpf. All details as in Fig. 8.

In A) and B) images of control and BPA-exposed larvae are reported, showing representative BPAinduced phenotypes and 5-HT immunoreactivity, respectively, scale bars: 20 µm.

In C) details are reported showing that BPA-induced phenotypic alterations are associated with a reduction in the number of 5-HT-ir cells (*); arrowheads indicate emerging neurites. Scale bar: 5 µm. (Millipore filtered seawater) (pH 7.9-8.0, 38 ppt salinity, 15°C). Gametes were obtained by spontaneous spawning; egg quality (shape, size) and sperm motility were checked using an inverted microscope. Sperm and eggs were kept at 16°C until fertilization, that was carried out with an egg:sperm ratio of 1:10. After 30 min, fertilization success (n. fertilized eggs/n. total eggs x 100) was verified by microscopic observation (usually > 90%) and fertilized eggs were transferred to culture vessels at a density of 200 larvae mL -1 .

Calcofluor/Calcein staining

Stock solutions of Calcein (4 M in Dimethyl-sulfoxide/DMSO) were diluted in MFSW at 4 mM (4x final concentration). Calcofluor (UV channel, Exc: 408 nm/Em: 450-490 nm) was added to each single well at the time of sampling and prior to fixation (final concentration 0.02 mM in 0.01% DMSO), as previously described [START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF]. In each well, aliquots of 0.5 mL of 4x

Calcein (FITC channel, Exc: 488 nm/Em: 520-560 nm) solution and 1 mL of 2x the BPA solution were added to 0.5 mL of fertilized egg suspension (0.01% DMSO in MFSW) to reach the final nominal concentrations (0.05 0.5, 5 µM BPA and 1 mM calcein -0.01% DMSO) in a 2 mL volume. Concentrations of BPA in test solutions were checked by solid-phase extraction SPE/LC/MS as previously described [START_REF] Fabbri | Adaptation of the bivalve embryotoxicity assay for the high throughput screening of emerging contaminants in Mytilus galloprovincialis[END_REF][START_REF] Balbi | Impact of bisphenol A (BPA) on early embryo development in the marine mussel Mytilus galloprovincialis: Effects on gene transcription[END_REF].

Larval growth at 48 hpf

At 48 hpf, the effect of BPA was also evaluated by scoring shell malformations and measuring shell lengths in larvae from 5 independent parental pairs and in at least 50 larvae for each parental pair.

The frequency of shell malformations (% values) and mean shell lengths (µm) were measured. A larva was considered normal when the shell was D-shaped (straight hinge) and the mantle did not protrude out of the shell, and malformed if the larva had not reached the stage typical for 48 h (trocophore or earlier stages) or when some developmental defects were observed (concave, malformed or damaged shell, protruding mantle) [START_REF] Fabbri | Adaptation of the bivalve embryotoxicity assay for the high throughput screening of emerging contaminants in Mytilus galloprovincialis[END_REF]. The acceptability of test results was based on control samples for a percentage of normal D-shell stage larvae >75% (ASTM, 2012).

qPCR

The larval suspension was centrifuged at 800x g for 10 min at 4°C and the obtained pellets were lysed in 1 mL of TRI Reagent (Sigma Aldrich, Milan, Italy). Total RNA was extracted following the manufacturer's instructions (SigmaAldrich, Milan, Italy). RNA concentration and quality were verified using the Qubit RNA assay (Thermo Fisher, Milan, Italy) and electrophoresis using a 1.5% agarose gel under denaturing conditions. First strand cDNA for each sample was synthesized from 1 μg total RNA [START_REF] Balbi | Impact of bisphenol A (BPA) on early embryo development in the marine mussel Mytilus galloprovincialis: Effects on gene transcription[END_REF]. Primers pairs employed for qPCR analysis and their accessio n was included in the qPCR analysis to determine the specificity of target cDNA amplification.

Amplifications were performed in a StepOne real time PCR system apparatus using a standard 'fast mode' thermal protocol (Thermo Fisher, Milan, Italy). Melting curves were utilized for each target mRNA in order to verify the specificity of the amplified products and the absence of artefacts.

Dilution series of cDNA were used to calculate the amplification efficiency of each primer pair.

HEL and EF-α1 were used for data normalization as the best performing combination of reference gene products (EF1/HEL) [START_REF] Balbi | Impact of bisphenol A (BPA) on early embryo development in the marine mussel Mytilus galloprovincialis: Effects on gene transcription[END_REF]. Analyses were performed on at least four independent mRNA samples. Calculations of relative expression of target mRNAs was performed by a comparative CT method (Schmittgen and Livak, 2008) using the StepOne software tool (Thermo Fisher). Data are reported as fold changes observed within each life stage with respect to unfertilized eggs in control and BPA-exposed samples.

Figures

  TYR and HOX1 were selected as possible targets for BPA on the basis of their localization in the area of the shell field[START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF] Pin Huan et al., 2020). Expression of tyrosinase (TYR)

  mental arrest at the highest concentration tested. The double Calcofluor/Calcein staining, previously utilized to characterize the process of first shell formation in larvae grown in physiological conditions[START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF] and in response to acidification(Kapsenberg et al., 2018), thus appears a suitable method to evaluate not only phenotypical changes induced by chemical exposure, but also to identify early contaminant-induced alterations in organic matrix deposition versus calcification.With regards to the mechanisms involved in the effects of BPA, we have previously shown that, among different genes involved in shell formation, tyrosinase was the most upregulated gene in mussel early larval stages; moreover, TYR expression paralleled the formation of the shell field and growth of the organic matrix from 24 hpf[START_REF] Miglioli | Characterization of the main steps in first shell formation in Mytilus galloprovincialis : possible role of tyrosinase[END_REF]. These data indicated a key role of this phenol oxidoreductase, in the early and progressive guiding of matrix deposition and shell growth in M. galloprovincialis. The results obtained here show that in BPA exposed larvae the effects on shell growth were associated with quantitative and qualitative changes in transcription of tyrosinase from 24 to 48hpf: from the lowest concentration tested, BPA affected both the time course of transcription and the spatial pattern of expression of TYR, as shown by qPCR and ISH, respectively. These data indicate expression of TYR, a key gene in matrix deposition, represents a significant target for BPA in Mytilus early larval development. However, tyrosinases participate in the cross-linking and maturation of shell matrix proteins by catalysing the quinoprotein oxidation[START_REF] Ren | A Novel Tyrosinase Gene Plays a Potential Role in Modification the Shell Organic Matrix of the Triangle Mussel Hyriopsis cumingii[END_REF]. Therefore, BPA may act on other process upstream of matrix deposition and more directly responsible for the initial shell patterning. Homeobox-containing genes play essential roles in the regulation of development and differentiation of many organisms. In particular, HOX genes, a subset of homeobox genes that encode transcription factors containing a highly conserved homeodomain-binding motif, are involved in anterior-posterior axial patterning and morphogenetic processes[START_REF] Gehring | Homeodomain-DNA recognition[END_REF]. Several HOX genes have been identified in Mollusca; however, due to the vast diversity of body plans in each group (from bivalves to cephalopods), their functional role in development in each group is still to be elucidated(Huan et al., 2020). In gastropods, expression of HOX genes is initially detected in the trochophora shell field. Later, during development, these genes are expressed in the mantle, demonstrating their continuous role in larval shell formation and differentiation of mantle edge that secretes the adult shell(Samadi and Steiner, 2009, 2010). In bivalves, including M. galloprovincialis, different HOX genes have been identified (Barucca et al., 2003; Perez-Parallè et al, 2005); however, their role in development is still unknown. It has been recently proposed that HOX expression in the ventral ectoderm likely contributes to ventral patterning, such as neurogenesis, whereas HOX expression on the dorsal side is strongly correlated with shell formation and exhibits lineage-specific characteristics in each class of mollusc (Huan et al., 2020).

(

  Voroneskaya et al., 2008 and refs. quoted therein). In M. trossulus, the earliest 5-HT immunoreactive (5-HT-ir) neuron was detected in the trochophora in the region of the developing apical organ (AO), the most conserved larval sensory structure, which later becomes a part of the emerging cerebral ganglion[START_REF] Voronezhskaya | Neuronal development in larval mussel Mytilus trossulus (Mollusca: Bivalvia)[END_REF]. In different bivalve species, the AO develops from early trochophora where it consists of both 5-HTir and FMRFamide ir cells[START_REF] Voronezhskaya | Neuronal development in larval mussel Mytilus trossulus (Mollusca: Bivalvia)[END_REF][START_REF] Yurchenko | Nervous system development in the Pacific oyster, Crassostrea gigas (Mollusca: Bivalvia)[END_REF][START_REF] Pavlicek | Towards a ground pattern reconstruction of bivalve nervous systems: neurogenesis in the zebra mussel Dreissena polymorpha[END_REF]. In M. trossulus and Crassostrea gigas, the AO consists of three and five 5-HTir cells in trochophorae and in D-veligers, respectively[START_REF] Voronezhskaya | Neuronal development in larval mussel Mytilus trossulus (Mollusca: Bivalvia)[END_REF][START_REF] Yurchenko | Nervous system development in the Pacific oyster, Crassostrea gigas (Mollusca: Bivalvia)[END_REF]. Our data show that in control larvae of M. galloprovincialis, from the appearance of two 5-HT-ir cells in early trocophora at 24 hpf, the number of serotonin positive neurons progressively increased up to seven at 48 hpf, with the appearance of tiny neurites. These are the first results on the identification of 5-HTir neurons in the AO of M. galloprovincialis early larval stages. The higher number of 5-HT-ir neurons detected in the present work with respect to previous studies in other bivalves is probably due to the utilization of the nuclear staining and 3D confocal imaging, which allows a more accurate cell counting on sequential z-stacks of 5-HT-ir and Hoechst merged fluorescence signals (Fig.S1).

  neurogenesis and muscle contraction (Joyce and Vogeler 2018; Kim et al., 2019; Yurchenko et al., 2019; Sharker et al., 2019); consequently, exposure to BPA during early life stages may interfere with the onset of the serotoninergic system with potentially dramatic outcomes in wildlife populations. Moreover, BPA may affect other components involved in development of the neuroendocrine systems, such as FMRFamide or other neurotransmitters.Overall the results indicate that in Mytilus early larval stages exposure to BPA can have a significant impact not only on initial shell formation but also on development of the nervous system, indicating that this compound can act through pleiotropic mechanisms. Further research is needed to investigate the mechanisms of action of BPA on different target cells of developing larvae and the possible relationship between the two processes. A recent study showed that, in early oyster larvae, shell formation was modulated by monoamine neurotransmitters 5-HT and dopamine (DA) through TGF-β smad pathway, triggering the expression of tyrosinase to form initial shell[START_REF] Liu | Ocean acidification inhibits initial shell formation of oyster larvae by suppressing the biosynthesis of serotonin and dopamine[END_REF]. In this light, it is tempting to hypothesize that exposure to BPA may directly affect the metabolism of 5-HT (or other monoamines like DA), leading to dysregulation of organic matrix deposition and consequent calcification. Thus BPA has a strong neurodevelopmental impact in bivalves. However, it remains to be shown whether the effects of BPA exposure could be reversed after subsequent recovery to clean conditions or by other environmental factors. Increasing knowledge on the first steps of physiological larval development in different bivalve species may help identifying early signs of impact of different environmental stressors, from exposure to emerging contaminants to ocean acidification, thus contributing to a better understanding of potential perturbation of developmental processes of marine invertebrates in a global change scenario.

Figure 1 -

 1 Figure 1 -Effect of Bisphenol-A (0.05, 0.5 and 5 µM) on the time course of early shell formation in M. galloprovincialis from the trochophora to the first D-veliger stage at different times postfertilization (from top to bottom: at 24, 28, 32, 48 hpf). Columns correspond to experimental conditions (from left to right: Control, 0.05, 0.5, 5 µM BPA). Representative images (lateral view) show merged fluorescent signals of calcofluor (blue) and calcein (green) staining, respectively, the organic matrix and CaCO3 deposition. (Scale bars: 10 µm).

Figure 2 -

 2 Figure 2 -Effect of BPA (0.05, 0.5 and 5 µM) on the growth of organic matrix (blue) and calcified shell (green) in mussel larvae. Areas of each shell component (µm 2 ) measured in a single valva of BPA-exposed larvae are reported as % values of control (unexposed) larvae at 24 (A), 28 (B) and 32 hpf (C). In D) ratios of areas matrix/calcified shell in different experimental conditions are reported. Measurements were made on 15 larvae for each condition obtained from 5 parental pairs (N=5). * p ≤ 0.05, BPA-exposed vs Controls; # Control 28 hpf vs 32 hpf (one way ANOVA Kruskall-Wallis followed by Tukey's test).

Figure 3 -

 3 Figure 3 -Effect of BPA (0.05, 0.5 and 5 µM) on shell morphology at 48 hpf. A) representative images of control larvae and of the main phenotypes observed in BPA-exposed larvae (scale bar: 20 µm). B) percentage of different phenotypes: normally developed D-Veligers (white), with hinge malformations (light gray), with extruding mantle (dark grey) and arrested to the trochophora stage (black). C) Shell length of D-veligers, i.e. the anterior-posterior dimension of the shell parallel to the hinge line normalized as % of controls (Inset), * p ≤ 0.05. Measurements were made on 70 larvae for each experimental condition obtained from 5 parental pairs (N=5).

Figure 4 -

 4 Figure 4 -Time course of tyrosinase (TYR) expression in control and BPA-exposed larvae of M.

Figure 5 -Figure 6 -

 56 Figure 5 -Effect of Bisphenol A on TYR expression pattern evaluated by ISH in mussel larvae from 24 to 48 hpf. Columns (from left to right: Control, 0.05, 0.5 and 5 µM BPA); rows (from top to bottom: 24, 28, 32, 48 hpf). Scale bar: 20 µm

Figure 7 -

 7 Figure 7 -Transcriptional profile of the serotonin receptor (5-HTR) during early larval development in M. galloprovincialis evaluated by qPCR at different times pf. Data, reported as foldchanges with respect to eggs, represent the mean ± SD (N=4). Data were analysed by 1-way nonparametric ANOVA plus Tukey's test and statistical differences between control and BPA-exposed samples at each developmental stage are reported (* p<0.01). Expression of all transcripts was significantly different at all times pf with respect to eggs (one way ANOVA followed by Tukey's test; p < 0.01, not shown).

Figure 8 -

 8 Figure 8 -Confocal images of serotonin immunoreactive cells (5-HT-ir) in early larval stages of M. galloprovincialis from 24 to 32 hpf. A) control; B) BPA-exposed (0.05 µM). 5-HT-ir cells are shown in Red/Pink (Ex/Em: 590/617 nm), DAPI stained nuclei are shown in blue (Ex/Em: 358⁄461 nm). Rows correspond to hours post fertilization: 24, 28, 32 hpf. Columns show: a) brightfield and 5-HT merged channels, scale bar: 20 µm; b) 5-HT and DAPI merged channels, scale bar: 20 µm. c) 4-5 X magnified images of column B: white asterisks indicate the nuclei of 5-HT-ir cells (*) and white arrowheads emerging neurites, respectively, scale bar: 5 µm.

Figure 3 revised

 3 Figure 3 revised

5 -

 5 HTR [AB526218], forward CAGCTGCAAGATCGAGGATT-reverse TGAAGCCATCTTGACTGACG. qPCR reactions were performed in triplicate in a final volume of 15 μL containing 7.5 μL iTaq universal master mix with ROX (BioRad Laboratories, Milan, Italy), 5 μL diluted cDNA and 0.3 μM specific primers. A control lacking cDNA template (no-template)

Figure S1 -

 S1 Figure S1 -Counting of immunoreactive cells stained with anti-serotonin antibody (5-HT-ir cells) in a 48 hpf D-veliger from control conditions. The figure shows a montage of sequential z-stacks of 5-HT-ir staining (in red) and Hoechst (in blue) merged on the image. Numbers indicate the nuclei and white arrowheads indicate the emerging neurites. Z-distance between 2 images is 0.9 µm. Scale bars: 5 µm. The nucleus of cell 1 is visible from z1 to z6, cell 2 from z3 to z12, cell 3 from z6 to z13, cell 4 from z12 to z15, cell 5 from z13 to z19, cell 6 from z15 to z16, cell 7 from z20 to z24. Emerging neurites are visible in z1-z2 and in z19.

Figure S2 -

 S2 Figure S2 -Immunolocalization of 5-HT-ir cells in a mussel larva at 24 hpf (trocophora, lateral view). 5-HT-ir cells are shown as red fluorescence, the perimeter of the shell field is indicated in blue. The trochophora larval body is divided into a pre-trochal (on top) and post-trochal region by the prototroch, the equatorial band of ciliated cells. The pre-trochal area is characterized by the

Figure S3 -

 S3 Figure S3 -Effect of BPA (0.5 µM) on serotonin immunoreactivity (5-HT-ir) in early larval stages of M. galloprovincialis from 24 to 32 hpf. For control images, refer to Fig. 8. All details as in Fig. 8. In BPA-exposed larvae only one 5-HT-ir cells could be observed at 24hpf and two cells at 28 and 32 hpf. Scale bars: 20 µm.

Figure S4 -

 S4 Figure S4 -Effect of BPA (0.5 µM) on serotonin immunoreactivity (5-HT-ir) in early larval stages of M. galloprovincialis at 48 hpf. All details as in Fig. 9. In BPA-exposed larvae phenotypic alterations were associated with a reduction in the number of 5-HT-ir neurons (three in indented hinge, four in protruding mantle and two in arrested trocophora, respectively). Scale bars: 20 µm.

  al., 2012; Canesi and Fabbri, 2015; Corrales et al., 2015; ECHA, 2017; Sousa et al., 2018; Staples et al., 2018; Wu

Acknowledgments

The authors would like to thank Laurent Gilletta, Alexandre Jean,Régis Lasbleiz and the Centre de Ressources Biologiques Marines of the CRBM-IMEV that is supported by EMBRC-France, whose French state funds are managed by the Agence Nationale de la Recherche (ANR) withi the 'Investissement d'Avenir' program (ANR-10-INBS-02); Sameh Benaicha; the Ascidian BioCell group members. The experiments performed in the laboratory of R.D. were financed by an ANR grant (Marine-EmbryoTox project, ANR-14-OHRI-0009-01-1).

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Yannick Bleu

Florent Bourquard

Carole Farre

Carole Chaix

Jules Galipaud

Anne-Sophie Loir

Vincent Barnier

Florence Garrelie

Christophe Donnet

Boron doped graphene synthesis using pulsed laser deposition and its electrochemical characterization

During the design of graphene materials, their electronic, chemical and electrochemical properties -among others-can be tuned by incorporating heteroatoms in the honeycomb structure. In this study, we investigated boron doped graphene synthesis and the effect of boron content on the electrochemical property of graphene. To this end, boron-doped graphene (BG) was synthesized using pulsed laser deposition (PLD) with carbon and boron as solid sources, followed by rapid thermal annealing (RTA) in the presence of a nickel based thin film catalyst at a growth temperature of 900°C. The BG samples were characterized using standard spectroscopic techniques. Raman spectroscopy revealed that the as-prepared BG films consisted of few-layer graphene. Boron doping increased the level of defects in the films and also affected the formation of the graphene layer. X-ray photoelectron spectroscopy (XPS) demonstrated that boron atoms can be successfully doped into the graphene structure with control of boron content of around 1 and 2.5 at.%. Electrochemical characterization of undoped graphene (G) and boron-doped graphene (BG) was also performed by cyclic voltammetry using redox ferrocene dimethanol. BG electrodes exhibited higher reversibility than the G electrode.

Moreover, the heterogeneous electron transfer constant of the BG electrode with higher boron content of 2.5 at.% was twice that of the G electrodes. This indicates that BG electrodes have better electrochemical properties with respect to the G electrode. All these results prove that the pulsed laser deposition method is a viable alternative way to produce BG films with excellent electrochemical properties.

Introduction

Incorporating dopants in the graphene structure with various heteroatoms can be used to modulate the bandgap and tune the electronic, physical-chemical and optical properties of graphene for many applications in electrochemistry, sensors, photovoltaics and catalysis. In the recent past, Wang et al. [START_REF] Wang | Heteroatom-doped graphene materials: syntheses, properties and applications[END_REF] reviewed graphene doping with different heteroatom elements including boron, nitrogen, phosphorus, group VIa (O, S) and group VIIa (F, Cl, Br, I) elements.

More recently, Agnoli et al. [START_REF] Agnoli | Doping graphene with boron: a review of synthesis methods, physicochemical characterization, and emerging applications[END_REF] reviewed boron-doped graphene (BG) films, and pointed to both theoretical and experimental studies on structure and properties, as well as methods for synthesis, and potential applications in many areas. Indeed, the incorporation of boron in the structure of graphene, which has been less frequently investigated than nitrogen doping, increases the concentration of holes, inducing a p-doping effect, with a downshift of the Fermi level toward the Dirac point [START_REF] Rani | Designing band gap of graphene by B and N dopant atoms[END_REF]. As boron atoms cause sp 2 hybridization in the carbon lattice, the planar structure of graphene is preserved. Likewise, lower strain energy is generated in the graphene network by substitutional boron doping because B-C bonds in graphene are slightly longer than C-C bonds. Due to the strong B-C bond energy, the mechanical properties of graphene are preserved but the thermal conductivity of boron-doped graphene (BG) is drastically reduced compared to pristine graphene [START_REF] Agnoli | Doping graphene with boron: a review of synthesis methods, physicochemical characterization, and emerging applications[END_REF]. Furthermore, the B-C bond introduces defects in the nearby sites because a boron atom possesses three valence electrons leading to uneven charge distribution, which can facilitate charge transfer between neighboring carbon atoms and therefore enhance their electrochemical performance [START_REF] Sheng | Synthesis of boron doped graphene for oxygen reduction reaction in fuel cells[END_REF]. Many routes for the production of boron-doped graphene (BG) films have been investigated including liquid state synthesis [5-8], chemical vapor deposition (CVD) synthesis [9-16], and solid-phase synthesis [START_REF] Panchakarla | Structure, and Properties of Boron-and Nitrogen-Doped Graphene[END_REF][START_REF] Wu | Doped Graphene Sheets As Anode Materials with Superhigh Rate and Large Capacity for Lithium Ion Batteries[END_REF][START_REF] Kim | Raman Spectroscopy of Boron-Doped Single-Layer Graphene[END_REF][START_REF] Sathe | Metal-free B-doped graphene with efficient electrocatalytic activity for hydrogen evolution reaction[END_REF][START_REF] Fang | Boron-doped graphene as a high-efficiency counter electrode for dye-sensitized solar cells[END_REF][START_REF] Peng | Flexible Boron-Doped Laser-Induced Graphene Microsupercapacitors[END_REF][START_REF] Yeom | High-concentration boron doping of graphene nanoplatelets by simple thermal annealing and their supercapacitive properties[END_REF][START_REF] Wang | Boron-Doped Graphene: Scalable and Tunable p-Type Carrier Concentration Doping[END_REF][START_REF] Jo | Boron and phosphorous-doped graphene as a metal-free electrocatalyst for the oxygen reduction reaction in alkaline medium[END_REF][START_REF] Telychko | Electronic and Chemical Properties of Donor, Acceptor Centers in Graphene[END_REF][START_REF] Norimatsu | Epitaxial growth of boron-doped graphene by thermal decomposition of B4C[END_REF]. CVD has been widely and successfully used for high-quality graphene synthesis over large areas, and using different sources of boron to produce BG films but they all usually require high-temperature treatment and metal boride compounds can form on the catalytic substrate [9-13]. Moreover, controlling the concentration of boron in CVD synthesized BG films is not simple. Solid-state synthesis using a solid carbon source, e.g. using the PLD method, has been shown to be capable of producing both pristine and doped graphene [START_REF] Bleu | Review of Graphene Growth From a Solid Carbon Source by Pulsed Laser Deposition (PLD)[END_REF][START_REF] Bourquard | Electroanalytical Performance of Nitrogen-Doped Graphene Films Processed in One Step by Pulsed Laser Deposition Directly Coupled with Thermal Annealing[END_REF][START_REF] Maddi | Nano-Architecture of nitrogen-doped graphene films synthesized from a solid CN source[END_REF]. In our most recent work [START_REF] Bleu | Boron-doped graphene synthesis by pulsed laser co-deposition of carbon and boron[END_REF], we demonstrated the use of this alternative route for the first time: it consists of using carbon and boron solid precursors ablated by pulsed laser in controlled conditions, to synthesize BG films. We also already demonstrated the ability of PLD to produce boron-doped diamond-like carbon films (a-C:B) [START_REF] Sikora | Effect of boron incorporation on the structure and electrical properties of diamond-like carbon films deposited by femtosecond and nanosecond pulsed laser ablation[END_REF], thereby providing a considerable baseline for advances in the synthesis of boron-doped graphene (BG) films. In the present work, we addressed the relationship between the chemical composition and nanostructure of BG films produced using PLD with their electrochemistry properties. To this end, after BG film synthesis using PLD and RTA, the as-grown samples were characterized using XPS, Raman, and cyclic voltammetry for chemical, structural, and electrochemical analysis.

Experimental details

B-Graphene growth. This stage involves three main steps: the deposition of nickel thin film catalyst by thermal evaporation, the deposition of a-C:B film on the cleaned SiO2(300nm)/Si substrates using pulsed laser deposition (PLD), and the thermal annealing process responsible for the diffusion-segregation of carbon through nickel.

Firstly, after being cleaned ultrasonically in acetone, ethanol, and deionized water baths, the SiO2(300nm)/Si substrate was placed in a vacuum chamber pumped to a base pressure of 10 -6 mbar. Next, a 50-nm thick nickel (Ni) film was deposited by thermal evaporation on the top of the SiO2(300nm)/Si substrate. Secondly, the Ni(50 nm)/SiO2(300nm)/Si was placed in the vacuum PLD chamber and pumped to a base pressure of 10 -7 mbar. The co-ablation was operated at room temperature by an excimer laser KrF with 248 nm wavelength, a pulse duration of 20 ns, a repetition rate of 10 Hz an energy per pulse of 160 mJ. The energy density (fluence) of the laser beam was kept constant at 6.2 J/cm 2 . In these conditions, the ablation rates of the carbon (99.997 %) and boron (99, 993 %) were respectively 26 nm.mn -1 and 1 nm.mn -1 .

The sequences of co-ablation were defined to deposit an a-C:B film 4 nm thick, with a boron concentration of 9 and 4.5 at.%. A pure a-C film of similar thickness was also deposited on Ni(50 nm)/SiO2(300nm)/Si for the purpose of comparison. Thirdly, thermal annealing of the a-C:B/Ni/ SiO2(300nm)/Si film was performed at 900 °C for 7 min at a pressure of 10 -2 mbar in a rapid thermal annealing (RTA) oven with a heating ramp of 15 °C/s and a cooling ramp of about 0.5°C/s, to obtain graphene and boron-doped graphene on the top surface.

Characterization. The as-grown samples were analyzed using Raman spectroscopy to identify the graphene structure and XPS to analyze chemical composition and bonding. Raman spectroscopy was performed using an Aramis Jobin Yvon spectrometer at 442 nm (2.81 eV), with a spectral resolution of 2 cm -1 and a lateral resolution of ~1 μm. The laser beam was focused on the sample with a ×100 objective and the laser power was kept below 3 mW to avoid damaging the surface of the film. Raman components were associated with a Lorentzian fit, except for the asymmetric G peak, which was fitted using a Breit-Wigner-Fano function. A custom fitting function was computed for all recorded spectra, and a simple linear function was added to eliminate the background. This enabled access to the exact values of the various peak positions, widths, and maximum peak height intensities. Raman maps were built from the acquisition of each 1 μm spectrum over probed surfaces measuring 20 × 20 µm². The XPS measurement was carried out using a ULVAC-PHI Versaprobe II spectrometer with a focused monochromatic AlKα source (hν = 1,486.68 eV, 200 μm spot size). For all the samples analyzed, narrow scans were recorded for C1s, B1s, and O1s with a step size of 0.1 eV and 23 eV pass energy. The data were processed using Casa XPS, and Origin software. The binding energy of all the samples was calibrated using the reference C1 binding energy (284.8 eV) [START_REF] Yao | N 8 -Polynitrogen Stabilized on Boron-Doped Graphene as Metal-Free Electrocatalysts for Oxygen Reduction Reaction[END_REF][START_REF] Li | N-doped TiO2 nanotubes/N-doped graphene nanosheets composites as high performance anode materials in lithium-ion battery[END_REF]. Quantification was carried out using the transmission function of the apparatus and angular distribution correction for an angle of 45°. Sensitivity factors were extracted from the work of Wagner et. al. [START_REF] Wagner | Empirical atomic sensitivity factors for quantitative analysis by electron spectroscopy for chemical analysis[END_REF] in which the cross section and escape depth correction are incorporated. The electrochemistry performance of these samples was recently examined using cyclic voltammetry curves. Electrochemical measurements were carried out using a conventional three-electrode system with an internal volume of 5 mL at room temperature (Figure 1). This electrochemical cell was designed to maintain a fixed distance between the electrodes. This feature prevented further manipulation or movement of the electrodes (thereby fixing the cell geometry and insuring the reproducibility of the measurements). An Ag/AgCl was used as the reference electrode (-0.014 V versus SCE), a planar platinum electrode (0.59 cm 2 ) was used as the counter electrode and the undoped graphene and boron-doped graphene samples were the working electrodes. This three-electrode system was connected to a Bio-Logic potentiostat. The results were recorded using EC-Lab software (v11.27) (Bio-Logic Science Instruments). To characterize the electron transfer rate for the different undoped and boron-doped graphene samples, cyclic voltammetry was performed three times in a 0.5 mM 1,1 ferrocene-dimethanol solution of 0.1M NaClO4 with a potential ranging between -0.2 and 0.6 V vs Ag/AgCl at a scan rate of 50 mV s -1 . Only the last cycles were used to interpret the data.

Results and discussion

3.1.Structural and chemical analysis of the synthesized films

We investigated the nanostructure and chemical composition of the synthesized graphene and boron-doped graphene using Raman spectroscopy and XPS. Particular attention was paid to the difference between the features of the undoped and boron-doped graphene films, as well to the boron content in both a-C:B precursors and BG films. For this reason, prior to BG films, in this section, we included detailed XPS analysis of the a-C:B precursors. We observed the presence of carbon, boron, and oxygen in the survey spectra (data not shown) of the a-C:B films. The nickel was probably not detected because the top surface a-C:B film masks its contribution. On the contrary, in the survey spectra of the BG films, we observed not only C1s, B1s, and O1s, but also Ni2p, Si2s and Si2p. This is consistent with the well-known growth mechanism of graphene from a solid carbon-based precursor in the presence of a metal catalyst [36,37]: the constituents of the solid carbon film diffuse into the catalyst and segregate as graphene layers on the top surface of the catalyst during the annealing and cooling process. Moreover, annealing at 900°C causes dewetting of the Ni film, which leads to the concomitant formation of Ni nodules and of the graphene layer [START_REF] Banno | Transferfree graphene synthesis on insulating substrates via agglomeration phenomena of catalytic nickel films[END_REF][START_REF] Bleu | Boron-doped graphene synthesis by pulsed laser co-deposition of carbon and boron[END_REF][START_REF] Bleu | Graphene synthesis on SiO2 using pulsed laser deposition with bilayer predominance[END_REF][START_REF] Miyoshi | Study on transferfree graphene synthesis process utilizing spontaneous agglomeration of catalytic Ni and Co metals[END_REF]. Those considerations may explain why Ni and Si were detected by XPS. Further, the presence of oxygen in the a-C:B precursor sample can be attributed to adventitious contamination and oxygen incorporation in the films due to residual water vapor in the PLD chamber (maintained at around 10 -7 mbar during deposition) or during storage in the air after deposition. After rapid thermal annealing, we observed that the a-C:B of 4.5 at.% of boron produces BG film containing 1 at.% of boron, and the a-C:B precursor of 9 at.% of boron produces BG film with 2.5 at.%. Note that these percentages are related to the ratio B/(B+C). When we compared the boron content of the BG films with their precursors, we therefore observed a systematic loss of boron after the annealing process, at both boron concentrations. This could be attributed to the thermal annealing process, which may provoke cause the diffusion of boron atoms in the nickel catalyst during the synthesis of BG films, via a mechanism that remains to be clarified.

Using high-resolution core-level spectra, we performed peak deconvolution to reveal the detailed configurations of the boron, carbon, and oxygen. Moreover, the peak deconvolutions and their exact interpretation are rather controversial due to the proximity of electronic configurations of carbon and boron with a slight difference in their electronegativity, as well as In the a-C:B film, the B1s contributions (Figure 2a), centered at 188. The high-resolution XPS spectra of C1s, B1s, and O1s for BG2.5% and BG1% are reported in Figure 3. Indeed, after the conversion of a-C:B films into BG films using rapid thermal annealing, the position of the C1 contributions was not significantly modified. A relative increase in the C-B contribution was observed that could be due to the good incorporation of boron atom in the carbon network upon annealing. However, the possibility that this contribution includes some species of carbon dissolved in nickel cannot be excluded, as reported in our previous work [START_REF] Bleu | Dynamics of carbon diffusion and segregation through nickel catalyst, investigated by insitu XPS, during the growth of nitrogen-doped graphene[END_REF]. Moreover, an increase in the C-O and C=O contributions was observed in BG2.5%, in agreement with the higher contribution of O1s. Concerning the boron signal, in BG2.5%, the B1 contribution fit three peaks centered at 188. 8, 191.1, and 192.2 eV, assigned to BC3, BC2O, and BCO2 respectively. In BG1%, the deconvolution of B1s rather gives two boron species BC3 (189.3 eV) and BCO2 (192.2 eV). In any case, the presence of BC3, BC2O and BCO2 bonds suggests carbon atoms were replaced by boron atoms in the graphene network, as reported in most of the references concerning BG films cited above. The O1 core level spectra (Figure 3c,f Here, the Raman mapping of 20 x 20 µm² (each integrating 400 Raman spectra) was performed on representative areas of the undoped and boron-doped graphene samples with 1 and 2.5 at.%, and the maps were processed for the ID/IG and I2D/IG intensity ratios, 2D peak FWHM, and crystallite size La, as well as G, and 2D peak positions as shown in It is worth noting that the crystallite size (La) was deduced from ID/IG intensity ratio using using the Tuinstra-Koenig relation [START_REF] Cançado | General equation for the determination of the crystallite size La of nanographite by Raman spectroscopy[END_REF]: La (nm) = (2,4 × 10 -10 )λ 4 (

I D I G ) -1
with λ the laser wavelength in nanometer units.

We observed that the average value of the ID/IG intensity ratio increased with an increase in boron doping from 0.16 to 0.45. In addition, a decrease in the average value of the crystallite size (La) from 75 to 25 nm was observed with an increase in the level of boron doping. These results suggest that boron doping increased the density of defects in the graphene structure and reduced the crystallite size of graphene, in agreement with the results of previous reported studies on boron doped graphene [START_REF] Sheng | Synthesis of boron doped graphene for oxygen reduction reaction in fuel cells[END_REF][START_REF] Panchakarla | Structure, and Properties of Boron-and Nitrogen-Doped Graphene[END_REF][START_REF] Sahoo | Green synthesis of boron doped graphene and its application as high performance anode material in Li ion battery[END_REF]. In addition, the average value of the I2D/IG intensity ratio decreased from 1.11 to 0.70 with boron doping, whereas the average value of FWHM (2D) increased from 75 to 93 cm -1 . It is therefore not easy to conclude that the upshift of the G peak is only due to the boron doping effect. However, we clearly observed that boron doping markedly influenced the structure of the graphene. Concerning the 2D peak, we also observed an upshift from 2,747 to 2,758 cm -1 with boron doping. All these effects are illustrated in Figures 5a-c with the plot of ID/IG and I2D/IG ratios and crystallite size La and FWHM (2D) as well as the positions of the G and 2D peaks as a function of the boron doping level. As observed for the average value of the different maps, the intensity of the ID/IG and I2D/IG ratios evolved in the opposite way with an increase in the level of boron doping. The crystallite size La and the FWHM (2D) also displayed the opposite trend with boron doping, whereas the position of the G and 2D peaks rather followed the same trend. Further, it is worth underlining that the high values of the standard deviations for all the maps demonstrate that the number of layers, the density of defects, and the size of the crystallites in the synthesized undoped and boron-doped graphene are rather heterogeneous. Figure 5d shows some typical Raman spectra extracted from the map of each of the samples detailed in Table 2. The main peaks characteristic of graphene D, G, and 2D are visible. We observed that the D peak became more intense with boron doping, suggesting the existence of a high density of defects in the BG films. Taking into consideration the results of XPS and Raman analysis and interpretations, we have demonstrated that an amorphous a-C:B film obtained by co-ablation of carbon and boron can be thermally converted into a boron-doped graphene film containing a lower boron content than in the precursor film. In the following section, we highlight the electrochemical performance of these BG films.

3.2.Electrochemistry response of our synthesized G and BG films a. Results of cyclic voltammetry

Cyclic voltammetry (CV) was performed on the synthesized graphene (sample G) and borondoped graphene containing the two boron doping levels 0,1 (BG1%) and 2.5 at.% (BG2.5%).

At this stage, it is worth mentioning that all the cyclic voltammetry measurements were performed on the as-grown undoped and boron-doped graphene. However, during the CV measurements, the signal of these nickel residuals was not observed with CV analysis, suggesting that the presence of these nickel residuals does not influence the electrochemical results. Cyclic voltammetry (CV) measurements obtained with ferrocene dimethanol on undoped graphene (G) and B-doped graphene (BG) at the scan rate of 50 mV s -1 are presented in Figure 6, and the comparative electrochemical parameters are listed in Table 3.

The values of the potential separation between the oxidation and reduction peak E for G, BG1%, and BG2.5% were respectively 0.35, 0.17, and 0.20 V, demonstrating that the two boron-doped graphene electrodes exhibit higher reversibility and better kinetic electronic transfer than the undoped graphene electrode. However, these measurements also show that the electronic transfers are quasi-reversible and not Nernstian (E ~ 59 mV). In addition, we observed an increase in both oxidation and reduction currents with an increase in the concentration of boron, in agreement with the results of a previous study on BG film [4]. The two boron-doped graphene electrodes present higher faradaic current than the undoped graphene due to the redox molecule. This also suggests that the electrochemical properties of BG electrodes are better than those of undoped graphene. To estimate the kinetic rate of interfacial electron transfer constant k0, the dimensionless kinetic parameter Ψ was first determined using the method developed by Lavagnini [61] for a quasireversible system with ΔE values higher than 200 mV using the following equation:

𝛹 = 2.18 [ 𝛽 𝜋 ⁄ ] 1 2 ⁄ exp [-( 𝛽 2 𝐹 𝑅 𝑇 ⁄ ) 𝑛 𝛥𝐸]
where Ψ is a kinetic parameter, n is the number of electrons involved in the process (n = 1), F is the Faraday constant (F = 96485 C.mol -1 ), R the gas constant (R = 8.314 J.mol -1 K -1 ), T the temperature (T= 298 K), β the transfer coefficient generally assumed to be 0. Consequently, ko was found to be equal to 2.3 × 10 -3 , 2.5 × 10 -3, and 4.9 × 10 -3 cm.s -1 for undoped graphene G, BG1%, and BG2.5% respectively. It is clear from these results that undoped graphene possesses the smallest ko thereby pointing to reduced electron transfer properties. Although the increase due to a boron doping level of 1% seems very limited, increasing the boron doping level up to 2.5% almost doubles the ko value. All these results show that the electrochemical performance of BG2.5% with respect to BG1% is better, probably due to the higher concentration of boron content defects of BG2.5%, which can facilitate the transfer of charge between neighboring carbon atoms in a graphene lattice [START_REF] Xu | Facile synthesis of boron and nitrogen-doped graphene as efficient electrocatalyst for the oxygen reduction reaction in alkaline media[END_REF][START_REF] Niu | Pyrolytic synthesis of boron-doped graphene and its application as electrode material for supercapacitors[END_REF].

b. Relation between boron doping and electron transfer capability

To correlate the nanostructure of the undoped and boron-doped graphene with the kinetic rate of interfacial electron transfer (ko), the average ID/IG intensity ratios calculated in the Raman analysis section above are linked to the electron transfer constant ko values and shown in Figure 3. Figure 8 shows the correlation between the ko and the average ID/IG values. When a boron concentration was introduced in the graphene structure, the ID/IG ratio increased from 0.16 to 0.39, indicating increased disorder in the graphene material. Nevertheness, there was no significant effect on the electron transfer constant k0 with very little change from 2.3. 10 -3 to 2.5. 10 -3 cm.s -1 . Then, by increasing the boron concentration from 1 to 2.5 at. %, we observed much more disorder and the electron transfer constant almost doubled from 2.5. 10 -3 to 4.9.10 - 3 cm.s -1 . This suggests that the interfacial electron transfer capability depends not only on the defects present in the graphene material in agreement with our previous work on CVD graphene

[64], but more importantly on the concentration of dopant. Indeed, it is well known that pristine graphene does not present good interfacial electron transfer kinetics, but that an appropriate concentration of dopant leading to an appropriate density of defects can significantly improve the electrochemical activity of graphene, as we observed in the present study. For further analysis, BG2.5% was used for the stability study, as described below. Figure 9a shows that the intensity of oxidation and reduction current signal decreases with time and the number of cycles, from 0 to 30 minutes of cycling. Indeed, initially, the fresh BG2.5% electrode presents a redox behavior with the fastest kinetics (highest peak currents) (red curve).

By increasing the length of time in the ferrocene dimethanol electrolyte, the peak currents were reduced, especially at 10, 30 min, and E values increased slightly except for the period lasting10 minutes. This could be due to the number of cycles, which can also affect the sensitivity of the BG film. It is worth mentioning that the values of potential and currents of BG2.5% in Table 4 differed slightly from those listed in Table 3 because the measurements

were not taken at exactly the same location, evidence for heterogeneous Raman mapping.

Figure 9b

shows the results of a complementary stability study in which the sample was left in the cell in case the curve (black curve) -that was initially very reversible -tended to widen and increasingly resemble the signal of undoped graphene. Indeed, the peak currents significantly decreased while the E value increased to approach that of undoped graphene. The cumulative effect of cycling (~ 20 cycles in the same area) and incubation in the solution for 24 hours

(1,440 min) seemed to affect the electrochemical response. However, even though the peak currents decreased, the peak potential of the BG2.5% electrode with 5, 10, 30, and 1,440 min duration only shifted slightly. This means the kinetic rate did not change significantly, and that the decrease in the peak current could be due to the reduction in the apparent geometric area of the BG2.5% electrode. These results thus demonstrate that the length and the number of cycles affect the preservation stability of BG2.5%. Adhesion of the conductive layer to the support is probaby not sufficient to resist the successive cycles of potential achieved during the measurements. Thus, the stability of the BG electrode over time requires further improvement.

Conclusion

The present study addresses the relationship between the chemical composition and nanostructure of BG films produced using PLD, with their electrochemical properties. The film nanoarchitecture and chemical composition were investigated by combining Raman, and XPS.

The synthesized boron-doped graphene films comprise a few layers with the control of boron content of about 1 and 2.5 at.%. and with boron mainly bonded to carbon in both BC3 and BCO2 Further, cyclic voltammetry measurements showed that boron doping enhances the electrochemical activity of the boron-doped graphene films. By increasing the doping level from 1 to 2.5 at.%, the electron transfer becomes much higher, meaning that the electron transfer capability depends on the concentration of dopant. However, preserving the stability of the BG2.5% electrode is time-dependent, and needs to be improved to achieve much greater stability over time. Finally, this work shows that boron doped graphene can be easily synthesized using PLD with promising applications in electrochemical devices such as fuel cells, batteries and biosensors.

Figure 1 .

 1 Figure 1. Schematic of the set up of our three electrodes system: the working electrodes are G and BG, the counter electrode is platinum, the reference electrode is Ag/AgCl, and the electrolyte is 0.5 mM 1,1 ferrocene-dimethanol solution of 0.1M NaClO4.

  to the existence of numerous chemical functions combining B, C and O species. Our interpretation of XPS is based on a compilation of previously published data related to borondoped graphene [21,42-47]. The XPS spectra depicted in Figure 2a-c show the B1, C1, and O1 core levels of the a-C:B film containing 9 at.% of boron, as deposited by the co-ablation process of carbon and boron (i.e. before annealing allows its conversion into boron-doped graphene).

Figure 2 .

 2 Figure 2. (a) XPS-B1s, (b) XPS-C1s spectrum, (c) XPS-O1s spectrum. All spectra are for a-C:B (9 at.%).

  8 and 191.2 eV, are assigned to BC3 and BC2O respectively. The C1 contributions (Figure 2b), centered at 283.8, 284.7, 286.4 and 287.6 eV, are respectively assigned to C-B (peak related to B-doped structure, indicating the successful doping of B), sp 2 carbon (typically observed in diamond-like carbon films), C-O and C=O species. The O1 core level spectrum (Figure 2c) shows the deconvoluted peaks at about 531, 532, and 533.9 eV are associated respectively with O-C, O=C, and O-B bonds.

Figure 3 .

 3 Figure 3. High resolution XPS spectra of BG (2.5 at.%): (a) C1s, (b) B1s, (c) O1s. High-resolution XPS spectra of BG (1 at. %): (d) C1s, (e) B1s, (f) O1s.

  ) show the same oxygen bonds O-C, O=C, and O-B as those observed in a-C:B films. The relative abundance of oxygen in boron-and carbon-containing chemical groups is typical of top-surface compositions. The positions and FWHM of the carbon, boron and oxygen components derived from our fittings are summarized in

  derived from our fittings. The Csp² was fitted using an asymmetric line whereas we used the Gaussian -Lorentzian (GL30) line for the other peaks.The samples were also characterized by Raman spectroscopy and mapped to study the effect of boron doping on the synthesized graphene. In graphene-based materials, the D, G, and 2D bands are the most significant features observed in Raman spectroscopy. The G band appears around 1 580 cm -1 and is associated with covalent C-C bonding vibrations in the graphite matrix and is /was present in every carbon material containing sp2 bonding. The D band, located around 1 350 cm -1 , is associated with the pulsation of aromatic circles, and only appeared in the presence of defects and dislocations in the graphitic matrix [48-50]. The intensity ratio between the D band and G band (ID/IG) is thus an indication of disorder in the graphene structure. The 2D band is situated around 2 700 cm -1 and is associated with a double resonance Raman scattering process. With graphene-based materials, the intensity ratio of the 2D band versus the G band (I2D/IG) is a good indicator to identify the number of graphene layers. It is generally accepted that an I2D/IG ratio >1.4 represents the formation of monolayer graphene. Bilayer graphene can be identified by an I2D/IG ratio of between 0.75 and 1.4, and graphene with three and more layers can be identified by an I2D/IG ratio <0.75 [51-53].

Figure 4 .

 4 Table 2 lists the average values plus standard deviations of the ID/IG, I2D/IG, 2D (FWHM), La, G, and 2D positions taken from the Raman maps of each sample.

Figure 4 .

 4 Figure 4. ID/IG, La, I2D/IG, 2D (FWHM), 2D and G positions on Raman maps of (a) undoped graphene, (b) boron-doped graphene 1%, (c) boron-doped graphene 2.5%, with their average values.

Figure 5 .

 5 Figure 5. Plots showing dependence on the boron doping level as a function of the average value of (a) ID/IG ratio, I2D/IG ratio, (b) crystallite size (La), the FWHM (2D), and (c) positions of the G and 2D peaks for the synthesized undoped and boron-doped graphene (1, 2.5 at%). (d) Typical experimental (black) and fitted (blue) Raman spectra of the synthesized undoped and boron-doped graphene films (1, 2.5 at.%).

Figure 6 .

 6 Figure 6. Cyclic voltammetry curves measured in a 0.5 M 1, 1 ferrocene-dimethanol solution of 0.1 M NaClO4 with a scan rate of 50 mV/s. (a) CV of undoped graphene. (b) CV of BG1%. (c) CV of BG2.5%.

  5 [62,63] and ΔE is the peak-to-peak separation in volts. A plot of Ψ versus  -1/2 (v being the experimental CV scan rate) then allows the electron transfer rate constants ko to be determined through the slope of the linear fit. Indeed, to calculate the kinetic parameter Ψ, the voltammetry curves were recorded for each sample at different scan rates ranging from 2 to 200 mV/s. Next, we plot the calculated kinetic parameter (Ψ) versus the reverse of the square root of the scan rate ( -1/2 ), as shown in Figure 7.

Figure 7 .

 7 Figure 7. The plot of Ψ versus ν -1/2 enabling the estimation of the kinetic rate of interfacial electron transfer constant ko.

Figure 8 :

 8 Figure 8: The correlation between the kinetic rate of interfacial electron transfer (ko) and the average intensity ratio of the D peak over the G peak (ID/IG) of the G, BG1% and BG2.5%.

  c. Stability study of the Boron doped graphene with 2.5 at.% For the stability study, the CV curves were recorded for different lengths of time in the redox probe. Figures 9a-b show the CV curves of BG2.5% for the different lengths of time in ferrocene dimethanol. Several cycles were performed at 50 mV/s.

Figure 9 .

 9 Figure 9. Cyclic voltammetry on BG20 in a 0.5 M 1'ferrocene-dimethanol solution of 0.1 M NaClO4 (a) at 0 min (red), and after 5 min (blue), 10 min (green), and 30 min (black) of incubation; (b) at 0 min (red) and after 24 hours of incubation (purple). The scan rate is 50 mV/s.

Table 1 .

 1

	Samples		C functional groups		B functional groups	O functional groups
			C-B	Csp²	C-O	C=O BC3	BC2O BCO2 O-C	O=C O=B
	a-C:B	Position	283.8 284.7 286.4 287.6 188.8 191.2 /	531	532	533.9
		(eV)
		FWHM	1.4	1.7	1.8	1.9	2.2	3.3	/	1.9	1.9	1.9
		(eV)
	BG1% Position	283.8 284.8 286.3 287.3 189.3 /	192.2 531.2 532.3 533.2
		(eV)
		FWHM	1	1.8	1.1	1.3	2	/	3.7	1.6	1.6	1.6
		(eV)
	BG2.5% Position	283.8 284.7 286.1 287.5 188.8 191.1 192.2 531	532.2 533.2
		(eV)
		FWHM	1	1.6	1.4	1.4	1.3	1.2	2.8	1.7	1.7	1.7
		(eV)

Table 1 .

 1 Summary of the positions and FWHM of the carbon, boron and oxygen functional groups

Table 2 .

 2 Average values plus standard deviations of the Raman characteristics resulting from the 400 Raman spectra performed on representative areas of the synthesized undoped and boron graphene.

Table 3 .

 3 Results of electrochemical measurements on BG and undoped graphene films. *: Iox and Ired correspond to the integrated area under the peak and not the Imax.

Table 4 .

 4 Results of electrochemical measurements on BG2.5% for different lengths of time in ferrocene dimethanol at a scan rate of 50 mV/s.

  density of defects associated with a reduction in the size of the cluster.

	configurations and high
	Time (min) Eox (V)	Ered (V)	Iox (µA)	Ired (µA)	ΔE (V)
	0	0.37	0.15	0.43	0.56	0.22
	5	0.39	0.11	0.63	0.68	0.28
	10	0.30	0.06	0.34	0.56	0.24
	30	0.32	0.03	0.08	0.09	0.29
	1,440	0.315 -0.00536	0.168	0.198	0.320

Acknowledgments

This work was supported by the LABEX MANUTECH-SISE (ANR-10-LABX-0075) of University of Lyon, France, as part of the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR).