Camille Juin

Raimundo Gonçalves De Oliveira Junior

Audrey Fleury

Chloé Oudinet

Lior Pytowski

Jean-Baptiste Bérard

Elodie Nicolau

Valérie Thiéry

Isabelle Lanneluc

Laureen Beaugeard

Grégoire Prunier

Jackson Roberto

Guedes Da

Silva Almeida

Laurent Picot
email: laurent.picot@univ-lr.fr

Zeaxanthin from Porphyridium purpureum induces apoptosis in human melanoma cells expressing the oncogenic BRAF V600E mutation and sensitizes them to the BRAF inhibitor vemurafenib

Keywords: cancer, carotenoid, melanoma, microalgae, phytoplankton, zeaxanthin

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Introduction

Melanomas account for less than 2% of skin cancers but are responsible for 80% of their mortality [START_REF] Armstrong | The epidemiology of UV induced skin cancer[END_REF][START_REF] Grange | Epidemiology of cutaneous melanoma: descriptive data in France and Europe[END_REF][START_REF] Leiter | Epidemiology of melanoma and nonmelanoma skin cancer -the role of sunlight[END_REF][START_REF] Mackie | Epidemiology of invasive cutaneous melanoma[END_REF]. Melanoma cells are characterized by mutations that confer them a strong resistance to anticancer drugs-induced apoptosis and selective advantages for cell survival, proliferation and metastasis [START_REF] Locatelli | Recent Advances in the Biology, Therapy and Management of Melanoma[END_REF]. Particularly, mutations in the BRAF oncogen are found in 70% of malignant melanoma [START_REF] Davies | Mutations of the BRAF gene in human cancer[END_REF][START_REF] Haluska | Genetic alterations in signaling pathways in melanoma[END_REF][START_REF] Dankort | Braf(V600E) cooperates with Pten loss to induce metastatic melanoma[END_REF][START_REF] Dutton-Regester | A high-throughput panel for identifying clinically relevant mutation profiles in melanoma[END_REF][START_REF] Jang | Treatment of BRAF-mutant melanoma: the role of vemurafenib and other therapies[END_REF] and lead to over-activation of the MAP kinase pathway that stimulates cell proliferation. Most anticancer drugs only delay the early growth of melanoma tumors but fail to provide a long-terme cure because of the rapid acquisition of drug resistance [START_REF] Locatelli | Recent Advances in the Biology, Therapy and Management of Melanoma[END_REF][START_REF] Spagnolo | Overcoming resistance to BRAF inhibition in BRAFmutated metastatic melanoma[END_REF]. Additionally, melanoma cells display pronounced neoangiogenesis and a high ability to escape immune cell that explain why the 5-year survival rate for metastatic melanoma ranges from 5 to 10%, with a median survival of less than 8 months [START_REF] Marneros | Tumor angiogenesis in melanoma[END_REF][START_REF] Mathieu | Galectin-1 in melanoma biology and related neoangiogenesis processes[END_REF]. Brain metastasis are present in 75% of advanced melanoma patients and constitute a major cause of mortality because of the low permeability of the blood-brain barrier to chemotherapeutic drugs [START_REF] Hall | Long-term survival with metastatic cancer to the brain[END_REF]. The search for cytostatic, antimetastatic and antiangiogenic molecules in plants and algae has established that carotenoids have a great potential as natural antimelanoma compounds [START_REF] Hashimoto | Pharmacokinetics of fucoxanthinol in human plasma after the oral administration of kombu extract[END_REF][START_REF] Tanaka | Cancer chemoprevention by carotenoids[END_REF]Firdous et al., 2012;Gages et al., 2012;[START_REF] Baudelet | Antiproliferative activity of Cyanophora paradoxa pigments in melanoma, breast and lung cancer cells[END_REF][START_REF] Reboul | Absorption of vitamin A and carotenoids by the enterocyte: focus on transport proteins[END_REF][START_REF] Chung | Marine algal fucoxanthin inhibits the metastatic potential of cancer cells[END_REF][START_REF] Kim | Inhibition of tumor growth in vitro and in vivo by fucoxanthin against melanoma B16F10 cells[END_REF][START_REF] Xu | Effects of zeaxanthin on growth and invasion of human uveal melanoma in nude mouse model[END_REF][START_REF] Lu | Analysis and identification of astaxanthin and its carotenoid precursors from Xanthophyllomyces dendrorhous by high-performance liquid chromatography[END_REF][START_REF] Chen | Astaxanthin reduces MMP expressions, suppresses cancer cell migrations, and triggers apoptotic caspases of in vitro and in vivo models in melanoma[END_REF]. These pigments have no oral toxicity, are resorbed by enterocytes, transported in blood after per os consumption [START_REF] Burri | Serum carotenoid depletion follows first-order kinetics in healthy adult women fed naturally low carotenoid diets[END_REF][START_REF] Hashimoto | Pharmacokinetics of fucoxanthinol in human plasma after the oral administration of kombu extract[END_REF][START_REF] Reboul | Absorption of vitamin A and carotenoids by the enterocyte: focus on transport proteins[END_REF] and can integrate cell membranes [START_REF] Reboul | Absorption of vitamin A and carotenoids by the enterocyte: focus on transport proteins[END_REF]Oliveira-Júnior et al., 2016) and reach tumor cells where they exert cytotoxic, cytostatic, antimetastatic, anti-inflammatory and antiangiogenic activities [START_REF] Sugawara | Antiangiogenic activity of brown algae fucoxanthin and its deacetylated product, fucoxanthinol[END_REF][START_REF] Gagez | Epoxycarotenoids and cancer[END_REF]. Fucoxanthin, a carotenoid present in brown microalgae and seaweeds inhibits melanoma cells and tumor growth in vitro and in vivo [START_REF] Chung | Marine algal fucoxanthin inhibits the metastatic potential of cancer cells[END_REF][START_REF] Kim | Inhibition of tumor growth in vitro and in vivo by fucoxanthin against melanoma B16F10 cells[END_REF]. It also limits melanoma metastasis in murine models [START_REF] Chung | Marine algal fucoxanthin inhibits the metastatic potential of cancer cells[END_REF][START_REF] Kumar | Fucoxanthin: a marine carotenoid exerting anticancer effects by affecting multiple mechanisms[END_REF], suggesting that it also has a clinical efficacy in humans. We recently demonstrated that zeaxanthin (1), an abundant carotenoid found in various dietary sources (corn, spinach, saffron, seaweeds, microalgae) inhibits the in vitro growth of the highly invasive human melanoma cell line A2058 [START_REF] Baudelet | Antiproliferative activity of Cyanophora paradoxa pigments in melanoma, breast and lung cancer cells[END_REF].

Zeaxanthin also induced apoptosis in two human uveal melanoma cell lines (SP6.5 and C918) without impairing the cell viability of non cancer uveal melanocytes (Bi et al., 2013;[START_REF] Xu | Effects of zeaxanthin on growth and invasion of human uveal melanoma in nude mouse model[END_REF]. Zeaxanthin-induced apoptosis was associated to a decrease in the expression of the antiapoptotic proteins Bcl-2 and Bcl-xL and an increase in the expression of the proapoptotic proteins Bak and Bax (Bi et al., 2013). Zeaxanthin also evoked the release of mitochondrial cytochrome c in the cytosol and caspase-9 and -3 activation (Bi et al., 2013). In the present report, we performed additional experiments to further elucidate the molecular mechanisms of zeaxanthin pro-aptoptotic activity in melanoma cells and assessed its ability to sensitize melanoma cells to a BRAF inhibitor used to treat clinical melanoma. We selected the highly invasive A2058 human melanoma cell line as a relevant clinical model expressing the V600E BRAF oncogenic mutation (Dankort-Regester et al., 2012).

Material and methods

Microalgae culture, harvest and freeze-drying

Porphyridium purpureum (Bory) K.M.Drew & R.Ross CCAP 1380.3 (bangiophyceae, rhodophyte) (Fig. 1.) was grown at 120 μmol.m -2 . s -1 irradiance. Cells were grown in four units of 50-l column photobioreactors with 35‰ salinity seawater enriched by Walne medium [START_REF] Walne | Experiments in the large-scale culture of the larvae of Ostrea edulis (L.)[END_REF][START_REF] Juin | UPLC-MSE Profiling of phytoplankton metabolites: application to the identification of pigments and structural analysis of metabolites in Porphyridium purpureum[END_REF]. Batch cultures were maintained at 20 °C under continuous light provided by fluorescent lamps (Philips TLD 58 W 865) and bubbled with 0.22 μm filtered air containing 3% CO 2 (v/v).

Microalgae were harvested after 12-16 days of growth and separated from the culture medium by a two-step process. First step used a clarifier separator (Clara 20, Alfa Laval Corporate AB, Sweden) at 100 l.h -1 , 9,000 g, at room temperature.

Step two used a soft centrifugation at 4,000 × g, 20 mn, at 4 °C to separate the slurry. Algal paste was freeze-dried at -55 °C and P<1 hPa, on a freeze-dryer equipped with a HetoLyoPro 3000 condenser and a Heto cooling trap (Thermo, France).

Purification and characterization of Porphyridium purpureum zeaxanthin

Porphyridium purpureum pigments were extracted in ethanol using a mixer mill extraction process developed in our group [START_REF] Serive | Selection and optimisation of a method for efficient metabolites extraction from microalgae[END_REF]. Zeaxanthin (1) identification was confirmed after separation by analytical RP-HPLC [START_REF] Zapata | Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridinecontaining mobile phases[END_REF] and cross-check analysis of its polarity, absorption spectrum, maximal absorption wavelengths, band III/II ratio and fragmentation profile in UPLC-MS E [START_REF] Roy | Part VII. Data sheets aiding identification of phytoplankton carotenoids and chlorophylls, Chapter 3: Xanthophylls[END_REF][START_REF] Baudelet | Antiproliferative activity of Cyanophora paradoxa pigments in melanoma, breast and lung cancer cells[END_REF][START_REF] Juin | UPLC-MSE Profiling of phytoplankton metabolites: application to the identification of pigments and structural analysis of metabolites in Porphyridium purpureum[END_REF], in comparison with standard zeaxanthin (Sigma-Aldrich, France). Pure zeaxanthin was then collected by preparative RP-HPLC [START_REF] Pasquet | Antiproliferative activity of violaxanthin isolated from bioguided fractionation of Dunaliella tertiolecta extracts[END_REF] in glass vials, dried under reduced pressure in a Buchi R-210 rotatory evaporator at 40 °C (Buchi, France) and stored at -80°C before use in cell culture experiments.

Cell culture

A2058 (ATCC ® CRL-11147™, LGC ATCC Standards, France) is a melanoma cell line established from metastatic cells removed from the lymph node of a 43 years old male caucasian patient. It constitutes a clinically relevant model to assess the cytotoxicity of new antimelanoma drugs as it combines high invasive, metastatic and chemoresistance potentials with a gene mutation profile often encountered in human melanomas (V600E mutation in BRAF and mutations in the PTEN and P53 genes) [START_REF] Dankort | Braf(V600E) cooperates with Pten loss to induce metastatic melanoma[END_REF]. Cells were routinely grown as monolayers, at 37 °C in a 5% CO 2 -95% air humidified atmosphere, in DMEM (Fischer scientific, France) supplemented with 10% heat-inactivated (56 °C, 30 min) FCS (Dutscher, France) to which were added penicillin 100 U•ml -1 and streptomycin 100 µg•ml -1 .

Determination of zeaxanthin IC 50 in A2058 melanoma cells

Purified zeaxanthin (1) was solubilized in DMSO at 6 mM (stock solution) and diluted in cell culture medium to obtain 5 to 60 µM solutions. The final DMSO concentration in the cell culture medium was lower than 1%, tested as a negative control and validated as a non cytotoxic concentration. The antiproliferative activity of zeaxanthin was determined using the MTT assay (Sigma-aldrich, France) as previously described [START_REF] Putey | Synthesis of latonduine derivatives via intramolecular Heck reaction[END_REF][START_REF] Baudelet | Antiproliferative activity of Cyanophora paradoxa pigments in melanoma, breast and lung cancer cells[END_REF][START_REF] Hedidi | Synthesis of N-pyridyl azoles using a deprotometalation-iodolysis-N-arylation sequence and evaluation of their antiproliferative activity in melanoma cells[END_REF]. IC 50 was determined using the free Graph pad Prism software using the "sigmoidal dose response" (variable slope) function.

Nuclear membrane modification, chromatin condensation and DNA fragmentation

Sub-confluent A2058 melanoma cells were trypsinized and 2.10 5 cells were seeded in 6-well plates, in a final volume of 3 ml of control medium or culture medium containing zeaxanthin 40 µM (IC 50 ) or staurosporine 2 µM. Cells were grown for 72 h at 37 °C and washed in PBS 0.1M pH 7.4, before being fixed with formaldehyde 3% for 30 min at 37 °C. Cells were then rinsed in PBS, permeabilized with Triton X-100 1% in PBS and stained with DAPI 2 µg.ml -1 for 1 h at 37 °C. Cells were rinsed, mounted on glass microscope slides and observed using a Leica epifluorescence microscope equipped with an epifluorescence A filter block (excitation 340-360 nm) and a numeric camera.

Annexin V-Cy3 and 6-CFDA detection assay

Apoptosis was evaluated by using double staining with Annexin V-Cy3 (red) and 6carboxyfluorescein diacetate (6-CFDA, green) (Sigma-Aldrich ® , France). Cells (5 x 10 3 cells/well) were incubated in conventional culture conditions for 24 h. Then, cells were treated with zeaxanthin (IC 50 , 40 µM) for 72 h and staurosporine (positive control, 1 µM) for 24 h. Cells were further washed with PBS, suspended in binding buffer and stained with Annexin V and 6-CFDA solution for 10 min. DAPI solution was also added to the wells for DNA labelling. Finally, cells were observed under fluorescence microscope (ZEISS Axio Observer).

Flow cytometric detection of apoptotic cells

Melanoma cells were grown in control culture medium or treated with zeaxanthin 40 µM for 72 h before being stained for 30 min at 37 °C in PBS containing propidium iodide (PI 100 µg.ml -1 ) and Rnase A (100 µg.ml -1 ) (Molecular Probes, France). Cells were washed and suspended in 1 ml PBS before being analyzed using a FACS Cantoll flux cytometer (BD Biosciences, France) equipped with an air cooled blue LASER (=488 nm, 20 mW). Light diffusion parameters (forward and lateral scatter lights) were optimized to define the size threshold excluding cellular debris and cell clusters for single-cell fluorescence analysis. PI fluorescence was measured using a FL3 filter (nmand analyzed using the BD FACS Diva Software (BD Biosciences, France).

Distribution of melanoma cells in the different cell cycle phases and hypodiploidy was determined according to their DNA content as measured by the fluorescence intensity of PI: Diploid cells (2n): G0/G1 phase; Replicative cells (2n<DNA content<4n): S Phase; Tetraploid cells (4n): G2/M phase; hypodiploid cells (DNA content < 2n): apoptotic Sub-G1 Phase).

DNA internucleosomal fragmentation

After treatment with control culture medium or zeaxanthin 40 µM, 10 6 melanoma cells were washed with PBS and lysed in 400 µl lysis buffer (Tris HCl 10 mM pH 8 NaCl 150 mM, EDTA 40 mM, SDS 1%, proteinase K 0.2 mg.ml -1 ) for 3 h at 56 °C. The cell lysate was centrifuged (11000 × g, 15 min, 4°C) and the DNA contained in the supernatant was extracted for 15 min using a mix of phenol/chloroform/isoamyl alcool (PCI) (25/24/1 v/v/v) at pH 9. The mix was centrifuged (11000 × g, 15 min, 4°C), and the aqueous phase was collected to precipitate DNA using sodium acetate 3M in 1 ml absolute ethanol (1 night, -20°C). The precipitated DNA was centrifugated (11000 × g, 30 min, 4 °C), the supernatant was discarded and the pellet was dried 5 min at 60 °C. The DNA pellet was suspended in 50 µl Ultrapure water containing RNase A 100 µg.ml -1 for 30 min at 37 °C.

Twenty microliters of the DNA extract were loaded and separated on an agarose/tris-borate-EDTA 1% gel for 30 min at 100V. Gels were stained with ethidium bromide, and observed using a UV transilluminator.

Caspase-3 colorimetric assay

Caspase-3 activation was quantified using a commercial assay based on the hydrolysis of Ac-DEVD-pNA (CASP3C kit, Sigma-Aldrich, France).

Western-blot

A2058 cells were incubated in control culture medium or in the presence of zeaxanthin (1) 40 µM for 72 h. The cells were collected and lysed in a lysis buffer (HEPES 50 mM pH 7.4 CHAPS 5 mM DTT 5 mM). Total proteins were separated by 10% SDS-PAGE and then transferred to a nitrocellulose membrane. The membranes were blocked with 5% (w/v) nonfat dry milk in TBS for one hour and changed to an appropriate dilution of specific primary antibodies against Bid, Bim, Bak, Bcl-xL, IB, NF-B p65, p-NF-B (Ser536), IKK, IKK and β-actin (Ozyme, France) in milk overnight at 4 °C. The secondary antibodies were horseraddish peroxidase-conjugated antirabbit IgG (1:5000). Signals were detected using the ChemiDoc™ imaging system (Biorad, France).

Sensitization of A2058 melanoma cells to the BRAF inhibitor Vemurafenib

Vemurafenib was obtained from Selleckchem, France, diluted to a 10 mM stock solution in PBS 0.1 M pH 7.4, before further dilution in cell culture medium. A2058 cells were incubated for 72 h in control culture medium or in the presence of zeaxanthin 40 µM, Vemurafenib (0.1, 1 and 5 µM) or in a mix of zeaxanthin 40 µM and Vemurafenib (0.1, 1 and 5 µM). Antiproliferative activity was calculated using the MTT assay and potentiation of the Vemurafenib antiproliferative activity by zeaxanthin was expressed as the percentage of growth inhibition increase as compared to Vemurafenib alone.

Statistics

Antiproliferative activity of zeaxanthin was expressed as percentage growth inhibition ± SEM from three independent assays. The normal distribution of absorbance data in control and treated cells was demonstrated using the Hartley's Fmax test to confirm homogeneity of absorbances variances. The statistical significance of proliferation differences between control and treated cells was then investigated by an unpaired Student t test, using a free online calculator developed by Institut Pierre Louis d'Epidémiologie et de Santé publique UMR S 1136 INSERM University Pierre et Marie Curie Paris (http://marne.u707.jussieu.fr/biostatgv/?module=tests).

Results

3.1.Purification of zeaxanthin from Porphyridium purpureum

RP-HPLC of the ethanolic extract of Porphyridium purpureum gave a chromatogram containing eight major peaks at 436 nm (Fig. 1). The first pigment eluting as a major peak at 24.582 min was identified as zeaxanthin (1) as it presented a median polarity with maximal absorption wavelengths at 452 and 481 nm (Fig. 2.), a III:II band ratio of 29.03% (Fig. 2), a high resolution molecular weight of 568.4294 (Fig. 3) and a MS E fragmentation pattern characteristic of zeaxanthin (Fig. 3.) [START_REF] Juin | UPLC-MSE Profiling of phytoplankton metabolites: application to the identification of pigments and structural analysis of metabolites in Porphyridium purpureum[END_REF].

Exposure to zeaxanthin inhibits the proliferation of A2058 melanoma cells

A 72 h exposure to increasing concentrations of zeaxanthin [0-60 µM] induced a dosedependent reduction in the number of A2058 cells as compared to the untreated control, reaching 60% growth inhibition. The IC 50 value of zeaxanthin was determined as 40 µM using the free Graph pad Prism software "sigmoidal dose response" (variable slope) function. (Fig. 4.).

Exposure to zeaxanthin evoked cytotoxicity and nuclear fragmentation in A2058 cells

A2058 cells incubated in control medium showed a regular epithelial shape, except for mitotic cells exhibiting a round shape and weak adhesion to the culture dish (Fig. 5A.). The nucleus of control cells was round and showed no sign of DNA condensation, blebbing or shrinkage (Fig.

Zeaxanthin induces apoptosis in A2058 cells

To evaluate the effect of zeaxanthin on cell death, double fluorescence staining with Annexin V and 6-CFDA was performed to differentiate live and apoptotic cells. 6-CFDA is used to measure viability. In this sense, live cells will only stain with 6-CF (green), cells in early apoptosis will stain both with Annexin V (red) and 6-CF (green), and cells in late apoptosis will only stain with Annexin V and DAPI. After 72 h of treatment, zeaxanthin 40 µM increased the number of Annexin V and 6-CFDA double-stained cells, and Annexin V and DAPI stained cells compared to control, indicating enhancement of apoptosis (Fig. 6.). A similar result was observed for staurosporine (1 µM), a known pro-apoptotic agent. Scale bar : 50 µm.

Exposure to zeaxanthin evoked caspase-3 activation, internucleosomal fragmentation and hypopolyploidy in A2058 cells

Activation of caspase-3 is considered as a central event for integrating pro-apoptotic stimuli and activating downstream effector caspases and DNAses during cancer cell apoptosis. The basal activity of caspase-3 was low in A2058 cells grown for 72 h in control cell culture medium (Fig. 7).

Treatment with zeaxanthin 40 µM for 72 h induced a very significant increase in caspase-3 activity (Student t test, p < 0.01), as demonstrated by hydrolysis of the specific chromogenic substrate Ac-DEVD-pNA (Fig. 7). Treatment with staurosporine 2 µm, used as a positive control for caspase-3 activation, induced a highly significant increase in caspase-3 activity (Student t test, p < 0.001). Agarose gel electrophoresis confirmed the internucleosomal fragmentation of DNA extracted from A2058 cells treated with zeaxanthin 40 µM or staurosporine 2 µM (Fig. 9.), demonstrating the activation of caspase-activated DNase by zeaxanthin.

Zeaxanthin stimulates the expression of the pro-apoptotic factors Bim and Bid and inhibits the nuclear translocation of NF-B in A2058 melanoma cells

It was previously reported that zeaxanthin decreased the expression of antiapoptotic proteins (Bcl-2 and Bcl-xL) and increased the expression of proapoptotic proteins (Bak and Bax) in zeaxanthin-treated uveal melanoma cells (Bi et al., 2013). To complete the identification of signaling pathways involved in zeaxanthin-induced apoptosis of melanoma cells (Bi et al., 2013), the expression of pro-apoptotic, anti-apoptotic and pro-inflammatory factors were investigated by western-blot analysis. Zeaxanthin 40 µM respectively induced a high and moderate expression increase of the pro-apoptotic factors Bim and Bid (Fig. 10.) that could be involved in the onset of apoptosis. The expression of the pro-apoptotic factor Bak was unchanged. The level of phosphorylated NF-B p65 (Ser 536) was decreased while IBand Ikk were up-regulated, indicating that zeaxanthin inhibited the nuclear translocation of NF-B and subsequently downregulated the expression of pro-inflammatory genes. Ikkand unphosphorylated NF-kB expressions were unchanged (Fig. 10.).

Discussion

Advanced melanoma have a bad prognosis as most molecules used in cancer chemotherapy are ineffective in killing metastatic melanoma cells which are constitutively or adaptatively resistant to pro-apoptotic drugs. The development of targeted therapies using BRAF inhibitors has significantly improved the treatment of metastatic melanomas as the V600E BRAF oncogenic mutation is found in more than 70% of clinical cases. However most patients eventually develop resistance mechanisms that ultimately lead to therapeutic impasses. In this view, many research projects aim to identify natural molecules with cytostatic, antimetastatic and anti-angiogenic activities that do not weaken the immune system and could be used to potentiate the efficiency of chemotherapy and immunotherapy and slow the emergence of resistance mechanisms [START_REF] Cragg | Natural products in drug discovery and development[END_REF][START_REF] Caltagirone | Flavonoids apigenin and quercetin inhibit melanoma growth and metastatic potential[END_REF][START_REF] Niles | Resveratrol is a potent inducer of apoptosis in human melanoma cells[END_REF][START_REF] Mesquita | Cytotoxic activity of Brazilian Cerrado plants used in traditional medicine against cancer cell lines[END_REF][START_REF] Van Goietsenoven | Targeting of eEF1A with Amaryllidaceae isocarbostyrils as a strategy to combat melanomas[END_REF][START_REF] Pasquet | Antiproliferative activity of violaxanthin isolated from bioguided fractionation of Dunaliella tertiolecta extracts[END_REF][START_REF] Ahmad | Thymoquinone suppresses metastasis of melanoma cells by inhibition of NLRP3 inflammasome[END_REF][START_REF] Zhang | Novel mechanisms of anticancer activities of green tea component epigallocatechin-3-gallate[END_REF]Alqathama and Pietro, 2015;[START_REF] Mirzaei | Curcumin: A new candidate for melanoma therapy?[END_REF]. Many carotenoids meet all these activities, as they display high cytotoxicity in tumor cells from various histological origins, including chemoresistant melanoma cell lines, and exert significant antitumoral activity in vivo by inhibiting tumor growth, angiogenesis and invasivity [START_REF] Gagez | Epoxycarotenoids and cancer[END_REF][START_REF] Kumar | Fucoxanthin: a marine carotenoid exerting anticancer effects by affecting multiple mechanisms[END_REF][START_REF] Chen | Astaxanthin reduces MMP expressions, suppresses cancer cell migrations, and triggers apoptotic caspases of in vitro and in vivo models in melanoma[END_REF]. Moreover, most carotenoids have no oral toxicity and do not weaken the immune system [START_REF] Chew | Carotenoid action on the immune response[END_REF][START_REF] Pechinskii | The impact of carotenoids on immunity (Review)[END_REF][START_REF] Ghodratizadeh | Effect of carotenoid β-cryptoxanthin on cellular and humoral immune response in rabbit[END_REF]. Microalgae constitute an optimal source to produce carotenoids for pharmaceutical applications as they combine the advantages of synthesizing the wide chemodiversity of carotenoids, with high production yields, without the need of freshwater, agricultural surfaces or pesticides to be grown [START_REF] Mimouni | The potential of microalgae for the production of bioactive molecules of pharmaceutical interest[END_REF]. Selection of hyper-producing strains combined to optimization of their growth conditions and purification processes allow the recovery of high amounts of carotenoids devoid of chemicals or endotoxins. In the present report, we demonstrate that zeaxanthin, an abundant carotenoid present in microalgae, that can be easily obtained in high amounts from the rhodophyte Porphyridium purpureum, induces apoptosis in human melanoma cells expressing the oncogenic BRAF V600E mutation and potentiates the antiproliferative activity of vemurafenib, a BRAF inhibitor used in patients with advanced metastatic melanomas. Zeaxanthin (1) was previously reported to have no oral toxicity and decrease the incidence of various cancers after oral ingestion [START_REF] Thurnham | Studies on meso-zeaxanthin for potential toxicity and mutagenicity[END_REF][START_REF] Xu | Safety evaluation of meso-zeaxanthin[END_REF].

It was also reported to exert pro-apoptotic activity in human uveal melanoma cells (SP6.5 and C918) and limit uveal melanoma invasivity without impairing the viability of non cancer uveal melanocytes [START_REF] Xu | Effects of zeaxanthin on growth and invasion of human uveal melanoma in nude mouse model[END_REF]Bi et al., 2013). In A2058 melanoma cells, zeaxanthin IC 50 was determined as 40 µM, a concentration inducing cell rounding, chromatin condensation, nuclear fragmentation, hypodiploidy, cell apoptosis in early and late stages, accumulation of cells in sub-G1 phase, DNA internucleosomal fragmentation and activation of caspase-3. Zeaxanthin-induced apoptosis was accompanied by inhibition of NF-B and up-regulation of the pro-apoptotic factors Bim and Bid, demonstrating the involvement of the mitochondrial signaling pathway in apoptosis triggering, as previously reported in uveal melanoma models (Bi et al., 2013). The observation that zeaxanthin was able to moderately potentiate the in vitro antiproliferative activity of vemurafenib is a promising result of our study as it suggests its potential interest as a nutritional adjuvant increasing the sensitivity of tumor cells to BRAF inhibitors. By potentiating the antiproliferative effect of BRAF inhibitors, zeaxanthin may allow to decrease BRAF inhibitors effective doses, limit their adverse effects in patients and delay the emergence of resistance mechanisms [START_REF] Chu | Diverse cutaneous side effects associated with BRAF inhibitor therapy: A clinicopathologic study[END_REF][START_REF] Zimmer | Panniculitis with arthralgia in patients with melanoma treated with selective BRAF inhibitors and its management[END_REF][START_REF] Anforth | Cutaneous adverse events in patients treated with BRAF inhibitor-based therapies for metastatic melanoma for longer than 52 weeks[END_REF][START_REF] Welsh | Management of BRAF and MEK inhibitor toxicities in patients with metastatic melanoma[END_REF]. The molecular mechanisms involved in this increase of sensitivity as well as preclinical and clinical relevance of combining carotenoids with BRAF inhibitors will have to be further explored, as this strategy could extend the duration of chemotherapy efficiency. In summary, this study confirms the potential of zeaxanthin to limit the growth of chemoresistant melanoma cells, confirms the major interest of phytoplankton carotenoids as natural anticancer molecules devoid of oral toxicity, and suggests for the first time the interest of combining a carotenoid to BRAF inhibitors to potentiate their chemotherapeutic efficiency in melanoma cells.

Fig. 1 .

 1 Fig. 1. Porphyridium purpureum strain CCAP 1380.3. © Raymond Kaas. IFREMER.

Fig. 1 .

 1 Fig.1. RP-HPLC Chromatogram of Porphyridium purpureum ethanol extract at 436 nm obtained using the Van Heukelem and Thomas analysis (van Heukelem et al., 2001). The pigment profile was identical to that previously reported in Serive et al. (2017).

Fig. 2 .

 2 Fig.2. Identification of the pigment eluting at 24.582 min as zeaxanthin (1) based on absorption spectrum, maximal absorbance wavelengths and III:II band ratio.

Fig. 3 .

 3 Fig.3. MS E fragmentation pattern of zeaxanthin (1) isolated from Porphyridium purpureum.

Fig. 4 .

 4 Fig. 4. Growth inhibition of A2058 cell in the presence of zeaxanthin (1). A2058 cells were grown for 72 h in a cell culture medium containing increasing concentrations of zeaxanthin. The antiproliferative activity of zeaxanthin was observed for concentrations superior to 5 µM and IC 50 was determined as 40 µM.

  5B.). The treatment with staurosporine 2 µM (non selective kinases inhibitor, control apoptosis inducer) evoked a high cytotoxicity evidenced by cell shrinkage (Fig. 5C.) and DNA condensation in the nucleus (Fig. 5D.). A 72 h treatment with zeaxanthin 40 µM half lowered the cell density, evoked rounding of the cells (Fig. 5E.) and nuclear fragmentation (Fig. 5F.), suggesting a blockade in the cell cycle and apoptosis induction.

Fig. 5 .

 5 Fig. 5. Cytotoxicity and nuclear fragmentation in A2058 cells exposed to zeaxanthin 40 µM. A2058 cells were grown for 72 h in a control cell culture medium (A and B) or in a medium containing staurosporine 2 µM (C and D) or zeaxanthin 40 µM (E and F). Treatment with staurosporin evoked cell rounding and chromatin condensation (C and D) while zeaxanthin evoked cell rounding (E), chromatin condensation and nuclear fragmentation (F).

Fig. 6 .

 6 Fig. 6. Zeaxanthin induces apoptosis of A2058 melanoma cells. Annexin V (red) and 6-CFDA (green) double staining of apoptotic cells was examined by fluorescence microscopy. A2058 cells in early apoptosis showed both green and red stains; A2058 cells in late apoptosis showed red and blue stains; and control (untreated) cells stained green only. Cells treated with zeaxanthin (40 µM, for 72 h) or Staurosporine (1 µM, for 24 h) were considered in early or late apoptosis compared to control.

Fig. 7 .

 7 Fig. 7. Zeaxanthin induces caspase-3 activation in A2058 melanoma cells. A2058 cells were grown for 72 h in a control cell culture medium or in a medium containing zeaxanthin 40 µM or staurosporine 2 µM (positive control for apoptosis induction).

Fig. 8 .

 8 Fig. 8. Cell cycle analysis of A2058 cells grown for 72 h in control cell culture medium (A) or cell culture medium containing zeaxanthin 40 µM (B). The majority of control or treated cells were in the quiescence (G0) or pre-replicative (G1) phases. Zeaxanthin induced the appearance of a sub-G1 peak, characteristic of hypodiploidic cells undergoing cell deaths.

Fig. 9 .

 9 Fig. 9. Agarose gel electrophoresis of DNA extracted from A2058 cells incubated for 72 h in the absence (-) or presence of zeaxanthin 40 µM (zea) or staurosporine 2 µM (stau). Observation of a smear in the zeaxanthin and staurosporine lanes revealed the activation of caspase-activated DNases and internucleosomal fragmentation of DNA in apoptotic cells.

Fig. 10 .

 10 Fig. 10. Effect of zeaxanthin on the expression of pro-apoptotic and anti-apoptotic factors and activation of pro-inflammatory pathways in A2058 melanoma cells. Cells were incubated in control culture medium or in the presence of zeaxanthin 40 µM for 72h before protein extraction, SDS-PAGE and western blot.

Fig. 11 .

 11 Fig. 11. Zeaxanthin sensitizes A2058 melanoma cells to the BRAF inhibitor vemurafenib. Two thousands A2058 melanoma cells were grown for 72 h in control cell culture medium (A), medium containing zeaxanthin 40 µM (B), vemurafenib 5 µM (C), or a mix of zeaxanthin 40 µM and vemurafenib 5 µM (D). Zeaxanthin induced a 37.98, 25.75 and 10.70% increase of the antiproliferative activity of vemurafenib 0.1, 1 and 5 µM, respectively (E).

Acknowledgements

We are grateful to the French cancer league (Comité 17 de la Ligue Nationale contre le Cancer) for financial support and to the Poitou-Charentes region for CJ's PhD grant. We also thank the "Cancéropôle Grand Ouest, axe Valorisation des produits de la mer en cancérologie" for scientific support. LP is grateful to the Brazilian Society of Pharmacognosy for its kind invitation to the X th Brazilian Symposium Pharmacognosy in Petrolina in September 2015. We thank Dr Raymond Kaas from IFREMER for his kind permission to use the microphotography of Porphyridium purpureum and Antoine Bonnet (Platform for the High Resolution Analysis of Biomolecules, University of La Rochelle, France) for excellent technical assistance with HRMS.

Authors' contribution and responsibility

Competing interests

The authors declare they have no competing interests.


D Pérez Galacho

L Bramerie

C Baudot

M Chaibi

S Messaoudène

N Vulliet

L Vivien

C Peucheret

D Marris-Morini

Advanced modulation format using silicon modulators in the O-band

Keywords: silicon modulator, advanced modulation format, QPSK, Mach Zehnder

This paper review our recent work on silicon modulators based on free carrier concentration, working in the O-band of optical communications (1260 nm -1360 nm) for short distance applications. 25 Gbit/s OOK modulation is obtained using a driving voltage of 3.3 Vpp , and QPSK dual-drive Mach-Zehnder modulator (DDMZM) operating in the O-band is demonstrated for the first time.

INTRODUCTION

Significant research effort has been dedicated over the past years to the development of silicon-based photonic technologies [START_REF] Boeuf | Silicon Photonics R&D and manufacturing on 300-mm wafer platform[END_REF][START_REF] Dong | Silicon photonic devices and integrated circuits[END_REF]. Developing high performance silicon modulators based on the free-carrier plasma dispersion (FCPD) effect has been an important milestone. These modulators are commonly based on carrier depletion in PN junctions and outstanding performances have already been demonstrated [START_REF] Dong | High-speed low-voltage single-drive push-pull silicon Mach-Zehnder modulators[END_REF][START_REF] Thomson | 50-Gb/s silicon optical modulator[END_REF]. However, in order to keep up with the growing bandwidth demand in datacom applications, it is necessary to move from binary on-off keying (OOK) modulation format to more advanced formats such as 4-level pulse amplitude modulation (PAM-4) or quadrature phase-shift keying (QPSK). Advanced modulation formats using silicon modulators have already been demonstrated [START_REF] Dong | 112-Gb/s monolithic PDM-QPSK modulator in silicon[END_REF][START_REF] Dong | 224-Gb/s PDM-16-QAM modulator and receiver based on silicon photonic integrated circuits[END_REF][START_REF] Ding | Silicon 16-QAM optical modulator driven by four binary electrical signals[END_REF][START_REF] Samani | Experimental parametric study of 128 Gb/s PAM-4 transmission system using a multielectrode silicon photonic Mach Zehnder modulator[END_REF]. However, most of these demonstrations have been carried out in the C-band of optical communication systems (1530 nm -1570 nm) where the FCPD is stronger. Nevertheless, it is believed that silicon photonics is the best suited for next generation of short range optical fiber communications, where current standards favor the use of the O-band (1260 nm -1360 nm) in order to exploit the zero-dispersion region of standard single-mode fibers. In this work we present results on high performance silicon modulators operating in the O-band, including efficient OOK modulation and the first demonstration of a QPSK siliconbased dual-drive Mach-Zehnder modulator (DD-MZM) operating in the O-band.

SILICON MODULATOR DESIGN

The active region of the Mach Zehnder modulator is illustrated in Fig 1 . (a). It is based on a PN diode embedded in a silicon-on-insulator (SOI) waveguide. The waveguide width and eight are 400 nm and 300 nm, respectively, while its slab height is 50 nm. The PN diode is obtained by boron and phosphorus implantation of the waveguide. Targeted doping concentrations are P = 5 × 10 17 cm -3 and N = 1.4×10 18 cm -3 . The junction interface is 25 nm shifted from the center of the waveguide to benefit from larger effective index variation. Highly doped P ++ and N ++ regions are used away from the optical mode to reduce access resistance without increasing optical losses. The modulators were fabricated using the 300 mm DAPHNE technological platform from STMicroelectronics [START_REF] Baudot | Daphne silicon photonics technological platform for research and development on wdm applications[END_REF].

The phase shifter efficiency was estimated by measuring the transmission of ring resonators embedding the phase shifter inside the ring, as a function of the applied voltage. The results was a Vπ Lπ product between 0.95 Vcm and 1.15 Vcm for a reverse applied voltage range from 1 V to 4 V [START_REF] Perez-Galacho | Low voltage 25Gbps silicon Mach-Zehnder modulator in the O-band[END_REF]. These values are the highest reported modulation efficiencies of carrier depletion based modulators in the O-Band.

Mach-Zehnder modulators are using a dual drive configuration, as schematically illustrated in

OOK MODULATION

The set-up used for OOK modulation characterization is schematically reported in Fig 2 . Both arms of the 1 mm long DD-MZM were driven in push-pull configuration, i.e. one arm is driven with the PRBS data signal and the other with its complementary. The heaters were used to set the interferometer to the quadrature point. Thanks to the push-pull configuration and the high efficiency of the active region, it has been possible to reduce the voltage swing down to 3.3 Vpp, with a DC bias of 1.7 V. The corresponding eye diagram at 25 Gbps is reported in Fig. 3. An extinction ratio (ER) of 5 dB was obtained, while the total on-chip losses were 3.6 dB at this operating point.

QPSK MODULATION

DD-MZMs can also be used to generate QPSK signals with a simpler structure than standard IQ modulators based on nested MZMs [START_REF] Ho | Generation of arbitrary quadrature signals using one dual-drive modulator[END_REF][START_REF] Krause | Demonstration of 20-Gb/s DQPSK with a single dual-drive Mach-Zehnder modulator[END_REF]. Furthermore, this scheme allows a reduction of the driving voltage applied to the transceiver and thus to the power consumption to generate the QPSK constellation, offering a better power efficiency and scalability that are key advantages for short distance intra data center communications. The set-up used for QPSK modulation characterization at 20 Gbps (10 Gbaud) is reported in Fig 4. Two 2 15 -1 long de-correlated pseudo random binary sequences (PRBSs) at 10 Gbps were amplified to reach a swing voltage Vpp of 7 V and applied to each of the phase shifters in order to achieve a peak-to-peak phase modulation of π in each arm, while the phase-shift introduced by the heater is adjusted to operate the DD-MZM at the quadrature point (Φ=π/2). TE-polarized light from an O-band external cavity laser followed by a fiber polarization controller was coupled to the photonic integrated circuit using a single polarization grating coupler (GC). The same laser was also used as local oscillator (LO). The modulated optical signal was then sent to the coherent receiver through a variable optical attenuator in order to adjust the received power. The I/Q signals of the receiver were fed to a 40 Gsps digital sampling oscilloscope (DOS) with 16 GHz electrical bandwidth. Blocks of 10 5 symbols were acquired and then processed offline. An adaptive finite impulse response (FIR) filter with decision-directed least mean square (DD-LMS) tracking was used as digital signal processing (DSP). The processed samples were used to obtain the constellation diagrams and to compute bit error rate (BER). The BER was calculated over five blocks of 10 5 symbols.

For QPSK modulation characterization, a 2 mm-long MZM was used, as it is necessary to achieve a  phase variation on each arm of the MZM. On-chip optical losses are estimated below 3.8 dB, corresponding to 2.6 dB for the 2 mm-long active region, while losses below 0.6 dB are estimated for each MMI couplers. After DSP, a classical QPSK constellation diagram was obtained, as illustrated in Fig 5 . (a) for a receiver input power of -22 dBm. The measured BER performance of the Si DD-MZM is reported in Fig 5 . (b), and compared with that obtained with a LiNbO3 modulator in a standard IQ configuration and a LiNbO3 modulator in a DD-MZM configuration. It can be seen that a penalty of 5 dB occurs between the LiNbO3 IQ modulator and the LiNbO3 DD-MZM for a BER of 10 -3 .This power penalty is thus inherent to this generation scheme. Interestingly the power penalty of the silicon DD-MZM is only 1.5 dB with respect to its LiNbO3 counterpart.

CONCLUSION

In this paper we reported our recent work on Si modulators working in the O-band, targeting short range communications. The design of high performance silicon modulators is challenging at this wavelength because the free carrier plasma dispersion effect is weaker than in the C-band. However 25 Gbit/s OOK operation is demonstrated using a push pull configuration, with a reduced peak-to-peak voltage of 3.3 V, and on chip optical loss of only 3.6 dB at the operating point. A DD-MZM can also be used to generate QPSK modulated signals. We thus demonstrated for the first time the use of a silicon DD-MZM to generate a QPSK signal in the O-band.

  Fig 1. (b). Phase shifter lengths of 1 mm or 2 mm have been used in both arms of symmetric Mach-Zehnder structures. Multi-mode interference (MMI) couplers are used as input/output splitter and combiner. Metallic heaters form low speed/high efficiency static phase shifters, in order to tune the direct current (DC) phase shift Φ between both arms. The radio frequency (RF) signals are applied on each PN phase shifter independently, using GSGSG coplanar waveguides, while DC tips are used to apply the static phase shift (Fig 1. (c)).

Figure 1 .

 1 Figure 1. (a) Phase shifter design: a PN diode is located in the SOI rib waveguide; (b) layout of the dual-drive Mach Zehnder modulator; (c) picture of the device under test.

Figure 2 .

 2 Figure 2. Schematic representation of the set-up used for OOK-modulation characterization.

Figure 3 .

 3 Figure 3. Measured eye diagram at 25 Gbps of the 1 mm long DD-MZM with an applied voltage swing of 3.3 Vpp and 1.7 V DC bias.

Figure 2 .

 2 Figure 2. Schematic representation of the set-up used for QPSK-modulation characterization. Two 2 15 -1 long de-correlated pseudo random binary sequences at 10 Gbps are applied on each of the MZ arm.

Figure 5 .

 5 Figure 5. (a). Typical QPSK constellation diagram after DSP of the Si DD-MZM modulator at 10 Gbaud (20 Gbps), (b) Measured BER as a function of the average received power for the Si DD-MZM under investigation, and comparison with LiNbO3 modulator in DD-MZM configuration and LiNbO3 IQ modulator.

ACKNOWLEDGMENTS

This work was supported by the European project Plat4m (FP7-2012-318178) and the European project Cosmicc (H2020-ICT-27-2015-688516). Support of French Industry Ministry/DGE through the Nano2017 program is also acknowledged.


Christine Lors
email: christine.lors@imt-lille-douai.fr

Jean-François François Ponge

Denis Damidot

Environmental hazard assessment by the Ecoscore system to discriminate PAH-polluted soils

Keywords: PAHs, Contaminated soils, Ecotoxicity, Solid bioassays, Liquid bioassays, Bioavailability, Ecoscores

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

Introduction

Industrial activities led to the discharge of a wide range of hazardous chemicals in soils. These pollutants include mostly hydrocarbon aromatic hydrocarbons (PAHs) and heavy metals. PAHs are a group of persistent hydrophobic organic pollutants which contains two or more fused aromatic rings [START_REF] Cerniglia | Biodegradation of polycyclic aromatic hydrocarbons[END_REF]. PAHs are mainly generated from the incomplete combustion and pyrolysis of organic materials (wood, coal, oil, petrol and plastics), coal liquefaction and gasification, creosote production, petroleum refining and other high-temperature industrial processes [START_REF] Cerniglia | Biodegradation of polycyclic aromatic hydrocarbons[END_REF][START_REF] Bispo | Toxicity and genotoxicity of industrial soils polluted by polycyclic aromatic hydrocarbons (PAHs)[END_REF]). PAHs are a major environmental and health concern because of their potentially toxic, mutagenic and carcinogenic properties [START_REF] White | Mutagens in contaminated soil: a review[END_REF] PAHs can adversely affect not only human health but also terrestrial and aquatic ecosystems. Hazard risk assessments of polluted soils are usually performed by chemical analysis to determine concentrations of target compounds. However, chemical analysis does not allow to identify all the compounds but only to quantify those which are analysed. Moreover, it does not provide information on the bioavailability of pollutants, neither on synergic or antagonistic phenomena between pollutants, nor on their effect on living organisms [START_REF] Juvonen | A battery of toxicity tests as indicators of decontamination in composting oily waste[END_REF]. In the case of PAHs that are hydrophobic organic pollutants, they may be adsorbed onto soil matrices and thus be less bioavailable [START_REF] Semple | Bioavailability of hydrophobic organic contaminants in soils: fundamental concepts and techniques for analysis[END_REF]. Soil properties, such as organic matter content and ageing, also play an important role in the bioavailability of PAHs [START_REF] Chung | Effect of soil properties on bioavailability and extractability of phenanthrene and atrazine sequestered in soil[END_REF][START_REF] Riding | Chemical measures of bioavailability/bioaccessibility of PAHs in soil: fundamentals to application[END_REF]). These parameters can significantly affect their fate in ecosystems and their impact on species and populations [START_REF] Peijnenburg | Implementation of bioavailability in standard setting and risk assessment[END_REF]. Thus, chemical analysis needs to be complemented with an ecotoxicological approach allowing integrating the effects of all bioavailable contaminants [START_REF] Fernandez | Ecological risk assessment of contaminated soils through direct toxicity assessment[END_REF][START_REF] Leitgib | Comparison of bioassays by testing whole soil and their water extract from contaminated sites[END_REF]). As a species sensitive to all environmental contaminants does not exist, a battery of bioassays involving organisms at different levels of biological organization and accounting for acute/chronic ecotoxicity and genotoxicity is recommended to evaluate environmental hazards of contaminated soils [START_REF] Rila | Application of bioassays for risk characterization and remediation control of soils polluted with nitroaromatics and PAHs[END_REF][START_REF] Fernandez | Ecological risk assessment of contaminated soils through direct toxicity assessment[END_REF].

Different approaches were performed to assess soil ecotoxicity: bioassays on whole soil using bacteria, earthworms, collembolans and plants [START_REF] Lors | Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils[END_REF], liquid bioassays applied on both water and organic extracts [START_REF] Bispo | Toxicity and genotoxicity of industrial soils polluted by polycyclic aromatic hydrocarbons (PAHs)[END_REF] and combinations of both bioassays on whole soil and on soil water extracts (Mendoça and Picado 2002;[START_REF] Eom | Ecotoxicity of a polycyclic aromatic hydrocarbon (PAH)-contaminated soil[END_REF]). Direct toxic effects on terrestrial organisms may reflect the ecotoxicological potential of contaminated soils [START_REF] Fent | Ecotoxicological problems associated with contaminated sites[END_REF][START_REF] Lors | Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils[END_REF], whereas soil organic extracts may lead to an overestimation of the real bioavailability of organic pollutants [START_REF] Alexander | Research needs in bioremediation[END_REF]. A combined approach using terrestrial and aquatic bioassays gave satisfactory results from the viewpoint of ecological relevance [START_REF] Lors | Comparison of solid and liquid-phase bioassays using ecoscores to assess contaminated soils[END_REF].

Different bioassay battery-integrated indexes were reported to characterize the ecotoxicological risk of contaminated sites, particularly contaminated sediments: a Test Battery integrated Index (TBI) is reported in the handbook of ISPRA (2011) and a Toxicity Classification System (TCS) in [START_REF] Persoone | A practical and user-friendly toxicity classification system with microbiotests for natural waters and wastewaters[END_REF]. These indexes were used to integrate the results of several tested bioassays. TCS is based on two values: a ranking in 5 acute hazard classes (no, slight, acute, high acute, very high acute hazards) and a weight score for each hazard class (0 to 3). TBI allows ranking five levels of ecotoxicological risk (not significant, low, medium, high, very high), which differ from those of TCS for the first three classes. The quality of sediments of Taranto seas (Mar Grande and Mar Piccolo) was evaluated by these two integrated systems from a battery of five test species representing different trophic levels [START_REF] Prato | Ecotoxicological evaluation of sediments by battery bioassays: application and comparison of two integrated classification systems[END_REF]. The results obtained showed a similarity between TCS and TBI for the high levels of ecotoxicological risk.

Nevertheless, for the lower levels of toxicity, TCS gave a more severe assessment of risk, classifying sediment samples as high acute toxic whereas TBI indicated a low toxicity. In the present study, the Ecoscore System (ES, [START_REF] Lors | Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils[END_REF]) was applied to contaminated soils providing from historical industrial sites.

The first goal of this work is to demonstrate through two historical contaminated soils presenting a similar total concentration of PAHs that chemical analyses are not sufficient to evaluate the hazard of polluted soils and these analyses have to be complemented by ecotoxicological bioassays.

The second goal is to confirm the robustness of a method previously defined by [START_REF] Lors | Comparison of solid and liquid-phase bioassays using ecoscores to assess contaminated soils[END_REF], in order to evaluate the environmental risk through the calculation of an ecoscore. It was thus applied in the present article to two polluted soils, in order to check its robustness. Additionally, the combination of chemical and toxicological data was used to bring some new insights on the ecotoxicity of different types of PAHs.

Data on the factory soils used for the present study have been already presented in [START_REF] Lors | Comparison of solid and liquid-phase bioassays using ecoscores to assess contaminated soils[END_REF], but they have been reanalysed for the present article, in order to standardize the Ecoscore System (ES) for more user-friendly comparisons between soils. In order to allow a better appraisal of the interest of the ES method to ESPR readers and to facilitate comparison with similar studies, data obtained on the soils under study, as well as the methods we used to analyse them from a chemical and ecotoxicological point of view, will be presented in the foregoing chapters.

Materials and methods

As mentioned above, analytical and ecotoxicological data have been already presented in [START_REF] Lors | Comparison of solid and liquid-phase bioassays using ecoscores to assess contaminated soils[END_REF]. They have been briefly summarized below.

Soil samples

Experiments were carried out on two contaminated soils, named Soil A and Soil B, sampled from two historical industrial sites located in the North of France. The distillation of coal tar was the main activity on these sites, taking place from 1923 to 1987 and 1925 to 1973, respectively. Soil A was sampled in the untreated industrial site whereas Soil B underwent a windrow treatment for 18 months from October 1995 to June 1997. Despite bioremediation, this soil was still polluted with PAHs, with a total concentration still around that of Soil A.

Unpolluted soils were sampled in the two studied sites in uncontaminated areas. Chemical characteristics of these control soils, named Control A and Control B, are reported in Table 1. These soils were used as controls in the avoidance test and as a dilution matrix in solid-phase bioassays. The ecotoxicological characterization of control soils did not show any ecotoxicity. The procedure used for soil sampling has been described by [START_REF] Lors | Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils[END_REF].

Analytical data

Soil water extraction was carried out according to ISO 21268-2 (2007). PAH-releasing capacity was expressed by the ratio between PAH-concentration in water extract (per unit mass of soil for water extraction) and PAH-concentration in soil.

Soil pH water was determined using a Consort C83 pH-meter fitted with a glass electrode corrected for temperature. Total organic carbon concentration was obtained from total carbon and inorganic carbon contents, determined with a TOC-5000A Shimatzu ® analyser, according to ISO 10694 (1995).

Concentrations of the 16 PAHs of the US-EPA list [START_REF] Verschueren | Handbook of environmental data on organic chemicals, 4th edn[END_REF] The particle size distribution of soils was determined by separating soil samples into six fractions : > 2000, 2000-200, 200-50, 50-20, 20-2, and < 2 µm. The first three fractions were obtained by dry sieving using sieves between 50 and 200 µm. The smaller fractions (< 50 µm) were obtained by moist procedure, at first by sieving the soil under water through a 20 µm sieve. The fraction retrieved on the sieve, corresponding to the fraction 20-50 µm, was dried to 20°C.

The suspension, corresponding to the fraction < 20 µm, was centrifuged to 1000 rpm during 3.5 min. The recovered fraction was dried at 30°C.

Ecotoxicological data

Bioassays were performed to assess the direct toxicity of soils and soil water extracts to terrestrial and aquatic organisms, respectively. The set of bioassays included acute, chronic and genotoxicity effects, using organisms representative of a variety of trophic levels.

Toxicity end-points were the responses of test organisms to soils or water extracts in test media (%, w/w).

Results were calculated as concentrations producing no significant effect (NOEC), percent inhibition at the highest concentration of the tested sample or as concentrations decreasing the measured endpoint by 10%, 20% and 50%

[E(L)C 10 , E(L)C 20 and E(L)C 50 , respectively] compared to controls. E(L)C x values were calculated following adjustment of data to a log-probit logistic model [START_REF] Litchfield | A simplified method of evaluating dose-effect experiments[END_REF].

The toxicity of soils was evaluated by [START_REF] Lors | Comparison of solid and liquid-phase bioassays using ecoscores to assess contaminated soils[END_REF] from nine bioassays tested on the basis of their best sensitivity to PAH pollution. This set of bioassays included both solid and liquid phases and addressed acute and chronic toxicities and genotoxicity: two rapid bioassays (Microtox ® and springtail avoidance), a micronucleus test and three bioassays of a longer duration (algal growth, lettuce germination and springtail reproduction).

Bioassays applied directly on the soil included an acute phytotoxicity bioassay on Lactuca sativa (ISO 11269-2 2005). A chronic toxicity bioassay based on springtail (Folsomia candida) reproduction was conducted according to ISO 11267 (1999) modified by Martínez [START_REF] Aldaya | Avoidance bio-assays may help to test the ecological significance of soil pollution[END_REF]. An avoidance test was conducted on Folsomia candida according to Martínez [START_REF] Aldaya | Avoidance bio-assays may help to test the ecological significance of soil pollution[END_REF] and [START_REF] Lors | Use of an avoidance test for the assessment of microbial degradation of PAHs[END_REF]. Terrestrial toxicity bioassays were performed by using Control A and Control B as a dilution matrix for Soil A and Soil B respectively. The pH of both soils was around 8 and thus was compatible with requirements of test organisms.

The toxicity of water extracts to aquatic organisms was assessed through both acute and chronic effects. An acute ecotoxicity test was performed by measuring the inhibition of bioluminescence of the bacterium Vibrio fischeri according to ISO 11348-3 (1998). Chronic ecotoxicity was determined on the growth of the fresh water alga

Pseudokirchneriella subcapitata according to ISO 8692 (2004). Each concentration was tested in six replicate microplates. The % inhibition of the population growth was determined for each concentration by comparison with the control.

The genotoxicity of water extracts was evaluated with the in vitro micronucleus assay applied on mouse lymphoma cells L5178Y according to the procedure described by [START_REF] Nesslany | A micromethod for in vitro micronucleus assay[END_REF]. The criteria for determining a genotoxic effect were a concentration-related increase in the number of micronucleated cells and a statistically significant increase over the spontaneous level in at least one treatment schedule.

Calculation of ecoscores

Toxic effects were calculated as percentages of inhibition at a given concentration or as LE Cx values. Percent inhibition was determined with respect to the control soil. LE Cx values were calculated following adjustment of data to a logprobit logistic model [START_REF] Litchfield | A simplified method of evaluating dose-effect experiments[END_REF]. NOEC was the highest concentration tested that did not significantly differ from control with a type I error (α) of 5%. LOEC was not used and was replaced by EC 10 or LC 10 .

Toxicity values were also expressed in Toxic Units (TU), using the formula TU = 100/EC(or LC) 50 .

From five ecotoxicological parameters, E(L)C 50 , E(L)C 20 , E(L)C 10 , NOEC, % inhibition, scores were calculated by assigning to each endpoint value a score between 0 and 100 as a function of its intensity. It was noticed that the scale of the scores was modified compared to that defined by [START_REF] Lors | Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils[END_REF] (0 < score < 3), in order to normalize to 100 the maximum effect.

Results and discussion

Chemical and toxicological characteristics of the soils

The particle size distribution of the studied soils was reported on Figure 1. Soil B was composed almost exclusively of particles between 2000 and 50 μm (79%), corresponding to sand. Within this class, the fraction 2000-200 μm (coarse sand) was dominant (56%) compared with fine sand (23%). Silt and clay fractions were present in very small proportion, 3.3% and 0.2%, respectively. The particle size distribution of soil A was comparable to that of soil B: the sand fraction was dominant (86%), with a similar dominance of coarse sand (68%). Finer textural classes were distributed with proportions almost similar to those of soil B: 4.4% and 0.3% for silt and clay fractions, respectively.

Soil A was mainly contaminated with organic compounds. This soil was heavily polluted with PAHs, with a global content of the 16PAHs of the US-EPA list around 3 g kg -1 dry soil, mainly represented by 2-, 3-and 4-ring compounds (Table 1). Three-ring PAHs were the most represented (44% of  16PAHs -1279.2 mg kg -1 dry soil), followed by 2-and 4-ring compounds (28 and 20 %, 594.2 and 808.9 mg kg -1 dry soil, respectively) (Fig. 2). Five-and 6-ring PAHs were hardly present in this soil (5 and 2 %, 149.2 and 63.2 mg kg -1 dry soil, respectively).

Soil B was highly polluted with PAHs, to a level similar to Soil A ( 16PAHs= 3687.2 mg kg -1 ) (Table 1).

However, the PAH distribution pattern was different from Soil A. Four-ring PAHs were the most represented (50% of  16PAHs -1726.9 mg kg -1 dry soil) followed by 5-and 6-rings PAHs (22 and 11% -796.1 and 389.8 mg kg -1 dry soil) (Fig. 2). Contrary to soil A, 3-ring PAH content was lower, 623.5 against 1279.2 mg kg -1 dry soil for Soil A. This difference of distribution is probably the consequence of the partial biodegradation of PAHs during biotreatment that was more efficient to decrease 2-and 3-rings PAHs, as expected [START_REF] Lors | Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils[END_REF]). Thus, the concentrations of 4-, 5-, and 6-rings PAHs were higher for Soil B contrary to 2-and 3-rings PAHs.

A higher amount of organic carbon was also detected in Soil B (442 g kg -1 dry soil) compared to Soil A (90 g kg -1 dry soil). This difference could be explained by the addition of compost during the windrow treatment of Soil B.

This can be confirmed by the high N and P amounts detected in this soil, amounting to 5600 and 1900 mg kg -1 , respectively (Table 1). As the dominant fraction of the two soils was coarse sand, it is possible that the organic part could be contained in this fraction.

Soil A induced a strong inhibition of lettuce germination. At the highest dose tested, lettuce germination was inhibited to about 70.9% (Table 2) and the bioassay-score was to 93 (Table 4). Conversely, Soil B showed a significantly lower phytotoxicity towards lettuce germination with an inhibition of 21.4% (Table 3) and a bioassayscore of only 47 (Table 4).

The Folsomia population reproduction bioassay showed that Soil A had a high chronic ecotoxicity, with an inhibition rate of 100% and a bioassay-score of 100 (Tables 2 and4) whereas Soil B did not elicit any response by

Folsomia candida populations (Table 3). For Soil B, the behavioural test seemed to be more sensitive than ecotoxicity tests since it allowed to detect a significant repellence (bioassay-score = 67, Table 4). Nevertheless, the repellence level was lower than that of Soil A (bioassay-score = 100, Table 4).

Soil A presented a strong ecotoxicity towards terrestrial organisms whereas the ecotoxicity of Soil B was moderate.

Ecoscores of Soil A and Soil B, obtained with this battery of bioassays, were 97.7 and 38.0, respectively (Table 4).

The fact that these two soils showed the same high level of organic pollution whereas their ecotoxicity assessed by solid bioassays was different demonstrates that the global content of PAHs is not the pertinent parameter to evaluate soil pollution hazards. Conversely, the distribution of PAHs gave a better picture of soil pollution and it can explain partly the ecotoxic responses obtained. In fact, the high ecotoxicity of Soil A to terrestrial organisms is probably related to its high concentration in 3-ring PAHs. Indeed, a positive and significant relationship between ecoscores pooled over the three solid bioassays and the concentration of 3-ring PAHs was found by [START_REF] Lors | Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils[END_REF]. Soil A exhibited a high ecotoxicity on organisms tested (plants and Collembola), with bioassay-scores of 93, 100 and 100 to Lactuca germination, Folsomia population growth and Folsomia avoidance, respectively (Table 4). These results Moreover, Soil B exhibited a weak acute ecotoxicity towards lettuce and no chronic ecotoxicity towards Collembola. Comparatively to Soil A, Soil B contains lesser concentrations of 3-ring PAHs but higher concentrations of 4-ring PAHs. If we consider that 3-and 4-rings PAHs were responsible for the impact of PAHs on Collembola and earthworms [START_REF] Eom | Ecotoxicity of a polycyclic aromatic hydrocarbon (PAH)-contaminated soil[END_REF][START_REF] Sverdrup | Soil ecotoxicity of polycyclic aromatic hydrocarbons in relation to soil sorption, lipophilicity, and water solubility[END_REF], the results might indicate that 3-and 4-rings PAHs in Soil B were less bioavailable to the exposed organisms. Indeed, Soil B had 50% less 3-ring PAHs and these compounds were very weakly dissolved by water during extraction (concentration in water extract B equal to 4.1 µg L -1 ) (Table 5). In the same way, although the content of 4-ring PAHs was 50% higher, a weak fraction was dissolved in water after extraction (concentration in water extract B equal to 13.7 µg L -1 ). As a consequence, these compounds should be sequestered in other insoluble organic compounds in this soil.

Although any effect was detected on Folsomia candida population growth, Soil B was repellent to the same organism at a lower level than Soil A (Bioassay-scores of 100 and 67 for Soils A and B, respectively -Table 4), confirming the better sensitivity of behavioural tests (avoidance by Folsomia) compared to ecotoxicity tests.

Chemical and toxicological characteristics of water extracts

The water extraction of soils gives an information on the soluble fraction and thus on the fraction directly accessible to organisms through diffusion. Aqueous leaching tests showed that Soil A had a strong capacity to release PAHs. In fact, the ratio PAH concentration in water extract / soil was equal to 2.2 10 -3 . Water extract A mainly contained 3-ring PAHs (82% of  16PAHs -531.1 µg L -1 ) (Table 5). Their high concentration in solution was linked to their amount in Soil A (1279.2 mg kg -1 dry soil) and the higher solubility of PAHs of lower molecular weight (Table 1). Naphthalene was present at a small concentration (0.2 µg L -1 ) despite its high amount in Soil A. This was probably due to its volatilization during water extraction, given its high vapour tensile strength (37 Pa).

The leaching rate of PAHs decreased with the number of rings (Fig. 3). PAH concentrations in water extracts were correlated to the water-solubility of these pollutants. More soluble PAHs, such as acenaphthene, fluorene and phenanthrene, were found at the highest concentrations in Water extract A (Table 5). Conversely, 4-ring PAHs were present in a lesser proportion (15% of  16PAHs -96.9 µg L -1 ), whereas 5-and 6-rings PAHs were not significantly represented in water extract A (1.8% and 0.8% -11.3 and 5.3 µg L -1 , respectively) (Table 5).

Soil B had a low capacity for PAHs remobilization: this soil released 10 times lower amounts of PAHs than Soil A (1.6 10 -4 ). Five-and 6-rings PAHs were the most represented (respectively 37 and 30 % of  16PAHs -21.4 and 17.5 µg L -1 ) (Table 5). Among 5-ring PAHs, benzo(b)anthracene and benzo(a)pyrene were the major compounds in water extract B. Water extract B also contained 4-ring PAHs in a lesser proportion (23.9% of  16PAHs -13.7 µg L -1 ).

Three-ring PAHs were also hardly released and thus their concentrations were about 100 times less than Water extract A. The windrow treatment applied to this soil was efficient because it removed low molecular weight PAHs and remaining PAHs were the compounds most recalcitrant to microbial degradation. PAH degradation may also be limited by the slow desorption of PAHs from the soil matrix [START_REF] Juhasz | Pilot scale bioremediation of creosote-contaminated soil: efficacy of enhanced natural attenuation and bioaugmentation strategies[END_REF]. In fact, Soil B contains essentially 4-, 5-and 6-ring PAHs, which were characterized by a weak water-solubility and a high lipophily.

Water extract A exhibited a strong acute ecotoxicity to Vibrio fischeri (bioassay-score = 100) (Table 4).

Conversely, Water extract B did not show any acute ecotoxicity with the bioassay tested (bioassay-score = 0) (Table 4).

These results seemed to be linked to the 3-ring PAH content of these water extracts: high amount in water extract A ( 3-ring PAHs = 531.1 µg L -1 ) and small amount in water extract B (( 3-ring PAHs = 4.1 µg L -1 ) (Table 5). This confirms the strong correlation between acute ecotoxicity and 3-ring PAHs observed by [START_REF] Lors | Comparison of solid and liquid-phase bioassays using ecoscores to assess contaminated soils[END_REF], which is linked to their higher water solubility compared to PAHs of higher molecular weight [START_REF] Bispo | Toxicity and genotoxicity of industrial soils polluted by polycyclic aromatic hydrocarbons (PAHs)[END_REF].

However, the effects of Water extracts A and B on algal growth were comparable on the basis of ecoscores.

Water extracts A and B showed a high chronic response towards Pseudokirchneriella subcapitata (bioassay-scores of 80 for both water extracts) (Table 4). These results indicate that both soils had a potential impact to aquatic fauna in the long term and that ecotoxicological effects were due to 5-and 6-rings PAHs, although these were present in lower contents in both water extracts (Fig. 3). Nevertheless, the concentration in solution of 5-and 6-rings PAHs was above their hydrosolubility level (Table 5). The ratio between the concentration of each PAH in water extract and its watersolubility was higher than 1 for all 5-and 6-rings PAHs to the exception of benzo[g,h,i]perylene. This suggests that these high molecular-weight PAHs were associated with other compounds, such as suspended organic matter or were in colloidal form, making them bioavailable to aquatic organisms. Although it had been biotreated, Soil B contained a pool of PAHs that were potentially leachable. The battery must include bioassays taking into account different effects, acute, chronic toxicity and behavioural endpoints, in order to prevent potential hazards for ecosystems in the long term [START_REF] Prato | Ecotoxicological evaluation of sediments by battery bioassays: application and comparison of two integrated classification systems[END_REF].

The micronucleus test applied to mouse lymphoma cells showed a high genotoxicity only without S9 activator (bioassay-score = 80) for Water extract A, indicating the presence of directly genotoxic compounds in this water extract. Conversely, not any genotoxic effect was observed for Water extract B. The fact that Water extract A is genotoxic only without S9 activator suggests that its genotoxicity response is not due to PAHs but certainly to other compounds present in this water extract. In fact, PAHs are known to be activated into genotoxic metabolites by S9 rat liver enzymes [START_REF] Otto | A novel adrenocorticotropin&inducible cytochrome P450 from rat adrenal microsomes catalyzes polycyclic aromatic hydrocarbon metabolism[END_REF]IARC 2010). The ecotoxicological approach gives a global response and takes into account all potentially toxic compounds and not only those that have been determined by chemical analysis.

Comparison of ecoscores of soils and soil water extracts

The comparison of averaged ecoscores for solid and liquid phase bioassays showed that Soil A was classified as strongly toxic by both types of bioassays. Ecoscores were to 97.7 and 86.7 for solid and liquid phase bioassays, respectively. Conversely, Soil B, which was at the limit between weak and moderate effects, appeared weakly toxic by liquid phase bioassays (ecoscore = 26.7) and moderately toxic by solid phase bioassays (ecoscore = 38.0). This was due to a lower sensitivity of liquid phase bioassays compared to that of solid phase bioassays as showed by [START_REF] Lors | Comparison of solid and liquid-phase bioassays using ecoscores to assess contaminated soils[END_REF], an observation already made on plant growth tests by [START_REF] Ferrari | Ecotoxicological assessment of solid wastes: a combined liquidand solid-phase testing approach using a battery of bioassays and biomarkers[END_REF]. Chemical and ecotoxicological analyses of water extracts allowed us to know which water-soluble fraction was directly accessible to organisms. The water extract was complementary to the bulk soil in the procedure of hazard assessment. The weak toxicity of soil B compared to the strong toxicity of soil A was also confirmed by the calculation of ecoscores using both solid and liquid phase bioassays: ecoscores were 32.3 and 92.2, respectively (Table 4).

Our method of selection based on ecoscores was comparable to the Test Battery integrated Index (TBI).

However, TBI has an additional class, named 'very high' and this level corresponds to the 'high level' in the Ecoscore System (ES). Moreover, the high level in TBI corresponds to the medium level in ES. TBI thus overestimates the ecotoxicological risk in comparison with ES. In fact, [START_REF] Manzo | Application of a toxicity test battery integrated index for a first screening of the ecotoxicological threat posed by ports and harbors in the southern Adriatic Sea (Italy)[END_REF] showed that TBI did not allow highlighting differences among the sites studied and showed a general high ecotoxicological risk. Nevertheless, while overestimating the ecotoxicological risk compare to ES, similar discriminating capacity could be obtained when using TBI ecotoxicological risk levels: according to TBI Soils A and B are classed very highly and highly toxic, respectively.

Conclusions

Two coke factory soils contaminated with similar total PAH concentration were characterized by chemical and ecotoxicological approaches applied to the whole soil and to the water extract. The battery of bioassays including solid and liquid-bioassays took into account acute, chronic and genotoxic effects. The chemical analysis of soil water extracts gave information on the soluble fraction corresponding to the pollutants directly accessible to organisms. Nevertheless, it was necessary to complete chemical analyses by ecotoxicity bioassays which proved to be more sensitive indicators of soil quality. The Ecoscore System (ES), based on ecoscores calculated from a battery of liquid and solid phase bioassays, proved to be a robust method which allowed us to evaluate the sensitivity of bioassays and to classify soils according to their toxicity level and particularly to differentiate the two studied soils despite their similar total concentration of PAHs. The total PAH content of soil was not the more pertinent parameter to assess the hazard of polluted soils, contrary to the distribution of PAHs: the combination of chemical and toxicological approaches highlighted the relationship between acute ecotoxicity and 3-ring PAHs (most soluble compounds). In the same way, the chronic effect of both water extracts on algal growth can be explained by high molecular weight PAHs, such as 5and 6-ring PAHs. A procedure involving the battery of bioassays proposed (two rapid bioassays, Microtox and springtail avoidance, a micronucleus test and three bioassays of a longer duration, algal growth, lettuce germination and springtail reproduction) and the calculation of resulting ecoscores provides a discriminant assessment of soils contaminated by PAHs which could be implemented in bioremediation programs. It offers the advantage of allowing an easy comparison of various soils on the base of a wide array of acute and chronic solid-and liquid-phase toxicity tests, without resorting to complex data analyses. The proposed Ecoscore System proved effective to discriminate between weak and strong toxicity hazard of the two studied soils, despite their similar total PAH concentration. Table 1 Concentrations of PAHs in soils A and B and in their control soils (expressed in mg kg -1 dry soil ± SE) and concentrations of total organic carbon, total organic nitrogen, total phosphorus in soils A and B (expressed in mg kg -1 dry soil ± SE). NT not toxic, NM not mutagenic, N/A not applicable. The values of ecoscores noted in bold type correspond to the average of bioassay-scores

Figure captions

No. of rings

  For E(L)C 50 , E(L)C 20 , E(L)C 10 , NOEC, the following scale (x = endpoint value) was used: o 0 = no effect (x > 100) o 33 = weak effect (50 < x≤ 100) o 67 = medium effect (20 < x ≤ 50) o 100 = strong effect (x ≤ 20) For % inhibition, the following scale was used: o 0 = no effect (x ≤ 5) o 33 = weak effect (5 < x ≤ 20) o 67 = medium effect (20 < x ≤ 60) o 100 = strong effect (x > 60) For each bioassay, the five scores were summed up and divided by the number of endpoints, in order to calculate a bioassay-score. Bioassay-score values allowed evaluating the sensitivity of the different bioassays. An ecoscore was calculated for each soil by averaging the values of the different bioassay-scores. The following scale was used to define the environmental risk of PAH-polluted soils and classify soils in function of the intensity of toxicity: o no toxicity (ecoscore = 0) o weak toxicity (0 < ecoscore ≤ 33) o moderate toxicity (33 < ecoscore ≤ 67) o strong toxicity (67 < ecoscore ≤ 100)

  confirmed those ofSvrdrup et al. (2002) who showed that 2-, 3-and 4-rings PAHs significantly affected the survival and reproduction of Collembola and earthworms while PAHs with a high lipophily did not. The same results were obtained byČvančarová et al. (2013) who indicated a better sensitivity of the earthworm Eisenia fetida to PAHs with 3-4 rings aromatic rings. The growth inhibition of this organism is linked to the accumulation of PAHs in earthworm tissues. Although Soil A contains 4-ring PAHs at a lesser concentration than 3-ring PAHs, 4-ring PAHs also contribute to the ecotoxicological effects measured.

Fig. 1

 1 Fig. 1 Distribution of soil textural fractions (in mass %) for each studied soil.

Fig. 2 Fig. 3

 23 Fig. 2 Concentrations of 2-6 ring PAHs in the two studied soils. Values are means of three replicate dosages, with S.E. as error bars

  Fig. 1

  . The U.S. Environmental Protection Agency (US-EPA) has

	established	that	7	compounds	among 16	PAHs	are	potentially	carcinogenic	to	humans
	[http://www.epa.gov/region5/cleanup/indianaharbor/pdfs/supplementalriskassessmentchapt6.pdf].

  were dosed in soil and water extracts according to ISO 13877 (1998). However, PAH concentration in water extracts did not include acenaphthylene. The extraction of PAHs from soil samples was carried out with the solvent extractor system Dionex ® ASE 200 (Dionex Corporation ® , Sunnyvale, CA). Concentrations of the 16 PAHs were dosed in the extracts by High Performance Liquid Chromatography (Waters ® HPLC 2690, Milford, MS), coupled to a UV photodiode array detector (Waters ® 996). The ratio between PAH concentration in water extract and PAH concentration in soil allowed determining the PAH water extraction capacity of the studied soils. All chemical analyses were done in triplicate.

Table 2

 2 Ecotoxicological characteristics of Soil A according to a restricted battery of solid and liquid bioassays

			Soil A	Soil B	Control A	Control B
	Naphthalene	2	594.2  13.8	150.9  10.6	0.13  0.01	0.41  0.01
	Acenaphthylene	3	3.1  0.1	23.5  1.1	0.04  0.001	0.01  0.00
	Acenaphthene	3	217.4  1.2	2.0  0.1	0.10  0.11	0.01  0.00
	Fluorene	3	226.8  2.8	83.1  3.7	0.02  0.001	0.06  0.00
	Phenanthrene	3	629.3  4.2	308.2  17.7	0.08  0.01	1.01  0.03
	Anthracene	3	202.5  31.7	206.7  7.0	0.01  0.00	0.14  0.01
	Fluoranthene	4	414.3  1.2	625.2  30.7	0.16  0.03	2.01  0.05
	Pyrene	4	233.4  0.4	299.4  10.9	0.13  0.01	0.55  0.04
	Benzo[a]anthracene	4	85.7  0.9	391.9  13.1	0.08  0.00	0.37  0.03
	Chrysene	4	75.4  0.9	410.4  8.1	0.08  0.01	0.70  0.02
	Benzo[b]anthracene	5	56.2  0.3	210.8  5.8	0.02  0.001	1.13  0.25
	Benzo[k]fluoranthene	5	25.8  0.3	161.9  2.6	0.02  0.001	0.25  0.00
	Benzo[a]pyrene	5	60.4  6.7	364.1  2.9	0.03  0.001	0.92  0.17
	Dibenzo[ah]anthracene	5	6.9  0.2	59.3  0.3	0.02  0.001	0.09  0.00
	Benzo[ghi]perylene		32.5  1.0	196.1  6.5	0.10  0.05	0.47  0.01
	Indeno[123-cd]pyrene		30.8  0.3	193.7  1.6	0.10  0.05	0.42  0.02
	2-ring PAH		594.2  8.0	150.9  6.1	0.13  0.01	0.41  0.01
	3-ring PAHs		1279.2  20.3	623.5  17.1	0.26  0.12	1.23  0.05
	4-ring PAHs		808.9  1.0	1726.9  36.2	0.44  0.04	3.63  0.14
	5-ring PAHs		149.2  3.7	796.1  6.7	0.09  0.003	2.40  0.43
	6-ring PAHs		63.2  0.4	389.8  4.6	0.19  0.10	0.88  0.02
	 16 PAHs		2894.8  38.1	3687.2  48.8	1.10  0.70	8.56  0.65
	Total organic carbon		90000	442000	-	-
	Total organic nitrogen		1700	5600	-	-
	Total phosphorus		620	1900

Table 3

 3 Ecotoxicological characteristics of Soil B according to a restricted battery of solid and liquid bioassays

		EC 50	TU	EC 20	EC 10	NOEC	Inhibition
		(g.100 g -1 )	100/EC 50	(g.100 g -1 )	(g.100 g -1 )	(g.100 g -1 )	(%)
	Lactuca germination	>100	<1	80.8 (62.9->100)	41.7 (29.9-58.1)	<35	21.4
	Folsomia population growth	NT	NT	NT	NT	NT	0
	Folsomia avoidance	>100	<1	1.7 (0.6-5.1)	0.04 (0.004-0.403)	<0.35	12.5
	Microtox ® test	NT	NT	NT	NT	NT	0
	Algal growth	80.5 (51.7->100)	1.2	8.4 (5.3-13.5)	2.6 (1.2-5.7)	<6.25	56.5
	Micronucleus test (-S9)	NM	NM	NM	NM	NM	N/A
	Data from Lors et al. (2011)

Table 4

 4 Ecoscores of the two studied soils calculated from the bioassay scores obtained with the battery of solid and liquid bioassays

				Scores (%)
		Bioassays	Effects	Soil A	Soil B
		Lactuca germination	Acute ecotoxicity	93	47
	Solid phase	Folsomia population growth	Chronic ecotoxicity	100	0
	bioassays	Folsomia avoidance	Behaviour	100	67
		Solid phase bioassays battery		97.7	38.0
		Vibrio fischeri inhibition	Acute ecotoxicity	100	0
	Liquid phase	Pseudokirchneriella growth	Chronic ecotoxicity	80	80
	bioassays	Micro-nucleus test	Genotoxicity	80	0
		Liquid phase bioassays battery		86.7	26.7
	Solid and liquid	Bioassays battery		92.2	32.3
	phase bioassays

Table 5

 5 Concentration of PAHs in water extracts A and B (expressed in µg L -1 ) and water solubility of PAHs at 25°C (expressed in µg kg -1 )

		No. of rings	Water extract A	Water extract B	Water-solubility
	Naphthalene	2	0.20  0.02	0.6  0.2	3.2 10 4
	Acenaphthylene	3	-	-	3.4 10 3
	Acenaphthene	3	154.1  1.5	0.6  0.1	3.9 10 3
	Fluorene	3	198.7  3.7	0.7  0.1	2.0 10 3
	Phenanthrene	3	151.4  2.8	1.4  0.2	1.3 10 3
	Anthracene	3	26.9  0.4	1.3  0.3	7.3 10 1
	Fluoranthene	4	51.2  0.9	4.1  0.7	2.6 10 2
	Pyrene	4	28.9  0.5	3.1  0.6	1.4 10 2
	Benzo[a]anthracene	4	8.0  0.1	3.1  0.6	1.4 10 1
	Chrysene	4	8.8  0.1	3.4  0.6	2.0 10 0
	Benzo[b]anthracene	5	3.85  0.05	8.4  1.4	1.2 10 0
	Benzo[k]fluoranthene	5	2.30  0.03	2.7  0.5	7.6 10 -1
	Benzo[a]pyrene	5	4.41  0.02	6.9  1.4	3.8 10 0
	Dibenzo[ah]anthracene	5	0.72  0.01	3.4  0.6	2.6 10 -1
	Benzo[ghi]perylene		3.00  0.03	8.4  1.7	6.2 10 1
	Indeno[123-cd]pyrene		2.30  0.04	9.0  1.8	5.0 10 -1
	2-ring PAH		0.20  0.02	0.6  0.2	-
	3-ring PAHs		531.1  8.3	4.1  0.7	-
	4-ring PAHs		96.9  1.7	13.7  2.4	-
	5-ring PAHs		11.3  0.1	21.4  3.8	-
	6-ring PAHs		5.30  0.05	17.5  3.5	-
	 16 PAHs		644.8  12.0	57.3  11.2	-

--Values are means of three replicate measures. Data from[START_REF] Lors | Comparison of solid and liquid-phase bioassays using ecoscores to assess contaminated soils[END_REF]

Values are means of three replicate measures. Data from[START_REF] Lors | Comparison of solid and liquid-phase bioassays using ecoscores to assess contaminated soils[END_REF]

Acknowledgements

The present study was performed with a financial support from the ADEME (Agence de l'Environnement et de la Maîtrise de l'Énergie, France), which is greatly acknowledged. We thank Total (France) and Charbonnages de France (France) to put industrial sites at our disposal.