Yacine Cherifi

Alexandre Barras

Ahmed Addad

Baghdad Ouddane

Pascal Roussel

Ahcène Chaouchi

Sabine Szunerits

Rabah Boukherroub
email: rabah.boukherroub@univ-lille.fr

Simultaneous photocatalytic Cr(VI) reduction and phenol degradation over copper sulphide-reduced graphene oxide nanocomposite under visible light irradiation: Performance and reaction mechanism

Keywords: rGO/CuS nanocomposites, Photocatalysis, Phenol degradation, Cr(VI) removal

may come     Simultaneous photocatalytic Cr(VI) reduction and phenol degradation over copper sulphide-reduced graphene oxide nanocomposite under visible light irradiation: Performance and reaction mechanism

Introduction

Photocatalysts are nowadays increasingly used to treat environmental problems, in particular excessive discharges of industrial waste water. Industrial wastewater is often loaded with traces of toxic metal ions (e.g., Cr(VI), Cd(II), As(V)), Hg(II)) as well as with harmful organic pollutants (e.g., organic dyes, polychlorinated biphenyls, phenol ) at the same time [START_REF] Liu | Adsorption of Pb 2+ , Cd 2+ , Cu 2+ and Cr 3+ onto titanate nanotubes: Competition and effect of inorganic ions[END_REF][START_REF] Nezar | Efficient reduction of Cr(VI) under visible light irradiation using CuS nanostructures[END_REF][START_REF] Bai | Synergy removal of Cr (VI) and organic pollutants over RP-MoS2/rGO photocatalyst[END_REF]. Among the metal ions, hexavalent chromium, Cr(VI), is a highly toxic and nonbiodegradable pollutant, which can cause different health problems such as cancer pulmonary congestions, and liver damage. Owing to its high toxicity, Cr(VI) has been classified as a priority pollutant by the United States Environmental Protection Agency (USEPA) and many other countries [START_REF] Testa | Litter, Heterogeneous Photocatalytic Reduction of Chromium(VI) over TiO2[END_REF][START_REF] Nanda | Fabrication of mesoporous CuO/ZrO2-MCM-41 nanocomposites for photocatalytic reduction of Cr(VI)[END_REF]. Therefore, several methods have been developed for Cr(VI) removal, including ion exchange, chemical precipitation, adsorption and membrane separation techniques [START_REF] Deng | Application of porous N-methylimidazolium strongly basic anion exchange resins on Cr(VI) adsorption from electroplating wastewater[END_REF][START_REF] Abdullah | Recent trends of heavy metal removal from water/wastewater by membrane technologies[END_REF]. Currently, the reduction of Cr(VI) to Cr(III) using photocatalysis is considered to be the most promising approach, as it reduces the highly toxic Cr(VI) to less harmful Cr(III), a trace element necessary for human beings [START_REF] Deng | Insight into highly efficient simultaneous photocatalytic removal of Cr(VI) and 2,4-diclorophenol under visible light irradiation by phosphorus doped porous ultrathin g-C3N4 nanosheets from aqueous media: Performance and reaction mechanism[END_REF]. On the other hand, organic pollutants like phenolic compounds are major pollutants of the aquatic environment with carcinogenic and mutagenic characters, causing a risk to human health [START_REF] Lam | Enhanced sunlight photocatalytic performance over Nb2O5/ZnO nanorod composites and the mechanism study[END_REF][START_REF] Liu | Carbon nitride coupled Ti-SBA15 catalyst for visible-light-driven photocatalytic reduction of Cr(VI) and the synergistic oxidation of phenol[END_REF].

Hence, the development of catalysts for the simultaneous removal of organic pollutants and metal ions from wastewater represents an appealing approach. The strategy consists of using semiconductor catalysts, which upon light irradiation, the photogenerated electron-hole pairs are consumed separately in the corresponding oxidation and reduction half-reactions. This process not only prevents the recombination of the electron-hole pairs, but also improves the photocatalytic efficiency [START_REF] Sajjad | Bismuth-doped ordered mesoporous TiO2: Visible-light catalyst for simultaneous degradation of phenol and chromium[END_REF][START_REF] Wang | Recyclable nanoscale zero valent iron doped g-C3N4/MoS2 for efficient photocatalysis of RhB and Cr(VI) driven by visible light[END_REF].

Although semiconductors such TiO2, ZnO and their nanocomposites have been used for the simultaneous Cr(VI) reduction and phenol oxidation [START_REF] Sajjad | Bismuth-doped ordered mesoporous TiO2: Visible-light catalyst for simultaneous degradation of phenol and chromium[END_REF][START_REF] Sun | Visible Light Cr(VI) Reduction and Organic Chemical Oxidation by TiO2 Photocatalysis[END_REF][START_REF] Huang | The simultaneous photocatalytic degradation of phenol and reduction of Cr(VI) by TiO2/CNTs[END_REF][START_REF] Jin | Facile synthesis of porous single crystalline ZnO nanoplates and their application in photocatalytic reduction of Cr(VI) in the presence of phenol[END_REF], these photocatalytic processes have been conducted under UV light irradiation due to the wide band gaps of TiO2 and ZnO, which hinders their widespread application. To overcome this limitation and extend photocatalyst absorption to the visible range, coupling TiO2 with other semiconductor materials has been adopted as an effective strategy. For instance, TiO2/Co-g-C3N4 [START_REF] Zhou | Fabrication of TiO2/Co-g-C3N4 heterojunction catalyst and its photocatalytic performance[END_REF], TbxOy loaded TiO2-oriented nanosheets [START_REF] Lu | Enhanced photocatalytic degradation of aqueous phenol and Cr(VI) over visible-light-driven TbxOy loaded TiO2-oriented nanosheets[END_REF], N-doped TiO2-carbon composite fibres [START_REF] Zhang | Dual functional N doped TiO2 carbon composite fibers for efficient removal of water pollutants[END_REF], Cu-doped TiO2 nanoparticles modified with 8-hydroxyquinoline [START_REF] Zhang | Copper doping and organic sensitization enhance photocatalytic activity of titanium dioxide: Efficient degradation of phenol and tetrabromobisphenol A[END_REF], carbon nitride loaded into titanium incorporated SBA-15 mesoporous silica (Ti-SBA15-CN) [START_REF] Liu | Carbon nitride coupled Ti-SBA15 catalyst for visible-light-driven photocatalytic reduction of Cr(VI) and the synergistic oxidation of phenol[END_REF], and CdS/TiO2 [START_REF] Deng | A structural engineering-inspired CdS based composite for photocatalytic remediation of organic pollutant and hexavalent chromium[END_REF] have been successfully applied for simultaneous removal of metal ions and organic pollutants. For a more efficient use of visible light, various narrow-band gap semiconductor photocatalysts, such as BiVO4 [START_REF] Xie | Simultaneous photocatalytic reduction of Cr(VI) and oxidation of phenol over monoclinic BiVO4 under visible light irradiation[END_REF], bismuth oxyiodide/reduced graphene oxide/bismuth sulphide (BiOI/rGO/Bi2S3) [START_REF] Chen | Simultaneous removal of Cr(VI) and phenol contaminants using Z-scheme bismuth oxyiodide/reduced graphene oxide/bismuth sulfide system under visible-light irradiation[END_REF], PANI-sensitized g-C3N4/ZnFe2O4 heterostructure [START_REF] Patnaik | Enhanced photo catalytic reduction of Cr (VI) over polymer-sensitized g-C3N4/ZnFe2O4 and its synergism with phenol oxidation under visible light irradiation[END_REF] 2O3 -Bi2O2CO3 composite [START_REF] Wang | Fabrication of b-phase AgI and Bi2O3 co-decorated Bi2O2CO3 heterojunctions with enhanced photocatalytic performance[END_REF], and Au-Pd nanoparticles loaded on g-C3N4/MCM-41 [START_REF] Patnaik | Bimetallic co-effect of Au-Pd alloyed nanoparticles on mesoporous silica modified g-C3N4 for single and simultaneous photocatalytic oxidation of phenol and reduction of hexavalent chromium[END_REF] have been investigated for this photochemical process. Nevertheless, it is well-established that some of these photocatalysts have certain disadvantages such as photo-corrosion and instability. Therefore, it is important to develop highly efficient and robust photocatalysts for the simultaneous treatment of mixed pollutants.

A huge effort has been devoted to the development of visible light photocatalysts to maximize the use of clean, safe and abundant solar energy. In recent years, transition metal dichalcogenide (TMD) nanostructures have drawn enormous attention, owing to their outstanding electronic and optical properties as well as their wide applications in different fields, including energy conversion devices, photocatalysis, photovoltaics, biosensors, etc. [START_REF] Goel | Synthesis and biomedical applications of copper sulfide nanoparticles: From sensors to theranostics[END_REF][START_REF] Van Der Stam | Prospects of Colloidal Copper Chalcogenide Nanocrystals[END_REF][START_REF] Zhao | Development of plasmonic semiconductor nanomaterials with copper chalcogenides for a future with sustainable energy materials[END_REF]. Among them, the non-toxic, cheap and p-type semiconductor copper sulphide (CuS) exhibits excellent luminescence and photochemical properties with a narrow band gap (1.2 . CuS can harvest efficiently photons in the visible absorption window and thus makes it an interesting semiconductor for photocatalytic processes [START_REF] Nezar | Efficient reduction of Cr(VI) under visible light irradiation using CuS nanostructures[END_REF][START_REF] Estrada | An ionic liquid route to prepare copper sulphide nanocrystals aiming at photocatalytic applications[END_REF]. However, the catalytic activity of CuS is not satisfactory due to its poor absorption and low separation efficiency of photogenerated electron-hole pairs along with its anti-photo-corrosion stability. Therefore, improving the photo-activity and photostability of CuS remains a challenge and a priority for practical application in the environmental field [START_REF] Nie | A one-pot and in situ synthesis of CuS-graphene nanosheet composites with enhanced peroxidase-like catalytic activity[END_REF][START_REF] Zhao | Fabrication of a CuS/graphene nanocomposite modified electrode and its application for electrochemical determination of esculetin[END_REF].

Graphene-based materials have received a great deal of attention. Currently, constructing heterostructures between semiconductors and graphene can reduce the possibility of electronhole pair recombination to further improve the photocatalytic performance [START_REF] Ye | Facile one-pot solvothermal method to synthesize sheet-on-sheet reduced graphene oxide (RGO)/ZnIn2S4 nanocomposites with superior photocatalytic performance[END_REF][START_REF] Kusiak--Kozar | Graphene oxide-TiO2 and reduced graphene oxide-TiO2 nanocomposites: Insight in charge-carrier lifetime measurements[END_REF]. Due to the presence of electronic interactions and the charge equilibrium between semiconductor materials and graphene sheets, the potential of the conduction band of the materials is reduced, resulting in an increase in the photoinduced electron migration efficiency and a negative shift in the Fermi level of the materials [START_REF] Hu | Synthesis of flowerlike CuS/reduced graphene oxide (RGO) composites with significantly enhanced photocatalytic performance[END_REF][START_REF] Ding | Design of hierarchical CuS/graphene architectures with enhanced lithium storage capability[END_REF]. In addition, graphene acting as a carrier could enhance the degree of dispersion of the original samples to provide more reactive sites, which would lead to increased photocatalytic activity [START_REF] Hu | Synthesis of flowerlike CuS/reduced graphene oxide (RGO) composites with significantly enhanced photocatalytic performance[END_REF].

In the present study, we report a facile and practical strategy for the synthesis of reduced graphene oxide-copper sulphide (rGO-CuS) composites by a one-step hydrothermal approach.

Their catalytic activities are systematically investigated for the simultaneous photocatalytic reduction of Cr(VI) and oxidation of phenol to seek enhanced performance. In order to better understand the synergistic effect between Cr(VI) reduction and oxidation of phenol, systematic experiments were performed, including control experiments to reveal the roles of Cr(VI), phenol and rGO-CuS in the oxidation and reduction processes. The effects of the initial concentration of the substrates, pH and active oxygen species in the degradation process were investigated experimentally and the photocatalytic degradation mechanism was therefore proposed.

Experimental section 2.1. Synthesis of catalysts

The preparation of the rGO-CuS nanocomposites (2rGO-CuS, rGO-CuS and rGO-2CuS) was carried out by an easy hydrothermal method in a one-step process. To 40 mL of Milli-Q (MQ) water was added 40 mg of graphene oxide (GO) powder. A homogeneous solution was prepared by ultrasonic (464 W, 35 Hz) irradiation for 2 h. To this solution was added 20, 40 or 80 mg of copper sulphate (CuSO4) under magnetic stirring to obtain a solution (a). Then 25 mg of thiourea was dissolved in 25 mL of MQ water to obtain a solution (b). Solutions (a) and (b)

were mixed together and transferred into a 100 mL Teflon-lined autoclave and heated at 110 °C for 14 h. The suspensions of the obtained mixtures 2rGO-CuS, rGO-CuS and rGO-2CuS were centrifuged at 8000 rpm for 20 min for efficient separation from supernatant, washed repeatedly with MQ water and ethanol, and finally dried in a vacuum oven at room temperature For comparison, the pure CuS was synthesized by the similar experimental process in absence of GO powder. Similarly, rGO was prepared in absence of CuS precursors under the same hydrothermal process.

Photocatalytic activity test

The photocatalytic activities of the products were tested for the reduction of Cr(VI) under visible light irradiation. For all photocatalytic tests, a 50 mL aqueous solution containing a certain amount of Cr(VI) (K2Cr2O7) and 0.5 g L 1 of the catalyst in absence or presence of phenol was prepared. The pH of the suspension was adjusted by using 0.1 M HCl or 0.1 M NaOH solution. The resulting suspension was ultrasonicated for 1 min and magnetically stirred for 0.5 h in the dark to ensure adsorption desorption equilibrium.

The aqueous solution was irradiated under stirring at room temperature in air through with a cut off filter ( > 420 nm, to suppress the light with wavelength shorter than 420 nm) using a visible fiber lamp (Spot Light Source 400 700 nm, L9566-03, Hamamatsu, Japan). The intensity of the light was measured using a PM600TM Laser Fiber Power Meter (Coherent Inc, USA) and was determined as being 0.4 W cm -2 . During the visible light irradiation, about 2 mL of the suspension was taken from the reaction cell at given time intervals, and then centrifuged to remove the photocatalyst particles. The concentration of Cr(VI) was determined by the diphenylcarbazide (DPC) method [START_REF] Jin | Facile synthesis of porous single crystalline ZnO nanoplates and their application in photocatalytic reduction of Cr(VI) in the presence of phenol[END_REF]. The absorbance of sample solutions was measured using UV vis spectrophotometry at 540 nm after full color development. The concentration of phenol was measured by a HPLC system. Additionally, the total Cr content was also determined by ICP-AES.

Hydroxyl radical ( OH) formation measurement

The formation of OH radicals in presence of rGO, CuS, 2rGO-CuS, rGO-CuS and rGO-2CuS under visible light illumination was monitored by using terephthalic acid (TA) as the fluorescence probe. It is known that TA reacts with OH in basic solution to generate 2hydroxylterephthalic acid (TAOH), which fluoresces at approximately 426 nm upon excitation at 312 nm [START_REF] Hirakawa | Properties of O2and OH formed in TiO2 aqueous suspensions by photocatalytic reaction and the influence of H2O2 and some ions[END_REF]. In a typical process, 0.5 g/L of nanocomposite were dispersed in an aqueous solution (3 mL) containing 5 × 10 4 M TA and 2 × 10 3 M NaOH in a spectrometric quartz cuvette. The mixed solution was stirred in the dark for 10 min. After irradiation under the visible light, the reaction solution was centrifuged to remove the sample particles. The filtrate was analyzed using a PL spectrophotometer.

Results and discussion

Characterization

The phase composition and crystal structure of the CuS and CuS-rGO nanocomposites were studied with X-ray diffraction (XRD) (Fig. 1 and Fig. S1). The presence of sharp diffraction peaks evidences that the as-obtained products are crystalline. The XRD patterns of CuS and CuS-rGO nanocomposites comprise several peaks at 10.8°, 21.7°, 27.1°, 27.6°, 29.2°, 31.7°, 32.7°, 38.8°, 43.2°, 44.3°, 47.8°, 52.7°, 57.1° and 59.3° ascribed to the (002), (004), (100), ( 101), ( 102), ( 103), ( 006), ( 105), ( 106), (008), ( 110), ( 108), ( 202) and (116) diffraction planes, respectively [START_REF] Swaidan | Colorimetric sensing of dopamine in beef meat using copper sulfide encapsulated within bovine serum albumin functionalized with copper phosphate (CuS-BSA-Cu3(PO4)2) nanoparticles[END_REF]. All the diffraction peaks can be perfectly indexed as hexagonal covellite CuS (space group: P63|mmc), which are in good agreement with the JCPDS no. 78-2391, with cell parameters a=3.796 Å and c=16.36 Å. The absence of GO diffraction peak and the appearance of broad diffraction peak at ~22.4° in the XRD patterns of CuS-rGO nanocomposites indicates the effective reduction of GO to rGO [START_REF] Saranya | A template-free facile approach for the synthesis of CuS rGO nanocomposites towards enhanced photocatalytic reduction of organic contaminants and textile effluents[END_REF]. The XRD patterns reveal that the products were devoid of any detectable impurities or any other phases. Moreover, the addition of GO did not affect the crystal orientation of the CuS nanocomposites.

Raman spectroscopy is a powerful and non-destructive technique for investigating the structural nature of carbon-based materials such as graphene [START_REF] Cherifi | PMS activation using reduced graphene oxide under sonication: Efficient metalfree catalytic system for the degradation of rhodamine B, bisphenol A, and tetracycline[END_REF][START_REF] Stankovich | Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide[END_REF]. Figure S2 depicts the Raman spectra of GO, rGO, CuS and rGO-CuS nanocomposites. The Raman spectrum of GO exhibits two main characteristic peaks, the D band at ~1368 cm -1 attributed to defects and disorder in the hexagonal graphitic layers, and the G band at ~1589 cm -1 ascribed to the vibration of sp 2 carbon atoms in the 2D hexagonal lattice. Similarly, the Raman spectrum of rGO comprises the characteristic D and G bands at respectively 1368 and 1589 cm -1 .

Meanwhile, the Raman spectrum of the CuS displays a strong and sharp band at ~468 cm -1 assigned to the S-S stretching mode of A1g symmetry of CuS with a hexagonal structure [START_REF] Zhan | Photoresponse of multi-walled carbon nanotube copper sulfide (MWNT CuS) hybrid nanostructures[END_REF][START_REF] Zhang | Biomolecule-Assisted, Environmentally Friendly, One-Pot Synthesis of CuS/Reduced Graphene Oxide Nanocomposites with Enhanced Photocatalytic Performance[END_REF]. For rGO-CuS nanocomposites, all the Raman spectra consist of the D and G bands of rGO along with the peak of CuS. It is important to notice a red-shift of the D and G bands to 1353 and 1584 cm 1 , respectively, indicating the high ability for recovery of the hexagonal network of carbon atom and the presence of charge transfer between rGO and CuS [START_REF] Zhang | Biomolecule-Assisted, Environmentally Friendly, One-Pot Synthesis of CuS/Reduced Graphene Oxide Nanocomposites with Enhanced Photocatalytic Performance[END_REF].

The intensity ratio of the D and G bands (ID/IG) for rGO-2CuS increases to 1.31 (Table S1), as compared to 0.85 recorded for GO. These observations further confirm the formation of new and smaller sp 2 domains and testifies the reduction of GO to rGO during the hydrothermal reaction [START_REF] Stankovich | Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide[END_REF]. The results are in good agreement with XRD patterns.

Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) were further performed to assess the mesoporous structure and morphology of CuS and rGO-2CuS nanocomposite. Figure 2a depicts the TEM image of rGO-2CuS nanocomposite. It can be seen that all rGO sheets are densely decorated with a large quantity of small nanoparticles, and the rGO sheets in the composite are made of a few layers of graphene sheets. A close look at these nanoparticles reveals that they are randomly distributed on the surface with size ranging from 20 to 30 nm, as shown in Figure 2c. The HRTEM image, conducted on the scattered nanosheets, is displayed in Figure 2b. The regular spacing of the clear lattice planes is hexagonal phase of CuS [START_REF] Nezar | Efficient reduction of Cr(VI) under visible light irradiation using CuS nanostructures[END_REF]. Furthermore, the selected area electron diffraction (SAED) pattern demonstrated the growth of single crystals of CuS (Fig. 2d). Thus, these results are in good agreement with XRD analysis.

In addition, the corresponding EDX plot is shown in Figure S3. It clearly confirms the presence of Cu, S, and C elements with an atomic ratio of elemental copper and sulfur of approximately 1:1 (other peaks originate from the substrate used). This result indicates that individual CuS nanoparticles are well anchored onto the graphene sheets during the hydrothermal synthesis and prevent the restacking of rGO sheets during the reduction process.

The chemical composition of the rGO-2CuS composite was further confirmed by X-ray photoelectron spectroscopy (XPS) analysis (Fig. S4). The XPS survey spectrum in Figure S4a clearly shows the peaks of Cu2p, S2p, O1s, and C1s [START_REF] Nezar | Efficient reduction of Cr(VI) under visible light irradiation using CuS nanostructures[END_REF][START_REF] Zhang | Biomolecule-Assisted, Environmentally Friendly, One-Pot Synthesis of CuS/Reduced Graphene Oxide Nanocomposites with Enhanced Photocatalytic Performance[END_REF]. The O1s and C1s originate from rGO.

Figure S4b displays the core level spectrum of the Cu2p, comprising two peaks at 933.75 and 953.54 eV, which can be appropriately curve-fitted with four bands. The peaks at 933.75 and 952.25 eV are assigned respectively to Cu2p3/2 and Cu2p1/2 of Cu + , while the peaks at 935.75 and 954.38 eV are attributed to the characteristic peaks of Cu2p3/2 and Cu2p1/2 of Cu 2+ , in accordance with the literature data [START_REF] Karikalan | Sonochemical synthesis of sulfur doped reduced graphene oxide supported CuS nanoparticles for the nonenzymatic glucose sensor applications[END_REF]. The Auger line of Cu (Cu LMM) at 568.4 eV is the typical binding energy value of CuS [START_REF] Zhang | Biomolecule-Assisted, Environmentally Friendly, One-Pot Synthesis of CuS/Reduced Graphene Oxide Nanocomposites with Enhanced Photocatalytic Performance[END_REF][START_REF] Ghijsen | Electronic structure of Cu2O and CuO[END_REF]. Also, the two weak shake-up satellite lines at 943.0 eV and 962.6 eV indicate the presence of the paramagnetic chemical state of Cu 2+ [START_REF] Nezar | Efficient reduction of Cr(VI) under visible light irradiation using CuS nanostructures[END_REF][START_REF] Zhang | Biomolecule-Assisted, Environmentally Friendly, One-Pot Synthesis of CuS/Reduced Graphene Oxide Nanocomposites with Enhanced Photocatalytic Performance[END_REF][START_REF] Ghijsen | Electronic structure of Cu2O and CuO[END_REF]. In the case of S2p region (Fig. S4c), the deconvolution of the S2p spectrum leads to two main doublets at (161.5, 163.8 eV) and (164.6, 165.4 eV), that are typical values for metal sulphides and can be attributed to sulphide and disulphide, respectively [START_REF] Karikalan | Sonochemical synthesis of sulfur doped reduced graphene oxide supported CuS nanoparticles for the nonenzymatic glucose sensor applications[END_REF]. Moreover, the peaks at (166.4, 168.13 eV) and (168.97, 170.11 eV) are assigned to polysulfides, sulfate or thiosulfate (SO4 or S2O3 ) due to some surface oxidation [START_REF] Karikalan | Sonochemical synthesis of sulfur doped reduced graphene oxide supported CuS nanoparticles for the nonenzymatic glucose sensor applications[END_REF]. The core level C1s spectrum of the rGO-2CuS composite can be curve-fitted with three Gaussian peaks (Fig. S4d). The peak centered at 284.4

eV is attributed to the conjugated sp 2 -type C=C bonding in graphitic structure and the other two peaks located at 285.0 and 288.7 eV are ascribed to C-C and C=O, respectively [START_REF] Cherifi | PMS activation using reduced graphene oxide under sonication: Efficient metalfree catalytic system for the degradation of rhodamine B, bisphenol A, and tetracycline[END_REF][START_REF] Yuan | Facile synthesis of CuS/rGO composite with enhanced electrochemical lithium-storage properties through microwave-assisted hydrothermal method[END_REF].

Overall, the XPS data suggest that rGO-2CuS is well formed.

Figure S5a displays the UV vis absorption spectra of the synthesized rGO, CuS and CuS-rGO composites dispersed in water. The rGO exhibits an absorption peak at 245 nm, which can be assigned to the -* transition of aromatic C-C bonds along with a band at 410 nm due to the n-* transition of C=O bonds [START_REF] Cherifi | PMS activation using reduced graphene oxide under sonication: Efficient metalfree catalytic system for the degradation of rhodamine B, bisphenol A, and tetracycline[END_REF]. The UV vis absorption spectrum of pure CuS displays a characteristic covellite CuS-like absorption band in the near-IR region due to inter-band transitions between valence and unoccupied states [START_REF] Nezar | Efficient reduction of Cr(VI) under visible light irradiation using CuS nanostructures[END_REF][START_REF] Zhang | Biomolecule-Assisted, Environmentally Friendly, One-Pot Synthesis of CuS/Reduced Graphene Oxide Nanocomposites with Enhanced Photocatalytic Performance[END_REF][START_REF] Yuan | Facile synthesis of CuS/rGO composite with enhanced electrochemical lithium-storage properties through microwave-assisted hydrothermal method[END_REF]. The strong absorption in the visible range holds promise for the potential application of CuS-based materials in photocatalytic processes. Following the introduction of rGO, the absorbance of rGO-CuS composites increased throughout the spectrum compared to the CuS sample as a result of the absorption effect of rGO and the quantum effect of CuS. Moreover, it can be seen that a larger blue absorption profile has occurred, which might be attributed to quantum confinement [START_REF] Zhao | Plasmonic Cu S Nanocrystals: Optical and Structural Properties of Copper-Deficient Copper(I) Sulfides[END_REF]. The optical band gap was calculated using Eq. ( 1) by plotting 2 vs.

g depends on the type of band gap of the semiconductor (for a direct gap, , while for an indirect band gap, ). A 2 vs.

gives a line whose interception on the energy axis corresponds to the energy band gap. According to the above equation and based on a direct transition, as shown in Fig. S5b, the calculated optical band gap of pure CuS was 2.13 eV. Upon hybridization with rGO, the optical band gap of rGO-CuS nanocomposites changed from 2.06 to 1.96 eV when GO content increased from 1 to 2. These band gaps are higher than the bulk value, which could be due to quantum confinement in the small crystalline CuS nanostructures. photoreduction was improved through hybridization with rGO. Indeed, it could be seen that the time for the complete reaction was reduced to 60 and 50 min in the presence of rGO-CuS and rGO-2CuS, respectively. This contrasts with 2rGO-CuS catalyst for which a decrease of the photocatalytic activity was observed (80% reduction after 70 min, as compared to 100% achieved by CuS alone under otherwise identical experimental conditions). This decreased activity can be ascribed to the reduced amount of CuS within 2rGO-CuS. All together, these findings suggest that the visible light-induced photocatalytic activity of CuS nanoparticles can be enhanced through hybridization with rGO, which is most likely due to increased efficiency of light absorption and enhanced electron-hole separation of rGO-CuS nanocomposites [START_REF] Lee | Highly photoactive, low bandgap TiO2 nanoparticles wrapped by graphene[END_REF].

Effect of light intensity

The influence of light intensity on Cr(VI) photoreduction process was investigated for light intensity ranging from 0.1 to 0.5 W/cm 2 (Fig. S6). It was found that the photoreduction kinetics were significantly affected by the light intensity; decreasing of the light intensity from 0.4 to 0.1 W/cm 2 led to a clear decrease of the reduction rate from 100 to 85% after 70 min. In contrast, increasing the light intensity to 0.5 W/cm 2 had no effect on Cr(VI) reduction rate. Therefore, the optimal irradiation intensity of visible light was fixed at 0.4 W/cm 2 .

Effect of pH on photoreduction of Cr(VI)

The pH of the solution is a critical parameter in the photocatalytic process. The reduction of Cr(VI) at different initial pH values is summarized in Figure S7. The kinetic of Cr(VI) photoreduction increased slightly after 50 min of visible light illumination upon decreasing the initial pH value from 5.8 (the pH of the initial solution) to 2.5, whereas only 66.3% and 33.7%

of Cr(VI) were photocatalytically removed at pH 7 and 8.5 within the same period, respectively.

The effect of the acidified pH is consistent with the reported results [START_REF] Liu | Carbon nitride coupled Ti-SBA15 catalyst for visible-light-driven photocatalytic reduction of Cr(VI) and the synergistic oxidation of phenol[END_REF][START_REF] Jin | Facile synthesis of porous single crystalline ZnO nanoplates and their application in photocatalytic reduction of Cr(VI) in the presence of phenol[END_REF].

The dominant chromium species in the solution at pH 2.5 is HCrO4 or Cr2O7 [START_REF] Nanda | Fabrication of mesoporous CuO/ZrO2-MCM-41 nanocomposites for photocatalytic reduction of Cr(VI)[END_REF][START_REF] Liu | Carbon nitride coupled Ti-SBA15 catalyst for visible-light-driven photocatalytic reduction of Cr(VI) and the synergistic oxidation of phenol[END_REF].

Under these conditions, the photocatalyst surface can be highly protonated and thus leads to a strong electrostatic attraction towards the main species in solution. The pH could significantly influence the reduction of Cr(VI) to Cr(III) and a decrease in pH could increase the reduction kinetics of Cr(VI) [START_REF] Nanda | Fabrication of mesoporous CuO/ZrO2-MCM-41 nanocomposites for photocatalytic reduction of Cr(VI)[END_REF][START_REF] Muñoz | TiO2 catalysed reduction of Cr(VI) in aqueous solutions under ultraviolet illumination[END_REF]. Moreover, Cr(III) could form Cr(OH)3 which could precipitate on the surface of the photocatalyst at high pH solution, covering the activity sites of the catalysts [START_REF] Deng | Insight into highly efficient simultaneous photocatalytic removal of Cr(VI) and 2,4-diclorophenol under visible light irradiation by phosphorus doped porous ultrathin g-C3N4 nanosheets from aqueous media: Performance and reaction mechanism[END_REF][START_REF] Muñoz | TiO2 catalysed reduction of Cr(VI) in aqueous solutions under ultraviolet illumination[END_REF].

Synergistic effect of phenol on Cr(VI) photoreduction

It has been previously reported in the literature that the coexistence of metal-organic pollutants may enhance the photocatalytic reduction of Cr(VI) [START_REF] Liu | Carbon nitride coupled Ti-SBA15 catalyst for visible-light-driven photocatalytic reduction of Cr(VI) and the synergistic oxidation of phenol[END_REF][START_REF] Wu | Photocatalytic reduction of Cr(VI) with TiO2 film under visible light[END_REF]. In this work, the influence of variable initial phenol concentration on the reduction of 20 mg/L Cr(VI) over rGO-2CuS (0.5 g/L) was investigated (Fig. 4a). In the presence of phenol, the complete photoreduction time of Cr(VI) was reduced from 50 to 20 min when the Cr(VI)/phenol ratio was increased to 1:2, suggesting that the presence of phenol could improve Cr(VI) removal by acting as a scavenger of photo-generated holes. It can be seen that upon increasing the Cr(VI)/phenol ratio to 1:3, the time required to reach full reduction of Cr(VI) increased. This is maybe due to an increase in intermediates adsorbed on the rGO-2CuS nanocomposite surface with the increase in phenol concentration, which could cover the active sites of the catalyst [START_REF] Jin | Facile synthesis of porous single crystalline ZnO nanoplates and their application in photocatalytic reduction of Cr(VI) in the presence of phenol[END_REF].

Effect of Cr(VI) on phenol photodegradation

To assess the influence of Cr(VI) on phenol degradation, an aqueous solution of phenol (20 g/L), rGO-2CuS (0.5 g/L) and Cr(VI) (10 g/L) was irradiated under visible light for different time intervals. It could be observed that the extent of phenol degradation also increased greatly until complete degradation after 20 min of irradiation, following the addition of phenol/Cr(VI)

(1:2), as shown in Figure 4b. Furthermore, the improvement of phenol degradation indicates that Cr(VI) is an efficient scavenger of photo-generated electrons, which is similar to the observation reported in the literature [START_REF] Lu | Visible light induced photocatalytic removal of Cr(VI) over TiO2-based nanosheets loaded with surfaceenriched CoOx nanoparticles and its synergism with phenol oxidation[END_REF][START_REF] Wang | Synergistic photocatalysis of Cr(VI) reduction and 4-chlorophenol degradation over -Fe2O3 under visible light irradiation[END_REF]. The results confirmed the synergistic effect between Cr(VI) reduction and phenol oxidation.

The performance of the developed photocatalyst is comparable to and even better than that achieved by the most photocatalysts described in the literature for the simultaneous Cr(VI) photoreduction and phenol degradation (Table S2).

Kinetic study

The modeling of the kinetic data with an appropriate kinetic model allows to determine the apparent rate constant and the order of reaction of the photocatalytic process. It is wellestablished that the rate (r) of a photocatalytic reaction for a single component can be defined using equation ( 2) [START_REF] Lu | Visible light induced photocatalytic removal of Cr(VI) over TiO2-based nanosheets loaded with surfaceenriched CoOx nanoparticles and its synergism with phenol oxidation[END_REF][START_REF] Peyman | A sharp jump in photocatalytic activity of elemental sulfur for dye degradation in alkaline solution[END_REF]:

(2) where C0 is the initial concentration, C is the concentration of reactant, t is time, k is the rate constant of reaction, and kapp is the apparent rate constant.

Although this pseudo first-order rate equation has been extensively used in the literature for modeling photocatalytic kinetic data, it is based on the Langmuir classical isothermal model, which assumes a homogeneous surface. However, the surface of a catalyst is rarely homogeneous and several studies have been carried out in this regard suggested the possibility to use modified Langmuir Freundlich isotherm (an isotherm for adsorption onto heterogeneous surface from liquid phase), Equation 3 [START_REF] Peyman | A sharp jump in photocatalytic activity of elemental sulfur for dye degradation in alkaline solution[END_REF].

where the plot of ln(C/C0) versus t should give a line with a app [cat] .

s introduced recently in refs. [START_REF] Peyman | A sharp jump in photocatalytic activity of elemental sulfur for dye degradation in alkaline solution[END_REF][START_REF] Burés | A simple graphical method to determine the order in catalyst[END_REF]. By plotting the normalized concentration (C/C0) versus normalized time scale (t

[cat] ) at different concentrations of catalyst, the reaction order can be determined.

To study the kinetics of the photoreduction of Cr(VI) and phenol, the curves of ln(C/C0) vs.

time were plotted at different conditions (Fig. S8). All plots are linear, confirming the pseudo first-order reaction under the investigated experimental conditions. The obtained values of kapp are summarized in Tables S3 andS4.

Analysis of by-products of Cr(VI) and phenol

In order to better understand the photocatalytic process, the total residual concentration of Cr ions after photocatalytic reduction was assessed by ICP-AES. The experiment was carried out by visible light irradiation of a mixture of Cr(VI) (20 mg/L), phenol (20 mg/L) and GO-2CuS (0.5 mg/L) at pH 5.8 (Fig. 5). After 20 min of irradiation, a concentration of 0.015 mg/L of residual Cr(VI) ions was measured. This is far below the maximum allowable concentrations of 0.25 mg/L for industrial wastewater and 0.05 mg/L for drinking water [START_REF] Liu | Adsorption of Pb 2+ , Cd 2+ , Cu 2+ and Cr 3+ onto titanate nanotubes: Competition and effect of inorganic ions[END_REF][START_REF] Lu | Visible light induced photocatalytic removal of Cr(VI) over TiO2-based nanosheets loaded with surfaceenriched CoOx nanoparticles and its synergism with phenol oxidation[END_REF][START_REF] Vinu | Kinetics of simultaneous photocatalytic degradation of phenolic compounds and reduction of Metal Ions with nano-TiO2[END_REF].

Furthermore, XPS analysis was used to study the chemical states of the Cr species adsorbed on the catalyst surface after the photoreaction (Fig. S9). In the high-resolution spectrum of the Cr2p, the peaks located at 586.9 and 576.7 eV corresponding respectively to the 2p3/2 and 2p1/2 orbitals are attributed to Cr(III) [START_REF] Nezar | Efficient reduction of Cr(VI) under visible light irradiation using CuS nanostructures[END_REF][START_REF] Tadjenant | Graphene oxide chemically reduced and functionalized with KOH-PEI for efficient Cr(VI) adsorption and reduction in acidic medium[END_REF]. These results also confirm that rGO-2CuS nanocomposite is not only capable of simultaneously reducing Cr(VI) and oxidizing phenol, but also of removing Cr ions by adsorption [START_REF] Liu | Carbon nitride coupled Ti-SBA15 catalyst for visible-light-driven photocatalytic reduction of Cr(VI) and the synergistic oxidation of phenol[END_REF][START_REF] Jin | Facile synthesis of porous single crystalline ZnO nanoplates and their application in photocatalytic reduction of Cr(VI) in the presence of phenol[END_REF].

To investigate further the mechanism of phenol degradation using rGO-2CuS nanocomposite, the generated intermediate products were identified using high-performance liquid chromatography (HPLC). The results of HPLC (Fig. S10) revealed that two main intermediates identified as hydroquinone (7.4 min) and 1,4-benzoquinone (10.9 min) were formed. Their identification by HPLC was done based on their retention time, as compared to standard chemicals (Fig. S11). These by-products are formed following OH radical addition to the benzenic ring of phenol to yield hydroquinone and its subsequent oxidation to 1,4benzoquinone. It is not excluded that other by-products, derived from the ring-opening such as short-chain acids, are also generated during the photochemical degradation of phenol over rGO-2CuS [START_REF] Lam | Enhanced sunlight photocatalytic performance over Nb2O5/ZnO nanorod composites and the mechanism study[END_REF]. However, the products derived from the opening of the benzene ring could not be identified by HPLC. Instead, approximately 95% of the phenol was mineralized in 20 min (Fig. S12), suggesting that the phenol was not only decomposed into small organic compounds, but even mineralized into inorganic carbonaceous forms.

Stability of rGO-2CuS photocatalyst

Durability plays a key role in photocatalysis. Therefore, the repeatability and stability of the photocatalyst were investigated. Figures S13 and S14 depict the performance of the recycled rGO-2CuS nanocomposite for the mixed Cr(VI)/phenol system under visible light irradiation.

The photocatalyst was recovered by centrifugation after each cycle to regenerate the photocatalyst and reused in the next cycle without any additional treatment. As can be observed, even after 5 photocatalytic cycles, the photoreduction efficiency of Cr(VI) and phenol oxidation were as high as 92% and 88%, respectively, and no significant loss of photocatalytic activity can be seen during the cycling experiments. Furthermore, to demonstrate the stability of the rGO-2CuS, XRD analysis of the used photocatalyst wa performed (data not shown). The results revealed that no significant changes took place in the XRD patterns before and after the cycling test, indicating that the catalyst exhibits good visible-light photocatalytic stability.

Mechanism of the photocatalytic activity

Figure 6 displays the photocatalytic conversion rates of Cr(VI) and phenol over rGO-2CuO in the presence of different radical scavengers under visible light (> 420 nm) irradiation for 20 min. For reaction under N2 ambient conditions, before the start of the experiments, the reaction solution was purged with high-purity N2 (> 99.999%) for 1 h to remove dissolved O2 as much as possible, and continued to purge N2 through the photocatalytic process. The recorded rates of Cr(VI) reduction (Fig. 6a) and phenol degradation (Fig. 6b) in the mixed reaction system were higher in ambient air than in ambient N2. This could be due to the major role of O2 in the photocatalytic reaction process, through the reaction of dissolved O2 with photogenerated electrons to produce the O2 / OOH superoxide radicals [START_REF] Deng | Insight into highly efficient simultaneous photocatalytic removal of Cr(VI) and 2,4-diclorophenol under visible light irradiation by phosphorus doped porous ultrathin g-C3N4 nanosheets from aqueous media: Performance and reaction mechanism[END_REF][START_REF] Wang | Synergistic photocatalysis of Cr(VI) reduction and 4-chlorophenol degradation over -Fe2O3 under visible light irradiation[END_REF]. In the case of Cr(VI) reduction, the suppression effect was more evident, it is ca. 60% Cr(VI) reduction after 40 min irradiation in N2 gas ambient compared with 100% reduction achieved in air after 20 min photoreaction.

This can be attributed to important roles of O2 in the photocatalytic reaction process. Besides, it has been reported that O2 can reduce Cr(VI) to Cr(III) [START_REF] Dong | Synthesis and enhanced Cr(VI) photoreduction property of formate anion containing graphitic carbon nitride[END_REF][START_REF] Hu | Simultaneous photocatalytic Cr(VI) reduction and 2,4,6-TCP oxidation over g-C3N4 under visible light irradiation[END_REF]. Thus, in this study, O2 may efficiency of phenol was decreased from 100% to 56% after 20 min visible light irradiation.

The result clearly indicates that the phenol degradation mainly relies on the photogenerated holes [START_REF] Deng | Insight into highly efficient simultaneous photocatalytic removal of Cr(VI) and 2,4-diclorophenol under visible light irradiation by phosphorus doped porous ultrathin g-C3N4 nanosheets from aqueous media: Performance and reaction mechanism[END_REF][START_REF] Chen | Novel magnetically separable reduced graphene oxide (RGO)/ZnFe2O4/Ag3PO4 nanocomposites for enhanced photocatalytic performance toward 2,4-dichlorophenol under visible light[END_REF]. It is also observed that the photodegradation efficiency in air is higher than that in ambient N2. This can be attributed to dissolved oxygen, which promotes oxidation. However, the effect of dissolved oxygen was not so obvious in the presence of EDTA-Na2, indicating that h + are the most important reactive species for phenol oxidation.

In the photocatalytic reaction, the hydroxyl radical ( OH) is considered to be the most important active group. Thus, a study of OH radical formation can provide a quantitative index to estimate the number of charge carriers actually implicated in phenol degradation. It is wellestablished that the terephthalic (TA) fluorescence method is a very sensitive technique used to detect the amount of OH produced and whose increase implies an improvement in photocatalytic efficiency. The method relies on the reaction of TA with OH to generate a fluorescent TAOH (maximum emission at ~426 nm) [START_REF] Hirakawa | Properties of O2and OH formed in TiO2 aqueous suspensions by photocatalytic reaction and the influence of H2O2 and some ions[END_REF][START_REF] Lu | Visible light induced photocatalytic removal of Cr(VI) over TiO2-based nanosheets loaded with surfaceenriched CoOx nanoparticles and its synergism with phenol oxidation[END_REF]. From Figure S15a, the rGO-2CuS nanocomposite showed a considerably higher amount of OH production than CuS. This outcome is in good accordance with the results of photodegradation of phenol.

From the results of the experiments, we suggest a plausible mechanism for the photocatalytic reduction of Cr(VI) and oxidation of phenol over rGO-2CuS. Under visible light irradiation, rGO-2CuS could generate electrons and holes. Following this, the electrons in the CB of the rGO-2CuS in the ambient air are scavenged by O2 to generate O2 and migrate to Cr(VI) to reduce it. Meanwhile, the photogenerated holes left behind in the VB could produce OH from H2O. H2O2 could be produced by superoxide radical and H + . Besides, O2 not only could induce organics oxidation, but also could promote the Cr(VI) reduction by a two-step O2 mediated indirect reduction process [START_REF] Dong | Synthesis and enhanced Cr(VI) photoreduction property of formate anion containing graphitic carbon nitride[END_REF]. In addition, due to the high oxidation efficiency of H2O2, its existence would inevitably lead to the oxidation of reduced chromium to Cr(VI).

The existence of H2O2 in the catalytic system was highlighted by the absorption peak of o-tolidine at 446 nm in the UV-vis spectrum [START_REF] Wang | Synergistic photocatalysis of Cr(VI) reduction and 4-chlorophenol degradation over -Fe2O3 under visible light irradiation[END_REF][START_REF] Liu | Metal-free efficient photocatalyst for stable visible water splitting via a two-electron pathway[END_REF]. From Figure S15b, as the intensity of the absorption peak decreases, the H2O2 content of the simple solution of Cr(VI) and phenol decreases compared to that of the water under visible light irradiation. In case the Cr(VI)/phenol combined system, almost no H2O2 could be observed, suggesting that phenol degradation could be effective in consuming H2O2 and inhibiting oxidation of the lower valence states of chromium to improve Cr(VI) reduction activity [START_REF] Wang | Synergistic photocatalysis of Cr(VI) reduction and 4-chlorophenol degradation over -Fe2O3 under visible light irradiation[END_REF]. Moreover, the photogenerated holes and various active oxidants, such as H2O2, OH and O2 , could oxidize phenol. During this photocatalytic reaction system, Cr(VI) acts as an electron acceptor, which can react with the photogenerated CB electrons; and meanwhile, phenol acts as an electron donor, which can react with the photoexcited holes. Both processes can enhance the separation rate of the photogenerated electron holes, resulting in much more CB electrons for Cr(VI) reduction and VB holes for phenol oxidation.

The above results show the synergistic effect of mixed Cr(VI)/phenol system. The generation of photogenerated electron-hole pairs, radicals and the reaction process can be summarized according to the following equations: Therefore, based on the results and analysis of the experiments, a plausible mechanism was proposed and illustrated in Figure 7.

hv rGO-2CuS + ( + ) (4)

Conclusion

In

3. 2 . Photocatalytic performance 3 . 2 . 1 .

 2321 photocatalytic Cr(VI) reduction. CuS revealed high photoreduction activity of Cr(VI) to Cr(III)

  photogenerated electrons and holes, but also increases chromium reduction in Cr(VI)/phenol

Figure captions Figure 1 :

 captions1 Figure captions

Figure 2 :

 2 Figure 2: (a) Typical TEM images, (b) HRTEM image (inset is the high magnification TEM

Figure 3 :

 3 Figure 3: Photocatalytic reduction of Cr(VI) (20 mg/L) over rGO-CuS (0.5 g/L) under visible

Figure 4 :

 4 Figure 4: (a) Photocatalytic reduction efficiencies of Cr(VI) (20 mg/L) in presence and absence

Figure 5 :

 5 Figure 5: (a) Efficiency of removal of Cr, Cr(VI)+Cr(III) in the coexisting Cr(VI)-phenol

Figure 6 :

 6 Figure 6: (a) Cr(VI) reduction and (b) phenol degradation in presence of different scavengers

Figure 7 :

 7 Figure 7: Schematic illustration for the synergistic photocatalysis of Cr(VI) reduction and

Figure 1 31 Figure 2 32 Figure 3 33 Figure 4

 312323334 Figure 1

Acknowledgements

The authors gratefully acknowledge financial support from the Centre National de la Recherche Scientifique (CNRS), the University of Lille -Sciences and Technologies, and the Hauts-de-France region. Yacine Cherifi thanks the Algerian Ministry of Higher Education and Scientific Research (M.E.S.R.S) for a PNE fellowship. ICP analyses were performed in the «

also tend to reduce Cr(VI) during the photocatalytic reaction process. However, Cr(VI) can also be reduced in the absence of O2, as illustrated in Figure 6a. This indicates that CB electrons from rGO-2CuS also contribute to the reduction of Cr(VI). While the purging effect of N2 on phenol degradation is less obvious, suggesting that the phenol could be degraded by valence band (VB) holes when photogenerated electrons were scavenged by Cr(VI) in mixed Cr(VI)/phenol system. In the presence of Cr(VI), conduction band (CB) electron scavenging and electron-hole separation are enhanced, increasing the rate of phenol degradation, as shown in Figure 6b.

In Figure 6a, introduction of 1 mM EDTA-Na2, a hole scavenger, leads to a decrease in the photoreduction activity of Cr(VI). Furthermore, when the reaction process was carried out under N2 ambient, Cr(VI) reduction decreased to higher extent than in the presence of EDTA-Na2 only. This could be attributed to the decreased electron transportation of the O2/O2 mediated reduction in the absence of dissolved oxygen. Besides, it was previously reported that Cr(VI) could be reduced by either photogenerated electrons or other active species [START_REF] Dong | Synthesis and enhanced Cr(VI) photoreduction property of formate anion containing graphitic carbon nitride[END_REF][START_REF] Hu | Simultaneous photocatalytic Cr(VI) reduction and 2,4,6-TCP oxidation over g-C3N4 under visible light irradiation[END_REF].

The O2 is generated from the dissolved oxygen and photo-generated electrons of the rGO-2CuS under irradiation, and could then be transformed into H2O2 and/or hydroxyl radical ( OH) in water. Potentially, the O2/O2 mediator not only improves the electron transfer for CB to Cr(VI), but also promotes the formation of OH from H2O per photogenerated hole [START_REF] Dong | Synthesis and enhanced Cr(VI) photoreduction property of formate anion containing graphitic carbon nitride[END_REF].

In this study, in the event that the CB position of a semiconductor is more negative than the reduction potential of O2/O2 , trapping of photogenerated electrons with molecular oxygen is possible to produce O2 and the photocatalytic reduction is also enhanced by effective consumption of photogenerated holes in this mixed photocatalytic process [START_REF] Wang | Synergistic photocatalysis of Cr(VI) reduction and 4-chlorophenol degradation over -Fe2O3 under visible light irradiation[END_REF][START_REF] Dong | Synthesis and enhanced Cr(VI) photoreduction property of formate anion containing graphitic carbon nitride[END_REF].

The photocatalytic activity of phenol degradation with the addition of EDTA-Na2 was also examined (Fig. 6b). The results revealed that when EDTA-Na2 was introduced into the reaction system, it caused a rapid deactivation of the rGO-2CuS photocatalyst and the degradation


Marie-Pierre Brenier-Pinchart

Florence Robert-Gangneux

Isabelle Accoceberry

Simon Pichard

Cécile Garnaud

Hélène Fricker-Hidalgo

Maude F Lévêque

Gautier Hoarau

Hervé Pelloux

Patrick Bastien

Yvon Sterkers
email: yvon.sterkers@umontpellier.fr

Emmanuelle Varlet-Marie
email: emmanuelle.varlet@umontpellier.fr

Brenier-Pinchart Mp

Lévêque Mf

Varlet-Marie E J Mol

Multicenter Comparative Assessment of the TIB MolBiol Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis

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

Introduction

Toxoplasma gondii is a worldwide distributed protozoan parasite causing toxoplasmosis [START_REF] Montoya | Toxoplasmosis[END_REF] . Human transmission pathways include (i) the consumption of raw/undercooked meat containing cysts or food/water contaminated by oocysts shed in the cats' feces, (ii) vertical transmission of tachyzoites or (iii) the transplant of organ/hematopoietic stem cells containing cysts. The infection is most often asymptomatic in healthy individuals. However, there are two severe clinical issues in Toxoplasma infection: (i) a primary infection occurring in pregnant women may infect the fetus and cause congenital toxoplasmosis (CT), leading to miscarriage, chorioretinis or malformative syndrome; (ii) the reactivation of cysts in previously infected immunocompromised hosts (solid organ transplants, hematopoietic stem cell transplants, AIDS patients) may cause lifethreatening complications such as cerebral or disseminated toxoplasmosis. In both situations, the biological diagnosis is challenging and has greatly beneficiated from molecular methods [START_REF] Sterkers | Novel interpretation of molecular diagnosis of congenital toxoplasmosis according to gestational age at the time of maternal infection[END_REF][START_REF] Robert-Gangneux | Molecular Diagnosis of Toxoplasmosis in Immunocompromised Patients[END_REF] . Currently, molecular detection of Toxoplasma infection relies on laboratory-developed or 'in-house' real time PCR assays [START_REF] Roux | Evolution of Toxoplasma-PCR methods and practices: a French national survey and proposal for technical guidelines[END_REF] . Although they represent sensitive methods 2, 3 , they suffer from lack of standardization. Efforts towards Toxoplasma-PCR standardization and quality management include the use of commercialized PCR assays. Their performances, however, are variable and require proper evaluation by expert laboratories [START_REF] Morelle | Comparative assessment of a commercial kit and two laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis[END_REF][START_REF] Filisetti | Multicentric comparative assessment of the bio-evolution Toxoplasma gondii detection kit with eight laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis[END_REF][START_REF] Robert-Gangneux | Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis[END_REF][START_REF] Döşkaya | Comparison of an in House and a Commercial Real-Time Polymerase Chain Reaction Targeting Toxoplasma Gondii RE Gene Using Various Samples Collected From Patients in Turkey[END_REF] -the more so since Toxoplasma parasitic loads in clinical samples are often low, which requires that PCR assays show a high sensitivity. For example in CT, the median concentration in the amniotic fluid (AF) has been reported to be around 10 tachyzoites/mL [START_REF] Romand | Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii[END_REF] . In this study, the technical performances of the TIB MolBiol ® LightMix® Kit Toxoplasma gondii PCR assay (Roche®, Meylan, France) were compared with those of reference laboratory-developed real time PCR assays in three distinct clinical settings: CT (using amniotic fluid and placenta), toxoplasmic chorioretinitis (using aqueous humor), and toxoplasmic infection in immunocompromised patients (using blood, cerebrospinal fluid and miscellaneous samples).

Materials and methods

Study scheme

The study was conducted in accordance with the regulations of the local medical ethics committee of the Hospital University Center (CHU) of Montpellier, France, in line with the revised Helsinki Declaration. Written consent was obtained before any amniotic fluid sampling. This study was performed in the Departments of Parasitology-Mycology of four Academic Hospital Centers (Montpellier, Grenoble, Rennes and Bordeaux), hereafter referred as center A, B, C and D respectively. Centers A, B and C are members of the "Molecular Biology Pole" of the French National Reference Center for Toxoplasmosis (http://cnrtoxoplasmose.chu-reims.fr/, last access April 07, 2021). Center D uses the TIB MolBiol® method in routine practice. All four centers are proficient centers for the molecular diagnosis of toxoplasmosis.

The study scheme comprised four parts. The first part aimed at testing the TIB MolBiol® method using simulated calibrated specimens from external quality assessments and was performed by center A. The second part of the study consisted in determining PCR efficiencies and analytical sensitivities based on serial dilution assays using two standards for Toxoplasma detection and was performed by centers A and B. The third part aimed at testing clinical samples and involved the four centers. The results obtained using TIB MolBiol ® were compared with those obtained in routine in each center. The fourth part is a prospective study performed by center D.

Lyophilized standards and external Quality Control samples

Two standards were used for the molecular detection of Toxoplasma. The first one was a standard described by Varlet-Marie et al. [START_REF] Varlet-Marie | Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays[END_REF] , based on a 10-fold serial dilution assay to include six concentrations ranging from 10,000 to 0.1 T. gondii per mL. The second one was the DNA standard provided by the commercial kit, which includes a row of six standards ranging from 10 6 to 10 target molecules per PCR tube. In both cases, each serial dilution concentration was tested in triplicate. The TIB MolBiol ® assay was performed with and without the internal control provided by the manufacturer. Twenty external Quality Control samples, 13 positive and 7 negative samples, were also included.

Clinical samples

In total, 133 clinical samples were included in the study, collected within the frame of routine practice of the four participating centers. They comprised 76 AF, 16 [START_REF] Martino | Toxoplasmosis after hematopoietic stem cell transplantation[END_REF] , allowing the distinction between Toxoplasma infection and disease. Ocular toxoplasmosis was also confirmed using a comparative Toxoplasma western blot (LDBio Diagnostics, Lyon, France) [START_REF] Greigert | When Biology Supports Clinical Diagnosis: Review of Techniques to Diagnose Ocular Toxoplasmosis[END_REF] .

DNA extraction

The DNA extraction procedure was adapted to the specimens. Center A used the Tween-Nonidet-NaOH method for the paucicellular fluids (AF, aqueous humor and CSF), as previously described [START_REF] Hohlfeld | Prenatal diagnosis of congenital toxoplasmosis with a polymerase-chain-reaction test on amniotic fluid[END_REF] and a protein precipitation method for tissues and cellular specimens (whole blood, cord blood, and placenta). Briefly, placenta/tissues were ground, trypsinized, filtered, washed, and lysed with proteinase K (Euromedex®, Souffelweyersheim, France) in Tris-SDS (2-3µL/100µL of final solution); buffy coat fractions (300µL), isolated from cord blood and whole blood specimens after centrifugation for 10 min at 1750g, were also lysed with proteinase K. DNA was prepared with the protein precipitation solution kit (A795A; Promega®, Charbonnières-les-Bains, France) [START_REF] Sterkers | Novel interpretation of molecular diagnosis of congenital toxoplasmosis according to gestational age at the time of maternal infection[END_REF] . Centers B and C used DNA minikit (#51304; Qiagen®, Courtaboeuf, France) and center D used the Roche® MagNApure Compact as described by the manufacturers. According to previous published data, DNA extracts were kept frozen at -20°C until the study [START_REF] Delhaes | Freezing and storage at -20 degrees C provides adequate preservation of Toxoplasma gondii DNA for retrospective molecular analysis[END_REF] (Table 1).

Table 1. Routine PCR assays characteristics in the four participating centers

PCR

Reischl et al. [START_REF] Reischl | Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes[END_REF] Reischl et al. [START_REF] Reischl | Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes[END_REF] Robert-Gangneux et al. [START_REF] Robert-Gangneux | Clinical relevance of placenta examination for the diagnosis of congenital toxoplasmosis[END_REF] Costa et al. [START_REF] Costa | Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes[END_REF] Thermocycler

PCR methods

The reference PCR methods were based on hybridization probes (Fluorescence Resonance Energy Transfer technology) for centers A, B and D, and hydrolysis probe (TaqMan) for center C. Centers A, B and C used a laboratory-developed real-time PCR targeting the non-coding repetitive sequence rep529 [START_REF] Homan | Identification of a 200-to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR[END_REF] in the experimental conditions described by Reischl et al [START_REF] Reischl | Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes[END_REF] in centers A and B, Robert-Gangneux et al. [START_REF] Robert-Gangneux | Clinical relevance of placenta examination for the diagnosis of congenital toxoplasmosis[END_REF] in center C and Costa et al. [START_REF] Costa | Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes[END_REF] in center D. The experiments were performed on a Light Cycler 480 (Roche®) in center A and C and Light Cycler 2.0 (Roche®) in centers B and D (Table 1). In agreement with good laboratory practices, the laboratory-developed PCR assays included positive controls, negative controls, DNA extraction controls (human beta-globin gene revealed in SYBR green as described by Saiki et al. [START_REF] Saiki | Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia[END_REF]

Data and statistical analysis

Crossing point (Cp) values were calculated using the "absolute quantification/second derivative maxima" (LightCycler® 480 Software release 1.5.1.62, Roche®). The performances of the PCR were evaluated by calculating the linear measuring range (regression analysis, R 2 ) and the efficiency (Eff=10 (-1/slope) -1). Agreement and correlation between the two methods for the positive specimens were evaluated by Bland-Altman and correlation plots. Bland-Altman plots [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF] are a graphical representation of the difference of PCR crossing point (Cp) values in abscissa plotted against the mean of Cp values in ordinate, here for all samples analyzed using a laboratory-developed method (Center A-C) and the commercial kit [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF] .

Results

Comparative assessment using standards for PCR detection

Two centers (A and B) compared the TIB MolBiol® method and their reference diagnostic method by testing the standard of the NRC-T network ("TG25040, 1x10 4 parasites per mL, type II strain, Montpellier 2014") [START_REF] Varlet-Marie | Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays[END_REF] . Between three and six reactions were used for testing each of the standard's concentrations (10,000, 1000, 100, 10, 1 and 0.1 T. gondii genome equivalents (T)/mL) (Table 2). Results obtained using all methods were identical down to 1 T/mL, but slightly lower for the commercial kit when using the 0.1 T/mL concentration (Table 2). The lower threshold of Toxoplasma DNA detection (defined as the lowest concentration where >50% of the reactions remain positive) [START_REF] Sterkers | Multicentric comparative analytical performance study for molecular detection of low amounts of Toxoplasma gondii from simulated specimens[END_REF] was 0.1 T/mL for the laboratorydeveloped method of center A, and 1 T/mL for the laboratory-developed method of center B as well as the TIB MolBiol® method in both centers (Table 2). This threshold was not reached when the TIB MolBiol® DNA standard was included (Supplementary Table 1). The relationship between the Cp and the DNA concentration was linear over 6 log units (r²>0.99) for both standards. The PCR efficiencies of the commercial kit were equivalent for the two standards (Eff=93% and 99% respectively) (Figure 1). The TIB MolBiol® commercial kit includes an internal control (IC). The possibility of an internal competition between the IC and low concentrations of the target was investigated by testing the DNA standard both with and without the IC. In the 'with IC condition', IC was added in the PCR tube with the sample extracted DNA, and not added directly in the sample as an extraction control. No difference in sensitivity was observed whether using or not using the IC with the TIB MolBiol® method (Supplementary Table 1). Similarly, no difference was seen when testing the standard of the NRC-T with or without including this IC, still using the TIB MolBiol® method (Table 2). Of note, when testing the highest concentrations of Toxoplasma DNA, the IC was not amplified (Table 2 and Supplementary Table 1). This also occurred with clinical samples when the Cp was <24.

Figure 1. Linear measuring range and PCR efficiency from serial dilution assays. A. serial dilution assay from LightMix® Kit Toxoplasma gondii (EC). B. serial dilution assay from the NRC-Toxoplasma

Figure 2. Bland-Altman and correlation plots representing the concordance between the Cp values obtained using laboratory-developed methods and the commercial kit for the molecular detection of T. gondii. Bland Altman plots for center

Comparative assessment using External Quality Control samples

Centers A-C tested 20 External Quality Control samples produced by the NRC-T; these were lyophilized spiked AF samples, of which 7 negative and 13 tittered at different concentrations, ranging from 100±15 T/mL to 5±4 T/mL. The results obtained using TIB MolBiol® were in agreement with the expected results and each of the reference methods, with two exceptions. Indeed, Center C found two falsely negative results, one using the commercial kit and one using its own laboratorydeveloped method (Supplementary Table 2). The falsely negative result obtained by the routine method concerned a sample of spiked AF at 14±5T/mL sent in duplicate; the duplicate sample was found concordantly positive by the routine and the TIB MolBiol® methods. The other discrepant result, found falsely negative using TIB MolBiol®, was a spiked AF at 5±4T/mL that was sent in triplicate, the remaining three samples were found concordantly positive by the routine and the TIB MolBiol® methods. At such a low concentration, the Poisson law may apply and finding two out of three samples positive should be considered as acceptable.

Comparative assessment using clinical samples

In total, in the four centers, 133 clinical samples were included in the study, issued from DNA collections or prospectively tested (cf. Materials & Methods and Supplementary Table 2). The results were concordant between the commercial kit and the reference method for 130/133 (97.7%) samples. These included 59 negative and 74 positive samples (Table 3 and Supplementary Table 2). The three discrepant results were found in center A and D; they were made of two falsely negative results of TIB MolBiol® (center A and D) and one falsely positive result of TIB MolBiol® (center D). The first false negative of TIB MolBiol® were found inconstantly positive (1/6 PCR reactions) in routine and negative using TIB MolBiol® (0 positive PCR/1 PCR reaction performed). It concerned an aqueous humor from a chorioretinitis confirmed by a positive comparative immunoblot and by a concordant positive PCR using both assays in the vitreous. This case highlights how the parasite burden can be extremely low. The second case was also an aqueous humor with a low parasite burden (3/3 positive PCR reactions in routine vs 0/1 by TIB MolBiol®). Of note, in both cases, TIB MolBiol® was performed in simplicate due to the low volume of extracted DNA in this kind of samples. The third discrepancy was a BALF sample (center D), positive in 1 out of 3 reactions (Cp=35) using the kit but negative using the laboratorydeveloped method (0/4 PCR reactions); since toxoplasmosis was clinically ruled out in this patient, this likely represents a falsely positive result from the TIB MolBiol® assay. The concordance between the Cp values obtained using laboratory-developed methods and the commercial kit for the molecular detection of T. gondii was assessed. Ten concordantly positive samples analyzed in center A, 22 in center B and 19 in center C were included in the study. The Bland Altman plots and linear regression showed very good agreement with a single sample outside the ±1.96 standard deviation area and a high R 2 (Figure 2).

Table 3. Consistency between the TIB MolBiol® and reference methods using characterized clinical

Discussion

Commercial kits are now widely used for the molecular diagnosis of toxoplasmosis. Indeed, they most often offer convenient turnkey protocols which may be an advantage for the accreditation of medical microbiology laboratories. However their performances cannot be taken for granted and should be analyzed by independent expert networks. All participating centers belong to the "Molecular Biology Pole" of the NRC-T; they are regional reference centers for the molecular diagnosis of toxoplasmosis, and all laboratorydeveloped methods have been published [START_REF] Sterkers | Novel interpretation of molecular diagnosis of congenital toxoplasmosis according to gestational age at the time of maternal infection[END_REF][START_REF] Robert-Gangneux | Clinical relevance of placenta examination for the diagnosis of congenital toxoplasmosis[END_REF][START_REF] Costa | Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes[END_REF][START_REF] Sterkers | Diagnosis of congenital toxoplasmosis by polymerase chain reaction on neonatal peripheral blood[END_REF][START_REF] Brenier-Pinchart | Molecular Diagnosis of Toxoplasmosis: Value of the Buffy Coat for the Detection of Circulating Toxoplasma Gondii[END_REF] .

Participating centers compared the TIB MolBiol® method with their own laboratory-developed method used in routine diagnosis. For this, (i) the standard lyophilized preparation of T. gondii used as a common standard within the NRC-T network [START_REF] Varlet-Marie | Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays[END_REF] , and (ii) 133 patients samples obtained in various clinical settings were used. First of all, the respect by this PCR assay of essential rules was verified. In agreement with laboratory quality management, TIB MolBiol® included a negative control and an internal control (IC). Of note, we actually added this control in the PCR reaction mix rather than before DNA extraction. This attitude is frequently found in centers being part of a shared microbiology technical platform. Indeed, a single extraction control is used for all the PCR assays and target-specific positive controls are used as IC to check for the presence of inhibitors. The kit also included a standard row to estimate the quantity of the target sequence in clinical samples. TIB MolBiol® is the first Toxoplasma commercial kit including hybridization probes (FRET). The use of hybridization probes allows amplified fragments to be analyzed using melting curves, which is a major advantage over hydrolysis probes [START_REF] Navarro | Real-time PCR detection chemistry[END_REF] , but does not prove to be particularly useful in routine practice. This implies that this kit was developed solely for the Roche® LightCycler® devices. Regarding practicality, the major drawback of this kit is that it necessitated the reconstitution of several reagents, including MgCl2; in addition, a 10X PCR master mix had to be purchased separately. Finally, it did not include heat-labile Uracil-N-Glycosylase to limit carryover DNA contamination. However, since the master mix is not included, this step can be added using a master mix which contains uracil instead of thymidine; one must then check the absence of effect of this on the sensitivity of the assay.

In total, taking into account the twenty EQA samples and the 133 clinical samples, the sensitivity and specificity of the routine methods were respectively 86/87 (98.9%) and 66/66 (100%). Sensitivity and specificity of the TIB MolBiol® kit were respectively 84/87=98.1% and 65/66=98.5%. This was not statistically different from the figures obtained using the routine methods. The use of the commercial PCR assay TIB MolBiol LightMix® Toxoplasma gondii therefore proved satisfactory for the diagnosis of toxoplasmosis in diverse clinical settings: congenital, ocular and disseminated toxoplasmosis. Its performances, using a wide variety of clinical samples, were equivalent to those of reference laboratory-developed assays. Yet, the analytical sensitivity threshold determined here was 10-fold higher than that of the most sensitive method of the NRC-T (0.1 T/mL vs 1 T/mL).

For this reason, it must be noted that falsely negative results may be observed for very low parasitic loads, particularly if a single PCR reaction is performed. The supply with this commercial kit of an internal control which does not appear to compete nor interfere with the detection of low parasite concentrations is a positive point. Finally, this kit offers the possibility of an absolute quantification of the parasitic load, which, in view of the possible correlation with the severity of the disease [START_REF] Costa | Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes[END_REF][START_REF] Yamamoto | Association of Parasite Load Levels in Amniotic Fluid With Clinical Outcome in Congenital Toxoplasmosis[END_REF] , might be interesting; however, the DNA standard allowing quantifying parasitic loads is expressed as a number of target copies, and not in Toxoplasma per mL, which may hinder inter-laboratory comparisons and interpretation since the existing studies examined the parasitic loads in parasites per mL.

  ) for center A, quantification of DNA by spectrophotometry for center B and DiaControl IDNA YD (Diagenode s.a. Liège, Belgium) for center C, and PCR inhibition internal controls (low concentration T. gondii DNA in a separate tube for center A, BacterioPhiX for center B and DiaControl IDNA YD (Diagenode) for center C). The TIB MolBiol ® assay (LightMix® Kit Toxoplasma gondii (EC) order no. 40-0217-32) was used according to the manufacturer's recommendations. It targets a 134-bp fragment from a repeated element of T. gondii genome (not specified by the manufacturer); the resulting PCR fragment of T. gondii is analyzed with LightCycler® Red 640 labeled probes. This kit includes a negative control (NTC), an internal control (IC) also named extraction control target (ECT) or spiked extraction control (sEC), which can be added to the sample before extraction (10µL) or in the PCR reaction mix (0.5µL), and a standard row made of target DNA in six different concentrations. The control reaction generates a product of 300 bp, detected using LightCycler® Red 690 labeled hybridization probes. The use of a color compensation file generated with the LightMix® Color Compensation HybProbe 530/640/690 kit (order no. 40-0318-00) is a prerequisite to detect the control reaction.

  Figure 2. Bland-Altman and correlation plots representing the concordance between the Cp values obtained using laboratory-developed methods and the commercial kit for the molecular detection of T. gondii. Bland Altman plots for centerA-C. The pink lines represent the means and ±1.96 standard deviations. Correlation plots: Cp values of the laboratory-developed methods were plotted against the values obtained with the commercial kit (Center A-C). Linear regressions with their equation and R 2 values are presented for center A-C.

  for paucicellular fluids, b for tissues and cellular specimens, c Total_NA_plasma_100_400_V3_2 protocol with 200 and 100 µL for sample and elution volume respectively

		Light Cycler 480 II	Light Cycler 2.0	Light Cycler 480	Light Cycler 2.0 (Roche®)
		(Roche®)	(Roche®)		(Roche®)
	PCR mix	LightCycler® 480 Probes	Light	Cycler®	Taqman Universal	Light Cycler® FastStart
		Master (#04707494001)	FastStart DNA Master	Master	Mix	DNA Master HybProbe
			HybProbe		(#4305719; Applied	(#03003248001)
			(#03003248001)	Biosystems,
					Villebon-sur-
					Yvette)

a

Table 2 . Comparison of routine and TIB MolBiol® methods using serial dilutions of the standard of the National Reference Center for Toxoplasmosis

 2 Cp means" should not be compared between different centers but only within the same center. 2 IC: internal control supplied with the TIB MolBiol® kit. w/o: without. Cp: crossing point. Sd: standard deviation

			Concentration (T/mL)	10,000	1000	100	10	1	0.1
		Routine Eff=94% R 2 =0.97	N Cp mean ±sd 1	6/6 19.7 ±0.3	6/6 23.5 ±0.6	6/6 26.7 ±0.5	6/6 29.6 ±0.8	6/6 32.8 ±1.2	4/6 35.6 ±1.9
		TIB MolBiol with IC	N	6/6	6/6	6/6	6/6	5/6	1/6
		Eff=99%	Cp mean ±sd	20.0 ±0.1	24.0 ±0.1	27.4 ±0.1	30.6 ±0.3	33.1 ±2.1	35.9
	Center A	R 2 =0.96 TIB MolBiol w/o IC 2 Eff=90% R 2 =0.99	IC Cp mean ±sd N Cp mean ±sd	Neg 6/6 19.8 ±0.1	27.3 ±0.4 6/6 23.9 ±0.1	26.6 ±0.1 6/6 27.8 ±0.2	27.1 ±0.1 6/6 31.5 ±0.3	27.7 ±0.1 4/6 33.9 ±0.4	27.7 ±0.1 1/6 35.3
	B Center	Routine Eff=94% R 2 =0.99 TIB MolBiol with IC Eff=93% R 2 =0.98	N Cp mean ±sd N Cp mean ±sd IC Cp mean ±sd	3/3 21.0 ±0.2 3/3 20.7 ±0.2 Neg	3/3 24.1 ±0.2 3/3 23.7 ±0.4 Neg	3/3 27.1 ±0.1 3/3 26.6 ±0.8 27.0±0.1	3/3 30.4 ±0.5 3/3 30.1 ±0.7 28.9±0.1	3/3 33.8 ±1.1 3/3 33.4 ±1.6 29.5±0.2	1/3 35 1/3 33.5 29.7±0.2
	1 "

Acknowledgements

We thank Jean-Marc Costa for its contribution in the routine diagnosis assay of center D. We thank Sylvie Douzou, Ghyslaine Serres and Bounleth Sanichanch (Montpellier), Valérie Martin (Grenoble), Pauline Comacle (Rennes) and Géraldine Desserre (Bordeaux) for their technical assistance. We acknowledge the financial support of "Santé Publique France" through the French National Reference Center for Toxoplasmosis, and of the RSI Assurance Maladie Professions Libérales-Provinces, C.A.M.P.L.P. for funding the LightCycler 480 real-time thermocycler (Roche®) in Montpellier.

Supplementary materials

1 Supplementary Table S1