S Ahmad

W Sebai

M-P Belleville

Nicolas Brun

A Galarneau

José Sanchez-Marcano

Enzymatic monolithic reactors for micropollutants degradation

Keywords: enzymatic flow through reactor, monolith, laccase, antibiotic degradation, modeling, water treatment

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Introduction

Antibiotics are commonly used in human and animal medicine for controlling and treatment of infectious diseases. A considerable amount of antibiotics is not metabolized in human or animal body and is thus discharged in wastewater and ends up in rivers, lakes and landfills. Antibiotics are refractory micropollutants, their concentration in treated wastewaters depends on their origin (domestic, hospitals or industrial), it is ranged from 10 ng L -1 to 10 µg L -1 [START_REF] Danner | Antibiotic pollution in surface fresh waters: Occurrence and effects[END_REF]. Antibiotics, like tetracycline (TC) and oxytetracycline (OTC), are anti-microbial agents recalcitrant in nature; they are currently found in wastewaters and natural water bodies [START_REF] Szymańska | Presence of antibiotics in the aquatic environment in Europe and their analytical monitoring: Recent trends and perspectives[END_REF]. It has been found that TC and OTC increase the ecotoxicity and anti-microbial resistance [START_REF] Halling-Sørensen | Toxicity of Tetracyclines and Tetracycline Degradation Products to Environmentally Relevant Bacteria, Including Selected Tetracycline-Resistant Bacteria[END_REF], thus their presence in water can cause threat to human health.

Many pharmaceuticals present in wastewaters are not completely removed by conventional treatment plants [START_REF] Stackelberg | Persistence of pharmaceutical compounds and other organic wastewater contaminants in a conventional drinking-water-treatment plant[END_REF][START_REF] Bilal | Biocatalytic degradation/redefining "removal" fate of pharmaceutically active compounds and antibiotics in the aquatic environment[END_REF] and hence alternate removal techniques needs to be developed for the complete and efficient removal of these products. Biodegradation of antibiotics is recently been explored using oxidoreductases enzymes as a bio-catalyst [START_REF] Bilal | Biocatalytic degradation/redefining "removal" fate of pharmaceutically active compounds and antibiotics in the aquatic environment[END_REF].

Laccases are able to catalyze the oxidation of various aromatic compounds (particularly phenols) with the concomitant reduction of oxygen to water Their active site consists of 4 copper atoms, one responsible for the oxidation of phenolic groups and a cluster of 3 copper atoms responsible for the activation of oxygen. However, the mechanism of action of laccases, and in particular the role of the metal center, remains poorly known but many authors agree with the fact that it is a radical mechanism [START_REF] Varga | Enzymatic treatment and subsequent toxicity of organic micropollutants using oxidoreductases -A review[END_REF][START_REF] Cui | Optimization protocols and improved strategies of cross-linked enzyme aggregates technology: current development and future challenges[END_REF][START_REF] Barbosa | Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties[END_REF][START_REF] Boudrant | Parameters necessary to define an immobilized enzyme preparation[END_REF] (Figure 1). Laccase (EC 1.10.3.2) from Trametes versicolor, is an oxidoreductase enzyme which had been extensively studied for the biodegradation of antibiotics and found very promising due to its ability to oxidize large variety of these pharmaceuticals.

Nevertheless, generally enzymatic processes present some drawbacks like inactivation, lack of stability and reusability of enzymes, which can make the process uneconomical and unfeasible to be implemented on "large scale".

Immobilization of enzymes improves the reusability and sustainability of enzymatic process [START_REF] Cui | Optimization protocols and improved strategies of cross-linked enzyme aggregates technology: current development and future challenges[END_REF] and allows bioreactors operating in continuous mode. Immobilization may improve many enzymes properties like activity, stability, selectivity, specificity, inhibition [START_REF] Barbosa | Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties[END_REF][START_REF] Boudrant | Parameters necessary to define an immobilized enzyme preparation[END_REF][START_REF] Virgen-Ortíz | Desorption of Lipases Immobilized on Octyl-Agarose Beads and Coated with Ionic Polymers after Thermal Inactivation[END_REF][START_REF] Gonçalves | Trends on enzyme immobilization researches based on bibliometric analysis[END_REF].

Different Immobilization techniques have been used with different support materials [START_REF] Mohamad | An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes[END_REF], they include adsorption [START_REF] Laveille | Hemoglobin immobilized on mesoporous silica as effective material for the removal of polycyclic aromatic hydrocarbons pollutants from water[END_REF], encapsulation [START_REF] Laveille | Oxidation reactions using air as oxidant thanks to silica nanoreactors containing GOx/peroxidases bienzymatic systems[END_REF][START_REF] Cazelles | Reduction of CO 2 to methanol by a polyenzymatic system encapsulated in phospholipids-silica nanocapsules[END_REF][START_REF] Galarneau | Immobilization of lipase on silicas. Relevance of textural and interfacial properties on activity and selectivity[END_REF], covalent grafting [START_REF] Mureseanu | Selective Cu2+ adsorption and recovery from contaminated water using mesoporous hybrid silica bioadsorbents[END_REF], and adsorption followed by crosslinking [START_REF] Hartmann | Biocatalysis with enzymes immobilized on mesoporous hosts : the status quo and future trends[END_REF]. The best method for biomolecules immobilization is not universal and will depend on the biomolecule itself. For laccases it was shown that a covalent grafting via a crosslinking agent such as glutaraldehyde is very efficient. Immobilization support materials like ceramic mono or multichannel membranes [START_REF] De Cazes | Design and optimization of an enzymatic membrane reactor for tetracycline degradation[END_REF][START_REF] De Cazes | Characterization of laccase-grafted ceramic membranes for pharmaceuticals degradation[END_REF], nano-fibrous membranes [START_REF] Taheran | Covalent Immobilization of Laccase onto Nanofibrous Membrane for Degradation of Pharmaceutical Residues in Water, ACS Sustain[END_REF], metal nanoparticles [START_REF] Shi | Removal of sulfonamide antibiotics by oriented immobilized laccase on Fe3O4 nanoparticles with natural mediators[END_REF], carbon nanotubes [START_REF] Kadam | Supermagnetically Tuned Halloysite Nanotubes Functionalized with Aminosilane for Covalent Laccase Immobilization[END_REF] and mesoporous materials [START_REF] Wen | Immobilized laccase on bentonite-derived mesoporous materials for removal of tetracycline[END_REF], magnetic cross-linked enzyme aggregates (M-CLEAs) [START_REF] Yang | Degradation of tetracycline by immobilized laccase and the proposed transformation pathway[END_REF] have successfully demonstrated the immobilization of laccase for the degradation of antibiotics. However, many of these materials present a limited surface area available for grafting; this is especially the case of membranes. Indeed, enzyme immobilization yields and reactivity are relatively low [START_REF] Abejon | Design, economic evaluation and optimization of enzymatic membrane reactors for antibiotics degradation in wastewaters[END_REF][START_REF] Abejon | Large-scale enzymatic membrane reactors for tetracycline degradation in WWTP effluents[END_REF]. Silica monoliths prepared by a combination of spinodal decomposition and sol-gel process have a homogeneous structure of macropores interconnected with mesopores in the skeleton.

Macropores diameters are adjustable between 1 to 30 μm and mesopores diameters are adjustable between 8 to 20 nm leading to surface areas of 700 to 300 m 2 g -1 and a total porosity around 85% [START_REF] Galarneau | Hierarchical porous silica monoliths: A novel class of microreactors for process intensification in catalysis and adsorption[END_REF]. These monoliths are able to allow large water flows to transfer through their structure with low pressure drop due to their homogeneous network of large communicating macropores.

Enzymatic monoliths are interesting candidates to be applied in continuous flow through plug reactors. In this operating mode, the flow is forced to pass exclusively through the monolith porosity where the biocatalyst is immobilized and the contact between the reactants and the enzyme occurs during the mass transfer process. This configuration results in better control of the reaction through the "micro-reactor concept", where the distance between the biocatalyst and the substrate is considerably reduced, which increases the probability of reaction [START_REF] Westermann | Flow-through catalytic membrane reactors-Principles and applications[END_REF]. Moreover, monoliths have been successfully applied as a support material in chromatography [START_REF] Puy | Influence of the hydrothermal treatment on the chromatographic properties of monolithic silica capillaries for nano-liquid chromatography or capillary electrochromatography[END_REF], catalysis [START_REF] Fajula | Combining Phase Separation with Pseudomorphic Transformation for the Control of the Pore Architecture of Functional Materials: A Review[END_REF][START_REF] Sachse | Alumina-grafted macro-/mesoporous silica monoliths as continuous flow microreactors for the Diels-Alder reaction[END_REF] and for enzyme immobilization [START_REF] Wang | Immobilization of penicillin G acylase on macrocellular heterogeneous silica-based monoliths[END_REF]. All these studies concluded that these structures are very promising as a solid support to immobilize catalysts due to their highly interconnected and homogeneous porous structure with large surface area available for grafting. Therefore, the aim of this research study was to immobilize laccase on silica monoliths to further explore the applicability of enzymatic monoliths for the degradation of TC, an antibiotic chosen as a model substrate. In this research work, silica monoliths were prepared with uniform macropores of 20 μm and mesopores of 20 nm diameters, and grafted with a commercial laccase from Trametes versicolor (kinetic diameter 6 nm, with four cupper atoms as metallic active center and an isoelectric point around 4.5). Covalent binding through glutaraldehyde was chosen as immobilization method since it has been successfully used to grafted laccase on ceramic membranes [START_REF] De Cazes | Design and optimization of an enzymatic membrane reactor for tetracycline degradation[END_REF][START_REF] De Cazes | Characterization of laccase-grafted ceramic membranes for pharmaceuticals degradation[END_REF]. Enzymatic monoliths were then used as a plug flow reactor operating in recycling-mode for TC degradation. Finally, a model was built by coupling the hydrodynamics with the experimental reaction kinetics using computational fluid dynamics (CFD) in order to optimize and simulate the process of degradation.

Materials and methods

Polyethylene glycol (PEG) (99%, 100 kDa), tetraethoxysilane (TEOS) (99%), (3-aminopropyl) triethoxysilane (APTES) (99%), commercial powder of laccase from Trametes versicolor (activity ≥0.5 U mg -1 ), tetracycline (TC) (≥98.0%), glutaraldehyde (25% v/v) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (≥98.0%,) were purchased from Sigma-Aldrich.

Silica monoliths synthesis, functionalization and characterization.

Silica monoliths with hierarchical porosity (macro-/mesoporosity) were prepared by a controlled sol-gel process with TEOS and PEG according to a method previously reported [START_REF] Galarneau | Hierarchical porous silica monoliths: A novel class of microreactors for process intensification in catalysis and adsorption[END_REF]. The surface area of silica monolith before enzyme immobilization was 336 m 2 g -1 . Prior to enzyme immobilization, the monoliths were pre-activated by grafting amino groups. For this purpose, they were firstly dried at 250°C under vacuum for 4 h and then immersed in a solution of APTES in ethanol (in order to have an excess of 10 amino groups per nm 2 ). The activation was carried out under reflux (80 °C) overnight. The reactors were built by cladding the monoliths inside Teflon™ heat shrinkable gains at 180 °C for 2 h to be finally connected to stainless-steel tubing. This tubular plug-flow reactor configuration allowed the liquid feed penetrating the internal porosity of activated monoliths without preferential ways. The monoliths were named as "preactivated monoliths" before enzyme grafting and "laccaseactivated" or "enzymatic monoliths" after enzyme immobilization, for instance see section 2.3.

Monoliths were characterized structurally by scanning electron microscopy (SEM) (Hitachi S-4800 I FEG-SEM). Porosity was determined by mercury porosimetry (Micrometrics Autopore 9220) and nitrogen adsorption at 77 K (Micromeritics Tristar 3020). Before nitrogen adsorption measurements, monoliths were outgazed for 12h at 250°C and 80°C for raw and grafted silica monoliths respectively. The quantitative determination of the number of amine functions on the surface of the silica monolith was determined by TGA analysis (PerkinElmer STA 6000) using a high resolution program (from 40 to 900 °C) under air with a ramp of 10°C per minute. The number of grafted functions was determined by the loss of weight of the material from 200°C to 900°C and taking in account the dehydroxylation of the surface of the monolith.

Permeability tests

Permeability tests were carried out both before and after immobilization of enzymes by filtrating osmosed water through the monoliths. Monoliths of 0.6 cm diameter and 0.5 cm length were connected to a flow system consisting of a HPLC pump and a pressure gauge as shown in Figure 1. The flow rates were varied from 1 mL min -1 to 5 mL min -1 . The back pressure exerted by monoliths was measured thanks to a pressure gauge and then permeability (k) was calculated by Darcy equation as follows:

𝑘 = 𝑄 𝐴 𝑙 ∆𝑃 𝜇 (1)
With k Permeability coefficient (m 2 ), Q the flow rate (m 3 s -1 ), A the cross section of the monolith (m 2 ), μ the viscosity of the fluid (μ = 1.002 mPas at 20 °C for water), l the length of the monolith (m) and ΔP the difference of pressure at the outlet and inlet of the monolith (Pa).

Laccase immobilization

Covalent immobilization of enzymes through glutaraldehyde binding is a widely used immobilization method that prevents enzyme leakage. Moreover, previous works confirmed that this method can be successfully used to grafted this commercial laccase on ceramic membranes [START_REF] De Cazes | Design and optimization of an enzymatic membrane reactor for tetracycline degradation[END_REF][START_REF] De Cazes | Characterization of laccase-grafted ceramic membranes for pharmaceuticals degradation[END_REF]. Laccase immobilization was carried out by putting into contact the preactivated monoliths within a glutaraldehyde solution 4 % (v/v) prepared in a citrate phosphate buffer solution (pH 7, 0.1 M). For this purpose cladded monoliths were filled with 0.5 mL of glutaraldehyde solution and allowed to react for 30 minutes. Monoliths were then rinsed 5 times with 0.5 mL of the same buffer solution in order to remove unreacted glutaraldehyde. During the reaction between glutaraldehyde and APTES the white or pale yellow monoliths turn deep orange within minutes. This change in colour indicates that the reaction has occurred and has been explained by George et al [START_REF] George | Grafting of Acrylonitrile onto Gelatin in Zinc Chloride Medium[END_REF] as the result of the formation of an aldimine (Schiff linkage (=CHN=), between the free amino groups of APTES and glutaraldehyde.

Finally, monoliths were filled a volume of laccase solution (5±1 U mL sol -1 ) prepared in a citrate phosphate buffer (pH 7, 0.1 M). For this purpose the enzymatic solution was introduced at the entrance of the tube containing the monolith, allowing the penetration into the monolith porosity by capillarity. The tube was closed and turned several times during 1 hour, and then rinsed with the buffer solution. After immobilization, enzymatic monoliths were stored in the same buffer solution at 4°C. It is worth noting that grafted monoliths were stable under washings and tests, thus it can be assumed that enzymes were covalently immobilized.

The schematics of covalent bonding through the monolith and APTES and formation of aldimine are illustrated in Figure 2.

Activity measurement

Activity (U mL -1 ) of free laccase was measured using 2,2′-Azinobis-(3-ethylbenzthiazoline-6sulphonate) (ABTS) as substrate. 100 µL of diluted enzyme solution was added to 900 µL of 1 mM ABTS solution prepared in a citrate phosphate buffer solution (pH 4, 0.1 M), absorbance change was followed for 1 minute by spectrophotometry at 420 nm (ɛ = 3600 M -1 cm -1 ), activity (U mL -1 ) was then estimated by slope of absorbance vs time.

The measurement of the enzymatic activity of immobilized laccase was carried out with crushed laccase-activated monoliths; 5 mg of the active powder were added to 25 mL of ABTS solution (1 mM) prepared in the same citrate phosphate buffer mentioned above in a 100 mL flask at 25°C, under air and agitation. The activity (U mg monolith -1 ) was estimated as explained above by the change in absorbance of the ABTS solution.

The enzyme immobilization yield was calculated considering the activity of the enzyme stock solution used for immobilization before and after activation process taking into account the activity of rising solutions as expressed in Equation 2 and 3.

𝐴 𝑖𝑚𝑚𝑜𝑏𝑖𝑙𝑖𝑧𝑒𝑑 = 𝐴 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 -(𝐴 𝑙𝑒𝑓𝑡 + ∑ 𝐴 𝑟𝑖𝑛𝑠𝑖𝑛𝑔 ) (2)
𝜌 𝑖𝑚𝑚𝑜𝑏𝑖𝑙𝑖𝑧𝑎𝑡𝑖𝑜𝑛 = 𝐴 𝑖𝑚𝑚𝑜𝑏𝑖𝑙𝑖𝑧𝑒𝑑 𝐴 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 × 100 (3)

Determination of reaction kinetics parameters for free and immobilized laccase

Apparent reaction kinetic parameters for free and immobilized laccase (with crushed monoliths)

were determined using ABTS and TC as substrates. For this purpose an equivalent amount of 0.01U of enzyme (liquid solution of enzyme or crushed laccase-activated monolith) were added to 25 mL of ABTS solution (20 -100 µM in citrate phosphate buffer solution, pH 4). In the case of experiments with TC the amount of free (5.0 U) and immobilized enzyme (3.0 U) were added to 10 mL of TC solution (2 -20 ppm in osmosed water, pH 6). Runs for both substrates were carried out in stirred Erlenmeyer flasks under air atmosphere. Linear regression was applied on the obtained data and then apparent reaction kinetic parameters (V max and K M ) were determined by Lineweaver-Burke plot according to equation 4:

1 𝑉 = 𝐾 𝑀 𝑉 𝑚𝑎𝑥 1 [𝑆] + 1 𝑉 𝑚𝑎𝑥 (4)
Here V is the rate of substrate consumption (µmols -1 ); V max is the maximum rate of substrate consumption (µmols -1 ) and K M is the Michaelis-Menten constant (µmol) and S the substrate concentration (µmol).

Storage and operational stability of laccase-activated monoliths

The stability of enzymatic monoliths under storage conditions (citrate phosphate buffer (pH 7) at 4 °C was determined by measuring the residual activity over a 30-day period. For this purpose several enzymatic monoliths [START_REF] Virgen-Ortíz | Desorption of Lipases Immobilized on Octyl-Agarose Beads and Coated with Ionic Polymers after Thermal Inactivation[END_REF] were prepared under the same conditions, then they were stored and every 5 days samples were taken and their activity measured according to the protocol explained in section 2.4. The stability of laccase-activated monoliths under continuous operation was also studied for TC degradation. For this purpose TC (20 ppm) degradation tests were carried out following a sequential procedure: after 8 hours of reaction the pilot was emptied, rinsed with osmosed water and kept at room temperature overnight, then a new fresh TC solution was introduced and the run start again. The cycles were repeated for a total duration of 75 hours.

Modeling and Simulation

To setup the "steady-state model" a homogenous porous structure was considered, keeping in view a global porosity and permeability through all monolith volume. A Steady state modeling was based on the coupling of mass balances, hydrodynamics inside the monoliths' porosity and enzymatic kinetics [START_REF] Ameur | Development of a multichannel monolith large-scale enzymatic membrane and application in an immobilized enzymatic membrane reactor[END_REF]. Permeability and reaction kinetics parameters (K M and V max ) used in modeling were measured experimentally as discussed in section 2. Similarly reaction rate (Eq.7) was estimated by introducing Michaelis-Menten equation applied to the TC degradation in the model.

𝑅 𝑖 =

𝑉 𝑚𝑎𝑥 𝐶 𝑖 𝐾 𝑀 +𝐶 𝑖 [START_REF] Cui | Optimization protocols and improved strategies of cross-linked enzyme aggregates technology: current development and future challenges[END_REF] where C i is TC concentration.

The main assumptions taken for the model are:

-Isothermal operation.

-Global and homogeneous porosity through all monolith volume.

-Enzymatic activity and permeate flow rate are constant.

-Physical properties of reaction media (viscosity, density) are constant under different operating conditions.

Equations ( 5) to [START_REF] Cui | Optimization protocols and improved strategies of cross-linked enzyme aggregates technology: current development and future challenges[END_REF] were coupled and solved simultaneously with COMSOL Multiphysics ® software (version 5.3). Physics controlled fluid dynamic fine size mesh was used. Maximum and minimum element size was 0.06 and 0.32 mm. Further decreasing the element size had no effect on output TC concentration.

Dynamic modeling was then performed by applying mass transport balance in the reservoir and closed loop as explained by Younas et al. [START_REF] Younas | Extraction of aroma compounds in a HFMC: Dynamic modelling and simulation[END_REF]. For this purpose Matlab ® (version 18) was implemented to update the output results obtained from Comsol Multiphysics ® . In order to couple the both software, live-link application was used.

Mass transport modeling in the reservoir

As far as the TC concentration in the tank decreased gradually the dynamic variation of TC concentration with time was calculated by applying a transient differential equation around the tank:

𝑉 𝑑𝐶 𝑇𝐶 𝑑𝑡 = 𝑄(𝐶 𝑖𝑛 𝑛+1 -𝐶 𝑜𝑢𝑡 𝑛+1 ) (8)

Results and discussion

Structural characterization of monoliths

Nitrogen adsorption/desorption isotherms at 77 K and mercury porosimetry measurements shown that monoliths present a very high porous structure with a total porosity of ε = 0.83 (V macro = 3.0 mL g -1 , V meso = 0.8 mL g -1 ), a low density of 0.20 g cm -3 and mesopores with a mean diameter of 18 nm developing a large surface area of 336 m 2 g -1 . The macroporous surface determined by mercury porosimetry is around 1 m 2 g -1 . After the activation, amino-functionalized monoliths contain 0.8 mmol NH 2 g monolith -1 corresponding to a grafting of 1.5 NH 2 per nm 2 with 72% of tridentate silanes (28% of bidentate). After laccase immobilization by covalent grafting through glutaraldehyde bounds important textural changes occur with a decrease of the specific surface area (164 m 2 g -1 ), V meso (0.5 mL g -1 ) and mesopores diameter (15 nm). Figure 4 shows a SEM picture of the structure of a raw silica monolith (4a) and laccase-grafted monolith (3b). From Many hypotheses can be done to explain this behavior; on one hand laccase grafting was occurring more extensively in the mesoporosity, on the other hand a very thin layer of the immobilized enzyme was perhaps covering the internal surface of the surface of the solid (as is observed in Figures 4b and4d above). The very thin coating does not significantly affect the macroporosity but it can partially cover mesopores' mouth. Moreover, the decrease of the specific surface, mesopores volume and diameter discussed above are also indications that both hypotheses are possible.

Permeability coefficient

When water flow rate varied from 1 to 4.5 mL min -1 through a cladded monolith, the upstream pressure increased linearly from 10 to 40 mbar. These measured values of upstream pressure are very low and confirm that monoliths present interesting structure with very high porosity and communicating macropores. Permeability coefficient (k) of raw silica monoliths calculated from Equation1 was equal to 5.9 10 -12 m 2 . Other authors has reported the same order of magnitude of permeability (11 10 -12 m 2 ) [START_REF] Ciemięga | Continuous-Flow Monolithic Silica Microreactors with Arenesulphonic Acid Groups: Structure-Catalytic Activity Relationships[END_REF] but with monoliths with pore size of 30 -50 µm. After enzyme immobilization, no change was observed for the value of permeability coefficient; enzyme grafting does not affect the mass transfer.

Activity and immobilization yield

The activity of a single monolith (6 mm diameter and 5 mm length, mass of 50 mg) was determined using crushed monoliths as explained in section 2.4. The mean activity of enzymatic monoliths was of 5.0 ± 0.5 U. The immobilization yield of enzymes is an important factor for process efficiency and cost. Immobilization yield measured was equal to 80 ± 5%. This relatively high immobilization yield is certainly reached because the small volume of enzymatic solution employed together with the high specific surface with a high number of reactive sites (amino groups coupled with glutaraldehyde). The immobilization probably also favored by the structure of monoliths of extremely interconnected macroporous and mesoporous networks and the thin skeleton (6 µm), which reduces the diffusion path lengths facilitating the access to the numerous reactive sites. It is important to notice that grafted monoliths were stable under washings and after successive reactivity tests, thus it can be assumed that enzymes were covalently immobilized.

These initial results conclude a successful immobilization of laccase on silica monoliths and demonstrated very high immobilization yield.

Determination of apparent reaction kinetic parameters

Apparent kinetic parameters K M and V max for both free and immobilized enzymes (crushed monoliths) were determined with two different substrates ABTS and TC by the method discussed above in section 2.5. Measured values are shown in Table 1. It can be observed that after immobilization the apparent K M value decreased by 1.6 and 4 times for ABTS and TC, respectively. This result is surprising because generally structural conformation and diffusion limitations occur after immobilization. However, a similar trend has already been reported for laccase immobilized on membranes [START_REF] De Cazes | Design and optimization of an enzymatic membrane reactor for tetracycline degradation[END_REF][START_REF] De Cazes | Characterization of laccase-grafted ceramic membranes for pharmaceuticals degradation[END_REF] and lipase on mesoporous SiO 2 microparticles [START_REF] Kumar | SiO2 microparticles with carbon nanotube-derived mesopores as an efficient support for enzyme immobilization[END_REF].

V max value depends on the concentration of the enzymatic solution (for free enzymes) or the amount of immobilized enzymes on a support. In this work the kinetic experiments with ABTS were carried out in order to have in both cases, the same amount of enzymes in terms of activity towards ABTS (i.e. 0.25 U). It was observed that V max value was higher for immobilized enzymes indicating a better reactivity of immobilized biocatalyst. As in the case of K M , this result is in good agreement with previous results reported by De Cazes et al (2014) [START_REF] De Cazes | Design and optimization of an enzymatic membrane reactor for tetracycline degradation[END_REF][START_REF] De Cazes | Characterization of laccase-grafted ceramic membranes for pharmaceuticals degradation[END_REF]. For TC, since activity used for free and immobilized was not same (0.3 U mL -1 ) and (0.15 U mL -1 ), respectively, it was not possible to compare the V max . Some authors [START_REF] Westermann | Flow-through catalytic membrane reactors-Principles and applications[END_REF][START_REF] Puy | Influence of the hydrothermal treatment on the chromatographic properties of monolithic silica capillaries for nano-liquid chromatography or capillary electrochromatography[END_REF] have noticed that the velocity of oxidation of substrates by laccases can decrease very rapidly during the first hours of reaction because the oxygen concentration depletion near catalytic sites. Indeed, the conversion can be improved by stirring on air atmosphere or reach 100% by using pure oxygen [START_REF] Puy | Influence of the hydrothermal treatment on the chromatographic properties of monolithic silica capillaries for nano-liquid chromatography or capillary electrochromatography[END_REF]. In the case of experiments carried out in the monolithic system in continuous configuration no stirring under air or oxygen was done. In addition, the concentration of enzyme was relatively high (15.0 U for 30 mL of 20 ppm of TC solution) and even if the calculated total initial amount of oxygen (7.7 10 -7 mol) was 5.5 times the initial amount of TC (1.4 10 -7 mol) it is possible that the oxygen depletion near the catalytic sites caused the observed decrease of the degradation rate. This effect was already demonstrated in the literature [START_REF] Westermann | Flow-through catalytic membrane reactors-Principles and applications[END_REF][START_REF] Puy | Influence of the hydrothermal treatment on the chromatographic properties of monolithic silica capillaries for nano-liquid chromatography or capillary electrochromatography[END_REF]. Moreover, TC degradation results in many different by-products that can also be oxidized by laccases [START_REF] Llorca | Identification of new transformation products during enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass spectrometer LTQ-orbitrap[END_REF][START_REF] Yang | Degradation of tetracycline by immobilized laccase and the proposed transformation pathway[END_REF] (sometimes, first oxidation products are more reactive that the initial substrate). Indeed, the oxygen necessary for the degradation of TC can also be consumed by these side reactions. This point is certainly critical for the conversion reached with enzymatic reaction within the porosity of the monoliths. Additional experiments were carried out under the same conditions (volume and concentration of solution, amount of activated monoliths) of the continuous configuration but with crushed activated monoliths in a stirred batch reactor with air bubbling. The results showed a total TC depletion in 6 hours. Indeed, the hypothesis of inhibition by products formation can be omitted.

Storage and operational stability of laccase-activated monoliths

Storage and operational stability of immobilized laccase is an important step for its practical applicability and plays a key role in process cost. Storage stability was studied for 30 days as explained in section 2.7. The results obtained demonstrated that there was no loss of enzymatic activity during the first 5 days of storage at 4°C. Then during the other 25 days of storage the activity decreased slowly reaching approximately 85% of the initial one. Similarly, the activity of free enzymes was studied under same storage conditions and for same time period as immobilized enzyme. The results demonstrated that there was no noticeable change in the activity of free enzymes even after 30 days of storage at 4ºC.

The operational stability of enzymatic monoliths was studied during 75 hours with the cyclical procedure described in section 2.7; the results are displayed in Figure 4. We can observe that the evolution of TC concentration is very similar for all of the cycles. As it was discussed in section 4.5, TC concentration decreases very rapidly during the first minutes of operation and then the degradation rate slowdown. When the solution is changed with a fresh one containing the initial TC and oxygen concentration the same pattern of TC depletion is obtained. Indeed we can conclude that the enzyme immobilized in monoliths is stable during at least 75 hours of operation. The slowdown of the reaction rate could be avoided by using air or oxygen bubbling in the reservoir.

Simulation

As explained in section 3, the steady state model (Eq.5-7) was coupled with the dynamic mass

Conclusions

Silica monoliths with a homogenous mesoporous/macroporous structure featuring, high specific surface area and porosity were successfully prepared. These monoliths were preactivated with APTES and glutaraldehyde in order to covalently immobilize laccase from Trametes versicolor.

From reaction kinetic parameters V max and K M values, it was concluded that after immobilization laccase was more active without losing affinity to substrate. Enzymatic monoliths were successfully implemented at laboratory scale for the degradation of tetracycline as model micropollutant in a continuous plug flow mode with recycling. System of three monoliths, connected in series was able to degrade 40-50% of TC in 20 ppm solutions in 5 hours. The immobilized laccase on silica monoliths exhibited high stability during storage as well as good operational stability during 75 hours of sequential operation both factors are interesting for their applicability at large scale.

Modelling and simulation of the TC concentration evolution for the plug flow reactor coupling the hydrodynamics and kinetics was in good agreement with experimental results for the first 5 hours of reaction. However, at longer times of reaction it was observed experimentally that the degradation rate slowdown, this behavior may be the result of a high enzymatic activity which causes a rapid lack of oxygen, co-substrate essential for the oxidation reaction. Indeed, the actual configuration of the plug flow reactor doesn't allow a continuous aeration or feed of oxygen. In a future; the design and scale up the pilot unit will have to consider an aeration system in the reservoir or in between the monoliths and a continuous process without recycling. Moreover further experiments will be conducted in order to identify the parameters of a new kinetics considering tetracycline and oxygen concentration.

Notations and symbols

Symbol Defintion

A Cross-sectional area (m 2 )

A immobilized Activity immobilized (U g -1 )

A initial Activity of enzymatic solution before immobilization (U )

A left Activity of enzymatic solution after immobilization (U)

A

Figure. 1

 1 Figure. 1 General reaction mechanism of laccases.

Figure 2 .

 2 Figure 2. Schematic of enzyme covalent bonding through APTES loaded monoliths

2. 6

 6 Tetracycline degradation testsTC degradation tests were carried out with three laccase-activated monoliths connected in series (0.6 cm diameter and 0.5 cm length, each, equivalent to a total of 15.0 U ± 1.5 U of enzymatic activity). The recirculation of the flow was ensured by a HPLC pump (Gibson model: 321, France) as shown in Figure3. A pressure sensor was connected at inlet of monoliths to measure back pressure. TC solution (20 ppm) prepared in osmosed water pH 6, at 25°C was flowed through the enzymatic monoliths in continuous recycle-mode. The flow rate was kept at 1 mL min -1 . Samples were taken from the reservoir and analyzed by high-performance liquid chromatography coupled to triple-quadrupole mass spectrometry (HPLC-MS). Samples were injected through a Macherey-Nagel C18 column (50 mm x 2 mm) with a Waters e2695 Separations Module, and the 410 m/z fragment was detected with a Micromass Quattro micro API device.

Figure 3 .

 3 Figure 3. Schematic draw of pilot for TC degradation with recycling.

5 .

 5 From the results of steadystate modeling, TC concentration evolution along the length of reactor was computed. Dynamic model was setup by taking mass transfer balance on the feed tank considering monolith as a plugflow reactor. The concentration field (C) for TC was calculated from the mass balance and reaction rate taking into account the TC depletion. As far as the reaction extend was based on the reactant consumption for the modeling, the classical mass balance equation coupling diffusion, convective flow and reaction kinetics was be employed: 𝛻(-𝐷 𝑖 𝛻𝑐 𝑖 ) + 𝑢𝛻𝑐 𝑖 = 𝑅𝑖 (5) Eq. 5 is the basic mass transport equation which couples the reaction rate with the flow within the monolith geometry, it is applied to simulate the concentration gradient (𝛻𝑐 𝑖 ) along the length of the monolith geometry. It includes TC diffusion coefficient (Di), velocity within porous domain (u), and enzymatic reaction rate (Ri). Velocity was calculated within geometry by applying Brinkman equation (Eq. 6) which is used to describe flowing fluids though porous materials under laminar conditions [36-38]. Indeed the Re value calculated for the flow conditions in this study (i.e. 2) was less than 10 allowing the use of Brinkman equation. 𝑢)𝐼] -(𝜇𝑘 -1 + 𝛽 𝐹 |𝑢| + 𝑄 𝑚  𝑝 2 ) 𝑢 + 𝐹 = 0(6)where  p is the global porosity of the domain; k (m 2 ) is the permeability of the domain; u (m s -1 ) is the Darcy's velocity (the ratio of flow rate to monolith cross section) and µ (mPa s) the viscosity of the solution.

Figure

  Figure4awe can notice the interconnected macropores network of approximately 20 μm of average diameter and a skeleton thickness of around 6 μm. The aspect of grafted monolith presents a slight difference respect to the raw one, only the softness of the surface seems to be

Figure 4 .

 4 Figure 4. SEM of monoliths showing the macroporous structure. a) Raw monolith, b) Laccase-grafted monolith, c) and d) are respectively magnification figures a) and b).

  ppm) degradation results are displayed in Figure5. Control experiments (blank tests) were carried out to study the effect of TC self-degradation or adsorption on the evolution of TC concentration. For these tests, enzymatic monoliths were first thermally deactivated by heating in oven at 100°C for 2 hours. The results of control experiments show that less than 2% of initial TC was removed. The evolution of TC concentration with enzymatic monoliths shows that laccase-activated monoliths were able to degrade 40% of initial TC in first 5 hours and then the degradation rate decreases dramatically. From this point the TC concentration decreases slowly up to 50% at 26 hours of reaction. Many hypotheses can be done to explain this behavior; they include the decrease of substrate concentration, if the kinetics obeys to a Michaelis-Menten equation. However, the diminution of the oxygen content, or even the formation of inhibiting byproducts can also be considered.

Figure 5 .

 5 Figure 5. Degradation of TC. C/C 0 : variation of the TC concentration respect to the initial TC concentration (20 ppm).

Figure 6 :

 6 Figure 6: Operational stability of monoliths for 75 hours of sequential operation.

(Eq. 8 )

 8 transfer applied on feed tank, using the outlet results from monolith and running a closed loop in Matlab®. Results of the dynamic mass balance simulation in the reservoir tank allowed calculating the degradation rates of substrate. The evolution of residual TC concentration is shown in Figure5a. The results of the model simulate relatively well the experimental evolution of TC concentration in the tank during the first 5 hours, period in which we can consider that Michaelis-Menten kinetics is valid because we don't have deficiency of oxygen to carry out the degradation. Steady-state model was solved in COMSOL Multiphysics ® considering the three monoliths in series as a single plug flow reactor. The output concentration for single pass (residence time for single pass 45-50 seconds) was calculated from surface integration at the outlet of monolith. Results are shown in Figure5b. It can be seen that TC concentration decreases along the reactor's length, but the conversion is relatively low at each passage (0.02 µmol L -1 ).

Figure 7 .

 7 Figure 7. a) Evolution of TC concentration inside the reservoir. Comparison of experimental and modeling base results from a dynamic simulation,  experimental results, simulation. b) Steady state CFD simulation results showing TC concentration change along the length of the reactor for a single pass.

F

  rinsing Activity of the rinsing solution (U) Volume Force (Nm -3 )

Table 1 . Apparent reaction kinetic parameters for the oxidation of ABTS and tetracycline (TC) with free and immobilized laccase.

 1

Acknowledgments

This research was funded by the ANR French agency, project MUSE ANR-16-IDEX-0006 project DEMEMO. Mr. S. Ahmad acknowledges the Higher Education Commission, Pakistan for the PhD scholarship. The authors wish to acknowledge the support from the chemistry plateform of campus in Montpellier (Plateform MEA University of Montpellier), where SEM has been performed.

I

Identity vector k Permeability coefficient (m 2 )

l Length of the monolith (m)

R i

Reaction rate (µmol m -3 s -1 )

[S] Substrate concentration (µmol L -1 ) u Darcy velocity (m s -1 )

V Reaction rate (µmolmin -1 )

V max Maximum reaction rate (µmolmin -1 ) µ Fluid viscosity (mPa.s)

Forchheimier coefficient (kg m -4 ) ∑ Summation


Brenda Omaña-Sanz
email: brenda.omana-sanz@univ-lille.fr

Djadidi Toybou

Ludovic Lesven

Valérie Gaucher

Alexandre Fadel

Ahmed Addad

Philippe Recourt

Delphine Yeghicheyan

Devrah Arndt

Caroline Celle

Valerie Gaucher

Jean-Pierre Simonato

Christopher Vulpe

Laurent Charlet

Sophie Sobanska

Benjamin Gilbert

Annette Hofmann
email: annette.hofmann@univ-lille.fr

An electrochemical method to rapidly assess the environmental risk of silver release from nanowire transparent conductive films

Keywords:

An electrochemical method to rapidly assess the environmental risk of silver release from nanowire transparent conductive films

Introduction

Silver nanowires (AgNWs) are one of the most promising alternative engineered nanomaterials (ENMs) to replace indium tin oxide (ITO) in transparent electrodes (Sannicolo et al., 2016). Random networks of AgNWs are being used for transparent conductive films (TCFs) to be applied in a wide range of technological applications from optoelectronics to heatable films and biomedical devices [START_REF] Doganay | Silver nanowire decorated heatable textiles[END_REF][START_REF] He | Flexible Transparent Conductive Films on the Basis of Ag Nanowires: Design and Applications: A Review[END_REF][START_REF] Toybou | A toxicologyinformed, safer by design approach for the fabrication of transparent electrodes based on silver nanowires[END_REF][START_REF] Zhang | Recent Progress on the Fabrication and Properties of Silver Nanowire-Based Transparent Electrodes[END_REF]. The interconnection of metallic nanowires, e.g. AgNWs, creates electric conductive paths allowing electrical current flow.

It is known that metallic silver is corroded when exposed to atmospheric conditions; similarly silver nanowires are also easily corroded [START_REF] Graedel | Corrosion mechanisms for silver exposed to the atmosphere[END_REF][START_REF] Molleman | Time, pH, and size dependency of silver nanoparticle dissolution: the road to equilibrium[END_REF], thereby causing failures in conductive networks. To counter this problem, application of a nano to micrometer thick layer of a protective coating material over the conductive NW network, before its integration into a device, is often performed in order to stop the diffusion of water vapor and gases, thus to avoid corrosion. Other external damages like scratches and bends are also avoided by this protection.

Different materials have been considered for creation of a protective coating, including reduced graphene oxide [START_REF] Ahn | Improved Thermal Oxidation Stability of Solution-Processable Silver Nanowire Transparent Electrode by Reduced Graphene Oxide[END_REF][START_REF] Duan | Can insulating graphene oxide contribute the enhanced conductivity and durability of silver nanowire coating?[END_REF], metal oxides and carbonates [START_REF] Jeong | Enhancing Thermal Oxidation Stability of Silver Nanowire Transparent Electrodes by Using a Cesium Carbonate-Incorporated Overcoating Layer[END_REF][START_REF] Khan | Stability Enhancement of Silver Nanowire Networks with Conformal ZnO Coatings Deposited by Atmospheric Pressure Spatial Atomic Layer Deposition[END_REF], optical adhesive [START_REF] Miller | Silver Nanowire/Optical Adhesive Coatings as Transparent Electrodes for Flexible Electronics[END_REF], chitosan [START_REF] Jin | Removable Large-Area Ultrasmooth Silver Nanowire Transparent Composite Electrode[END_REF], and polymers [START_REF] Chen | Neutral-pH PEDOT:PSS as over-coating layer for stable silver nanowire flexible transparent conductive films[END_REF][START_REF] Kim | A one-step roll-to-roll process of stable AgNW/PEDOT:PSS solution using imidazole as a mild base for highly conductive and transparent films: optimizations and mechanisms[END_REF][START_REF] Miao | Epoxy-embedded silver nanowire meshes for transparent flexible electrodes[END_REF][START_REF] Moreno | Facile preparation of transparent and conductive polymer films based on silver nanowire/polycarbonate nanocomposites[END_REF]Xu and Zhu, 2012). Although the coatings could delay corrosion, 100 % of effectiveness on the long-term still remains an issue [START_REF] Deignan | The dependence of silver nanowire stability on network composition and processing parameters[END_REF][START_REF] Jiu | The effect of light and humidity on the stability of silver nanowire transparent electrodes[END_REF]. A noticeable factor is that most of the protective coating materials in use, especially polymers have some degree of permeability at the molecular level.

Besides the permeability aspect, wear and tear of the NW containing products throughout their life cycle, i.e. from their active use to end-of-life disposal, may degrade the mechanical and chemical quality of the conductive film and enhance the corrosion of the nanowires.

Corrosion is responsible for the release of ionic silver and the formation of nanoscopic wire debris. These forms of silver are toxic at low levels to aquatic organisms and microorganisms, and to certain types of human cells [START_REF] Lehmann | In Vitro Dermal Safety Assessment of Silver Nanowires after Acute Exposure: Tissue vs[END_REF][START_REF] Liao | Bactericidal and Cytotoxic Properties of Silver Nanoparticles[END_REF]. Corrosion of AgNW TCFs may lead to critical silver exposure of the device user and/or to pollution of the receiving environment.

According to the inventory of available databases e.g. the Nanodatabase, R-Nano and StatNano (R-Nano.fr, https://www.r-nano.fr; STATNANO, https://statnano.com/; The Nanodatabase, http://nanodb.dk/), the number of household products and biomedical devices incorporating nano-silver is rapidly expanding (Bodycap, http://www.bodycapmedical.com/fr/; [START_REF] Naidu | Nano Silver Particles in Biomedical and Clinical Applications[END_REF][START_REF] Rajski | Medical textiles with silver/nanosilver and their potential application for the prevention and control of healthcare-associated infectionsmini-review[END_REF][START_REF] Yao | A Wearable Hydration Sensor with Conformal Nanowire Electrodes[END_REF]. Despite this fact, there is currently a lack of information on toxicological [START_REF] Lehmann | Crumpling of silver nanowires by endolysosomes strongly reduces toxicity[END_REF] and ecotoxicological effects of this widespread occurrence of trace silver. Studies concerning silver release have mainly focused on textiles [START_REF] Part | A review of the fate of engineered nanomaterials in municipal solid waste streams[END_REF]. Standardized risk assessment methods for nanowires is still missing.

Very few predictive models of Ag-ENM fate in the environment have been conducted with data from provenances other than textiles [START_REF] Giese | Risks, Release and Concentrations of Engineered Nanomaterial in the Environment[END_REF][START_REF] Sun | Dynamic Probabilistic Modeling of Environmental Emissions of Engineered Nanomaterials[END_REF]. Concerning silver nanowires, previous studies mainly focused on technical quality control of AgNW transparent electrodes, in particular on lifetime stability of the devices [START_REF] Deignan | The dependence of silver nanowire stability on network composition and processing parameters[END_REF][START_REF] Elechiguerra | Corrosion at the Nanoscale: The Case of Silver Nanowires and Nanoparticles[END_REF][START_REF] Jiu | The effect of light and humidity on the stability of silver nanowire transparent electrodes[END_REF][START_REF] Khaligh | Failure of silver nanowire transparent electrodes under current flow[END_REF][START_REF] Lin | Effects of Environmental Factors on the Stability of Silver Nanowire Transparent Electrodes[END_REF][START_REF] Mayousse | Stability of silver nanowire based electrodes under environmental and electrical stresses[END_REF]. To our knowledge, this is the first investigation to evaluate the capacity of AgNW in TCFs to corrode (hereafter referred to as corrodibility), in order to assess the risk of metal release to users and the environment. In this work, we developed an electrochemical method where corrosive conditions are imposed on AgNW TCF under aqueous conditions, to evaluate the potential for formation of ionic silver and tiny debris of nanowires and their transport from inside to outside the conducting film. An accelerated corrosion test is developed, completed within 20 min. Moreover, it can be considered green methodology as only very low electric power is needed and no chemical reagents other than tiny amounts of standard electrolyte salt are used.

The paper reports corrosion experiments conducted with AgNW films, either uncoated or coated with a protective layer of anti-scratch varnish AE12® of different thicknesses. Some AgNW films underwent natural alteration prior to experiments. These samples were considered as "pre-aged" and representative of nanoproducts during their life cycle or at end of life. Two commercial products were also investigated.

Materials and Methods

AgNW film samples

AgNW networks were deposited on large dimension transparent flexible substrate (10 x 10 cm) of polyethylene naphthalate (PEN), called hereafter "AgNW large plate". These large plates were used to prepare the "AgNW film" samples used in the corrosion experiments. The synthesis of AgNWs was conducted following the polyol method, the preparation of the large plates is described in Mayousse et al. (2013). The initial sheet resistances of AgNW large plates varied from 8 to 26 Ω/□. The nanowires used had diameters in the range 50-90 nm and lengths between 6-26 µm (SI appendix, Table S1) according to control measurements conducted with ImageJ software on FESEM images.

The AgNW large plates, when received, had an age of several months to more than one year and thus showed different degrees of aging. A thorough electron microscopic investigation was conducted on each plate before use, in order to discard damaged specimens not suitable for the experiments, or amenable to misleading interpretations.

To prepare the AgNW film samples, the AgNW large plates described above were cut into 2.5 x 3.5 cm 2 pieces, hereafter referred to as AgNW TCF or AgNW film. At least 3 samples were prepared from each AgNW large plate in order to assure reproducibility of results. Part of the samples were coated with a polysiloxane polymer (anti-scratch varnish AE12®, from ISOCHEM) used as a non-conductive coating material to protect the AgNW films used in the experiments (see details in SI appendix). Coating thicknesses of 500 and 700 nm (mean values) were achieved by varying the spin coating parameters. Polysiloxane is one of the materials studied as potential candidate for TCF coatings. According the findings by [START_REF] Toybou | Nanofils d'argent à dimensions maîtrisées: synthèse, toxicité et fabrication d'électrodes transparentes[END_REF], this coating effectively preserves the AgNW TCF under ambient degradation conditions and seems to improve initial conduction performances of the samples.

In addition, AgNW films of 2.5 x 3.5 cm 2 size, readily coated (by dip coating) with a protective layer of 5µm thickness (mean value), were prepared from new materials that had not undergone any aging prior to the experiments in this study.

Two commercial products were also tested with the method developed here. A silver antibacterial textile (silver-coated fibers) and heatable glasses from a skiing goggle containing silver ENM. Both products were purchased specifically for the experiments. The skiing goggle glasses were composed of a layer containing the ENMs and protected with a very thick over-coating layer of a polymer called polycarbonite by the manufacturer. For the experiments, both materials were cut into pieces of 2.5 x 3.5 cm 2 .

Sample characterization

The effect of the corrosion experiments on the AgNW films were evaluated by Field Emission Scanning Electron Microscopy (FESEM) using a JEOL JSM-7800F LV equipped with EDS/EBSD system, and a FEI Quanta 200 environmental scanning electron microscope (ESEM). The observations by FESEM were performed at 5 kV on coated AgNW films and at 15 kV on uncoated AgNW films. The observations by ESEM were conducted at 20 kV. Both secondary electron (SE) and electron backscattering (BSE) modes of imaging were used. To avoid surface charging, samples were chromium coated (70 Å) by electro-sputting under vacuum prior to FESEM observations. Atomic force microscopy (AFM) topographic observations and roughness measurements were carried out on a Dimension 3100 apparatus from Digital Instruments operated in Tapping Mode, using a set-point amplitude ratio close to 0.9 in order to reduce indentation effects and thereby optimize the topographic contrast. The Nanoworld silicon SPM sensors had a tip radius less than 10 nm, the nominal spring constant and resonance frequency of the cantilever being around respectively 50 N m -1 and 250 kHz.

Corrosion cell

The corrosion experiments were carried out in an electrochemical cell in a three electrode configuration (Figure 1). The AgNW film of 2.5 x 3.5 cm 2 was used as the anode, a platinum grid of similar size as the cathode. A mercury/mercurous sulfate electrode was used as reference. The electrochemical cell, specially designed for the experiments, made of PTFE, consists of a rectangular chamber of 10 mL volume, with two facing walls of the size of the electrodes. In the chamber top, slots are foreseen for positioning cathode and anode, and a hole for insertion of the reference electrode. The inter-electrode distance between anode and cathode is 0.5 cm. The reference electrode (RE-2CP, ALS Co.) is located outside the anodecathode path. The electrolyte is introduced into the chamber through another small hole in the chamber top. An automatic titrator (Titroline 7000, SI Analytics) controls solution addition.

All experiments were performed in a Faraday cage. A potentiostat (VSP, Bio-Logic) served to set the cell potential and to record the current generated.

Experimental conditions

NaNO 3 at 0.01 M was used as electrolyte solution (SI appendix, Figure S1). This concentration is representative of environmental conditions of continental surface and subsurface waters [START_REF] Stumm | Aquatic Chemistry-chemical equilibria and rates in natural waters[END_REF]. The 0.01 M NaNO 3 salt solution established naturally at a pH of 5.0 ± 0.2. This pH was retained as the working pH because it was well within the range where polysiloxane is chemically stable (SI appendix) and because this pH is representative for environmental conditions.

We determined the free corrosion potential (E corr ) of a 2 mm silver rod (Advent Research Materials) and of commercial AgNW (2.23% wt/wt dispersion, Nanogap) dispersed onto a carbon paste electrode. The corrosion potential of these silver materials was 0.45 V, detailed results can be found in [START_REF] Lehmann | Crumpling of silver nanowires by endolysosomes strongly reduces toxicity[END_REF]. To study corrosion on the AgNW film electrodes, we applied an overpotential of 0.8 V vs. SHE. At this voltage, the nanowires were not subject to any Joule heating problem [START_REF] Khaligh | The Joule heating problem in silver nanowire transparent electrodes[END_REF] neither in the aqueous nor in the aerial part of the electrode. Also the water electrolysis reaction (E°(pH5) = 0.925 V) is negligibly small at a potential of 0.8 V, thus not interfering with the Ag redox reaction or the pH of the solution.

The NaNO 3 electrolyte solutions and the K 2 SO 4 0.6 M reference electrode solution were prepared with ultrapure water and analytical grade reagents.

Experimental procedure

The corrosion experiments were conducted in the electrochemical cell under constant addition of electrolyte solution. This allowed to continuously renewing the solution-air contact line on the film surface. The continuous shifting of this "active zone" allowed maintaining high corrosion conditions throughout the experiment. Some tests were also conducted under fixed electrolyte volume conditions (SI appendix, Figure S2 A-B).

An initial volume of around 2 mL of electrolyte solution was placed in the reactor chamber, just enough to allow the solution surface to touch the lower edge of the AgNW film. The electric potential (set to 0.8V vs. SHE) and the automatic titrator were switched on simultaneously, thus insuring constant addition of solution over the duration of the experiment and starting at time t initial (Figure 1). A total volume of 2 mL of electrolyte solution was added at a flow rate of 100 µL.min -1 (20 min). This flow rate corresponds to a displacement of the solution/air interface inside the reactor chamber of 0.04 cm/min. Preliminary experiments showed that the chosen rate was enough to ensure a constant shift of the interfacial line between solution and air on the AgNW film. Electron microscopic controls showed that the movement is rapid enough to contain the capillary ascent of solution along the nanowires and along the borders of the film. The solution inside the cell was not stirred but the continuous addition of solution induced some degree of turbulent mixing. The pH of the solution, initially at 5, was controlled before and after the experiment. After 20 min, the experiment was stopped, the electrodes were removed carefully and an aliquot of the electrolyte solution was taken for Ag + analysis. The AgNW film sample was removed from the reactor, the area that had been immersed was cut off, carefully rinsed and placed in a desiccator for later surface analysis. The remaining part of the film, which had not been immersed in the electrolyte solution, was used for a subsequent experiment on the same sample.

All the solution samples were filtered through 0.2 µm pore size polycarbonate filters (PCTE Sartorius, Fisher Scientific), and several samples were additionally filtered through 10 kDa (equivalent to 4 nm) ultrafiltration membranes (Ultracel, Amicon 25 mm, Merck) using a stirred ultrafiltration cell system (Amicon, model 8010, 10 mL, Merck) in order to separate Ag nanoparticles from the truly dissolved fraction. All filtrates were acidified with HNO 3 2% trace analysis/ultrapure quality. Analyses of silver concentrations were conducted by Inductively-Coupled Plasma Mass Spectrometry (ICP-MS Agilent 7700x).

Results and discussion

In the electrochemical cell (Figure 1), the oxidation of silver metal is promoted by using the AgNW TCF as the anode and applying an electric potential higher than the corrosion potential. The silver metal is oxidized and goes into solution in the form of ionic silver (eq. 1).

Reaction at the anode (WE):

Ag + (aq) + e - (eq. 1)

In this study, we investigated the corrodibility of polysiloxane coated AgNW films with coating thicknesses varying from thin (500 nm), medium (700 nm) to thick coated (5 µm).

Uncoated AgNW films were also studied. Each selected film was submitted to the electrochemical corrosion test. The current response to the applied voltage was recorded via the potenstiostat. The raw and qualitative data was then transformed by integrating the traces over the experiment time, to obtain Q the quantity of charge, i.e. a single number representative of a given experiment (calculation details in SI appendix).

Features of corrosion after experiments

As mentioned above, the AgNW large plates available for the preparation of the samples, i.e. the "AgNW films" were carefully studied by Field Emission Scanning Electron Microscopy (FESEM) before use to discard damaged specimens not suitable for the experiments. On the plates kept for the study, the signatures of natural pre-aging processes or of synthesis related defects were identified (SI appendix, Figures S3,S4) so that the effects of our corrosion experiment could be clearly recognized.

The characteristic signature of nanowire degradation by electrochemical corrosion (our experiments) appears mainly in the active zone, i.e. along a triple junction line between solution, air and the partly immersed sample. The active zone was continuously renewed by a constant addition of electrolyte solution, thereby maintaining high electrical conduction thus continuous corrosion conditions. Electrochemical corrosion signature was fragmentation of the nanowires into an alignment of nanoparticles and/or nanorods. This feature appeared similarly for uncoated (Figure 2 A-D) and thin-coated AgNW films (Figure 2 E-H).

Fragmentation creates discontinuities leading to electrical failure. It has been shown

previously that the degradation of an AgNW network is dependent on several network characteristics [START_REF] Elechiguerra | Corrosion at the Nanoscale: The Case of Silver Nanowires and Nanoparticles[END_REF][START_REF] Mayousse | Stability of silver nanowire based electrodes under environmental and electrical stresses[END_REF]. Our results show that in a given sample, nanowires with smaller diameters were corroded preferentially to thicker nanowires or silver nanoparticle clusters (Figure 2D). When investigating the parameters nanowire diameter and sheet resistance, we observed that small diameters and/or low initial sheet resistances produced higher quantity of charge than large diameters and/or high initial resistances (SI appendix, Figure S5). In other words, corrosion is enhanced in networks with thin nanowires and/or high wire densities (i.e. high initial sheet resistances). Similar trends were described in previous studies [START_REF] Toybou | Nanofils d'argent à dimensions maîtrisées: synthèse, toxicité et fabrication d'électrodes transparentes[END_REF]. In some spots of the AgNW film, roughening of the nanowire surface was observed together with a decrease in NW brightness. Although this suggested mineral deposition, no evidence of silver oxide, hydroxide or sulfide on the wire surface was detected by EDS (SI appendix, Figure S4) maybe due to low amounts of these products particularly in the case of sulfide, below the detection limits of the technique used (SEM-EDS) as discussed previously by other authors [START_REF] Lin | Effects of Environmental Factors on the Stability of Silver Nanowire Transparent Electrodes[END_REF][START_REF] Toybou | Nanofils d'argent à dimensions maîtrisées: synthèse, toxicité et fabrication d'électrodes transparentes[END_REF]. It is most probable that the observed roughening was due to overgrowths made essentially of elemental silver [START_REF] Lin | Effects of Environmental Factors on the Stability of Silver Nanowire Transparent Electrodes[END_REF]. No sign of melting was observed at NW junctions or at NW tips, discarding the hypothesis of Joule heating [START_REF] Khaligh | The Joule heating problem in silver nanowire transparent electrodes[END_REF] to contribute to wire failure under our experimental conditions.

D A A B A C A E A F A G A H A
In AgNW films with medium coating thickness, similar pattern were observed as described above for thin coated and uncoated films, i.e. highly fragmented networks in the active zone and numerous patches of darkened nanowire surfaces (Figure 2 G-H) [START_REF] Mayousse | Stability of silver nanowire based electrodes under environmental and electrical stresses[END_REF].

Films coated with a thick layer of polymer (5 ± 1µm) could not be investigated by SEM because of insufficiently electron-transparency of that thick layer. Tiny cubic nanoparticles were observed in some AgNW films (from plates 1 and 9). These nanocubes, precipitates of Ag and Cl, appeared on the surface of nanowires or the surface of the polymer coating in case of coated films. The presence of traces of chlorine in our experimental set-up is attributed to Cl -impurities contained in AgNW, inherited from the reagents used for NW synthesis and then released upon NW dissolution. Details of this unexpected occurrence of Ag-Cl nanocubes and speciation calculations are discussed in SI appendix, Figures S6-S8.

Corrosion of AgNWs as a function of coating layer thickness

The presence of a protective layer on AgNW films and the thickness of this layer play a crucial role in limiting corrosion and failure of AgNW films.

Corrosion current responses are presented for four categories of AgNW films (Figure 3.a), from uncoated films to films with a thick coating layer of AE12. Each category includes AgNW films prepared by us (see details in SI appendix Table S1). The results are consistent with expectations, i.e. the developed quantity of charge was higher in experiments with AgNW films with a thin layer of polysiloxane and decreased as the thickness of the protective layer increased (Figure 3 Polymers such as PEDOT:PSS, PVP, PVA, that have often been used as coating materials [START_REF] Jiu | The effect of light and humidity on the stability of silver nanowire transparent electrodes[END_REF], are hydrophilic polymers with a plurality of polar groups which tend to sorb water. Similarly, AE12 polysiloxane used in the present work is also hydrophilic according to fabricant communication and corroborating results from water contact angle measurements (SI appendix, Figure S9). [START_REF] Celle | Oxidation of copper nanowire based transparent electrodes in ambient conditions and their stabilization by encapsulation: application to transparent film heaters[END_REF] reported a water vapor transmission rate of 80 mg.m -2 .d -1 for a polysiloxane layer of 1.5 µm thickness. The polarity of these polymers confers them some degree of permeability, which explains why ion diffusion is possible through the protective layer, therefore why a corrosion current is measured by the potentiostat.

To control that the coating remained intact throughout the experiment, we conducted AFM surface roughness measurements on the surface of an AgNW thin coated film, on treated and untreated zones. Results (SI appendix, Figure S10) show no significant difference between roughness values in both zones, indicating that the polymeric material exhibits no textural alteration at a scale of tens of nanometers.

Electrochemical corrosion results of the two commercial products containing silver, a silver antibacterial textile and a skiing goggle with heatable glasses containing silver ENM (Figure 3.b) showed evidence of corrosion in both materials. The skiing goggle glasses (blue bar) generated low corrosion current compared to the quantity of charge measured in the AgNW films of the present study. This result may be related to the very thick layer of protective coating covering the conductive layer. In the case of silver antibacterial textile (green bar), the values were 2 orders of magnitude higher than for the AgNW films.

In the next section, the release of ionic silver during the corrosion test will be investigated quantitatively.

Quantification of dissolved silver (Ag + )

Silver released from AgNWs during the corrosion experiments was quantified by ICP-MS.

To distinguish between nanoparticulate Ag and truly dissolved Ag + , the filtration procedure included micro-filtration through a 0.2 µm pore size membrane and ultra-filtration through a 10 kDa membrane. Potential sources of silver contamination as well as interactions between Ag + and filter materials (i.e. sorption and desorption) were controlled but proved to be non significant under experimental conditions in this study.

Silver concentration was measured after each filtration step. Total silver was not quantified systematically due to the limitation of sample volume. Results showed no significant difference in concentration between nanoparticulate-dissolved (0.2µm) and truly dissolved (10 kDa) fractions (SI appendix, Figure S11). This strongly suggests that the main transfer path of silver from the AgNW film to the solution was via diffusion of Ag + through the polymer coating, or via direct diffusion in the case of uncoated AgNW film. In consequence, 0.2 um polycarbonate filtrated solutions were considered as true solutions.

In Figure 4.a, concentrations of dissolved silver (originating from AgNW films) are represented as a function of the thickness of the protective coating layer. Clearly, the amount of dissolved silver decreases as the thickness of the protection layer increases. For uncoated films, release of dissolved silver was not significantly higher than for coated films, corroborating the fact that corrosion is limited by detachment of silver nanowires, which thereby modulates the formation of ionic silver. While Figure 4.a shows mean values, single results specific to two given plates are presented in SI appendix, Figure S12.

To find out if a direct relation can be established between the amount of Ag + detected in solution (by ICP-MS) and the electrochemical results, we converted the parameters quantity of charge, resulting from the corrosion current measured by the potentiostat, and the amount of ionic silver released from AgNW films to comparable units (calculation details in SI appendix).

As seen in Figure 4.b, the data sets originating from coated films (blue dots) show a same general trend. A linear correlation can be drawn with a slope of almost 1, suggesting that silver corrosion feeds the electric current with almost no electron loss. Although data are somewhat scattered around the correlation line (R 2 = 0.79), this result supports the initial hypothesis that the amount of charge determined for an experiment is representative of the amount of silver released to solution. In contrast, the data sets for uncoated films (red dots) result in very scattered data, among which two sets are out of range with an Ag + /electron ratio close to 2:1. Here the relatively high value for the Ag + release may be attributed to loss of nanowire debris to solution, and these debris may have been included in the quantification of silver. Over all the results suggest that the measure of quantity of charge obtained in the corrosion experiment is a number representative of the amount of corrosion of silver in AgNW films. For coated films, it is directly proportional.

Concerning the quantification of results of the commercial products containing silver, the skiing goggle glasses released small amounts of dissolved silver (40.88 µg/L), comparable to the levels obtained in the thick coated AgNW films of the present study. Interestingly, indium was also released to solution (7.28 µg/L). EDS analyses suggest the presence of indium oxide in the skiing goggle glasses.

In the case of silver antibacterial textile, the concentration of dissolved silver reached 4.57 x10 4 µg/L. The high specific surface area of the silver used as a coating of the textile fibers may explain this very high release.

These first trials on commercial Ag containing household products demonstrate that the corrosion method developed here allows obtaining a rapid estimate on the corrodibility of a silver containing material and on the potential metal release. The application of the method to the antibacterial textile shows that it can be used as well for macro-scale corrodible silver containing compounds.

Anthropogenic silver release to the environment and potential impacts

When considering all categories of AgNW conductive transparent films investigated in this study, the range of silver concentrations released from the AgNWs films ranged from approximately 90 µg/L to 400 µg/L (0.83 µmol/L to 3.7 µmol/L). These concentrations can be confronted to toxicity data. Ionic silver has been shown to bioaccumulate in various aquatic organisms and to be highly toxic to bacteria, phytoplankton, and aquatic invertebrates and vertebrates with toxicity threshold values typically around 0.2 µg/L [START_REF] Croteau | Silver Bioaccumulation Dynamics in a Freshwater Invertebrate after Aqueous and Dietary Exposures to Nanosized and Ionic Ag[END_REF][START_REF] Davies | Toxicity of silver to rainbow trout (Salmo gairdneri)[END_REF][START_REF] Farkas | Effects of silver and gold nanoparticles on rainbow trout (Oncorhynchus mykiss) hepatocytes[END_REF]; US EPA, https://www.epa.gov/wqc/ambient-water-qualitycriteria-silver). These organisms have important roles in the trophic ecosystem as primary producers and consumers, and adverse impacts of silver to these organisms could have detrimental consequences to overall ecosystem health [START_REF] Glibert | Ecological stoichiometry and its implications for aquatic ecosystem sustainability[END_REF][START_REF] Vaughn | Biodiversity Losses and Ecosystem Function in Freshwaters: Emerging Conclusions and Research Directions[END_REF].

The toxicity of ionic silver to humans depends on the type of cells that are exposed, the liver cells being the most sensitive. According to the American Conference of Governmental Industrial Hygienists (ACGIH, https://www.acgih.org/), the occupational exposure limit for dissolved silver to prevent argyria is 0.01 mg/m 3 (0.01 µg/L).

Overall these limits demonstrate that the silver released during the corrosion test of the AgNW films is orders of magnitude above the levels acceptable for the natural environment and living species, even in the case of a thick protective coating (90 µg/L), thereby they point to the relevancy of this test.

Concerning anthropogenic input of silver to aquatic systems, Zhang et al. (2008) reported concentrations of dissolved silver in the surface water of Tokyo Bay ranging from 6.5x10 -4 µg/L to 1.7 x10 -3 µg/L, a level also obtained for the surface waters of the Japan Sea. [START_REF] Deycard | Inputs, dynamics and potential impacts of silver (Ag) from urban wastewater to a highly turbid estuary (SW France)[END_REF] found average dissolved concentrations of 1x10 -3 µg/L in river water of the "La Réole site" in France. They also reported that the daily silver concentration in an urban wastewater treatment plant located on the fluvial part of the Gironde estuary, ranged from 0.14 to 0.55 µg/L depending on weekday and meteorological conditions.

Estimations of nanosilver environmental release are primarily based on modelling approaches. [START_REF] Giese | Risks, Release and Concentrations of Engineered Nanomaterial in the Environment[END_REF] estimated environmental concentrations of Ag-ENM arising from medical applications to have reached 3.8x10 -4 µg/L in surface fresh waters in Germany in 2017 and predicted an increase to 8.9x10 -4 µg/L for 2030. In sewage treatment effluents, modelling calculations gave 1.89x10 -2 µg/L for 2017 and 3.06x10 -2 µg/L for 2030 respectively.

Silver released from AgNW films during the corrosion test may be put into the perspective of current background levels of Ag in anthropogenically influenced waters. Indeed the 5-10 mL of solution in the electrochemical cell would have to be diluted by an average factor of 2000 before "disappearing" in the general background. The diversification of (nano)silver containing products and the increase in their production will irrevocably increase the Ag background concentrations in natural systems and affect water quality.

The human body may come in direct contact with silver from anthropogenic source. [START_REF] Deycard | Inputs, dynamics and potential impacts of silver (Ag) from urban wastewater to a highly turbid estuary (SW France)[END_REF] reported that Ag concentrations in a typical household personal care product (deodorant) labeled as containing Ag, range from 0.2 to 0.4 mg/kg of solution (200 µg/L to 400 µg/L). These concentrations are within the interval of values generated during our corrosion test. The comparison is appealing because AgNW TCF are predestined to be in contact with skin. Thus, silver released from AgNW TCF may be significant compared to current silver levels in contact with the body.

Relevance of the developed corrosion test

The experimental results obtained in this work on silver corrosion in AgNW films allow us to estimate an average silver corrosion rate of about 3.7 x 10 -10 g/s (Calculation details in SI appendix, Table S3), which is a factor 30 larger than the rate measured for the natural dissolution of a noble silver foil in water saturated with air [START_REF] Graedel | Corrosion mechanisms for silver exposed to the atmosphere[END_REF]. This comparison highlights the strength of the method developed here, where a corrosion potential, slightly enhanced compared to natural conditions, leads to a fast and easily measurable response of the investigated sample. This method is therefore well adapted to assess the risk of metal release from everyday consumer products to their immediate environment (skin, corporal fluids, effluent waters, aquatic systems).

The potential application fields of the accelerated release test are wide, however the field of biosensors appears as a very promising one because the use of wearable electronic sensors containing metallic and in particular nanowire silver is growing and because in these devices, the film electrodes are often located in humid environment and in direct contact with the skin.

Moreover biocompatible capping agents e.g. citrate are often used in antibacterial Ag-ENMs, which can enhance silver solubility [START_REF] Liao | Bactericidal and Cytotoxic Properties of Silver Nanoparticles[END_REF].

An important aspect also is that the method can be applied at any time in the life cycle of the AgNW film. [START_REF] Nowack | Procedures for the production and use of synthetically aged and product released nanomaterials for further environmental and ecotoxicity testing[END_REF] discuss the importance of performing agingrelease tests in ENM-nanoproducts during use rather than in pristine materials, as consumers and the environment are mostly exposed to aged and transformed nanomaterials [START_REF] Baun | Regulatory relevant and reliable methods and data for determining the environmental fate of manufactured nanomaterials[END_REF]. The method may play a role in predicting the silver flux from household products, biosensor products and personal electronics containing nano-silver [START_REF] Steinhäuser | Reliability of methods and data for regulatory assessment of nanomaterial risks[END_REF] during life-time and at end of life. In the case these products come in contact with the environmental water compartment, the method will again provide valuable data for flux estimates.

While experiments presented in this work on AgNW coated films have been conducted with a polysiloxane coating (anti-scratch varnish AE12®), more research needs be perform with AgNW films with other matrices, other coatings, in particular hydrophobic polymers like epoxy or new materials such as optical adhesive or reduced graphene oxide layers, is necessary to work out the method limitations. Reference AgNW TCF materials would also need to be developed with known toxicological characteristics.

The current lack of standard methods to quantify the risk of exposure of ENM enabled devices to the environment, underline the importance of developing the accelerated corrosion method into a standard method for quantifying the releasability of metals from conductive thin films. The way forward will be to constitute a data base for reference conductive films to which new materials could be compared.

Conclusions

In this work, we developed an electrochemical method to evaluate the potential corrosion activity of silver in nanowire transparent conductive films (AgNW TCFs), with or without coating (polysiloxane varnish AE12 ®), and the possible release of ionic silver. We have demonstrated that there is a consistent link between the corrosion current response obtained from coated AgNW TCFs, Ag + release and the coatings' characteristics. The corrosion current in each experiment is transformed into quantity of charge, a single number representative of the corrodibility of a material. This corrosion indicator is established quickly, 20 minutes are sufficient for a diagnostic result. The method is thus seen as an accelerated corrosion test, with speed being a significant advantage compared to classical aging studies. Additionally, this method is free from harmful reagents and is low power consumption.

Previous works mainly focused on technical quality control of AgNW transparent electrodes, in particular on lifetime stability of the devices. To our knowledge, this is the first investigation of the corrodibility of a conductive nanomaterial under aqueous conditions, in order to assess the risk of metal release to users and the environment.

A significant result obtained with the accelerated corrosion test is the fact that protective coatings of AgNW films do not systematically provide full protection against corrosion of silver and the release of ionic silver to outside the material. Our results demonstrated that even a thick 5 µm layer of protective polysiloxane coating could not stop AgNW corrosion completely, still leaving a risk of silver (Ag + ) release.

The accelerated corrosion method is promising because it tests the degree of isolation of a material by the possibility of electron flow The measure of corrodibility depends not only on the presence of (nano) silver but also on textural parameters, in particular the degree of dispersion of the particles in the material and the characteristics of protective coatings. We suggest that the accelerated corrosion method could be elevated to the level of a standard material quality control method to quantify the risk of silver release by corrosion from (nano)silver containing products.

The method is not strictly limited to the study of AgNW TCFs, and can be adapted to products containing other oxidisable conductive nano-materials, in particular those with oxidation potential below that of water. Several technology-critical elements (TCE), increasingly applied as ENMs, with application fields where toxicology limits are even less well explored, could be targeted.

Figure 1 .

 1 Figure 1. Schematic of the electrochemical corrosion cell used in the corrosion tests. A threeelectrode configuration was used: the AgNW film was used as anode (working electrode WE), a platinum grid as cathode (counter electrode CE), a mercury/mercurous sulfate electrode as reference electrode (RE). The automatic titrator allowed constant addition of solution over the duration of the experiment (t final -t initial = 20 min). The arrow shows the direction of increase of the solution level, thus the progression of the provoked corrosion.

Figure 2 .

 2 Figure 2. SEM images (BSE) of AgNW films from plate 5 (NW diameter 57 ± 10 nm, NW length 7 ± 2 µm) unexposed and exposed to the corrosion test. Each pair of images (e.g. A-B; C-D, etc) are images of the same film at different magnifications. A-B) Pristine nanowires in an uncoated film, some silver clusters are seen, an evidence that natural corrosion took place prior to experiments. C-D) Fragmentation, nanowire discontinuities and formation of nanoparticles as a result of 20 min of corrosion test. E-F) Pristine nanowires in a film protected with a thin layer of AE12 coating. G-H) Same film after 20 min of corrosion test; fragmented nanowires with dissolution gaps appearing dark. Another example of electrochemical corrosion signatures on AgNW films (from plate 9: NW diameter 86 ± 13 nm nm, NW length ± 4 µm) can be found in SI appendix, Figure S13.

Figure 3

 3 Figure 3. a) Comparison between quantity of charge (mean values) generated in AgNW films with varying thicknesses of polysiloxane coating: uncoated, thin (500 nm), medium (700 nm) and thick (around 5 µm). The high standard deviation values (n= 5) observed in each bar are related to the variable qualities of the different plates used (see SI appendix, Figure S12). b) Quantity of charge for the commercial products tested: heatable glasses containing silver ENM (blue bar) and silver antibacterial textile (green bar). All the results correspond to experiments at 0.01 M ionic strength, imposed potential of 0.8 V vs. SHE and pH 5.

Figure 4 .

 4 Figure 4. a) Silver concentrations (mean values) in the reactor solutions of the experiments presented in Fig. 3.a. The high standard deviation values (n= 5) are related to the variable qualities of the different plates used (see SI appendix, Figure S12). b) For individual samples, correlation between quantity of electrons released (Er) derived from current measurements by potentiostat and dissolved silver from AgNW films (Ag + ) detected in solution by ICP-MS. Coated films (bleu dots) show good linear relation between both parameters with a slope of almost 1, while uncoated films (red dots) gave rise to more scattered data. Detailed calculations of both parameters in SI appendix.

Acknowledgements

This study was conducted in the framework of the project NanoWIR2ES (Nanowire Intelligent Re-Design and Recycling for Environmental Safety) supported by the European Union ERANET-SIINN program (European Research Area Network Safe Implementation of Innovative Nanoscience and Nanotechnology) and was funded by the « Region Hauts de France ». Many thanks to Guillaume Bai (University of Lille) who conducted water contact angle measurements on polysiloxane resin, and Sandra Ventalon (University of Lille) for her help in the spectroscopic observations. We greatly appreciated working with Christophe Vermander (Ecole Centrale de Lille) who realized the electrochemical reactor and Jean Claude Bruyere who helped elucidating the chemical properties of polysiloxane AE12.

Abbreviations

AgNW: silver nanowires Argyria: is a condition caused by excessive exposure to chemical compounds of the element silver, or to silver dust. Supplementary data contain complementary information regarding method and results: table of large plates used for preparation of AgNW films, description of coating material and sample preparation, effect of ionic strength on current generation, details on calculation of quantity of charge, experiment results under fixed electrolyte volume, images showing NW displacement on uncoated films, images highlighting characteristic features of natural alteration (artifacts) on NW films, effects of NW-network density and NW diameter on current, description of silver-chloride nanocubes and EDS spectra, table of stability constants for silver species, model calculations for aqueous silver speciation, water contact angle measurement on control samples, AFM roughness measurements on coated AgNW films, dissolved silver after micro and ultrafiltration, comparison between unaltered and altered large plates, conversion of Ag+ concentrations and electric current generated to a common unit (nmol.cm -2 ), details for calculations of corrosion rates, supplementary images illustrating corrosion signatures on AgNWs after corrosion test.