Alice Gimat
email: alice.gimat@orange.fr

Sebastian Schoeder

Mathieu Thoury

Anne-Laurence Dupont
email: anne-laurence.dupont@mnhn.fr

Sebastian Schöder

Impact of the Paper Degradation State and Constituents on Its Behavior During and After X-ray Exposure

Keywords: calcium carbonate, cellulose degree of polymerization, gelatin, iron gallate ink, yellowing, UV uorescence calcium carbonate, cellulose degree of polymerization, gelatin, iron gallate ink, yellowing, UV fluorescence

Paper is a complex biopolymer material which contains papermaking additives and often bears inks and other graphic media. Cultural heritage paper-based artefacts are most often deteriorated to some extent. This research explores how intrinsic factors such as constituents and degradation state can impact the modifications incurred in aged papers during and after X-ray examination. To this end laboratory model papers, artificially aged, and 18 th and 19 th century archival documents, with and without additives (gelatin, calcium carbonate) and iron gallate ink, were exposed to Synchrotron X-ray radiation at doses that were previously shown to incur damage in unaged cotton papers (0.7 to 4 kGy). Glycosidic scissions, hydroxyl free radicals, UV luminescence and yellowing were measured immediately after the irradiation, and were monitored over a period of three years. The depolymerization of cellulose was lower in the aged papers, as well as in the papers containing calcium carbonate and gelatin, than in the unaged fully cellulosic papers. Compared to the papers with no additives, there were more hydroxyl free radicals in the papers with calcium carbonate and slightly less in the gelatin sized papers. UV luminescence and yellowing both appeared post-irradiation, with a delay of several weeks to months, while the intensity of the responses was impacted by the various paper constituents. The papers with iron gallate ink showed limited degradation in the low doses range, most probably due to recombination of the free radicals produced. Doses below 4 kGy did not cause yellowing or UV luminescence of the archival papers within the whole monitoring period. At higher doses (26 to 36 kGy), a slight UV luminescence appeared after 21 months, as well as a slight yellowing after three years, in some of them. No clear correlation between the degradation induced by the irradiation and the constituents in the paper nor its conservation state could be made. The archival papers in good conservation state depolymerized to the same extent as the model papers, while the most degraded archival papers were less impacted than the latter.

Introduction

X-ray analytical techniques are used to examine historic documents and artworks on paper and gain insight into their materials, manufacturing techniques and history [START_REF] Creagh | Chapter 1 Synchrotron Radiation and its Use in Art, Archaeometry, and Cultural Heritage Studies[END_REF][START_REF] Albertin | Ancient administrative handwritten documents: X-ray analysis and imaging[END_REF]IAEA 2016;[START_REF] Kozachuk | Possible Radiation-Induced Damage to the Molecular Structure of Wooden Artifacts Due to Micro-Computed Tomography, Handheld X-Ray Fluorescence, and X-Ray Photoelectron Spectroscopic Techniques[END_REF][START_REF] Pouyet | Revealing the biography of a hidden medieval manuscript using synchrotron and conventional imaging techniques[END_REF]). Yet because they are ionizing, Xrays induce changes in organic (as well as inorganic) materials. Cellulose depolymerization, oxidation and changes in the optical properties have been observed under gamma-ray [START_REF] Ershov | Radiation-chemical degradation of cellulose and other polysaccharides[END_REF][START_REF] Bouchard | The effects of ionizing radiation on the cellulose of woodfree paper[END_REF][START_REF] Henniges | Electron Beam Irradiation of Cellulosic Materials-Opportunities and Limitations[END_REF][START_REF] Bicchieri | Effects of gamma irradiation on deteriorated paper[END_REF]) and X-ray exposures [START_REF] Mantler | Analysis of art objects and other delicate samples: Is XRF really nondestructive?[END_REF][START_REF] Kozachuk | Possible Radiation-Induced Damage to the Molecular Structure of Wooden Artifacts Due to Micro-Computed Tomography, Handheld X-Ray Fluorescence, and X-Ray Photoelectron Spectroscopic Techniques[END_REF][START_REF] Gimat | Short-and Long-Term Effects of X-ray Synchrotron Radiation on Cotton Paper[END_REF]. In quasi-pure cellulose paper, the impact of X-rays has been shown to be proportional to the dose [START_REF] Gimat | Short-and Long-Term Effects of X-ray Synchrotron Radiation on Cotton Paper[END_REF].

However, due to the large diversity in the components and in the degradation state of historic cellulosic artefacts, the global impact of X-ray photons is difficult to foresee.

Paper is made of plant fibers, which besides cellulose, most often also contain other biopolymers such as hemicelluloses and lignin. Additives, fillers and sizing, are usually added to writing and drawing quality papers to enhance usability parameters: e.g. reduce water permeability, increase opacity and enhance brightness. In cultural heritage collections, such papers also often bear media such as inks and pigments. The materials and chemicals used are diverse. In ambient conservation conditions, a number of these additives can impact the paper degradation rate. For instance gelatin (Dupont 2003a) and alkaline minerals [START_REF] Reissland | Ink Corrosion Aqueous and Non Aqueous Treatment of Paper Objects -State of the Art[END_REF][START_REF] Sequeira | Deacidification of paper using dispersions of Ca(OH)2 nanoparticles in isopropanol. Study of efficiency[END_REF][START_REF] Ahn | Sustainability of Mass-Deacidification. Part II: Evaluation of Alkaline Reserve[END_REF][START_REF] Poggi | Calcium hydroxide nanoparticles in hydroalcoholic gelatin solutions (GeolNan) for the deacidification and strengthening of papers containing iron gall ink[END_REF]) have been shown to decrease the cellulose depolymerization rate, whereas transition metals in inks and pigments promote degradation by producing acids and free radicals [START_REF] Selih | The role of transition metals in oxidative degradation of cellulose[END_REF][START_REF] Potthast | Iron gall ink-induced corrosion of cellulose: aging, degradation and stabilization. Part 1: model paper studies[END_REF]. If and how additives can impact the radiation-induced degradation of cellulosic paper is still unknown. The presence of absorbing elements, such as iron in the metalgallate ink or calcium in the fillers could have a shielding effect and decrease the nominal X-ray dose, thereby lowering the degradation impact. Such a shielding effect could also be counteracted by the free radicals formed via the transition metals, which are known cellulose degradation promotors [START_REF] Emery | Iron-catalyzed oxidation of wood carbohydrates[END_REF][START_REF] Jeong | Degradation of cellulose at the wet-dry interface. II. Study of oxidation reactions and effect of antioxidants[END_REF]. It has been shown that iron-containing pigments undergo a redox reaction under X-ray radiation [START_REF] Bertrand | Mitigation strategies for radiation damage in the analysis of ancient materials[END_REF]Gervais et al. 2015;Gimat 2016). Moreover, the structural modification of an additive under irradiation can also affect the paper degradation rate. For instance, X-rays were shown to produce defects inside calcium carbonate [START_REF] Kabacińska | Nanoscale Effects of Radiation (UV, X-ray, and γ) on Calcite Surfaces: Implications for its Mechanical and Physico-Chemical Properties Kocar D[END_REF], whereas polypeptide chains (e.g. gelatin) were shown to undergo hydrolysis [START_REF] Moini | Assessing the Impact of Synchrotron X-ray Irradiation on Proteinaceous Specimens at Macro and Molecular Levels[END_REF]. Bicchieri et al. have examined the combined impact of the degradation state and certain paper additives on the degradation incurred by ionizing radiation used for mold disinfection [START_REF] Bicchieri | Effects of gamma irradiation on deteriorated paper[END_REF]. The authors used gamma-rays at a dose of 3 kGy.

They tested cellulose paper Whatman n°1, as well as a commercial permanent paper (with CaCO3 filler and optical brighteners, and sized with alkyl ketene dimers), which they pre-degraded by an acid treatment. The acid treated samples showed less radiation induced depolymerization than the control samples, indicating that the degradation state played a role. The permanent paper yellowed more than Whatman n°1, which was attributed to structural modifications of calcium carbonate and optical brighteners under gamma radiation. To our knowledge, such study has not been conducted using X-rays, nor at lower doses used during synchrotron X-ray examination of cellulosic cultural heritage artefacts. This lack of research motivated the present study, which attempts at better understanding the mitigated impact of X-rays on paper, depending on the fiber deterioration level and on the presence of components other than the fibers.

Handmade linen rag papers from the 18 th and 19 th century and industrially-made cotton linters papers (Whatman n°1) to which various additives were incorporated (gelatin, calcium carbonate and iron gallate ink) were exposed to synchrotron X-ray radiation. The papers, some of which had been previously artificially aged, were irradiated at doses in the range 0.7-4 kGy. The samples were characterized immediately after the exposure using a multiscale analytical procedure developed in a previous study [START_REF] Gimat | Short-and Long-Term Effects of X-ray Synchrotron Radiation on Cotton Paper[END_REF], which encompasses the macroscopic (yellowing and UV luminescence) and the microscopic scales (glycosidic scissions and formation of hydroxyl radicals). The changes were monitored over a period of three years.

Materials and Methods

Paper samples

Laboratory-prepared samples

Two types of paper were used: Whatman n°1 (W), which is a commercial paper made of cotton linters (min. 98% alpha cellulose), and a linen rag paper (R) manufactured using traditional stamper beating at Moulin du Verger papermill (Puymoyen, France). W and R were used either with no further modification (control samples), or upon undergoing various artificial aging treatments (aged samples), in an attempt to approach the molecular degradation state of centuries old cultural heritage papers. Two artificial aging conditions, one predominantly hygrothermal (hyg) and the other predominantly oxidative (ox), were used to depolymerize and increase the carbonyl content of cellulose. The conditions were adjusted so as to achieve a similar degree of polymerization (DP) and a different degree of oxidation in the hyg and ox samples.

Hyg aging of W and R (samples called W_hyg and R_hyg) was performed according to the TAPPI method (TAPPI T573 sp-15 2015). Glass tubes (Wheaton, 35 mm internal diameter (ID) × 147 mm, 144 mL) were filled with 4.0 g of paper (dry weight), i.e. 4.23 g of paper conditioned at 50% relative humidity (RH) and 23 °C, based on the value of the equilibrium moisture content (EMC = 5.43 %wt) determined with the sorption isotherm. The tubes were hermetically closed and heated at 100 °C in an oven (Memmert UN 55 oven) during 10 days to reach a decrease in DP of about 50%. During hyg aging, both hydrolysis and oxidation occur. The weight-average degree of polymerization (DPw) of cellulose was measured using SEC-MALS-DRI (details in Physicochemical characterizations section) and the copper number (NCu) was determined using the standard method (TAPPI -T 430 cm-99 1999). The total carbonyl groups concentration was derived from NCu using the following formula proposed by Röhrling: [𝐶𝑂] = (N Cu -0.07) 0.06 [START_REF] Röhrling | A Novel Method for the Determination of Carbonyl Groups in Cellulosics by Fluorescence Labeling. 2. Validation and Applications[END_REF]. DPw for W_hyg and R_hyg was 1490 ± 2% and 1961 ± 1.6%, respectively. NCu of W_hyg was 0.11.

Oxidative degradation (ox) was carried out by immersing W in an aqueous solution of sodium hypochlorite (0.42-0.62% active chlorine) adjusted to pH 7 with HCl 6 N, during 15 min under gentle stirring. At this pH, NaClO is known to promote enhanced carbonyl groups formation (aldehyde, ketone, and carboxyl groups) on C2, C3 and C6, short chain organic acids as well as considerable glycosidic scissions [START_REF] Nevell | Cellulose Chemistry and its Applications[END_REF]. The samples (called W_ox) were abundantly rinsed with milli-Q water until neutral pH of the water, and were dried between blotters.

The DPw for W_ox was 1352 ± 2% and NCu was 0.42. Some of the W_ox samples additionally underwent a reduction treatment with Na(BH)4 to reduce the carbonyl groups produced during the aging (aldehyde and keton functions) to alcohol groups, and achieve a nearly null NCu. To this end, a solution made with anhydrous Na(BH)4 (Sigma)

(2.91g) dissolved in absolute ethanol (154 mL) was prepared, in which 1.54 g of paper was immersed and left under gentle stirring during 12 hours. After reduction, the papers were abundantly rinsed with milli-Q water until the water reached neutral pH, and were dried between blotters. The samples were called W_red.

A portion of the W and R control samples were sized by immersing the paper sheets in a 20 g L -1 aqueous solution of type B photographic grade gelatin from cattle bone (Gelita type restoration 1, Kind & Knox) at 30 °C during 10 minutes. The sheets were then dried vertically at ambient temperature. The sized samples were named W_G and R_G.

The dry gelatin uptake (UP) of W_G and R_G, determined as 𝑈𝑃 = 𝑚 𝑠𝑖𝑧𝑒𝑑 𝑝𝑎𝑝𝑒𝑟 𝑑𝑟𝑦 -𝑚 𝑢𝑛𝑠𝑖𝑧𝑒𝑑 𝑝𝑎𝑝𝑒𝑟 𝑑𝑟𝑦 𝑚 𝑢𝑛𝑠𝑖𝑧𝑒𝑑 𝑝𝑎𝑝𝑒𝑟 𝑑𝑟𝑦 , was 4.8% ± 0.2. Dry masses were calculated subtracting the EMC at 23 °C and 50% RH measured according to the standard method (TAPPI T 502 cm-07 1998). The UP value falls in the range of gelatin content in historical papers [START_REF] Barrow | Manuscripts and Documents: Their Deterioration and Restoration[END_REF][START_REF] Barrett | Evaluating the effect of gelatin sizing with regard to the permanence of paper[END_REF]) and corresponds to a substantial amount of size in the paper (qualified as with '+' in Table 1). Some of the W_G and R_G samples were used to apply the second compound of interest: iron gallate ink, also referred to as I (samples called W_GI and R_GI). The ink was prepared by mixing FeSO4, 7H2O (Sigma Aldrich, 99%) (40 g L -1 ), gallic acid monohydrate (Sigma Aldrich, 99%) (9 g L -1 ) and gum Arabic (Sigma Aldrich, G9752) (140 g L -1 ). The mixture was stirred during 3 days at room temperature. The amount of gum Arabic used was purposely high in order to limit the penetration of the ink inside the paper. The iron sulfate vs gallic acid ratio was adapted from a recipe used in previous work [START_REF] Rouchon | Room-Temperature Study of Iron Gall Ink Impregnated Paper Degradation under Various Oxygen and Humidity Conditions: Time-Dependent Monitoring by Viscosity and X-ray Absorption Near-Edge Spectrometry Measurements[END_REF]. Large inked strokes (1.5 cm wide each) were applied side by side with a flat-end metal pen ("Plakat", Brause) in order to cover the whole sample surface. This procedure was not intended to replicate a quill pen stroke, but to provide a large and homogeneous inked surface (21 cm²). The ink penetrated 30 to 112 microns into the paper, i.e. one third to one half of the sheet thickness, as observed with the optical microscope (Fig S1 in the Supplementary data file). The iron content determined by XRF using a previously established calibration curve (unpublished data) was similar in both samples: 97 (± 5) µmol g -1 in W_GI and 110 (±15) µmol g -1 in R_GI, values that are comparable to those in historical documents (36-179 µmol g -1 ) [START_REF] Rouchon | Room-Temperature Study of Iron Gall Ink Impregnated Paper Degradation under Various Oxygen and Humidity Conditions: Time-Dependent Monitoring by Viscosity and X-ray Absorption Near-Edge Spectrometry Measurements[END_REF].

The third compound added to the papers was CaCO3 (samples called W_Ca). W paper was immersed in a saturated aqueous solution of calcium hydroxide (95%, Sigma Aldrich) (approx. 1.4 gL -1 ) during 1 hour and was dried in ambient air. This was repeated four times successively in order to achieve a high calcium carbonate content. After each bath, the paper sheets were placed between two blotters, and the excess solution was removed by applying a 10 kg Cobb test metal roller once back and forth on the blotters. Then the sheets were dried under weight. The reaction of CO2 with the air when the paper is removed from the solution converts Ca(OH)2 to CaCO3, so-called alkaline reserve (AR). The AR determined according to the standard method (TAPPI T 553 om-00 2000)

was 1.18 ± 0.06 mol kg -1 , otherwise expressed as 6.0% ± 0.3 equivalent CaCO3. Additionally, a commercial permanent paper made of cotton linters, which contained 7.25% precipitated CaCO3 (Krypton parchment, Spexel Inc, formerly Domtar), was used (samples named K). Because it was manually prepared, Ca distribution inside W_Ca was less homogeneous than in K paper (Fig. S2 in supplementary data file). All the samples were conditioned prior to use at 50% RH, 23 ºC according to the standard method (TAPPI T 402 sp-08 2013).

Archival papers

Five archival paper documents from the 18 th and 19 th century manufactured with linen rags were chosen. They were named DCN, SE, LN1, LN5, and M. They seem to have different gelatin size content, varying from light to strong, and different DP (Table 1 andTable S1 in the Supplementary data file). SE is a page from an 18 th century printed volume and has a slightly brownish hue, which appears darker in the center inked area of the page, due to natural aging. DCN is a printed decree and has a very faint bluish hue. These two papers seem to have the lowest amount of sizing (Fig. S3 in the Supplementary data file). The three other documents (LN1, LN5 and M) are individual folios of notarial deed documents. M is a blank paper while both LN1 and LN5 are handwritten with iron gall ink. In SE, DCN, LN1 and LN5, only ink-free areas were used, in order to better compare with M. Additionally, to investigate the effect of the iron gall ink on ancient archival paper, the laboratory-made iron gallate ink was applied to some of the M samples using large strokes as previously described for papers W_GI and R_GI, which yielded a homogeneous inked area (sample called M_I).

X-ray radiation exposures 189

Synchrotron X-ray setup 190

The papers were cut into a few cm 2 samples, inserted in plastic photography slide frames, and 191 heated for 2 h at 40 °C (Memmert UN 55 oven) for gentle moisture desorption. They were then 192 placed 48 h in a climatic chamber at 23 °C and 50% RH for equilibrium moisture regain, after 193 which they were sealed in LDPE plastic bags where silica gel ProSorb (Atlantis) was added so as 194 to maintain 50% RH (± 5%). The EMC (23 °C, 50% RH) of the papers was determined according 195 to the TAPPI test method (TAPPI T 502 cm-07 1998) (Table 1). No moisture leakage was recorded 196 upon monitoring the RH inside the bags for at least 72 h prior to the synchrotron radiation (SR) 197 experiment with a temperature/humidity logger (Ibutton® Hygrochron,Measurement Systems 198 Ltd). The bags were themselves sealed with Escal® film also filled with silica gel to stabilize the RH to 50% for transportation from the laboratory to the synchrotron facility.

The irradiation was performed on the beamline PUMA (SOLEIL synchrotron, Saclay). A monochromatic beam (2×1 cm 2 ) from a double crystal monochromator (DCM) with Si(111) crystals was used at photon energies of 7.22 keV, 12.5 keV or 18 keV. The samples were exposed perpendicular to the beam, inside the LDPE bags. The irradiation duration varied to reach various doses in the range 7 Gy to 4 kGy. The dose D (Gy), i.e. the total energy deposited per mass unit of material, was calculated as follows:

D= F•E•t 𝑚 = I 0 •(1-e -μ•x )•E•t ρ•σ•x
with F, the absorbed photon flux (ph s -1 ) ; E, the energy of X-ray photons (J) ; t, the exposure time (s) ; I0, the incident flux (ph s -1 ) ; m, the mass of paper (kg) ; x, the thickness of paper (cm) ; ρ its density (g cm -3 ) ; σ, the beam imprint ; μ the linear attenuation coefficient (cm -1 ), which was determined by measuring the incident and transmitted flux impinging stacked sheets as previously described [START_REF] Gimat | Short-and Long-Term Effects of X-ray Synchrotron Radiation on Cotton Paper[END_REF].

Laboratory XRF spectrometer

W paper was irradiated using a Micro X-ray Fluorescence Spectrometer (M6 Jetstream, Brucker) with a 100 µm² polychromatic beam (0-50 keV). The beam is produced by an X-ray tube with a rhodium anode (50 kV, 600 μA). The instrument is equipped with a 100 μm-thick beryllium window and polycapillary optics are used to focus the X-ray beam. The X-ray detector is a 60 mm² SDD and has a Peltier cooler. For each energy of the X-ray source, the linear absorption coefficient μ was calculated using the coefficients, density and mass fraction of the most abundant components of paper, namely cellulose, water and air (Fig. S4 in the Supplementary data file).

The sample area was scanned in successive spots (area equal to the beam size) in one or repeated cycles. The signal to noise ratio of the XRF spectrum depends on the number of cycles and their duration. Two samples were thus exposed during 1 cycle for 500 ms and 3000 ms per spot and received doses of about 3.5 and 22 Gy, respectively. These doses were chosen to be in the same range as the two lowest doses used in the SR experiment at 7.22 keV (7 and 21 Gy). To study the dose-response reciprocity, one sample was exposed repeatedly for 30 cycles with 100 ms exposure per spot, thus being irradiated to 22 Gy.

Physico-chemical characterizations

After the irradiation, the samples were kept in the dark at 50% RH and 23 °C until analysis. The analyses were usually performed within 6 days, the latter being the shortest possible duration between the irradiation and the analysis. This allowed for immediate damage assessment. Postirradiation monitoring was carried out by regularly re-examining the samples.

Molar masses

The molar mass distribution and the number-and weight-average molar masses of cellulose Mn and Mw were determined using Size-Exclusion Chromatography (SEC), except for the inked samples which were analyzed using viscometry. For SEC, paper samples (3-5 mg) were prepared and analyzed as described previously (Dupont 2003b). The precision on Mw was between 0.2 and 4.0 RSD%, depending on the samples. S, the glycosidic scissions concentration, was calculated using DPn, with

DP n = 𝑀 𝑛 𝑀 𝐴𝐺𝑈 = N AGU N molecule 𝑡 ,
where NAGU is the total number of anhydroglucose units, i.e monomers (𝑀 𝐴𝐺𝑈 =162 g mol -1 ) and N molecule 𝑡 is the total number of cellulose molecules at any time t (µmoles). As each glycosidic bond scission increases by one the number of cellulose chains, the increase in the concentration of new ) µmoles g -1 paper (Whitmore and Bogaard 1994).

In order to avoid polluting the SEC columns with iron, DP of the gelatin-ink coated samples W_GI and R_GI was measured using viscometry in cupriethylene diamine (CED) (TAPPI T 230 om-19 1999) with a capillary viscometer Routine 100 (Cannon-Fenske). Irradiation was carried out four days after the ink application. Due to experimental constraints, the viscometry measurements were carried out 29 days after the irradiation. Before the viscometry analysis, the paper samples were chemically reduced with NaBH4 (same treatment as described above) in order to avoid solvent induced depolymerization. They were dried between blotters and conditioned at 50% RH and 23 °C. The viscometric DP (𝐷𝑃 𝑣 ) was calculated from the intrinsic viscosity [η] using the Mark-Houwink-Sakurada equation, by applying the coefficients proposed by Evans and Wallis [START_REF] Evans | Radiation damages during synchrotron X-ray microanalyses of Prussian blue and zinc white historic paintings: detection, mitigation and integration[END_REF]) : [𝜂] = 0.91 × 𝐷𝑃 𝑣 0.85 . 𝐷𝑃 𝑣 is assumed to be very close to 𝐷𝑃 𝑤 as 𝑀 𝑣 has been reported to be closer from 𝑀 𝑤 than 𝑀 𝑛 [START_REF] Sb | Properties and uses of cellulose solutions[END_REF]. This allows to parallel 𝐷𝑃 𝑣 with 𝐷𝑃 𝑤 with some confidence. The formula proposed by Dupont et al. [START_REF] Dupont | Accurate molar masses of cellulose for the determination of degradation rates in complex paper samples[END_REF]) was used for the conversion of 𝐷𝑃 𝑣 to 𝐷𝑃 𝑛 : 𝐷𝑃 𝑛 = 1575 𝑒 (𝐷𝑃𝑤 3536) ⁄ -1575.

Hydroxyl radicals

The paper samples were soaked for 3 minutes in a methanolic solution of TPA (98%, Sigma Aldrich) (1 mM). They were left to dry at ambient temperature for 24 h and conditioned at 23 °C at 50% RH. TPA reacts with hydroxyl free radicals (HO • ) in the paper and produces hydroxyterephthalic acid (HTPA), which accumulates in the paper. HTPA was extracted from the paper (2-3 mg) by soaking during three hours in 300 µl of phosphate buffer (KH2PO4 50 mM pH 3.2, 70% water:30% methanol), and was quantified by reverse phase liquid chromatography with UV and fluorescence detection (RP-HPLC/FLD-DAD) according to a previously established method [START_REF] Jeong | Degradation of cellulose at the wet-dry interface. II. Study of oxidation reactions and effect of antioxidants[END_REF]. HTPA (µmol g -1 ) was calculated with respect to the paper dry weight, subtracting the additives weight, to correlate sample behavior based on their cellulosic content only.

In order to ensure the quality of the results, it was verified that no HTPA was formed upon irradiating TPA powder.

Colorimetric and UV luminescence measurements

The diffuse reflectance and UV luminescence of the paper samples were measured with a noninvasive UV-Vis-NIR spectroradiometer (Specbos 1211UV, JETI). For UV luminescence, the excitation was centered at 365 nm (with full width half-maximum FWHM = 20 nm). Measurements

Results and discussion

Calculation of the X-ray dose

The extent of radiation damage usually depends on the X-ray dose absorbed by a sample. Different papers are expected to absorb X-ray differently, especially when heavy elements are present as the latter increase the absorption. Measuring the X-ray dose is thus essential in order to compare changes in paper samples after irradiation on a common basis. In order to do so, it is necessary to define the linear attenuation coefficient (μ) of each sample for each experimental setup and condition (see synchrotron X-ray setup for dose calculation). μ depends on the sample material, but also on the X-ray energy. For the laboratory instrument micro XRF irradiation, µ of the Whatman n° 1 paper (W) was calculated using the entire energy spectrum of the polychromatic beam (Fig. S4b in the Supplementary data file). For the experiments on PUMA beamline, the values of μ for W were measured at the three energies used for the irradiation. The values decreased with increasing energy: 4.29 ± 0.07 cm -1 at 7.22 keV, 1.02 ± 0.06 cm -1 at 12.5 keV and 0.31 ± 0.031 cm - 1 at 18 keV. The values for µ of the model and historical papers at 7.22 keV are given in Table 2.

Fe in W_GI and R_GI and the calcium carbonate in W_Ca and K both increase the photon absorption. Although they contain a similar amount of calcium carbonate and have a similar pH (~8.9), K has a higher µ than W_Ca. This value (9.56) was also the highest of all. This was explained by the higher paper density as well as smaller and more homogeneously distributed mineral particles. This observation also applies to the iron gallate ink coated papers, where the presence of iron on the surface increases the value of µ. The gelatin in W_G seemed to increase only slightly the value of µ, while the opposite was observed in R_G. The artificially aged samples W_hyg, W_ox, W_red and R_hyg also showed a higher X-ray absorption, which might be due to a slightly higher paper density.

The absorption coefficients of the archival paper samples varied from 5.60 (LN5) to 8.18 (DCN).

These values could again be related to the paper density, with the higher density papers DCN, MI and LN1 exhibiting the higher µ values. The distribution of the fibers and non-fiber components across the sheets also most probably played a role. DCN displayed the highest µ, which was attributed to the presence of calcium (Table 1), as well as copper and iron (Table 1, Table S1, Figs S3 & S5 in the Supplementary data file).

These results confirmed that the additives influenced the way paper absorbed X-rays and suggested that structural parameters such as fiber density may also play a role.

DP and hydroxyl radicals

Impact of the X-ray dose and photon energy A first experiment, carried out with a laboratory Micro X-ray Fluorescence Spectrometer, allowed assessing the impact of low doses in the range of those usually delivered by these laboratory instruments. All the unaged W samples, irradiated to a dose up to 22 Gy, had a similar DP to the Control sample (Fig. S6 in the Supplementary data file), indicating that no macromolecular degradation took place during the irradiation, whether the dose was delivered at once or in several stages. This is consistent with the lowest observable adverse effect dose (LOAED) for glycosidic scissions of 0.21 kGy defined in our previous work [START_REF] Gimat | Short-and Long-Term Effects of X-ray Synchrotron Radiation on Cotton Paper[END_REF].

Exposures to synchrotron X-ray radiation were carried out next to study (i) the variation of the photon energy and (ii) reach higher doses, similar to those used during spectroscopic examinations with this type of instrument. Synchrotron X-ray fluorescence experiments usually use energies in the range of 1 to 20 keV (Glaser and Deckers 2014), sometimes even higher if heavy elements are investigated. To investigate if the degradation was energy dependent, W samples were exposed to three photon energies: 7.22, 12.5 and 18 keV. Fig. 1 shows the glycosidic scissions concentration (S) as a function of the absorbed dose up to 3.9 kGy, at the three energy levels. S increased with the dose in the range of 0 to 6 μmol g -1 . The impact was very small below the LOAED (0.21 kGy)

and was followed by a linear increase from 0.5 kGy upwards. S increased steeply, similarly at 12.5 keV and 18 keV. At 7.22 keV, all the values of S were shifted up. A similar energy dependence has been observed for electron beam irradiation [START_REF] Bouchard | The effects of ionizing radiation on the cellulose of woodfree paper[END_REF]) at energies of several MeV.

While the photoelectrons created by the X-ray photons in our experiment have much lower energies, it seems like the inverse relation between kinetic energy and cellulose damage remains true in the keV regime. We are not sure why this is the case, but it is noteworthy that the inelastic mean free path (IMFP) varies considerably for electron energies in the range of our experiment compared to the typical average diameter of cellulose fibers. The IMFP for electrons in graphite changes from 9.2 nm at 7.3 keV to 19.5 nm at 18 keV [START_REF] Shinotsuka | Calculations of electron inelastic mean free paths. X. Data for 41 elemental solids over the 50 eV to 200 keV range with the relativistic full Penn algorithm[END_REF]. While the exact path lengths in cellulose will likely be slightly different, this shows that it is thus much more probable that a photoelectron produced by 18 keV X-rays escapes the cellulose fibers before causing damage than it is for one produced by 7 keV X-rays. Based on these results, all the following experiments were carried out at 7.22 keV, the most penalizing conditions, to enhance the chances of observing and characterizing damage.

Impact of the iron gallate ink

For the three samples coated with the iron gallate ink (W_GI, R_GI and M_GI), the DP of the nonirradiated samples was considerably lower than that of the Control samples (W, R and M) as represented by the arrows on Fig. 4. This was attributed to strong and almost instant acid hydrolysis and oxidation reactions due to the presence of iron gallate ink, which occurs within the period between sample preparation and analysis (33 days). This has been observed previously [START_REF] Rouchon | Room-Temperature Study of Iron Gall Ink Impregnated Paper Degradation under Various Oxygen and Humidity Conditions: Time-Dependent Monitoring by Viscosity and X-ray Absorption Near-Edge Spectrometry Measurements[END_REF](Rouchon et al. , 2016)). Indeed, a DP loss of 25% and 30% was measured for W_GI and R_GI, respectively, which is consistent with previous observations [START_REF] Rouchon | Room-Temperature Study of Iron Gall Ink Impregnated Paper Degradation under Various Oxygen and Humidity Conditions: Time-Dependent Monitoring by Viscosity and X-ray Absorption Near-Edge Spectrometry Measurements[END_REF] for inked

Whatman n 1 where a 24% DP loss was recorded within a similar timeframe. A striking observation was made in the low doses range: after irradiation (up to 0.29 kGy for W_GI, 0.36 kGy for R_GI and 2.4 kGy M_GI), the DP of the iron gallate ink coated samples was higher than the DP of their non-irradiated counterpart (Fig. 4, dashed lines). This was interpreted as having two possible causes. First, it has been shown that iron gallate ink containing papers produce free radicals, such as HO° and other reactive oxygen species (Gimat et al. 2016[START_REF] Gimat | Behavior of cellobiose in iron-containing solutions: towards a better understanding of the dominant mechanism of degradation of cellulosic paper by iron gall inks[END_REF]. This enhances the chances for free radicals recombination leading to the auto-oxidation termination reactions, and in turn lowers the concentration of radicals accumulated in the paper, thus, preserving cellulose from their attack. Secondly, the crosslinking induced by the recombination of cellulosic radicals could lead to an increase in DP which would be measurable if the radicals have high molar mass. This is consistent with the fact that in the low irradiation doses range, HTPA was produced in higher amount in the irradiated ink coated samples W_GI and R_GI than in the Control counterparts W and R (Fig. 3a, 3b), and in similar amount as in non-irradiated W_GI and R_GI. In the higher doses range (from 0.89 kGy for W_GI, 1.1 kGy for R_GI, and 6.3 kGy for M_GI), the samples reached a lower DP than the non-irradiated samples, and the HTPA concentration in the samples reached similar levels with and without ink. This was interpreted as an indication that the enhanced scissions induced by the irradiation at the high doses likely exceeded the supposed impact of the free radicals recombination reactions. Impact of the degradation state

The artificially degraded papers (W_hyg, W_ox and W_red) were irradiated at 7.22 keV to various doses to study if and how the degradation state modifies the impact of the X-rays exposure.

The intent was to possibly extrapolate the results to centuries-old cultural heritage paper. The three samples had a similar starting DP (DPw  1400, i.e about 50% lower than W) and a different oxidation state: NCu = 0.42 for W_ox (i.e. 5.83 µmol g -1 total carbonyl groups, as calculated according to [START_REF] Röhrling | A Novel Method for the Determination of Carbonyl Groups in Cellulosics by Fluorescence Labeling. 2. Validation and Applications[END_REF], NCu = 0.11 for W_hyg (i.e. 0.67 µmol g -1 total carbonyl groups)

and NCu = 0.02 for W_red (i.e. near-zero carbonyl groups besides the reducing ends) (Table 1).

Figures 5a and5b show the glycosidic scissions concentration in these samples as a function of the dose. In W_hyg, S increased linearly with the dose, yet slightly less than in W Control (Fig. 5a).

This suggests that lower DP and/or higher carbonyl groups concentration might lessen somewhat the impact of X-rays (slight "counter-degradation effect"). For samples that underwent oxidation (W_ox and W_red), S was in the same range (up to 6 μmol g -1 ) as for W and W_hyg at respective doses (Fig. 5b), yet with higher standard deviations. The main contrast between the two samples is in the low dose region. In the range -up to 0.5 kGy, while W_red underwent fewer glycosidic scissions, in W_ox S was higher than in the other samples, with a sharp increase to 1-2 μmol g -1 for doses below 1.4 kGy. This indicates that at low doses, a high concentration of carbonyl groups in the paper tended to enhance the X-ray induced depolymerization. In the higher doses range (≥ 1 kGy), the depolymerization of all the samples reached the same range, between 4 and 6 μmol g -1 .

The "carbonyl" ("pro-degradation") effect seemed overridden by the overall stronger depolymerization inflicted by the higher irradiation doses. Fig 6a shows that a similar amount of HO° free radicals was produced in W_hyg and in W, indicating that the free radicals were not fully responsible for the difference in the glycosidic scissions, and that the HO° were not significantly involved in the production of carbonyl groups.

Neither a pro-, nor a counter-degradation effect of low DP and high oxidation level was observed in the linen rag model papers. S extended higher (up to 8 μmol g -1 ) and increased linearly as a function of the dose, yet, in a similar way for the undegraded Control sample and for the degraded R_hyg, despite the DP of the latter being 34% lower (DPw  1961) (Fig. 5c). Moreover, above 0.1 kGy, slightly less HO° free radicals were detected in R_hyg than in R (Fig. 6b). This, and the large standard deviations on each data point of R samples muddles the interpretations. The extrapolation of the results from a simple machine-made cellulosic paper to a traditional handmade rag pulp paper is not straightforward. The next level of complexity, which was to test the response of archival papers, was thus anticipated as very challenging.

Archival paper documents

For the historic samples irradiated to various doses, S values were overall in the same range as those measured for the model samples. Only LN5 degraded more and showed S values at least twice as high at all the doses tested (Figs. 7a,7b). However, the depolymerization behavior with increasing dose was not progressive as observed for the model papers (except for LN1), especially in the low doses. For instance, SE did not undergo scissions below 3 kGy, and M had a constant degradation (plateau) response on the whole dose range, with S around 3.5 µmol g -1 (Fig. 7a). No correlation could be made with the DP (table 1), the paper constituents or the paper density. All the papers have similar iron and calcium content, except for DCN which has more calcium due to the calcium carbonate filler (Fig. S5 in the Supplementary data file). The other possible difference in composition would be the gelatin content, the latter being a factor that tends to lower the degradation in the model papers. Unfortunately, the gelatin content of the archival papers was unknown and could not be measured. However, an indirect indication of sizing was given by a water drop absorption test, which showed that M and LN5 were more hydrophobic than DCN (fig. S3 in the Supplementary data file). Even though there can be other reasons for paper hydrophobicity such as reduced porosity for instance, the former two showed higher S then the latter, which would tend to invalidate the aforementioned protective role of gelatin. The differences in S could thus arise from local heterogeneity and to samples' structural parameters such as porosity or constituents' composition. This was not unexpected as in handmade papers the additives are usually not as homogeneously distributed at the microscopic level as in industrial papers.

Very high doses, between 32 and 38 kGy, were then tested on the archival papers, as well as on W.

The results showed that DCN, LN1 and LN5 were similarly extensively degraded as W, with S comprised between 30 and 43 µmol g 7c). The production of HO° free radicals was measured in SE and M. In SE, HO° concentration was very low, but it was higher in M over the whole dose range reaching a similar amount as in the model papers containing Ca and ink (Fig. 7d). This difference was thus attributed to the slightly higher calcium and iron content in M than in SE (Fig. S5 in the Supplementary data file).

UV Luminescence and yellowing

Before the irradiation, all the samples, model and archival papers, exhibited luminescence under UV when excited at 365 nm, which is a common feature of paper (Fig. S7 in the Supplementary data file). The model samples W and R exhibited a luminescence maximum λmax at 432 nm, which is consistent with previous data [START_REF] Gimat | Short-and Long-Term Effects of X-ray Synchrotron Radiation on Cotton Paper[END_REF]. The intensity varied depending on the degradation state, luminophores being produced during the aging, and on the type of additive. For instance gelatin is expected to show a broad luminescence spectrum with λmax = 402 nm (unaged) and 414 nm (artificially aged at 50% RH and 80° C) [START_REF] Yova | Photochemical effects and hypericin photosensitized processes in collagen[END_REF][START_REF] Duconseille | Molecular changes in gelatin aging observed by NIR and fluorescence spectroscopy[END_REF].

After X-ray exposure, no change in the UV luminescence spectral distribution was observed during the three years monitoring period. The intensity at λmax [I(λmax)] of each spectrum was followed the data points for R on Fig 10a correspond to higher doses than for W samples and that R_hyg is the most strongly irradiated sample. This may indicate complex radiochemistry mechanisms of chromophore destruction and chromogens formation. As opposed to the observations after hygrothermal aging of gelatin sized papers (Dupont 2003a;[START_REF] Missori | Gelatine sizing and discoloration: A comparative study of optical spectra obtained from ancient and artificially aged modern papers[END_REF], the irradiation did not modify the yellowing in the gelatin sized papers. This may be related to the radical scavenging properties of gelatin, which could lower the kinetics of cellulose chromophores formation. To sum up, for the model samples, the additive that had the highest impact on luminescence was gelatin. This is most probably due to its own intrinsic luminescence properties and to maybe also to its chromogenic degradation products appearing post-irradiation. Yellowing did not appear to be linked to either the initial conservation state nor to the presence of additives, which indicates complex mechanisms at play of chromogenic structure formation and destruction. The initial UV luminescence spectra of the archival papers showed maxima with different intensities I(λmax) and positions (between 444 nm and 460 nm), which could be due to differences in the quantity and the type of UV-absorbing groups such as carbonyl compounds, respectively. This could also be due to a different moisture content, as the latter has been shown to affect the luminescence properties of paper (Kocar et al. 2005;[START_REF] Castellan | Studies on fluorescence of cellulosics[END_REF]. Before irradiation, no correlation between the state of degradation (DP) and the intensity of the luminescence of the papers could be made. Indeed, LN1 and M, both with similar DP around 1500, displayed more intense luminescence than the other historic samples, either more degraded (DPw LN5 = 1039 ± 5.7%, and DP SE =1000 ± 9.4%) or less degraded (DP DCN = 2869 ± 6.2%).The presence of additives such as gelatin could not be fully responsible of the luminescence intensity either, as the latter was not correlated to the hydrophobic properties used as an indication of the gelatin content (M and LN5 highly hydrophobic, LN1 medium hydrophobic, SE and DCN not hydrophobic) After irradiation at doses below 4.4 kGy no change was observed on the archival papers. Indeed, no differences in the UV luminescence (data not shown) nor the yellowing (b* < 1) were measured in the irradiated vs the non-irradiated areas (Fig. 9b). At the highest doses tested (26-36 kGy), a slight luminescence appeared on M and DCN twenty-one months after the irradiation (Fig. 10). It thus took almost two years for the luminophores to build up inside the archival papers.

Similarly, as with the model samples, no correlation between the luminescence and the DP, or the glycosidic scissions could be made. A test was made by irradiating M at a very high dose (290 kGy), which induced marginal luminescence, and only after ten months (data not shown). In conclusion, the behavior of historical papers under X-ray is multifactorial and difficult to predict. This study showed that the response of archival paper to X-ray radiation is very varied, in terms of DP losses (the largest being for DCN and LN1), luminescence (M and DCN exhibited the higher luminescence intensity), and yellowing (M and LN1 yellowed the most). These observations led to the conclusion that in the samples with additives and in the aged/degraded samples, optical changes (yellowing and luminescence) were mostly uncorrelated. Moreover, as observed with the model papers, these optical changes were also not directly correlated with the macromolecular state (depolymerization). These observations underline the complex chemistry triggered by the exposure to X-rays.

Conclusion

Synchrotron X-ray radiation at energies and doses most often applied for analytical purposes to paper-based cultural heritage has been shown to be detrimental to one-component cellulosic paper (Whatman n°1). However, field situations are complex as historic papers are multiparametric, which interferes with a precise prediction of the effect of X-ray radiation. They usually are degraded to some extent and they contain other constituents besides the biopolymers, such as papermaking additives, ink and their degradation by-products. The present research investigated how these parameters could influence the X-ray radiation-induced degradation when studied separately in model papers. The latter were artificially aged and/or supplemented with additives, which enabled to single out some of the influential parameters. The additives tested were gelatin as sizing agent, and calcium carbonate as filler. Iron gallate ink was applied on some of the gelatinsized papers, modeling writing/drawing medium. Following the same methodological approach as developed in a previous publication [START_REF] Gimat | Short-and Long-Term Effects of X-ray Synchrotron Radiation on Cotton Paper[END_REF], the changes were measured immediately after the irradiation at the microscopic level (macromolecular and molecular degradation) and the macroscopic changes embodied by the optical properties (UV luminescence and yellowing) were monitored time-delayed.

In the dose range from 0 to 4 kGy, gelatin-sized samples and samples with CaCO3 underwent a slightly reduced irradiation-induced depolymerization. Surprisingly, up to 0.89-1.1 kGy, the iron gallate ink coated papers had a higher DP than the Control samples, which was attributed to a decrease in the free radical initiated autooxidation reactions through radicals recombination and crosslinking. Above these doses, a higher rate of scissions induced by the irradiation prevailed. The production of hydroxyl free radicals was higher in all the samples containing CaCO3, maybe due to the increased lifetime of HO° at alkaline pH. The depolymerization behavior of the aged model samples was different in the industrially-made (Whatman no 1) and in the handmade papers (linen rags). Higher degradation state (lower DP) tended to stabilize Whatman n1 paper towards X-ray radiation, by lowering the macromolecular degradation. Conversely, the aged handmade paper showed a similar amount of glycosidic scissions as the unaged counterpart. Carbonyl groups in the artificially aged Whatman n1 papers increased the glycosidic scissions in the low doses range, below 0.5 kGy. Confirming previous results [START_REF] Gimat | Short-and Long-Term Effects of X-ray Synchrotron Radiation on Cotton Paper[END_REF], the optical changes appeared with considerable delay, often one year after the irradiation, and could not be directly correlated to the initial DP nor to the glycosidic scissions concentration that grew steadily during this postirradiation period (dark storage, room temperature). As expected, the archival papers made of linen rags had an overall more complex X-ray exposure behavior than the model papers. First, the DP was roughly constant in the low doses range below 4 kGy, which led us to increase the irradiation doses. The large dispersion of the data was attributed to the paper macroscopic and microscopic heterogeneity in terms of additives distribution degradation state and microstructure, but also to the presence of multiple possible pro-and counter-degradation constituents in historic papers acting synergistically. At very high doses (26-36 kGy), the archival papers reached the LODP immediately upon irradiation, similarly as Whatman n 1. No color change or UV luminescence were observed within one year after the exposure at those high doses. After twenty-one months, two archival papers showed a slight UV luminescence, but no clear connection with the depolymerization or with the constituents could be made. These contrasted results indicate that laboratory samples have their limitations to model archival/historic papers and that the radiochemistry at play is rather complex. However, the observation that, overall, the historic papers resisted better the X-ray exposures than modern papers is an important step forward that enables to consider analyzing historic papers with better confidence. This work focused on the paper material in chemical terms. Considering paper microstructure properties in the future may shed more light on the limitations encountered.

A significant outcome of this work was to show the importance of carefully choosing the analytical conditions that limit the exposure, thus the dose, when analyzing genuine artefacts using X-rays.

This can be achieved either by applying higher energy, or using low exposure times, and always maintaining some humidity, as demonstrated in our previous work. The mid-range relative humidity value recommended for paper-based cultural heritage storage is thus a good compromise.

(

  EMC) at 23 •C and 50% RH (TAPPI T 502 cm-07 1998), weight-average or viscometric-average (*) degree of polymerization (DP0), pH (TAPPI T 509 om-15 2002), copper number (NCu) (TAPPI -T 430 cm-99 1999), concentration of total carbonyl groups ([CO]) (Röhrling et al. 2002), ash content (measured at 525 °C) (TAPPI 211 om-02 2002), alkaline reserve (AR) expressed as % CaCO3 (TAPPI T 553 om-00 2000

  chains formed is equal to S. The number of scissions being equal to N molecule t -N molecule t0 and 𝑁AGU being equal to 6170 µmol g -1 of paper, hence S = 6170

  were normalized to a blue luminescent certified reflectance standard (USFS-461 Spectralon) to correct for light intensity change with time. The maximum intensity of each UV luminescence spectrum was used to monitor the global intensity change. To calculate the change the following formula was used: ∆I(λ max ) = I(λ max )[𝑖𝑟𝑟] -I(λ max )[𝐶𝑡𝑟𝑙] with I(λ max )[𝑖𝑟𝑟] and I(λ max )[𝐶𝑡𝑟𝑙] the intensities of luminescence at the wavelength maxima measured inside (irr) and outside (Ctrl) of the irradiated area, respectively. A spectrophotometer (Konica Minolta, CM-26d) was used to measure the chromaticity coordinates L*, a* and b* in the CIELAB 1976 system. The variation of the color coordinate b*, which spans on the blue-yellow scale, was used to quantify the yellowing expressed as 𝛥𝑏 * = 𝑏 * -𝑏 0 * (positive value).

Fig. 1 Fig. 2

 12 Fig. 1 Glycosidic scissions concentration S as a function of the X-ray dose in Whatman no. 1 irradiated at energy levels 7.22, 12.5 and 18 keV at 50% RH

Fig. 3

 3 Fig. 3 Impact of gelatin, iron gallate ink (a, b) and calcium carbonate (c) in W and R papers on the HTPA concentration as a function of X-ray dose. The logarithmic scale is used for easier visualization.

Fig. 4

 4 Fig. 4 Viscometric-average degree of polymerization (DPv) of iron gallate ink coated papers W_GI (a), R_GI (b), and M_GI (c) as a function of X-ray dose (kGy). The dose is represented on a logarithmic scale for easier visualization. Control samples without ink or gelatin are represented by the void data points. Arrows represent the DP drop due to the iron gallate ink. At low X-ray doses, irradiated samples have a higher DP than non-irradiated control sample (above the dotted lines)

Fig. 5

 5 Fig. 5 Glycosidic scissions concentration (S) in aged papers W (a, b) and aged rag papers R (c) as 459 a function of X-ray dose compared to respective control samples. 460

  -1 (Figs 7a & 7b). M and SE resisted surprisingly well, being the least degraded samples, with S close to 10 µmol g -1 . Despite the different kinetics, most samples approached LODP (Levelling Off Degree of Polymerization) with DPw W = 345; DPw M =318; DPw LN5= 426; DPw LN1= 495. The least degraded samples were DCN (DPw = 530) and SE (DPw = 708) (Fig.

Fig. 7

 7 Fig. 7 Glycosidic scissions concentration (S) (a, b), DPw (c) and HTPA concentration (d) in archival samples as a function of X-ray dose. Logarithmic scale (c-d) is used only for a better display of the data.

Fig. 9

 9 Fig. 9 Yellowing increase (b*) of papers measured three years after X-ray irradiation on W and R model papers (a) and on the archival papers (b, c).

Fig. 10

 10 Fig. 10 Photographs under UV light of W and archival papers exposed to X-ray radiation at doses between 26 and 33 kGy, 1 month (a) and 21 months (b) after the irradiation.

Table 1

 1 Samples characteristics: constituents, aging method, thickness (x), density (ρ), equilibrium moisture content

  ). Standard deviation (STD) is provided when possible. Cot: cotton papers; lin: linen rag pulp papers; ox: oxidative degradation; hyg: hygrothermal 184 aging; red: reduction treatment; nat. natural aging; Ca: calcium carbonate filler; I: iron gallate ink; Gel: gelatin sizing 185 (identified with hydroxyproline spot test, with ++: highest size content; +: medium size content; ~: lowest size content 186 (levels defined with a water drop absorption test) (Fig S3 in the Supplementary data file). n.d. stands for not determined.

	187
	188
	Sample	fiber Additive	aging	x µm	ρ g cm -3	EMC % wt	DP0	pH	NCu / [CO] μmol g -1	Ash %	AR %eq
	W	cot	none	none	170	0.51	5.43	2948±2%	6.90 ±0.02 0.05	<0.1
	W_hyg	cot	none	hyg	151	0.55	5.46	1490±2%	6.2 ±0.2	0.11 / 0.67
	W_ox	cot	none	ox	170	0.53	n.d.	1352±2%	6.73 ±0.02 0.42 / 5.83
	W_red	cot	none	ox/red	170	0.53	n.d.	1431±1.1%	6.41 ±0.05 0.02
	W_G	cot	Gel+	none	170	0.54	6.13	3021±1%	5.70
	W_GI	cot	Gel+, I	none	170	0.62	6.90	2225*	4.21
	K	cot	Ca	none	100	0.68	5.61	2566±2%	8.89		7.6	7.3
	W_Ca	cot	Ca	none	170	0.54	5.20	2803±2.2%	8.86		5.4	6.0
	R	lin	none	none	160	0.54	5.78±0.18 2980	7.90
	R_hyg	lin	none	hyg	141	0.60	n.d.	1961±1.6%	6.14
	R_G	lin	Gel+	none	160	0.53	6.57	3326±6.8%	7.68
	R_GI	lin	Gel+, I	none	150	0.55	7.21	2290*	5.15
	SE	lin	Gel~	nat	115	0.64	5.46	1000±9.4%	5.90
	LN5	lin	Gel++	nat	125	0.62	5.36	1039±5.7%	4.92		1.1
	M	lin	Gel++	nat	125	0.47	5.75±0.04 1490±12.8% 5.03		1.5
	M_I	lin	Gel++, I	nat	125	0.72	n.d.	702*	n.d.
	LN1	lin	Gel+	nat	165	0.68	5.12	1608±2.3%	5.28		0.8
	DCN	lin	Gel~, Ca	nat	90	0.72	6.01	2869±6.2%	7.61		2.5

Table 2

 2 Linear attenuation coefficient µ of Whatman n 1 papers (cot), linen rag papers (lin) and archival papers

	(archiv) at 7.22 keV, 50% RH.
					Model papers
	cot	W	W_hyg	W_ox	W_red	W_G	W_GI	K	W_Ca
	µ (cm -1 )	4.29±0.07	5.32±0.07 5.66±0.14	5.28±0.044	4.76±0.016 7.03±0.036	9.56±0.2	6.89±0.12
	ρ (g cm -3 )	0.51	0.55	0.53	0.53	0.54	0.62	0.68	0.54
	lin	R	R_hyg			R_G	R_GI
	µ (cm -1 )	5.01±0.25	5.69±0.28			4.77±0.07	7.99±0.08
	ρ (g cm -3 )	0.54	0.6			0.53	0.55
					Archival papers
	archiv	SE	LN5	LN1	DCN	M	M_I
	µ (cm -1 )	6.66±0.036	5.6±0.23	7.21±0.10	8.18±0.6	5.69±0.030 10.05±0.030
	ρ (g cm -3 )	0.64	0.62	0.68	0.72	0.47	0.72

Acknowledgements

This research was supported by Paris Seine Graduate School of Humanities, Creation, Heritage, Investissements d'Avenir ANR-17-EURE-0021-Foundation for Cultural Heritage Science. We are grateful to synchrotron Soleil for the access to the PUMA beamline within the proposal 20181723 and to Tülin Okbinoglu for her help on the beamline. We thank Samia Rebaa and Naomi Nganzami Ebale, undergraduate chemistry students (Sorbonne Université), for help with photography and spectroscopy. We also thank Sabrina Paris Lacombe for technical help with Size Exclusion Chromatography.

Documenting the exact location of the X-ray photons impact and implementing a long-term monitoring of the eventual changes through regular photographic follow-up under both UV and visible lights are also advisable.

Impact of calcium carbonate and gelatin

The glycosidic scissions and hydroxyl free radicals concentrations in the unaged papers, in the papers with calcium carbonate and in the papers with gelatin increased in a quasi linear fashion as a function of the X-ray dose, up to 6 μmol g -1 for W and up to 8 μmol g -1 for R (Figs. 2 & 3). S in W_G was slightly lower than in W Control (Fig. 2a). This observation is consistent with the fact that gelatin size tends to lower the depolymerization rate of cellulose during the degradation induced by aging (Dupont 2003a). Besides gelatin, this could also be partly due to the difference in moisture in unsized vs sized paper (EMC at 23•C of 5.43% and 6.13%, respectively), as moisture was shown to reduce cellulose depolymerization during synchrotron X-ray irradiation [START_REF] Gimat | Short-and Long-Term Effects of X-ray Synchrotron Radiation on Cotton Paper[END_REF]. Because ofa larger standard deviations on the data points, this was less clearly established for R and R_G, where S values were quasi similar (Fig. 2b) despite the EMC difference (5.78% vs 6.57%, respectively) (Table 1).

The samples with gelatin (W_G and R_G) produced less hydroxyl radicals than the Control samples (Fig. 3a and3b). This could be an indication that gelatin was able to scavenge the HO° produced during the irradiation.

In both W_Ca and K, S increased slightly less as a function of the dose than in W Control, which is especially visible at the high doses, as shown on Fig. 2c. This suggests that calcium carbonate can buffer the acids produced during the X-ray exposure, which is the expected role of the alkaline reserve in paper [START_REF] Whitmore | Determination of the Cellulose Scission Route in the Hydrolytic and Oxidative Degradation of Paper[END_REF][START_REF] Ahn | The influence of alkaline reserve on the aging behavior of book papers[END_REF]Rouchon and Belhadj 2016). On the other hand, W_Ca and K showed a larger HO° production than the Control samples (Fig. 3c). This, again, could be due to the pH, as an alkaline medium is known to enhance the lifetime of HO° radicals, and hence the probability that they react with TPA. These results also confirmed previous observations that the HO° concentration did not always correlate directly with the glycosidic scissions concentration, and indicate that other species and mechanisms are involved [START_REF] Jeong | Degradation of cellulose at the wet-dry interface. II. Study of oxidation reactions and effect of antioxidants[END_REF][START_REF] Gimat | Short-and Long-Term Effects of X-ray Synchrotron Radiation on Cotton Paper[END_REF]). over time. After eight months, the area irradiated at 7.22 keV of the model papers (dose range from 2.8 to 4 kGy, depending on the sample) exhibited an increase in the intensity of the UV luminescence compared to the respective Control samples (ΔI(max)), as shown Fig. 8 and Fig. S8 (Supplementary data file). All Whatman no.1 model papers (except for the sized samples), showed luminescence proportionally to the absorbed dose (Fig 8, white marks, linear trendline). The highest increase in luminescence was observed on the sized samples W_G and R_G with ΔI(max)R_G of 0.0042 and ΔI(max)W_G of 0.0036 (R_G data not shown), indicating that a large quantity of luminophores was produced post-irradiation. These samples were still the most luminescent samples after 11 months. The luminescence of W_hyg and K grew beyond that of the other samples between 8 and 11 months. The change in luminescence between unaged and aged R papers (with no additives) was slower than for unaged and aged W, with a smaller ΔI(max) of R and R_hyg (0.0011 and 0.00015, respectively) compared to W and W_hyg (ΔI(max) (0.0022 and 0.0025, respectively). Colorimetric measurements were carried out three years after the irradiation, on the irradiated areas and the non-irradiated control samples (Fig. 9). All the model samples showed very small b* values (Fig. 10a) and a global color change E* between 0.55 (for K) and 1.87 (for R), i.e. below the usually accepted level for a just noticeable difference. Among the W samples, the artificially aged W_hyg had the highest b* (1.35 ± 0.26). The opposite trend was observed with R samples, where R_hyg had a lower Δb* than R. This observed behavior difference is all the more valid since

Supplementary Files

This is a list of supplementary les associated with this preprint. Click to download.

GimatXrayadditiveSupplementarydata.docx


Séverine Toussaint

Jasper Ponstein

Mathieu Thoury

Rémi Métivier

Daniela Kalthoff

Benoît Habermeyer

Roger Guilard

Steffen Bock

Peter Mortensen

Sverre Sandberg

Fur glowing under UV: a widespread consequence of porphyrin accumulation in mammals

Keywords: UV Photoluminescence, Fluorescence, Pelage, Mammal, Spectroscopy, Multispectral imaging, Porphyrin

Spectacular photoluminescence (PL) phenomena have been increasingly reported in various organisms from diverse ecosystems. However, the chemical basis of this PL remains poorly de ned, and its potential ecological function is still blurry, especially in mammals. Here we used state-of-the-art spectroscopy and multispectral imaging techniques to document new cases of mammalian ultraviolet-induced PL (UV-PL) and to identify free-base porphyrins and natural derivatives as the organic compounds responsible for the reddish luminescence observed in the hairs and spines of distantly related species. We argue that pink to red UV-PL is predominantly observed in crepuscular and nocturnal mammals because porphyrins are photodegradable, and that this phenomenon might not have a speci c function in intra-or interspeci c communication but consists of a byproduct of a widespread physiological condition, overlooked in mammals.

Introduction

Ultraviolet-induced photoluminescence (UV-PL) has been observed in the external organs of plants 1 , 'invertebrates' 2 and in diverse vertebrates including ' shes', lissamphibians, squamates, birds, and mammals 3 . Concerning mammals, a handful of predominantly opportunistic observations have been reported, notably in the platypus [START_REF] Anich | Bio uorescence in the platypus (Ornithorhynchus anatinus)[END_REF] , some marsupials (opossums [START_REF] Meisner | Psychedelic opossums: uorescence of the skin and fur of Didelphis virginiana[END_REF]6 and bandicoots [START_REF] Reinhold | Mammals with uorescent fur : Observations from the Wet Tropics[END_REF] ), and rodents ( ying squirrels 8 and springhares [START_REF] Olson | Vivid bio uorescence discovered in the nocturnal Springhare (Pedetidae)[END_REF] ). Interestingly, this UV-PL can occur in different tissues, and the observed emitted colors vary a lot within the visible spectrum depending on the species. Reported observations include shades of dark blue from bone [START_REF] Bachman | Fluorescence of bones[END_REF][START_REF] Goutte | Intense bone uorescence reveals hidden patterns in pumpkin toadlets[END_REF][START_REF] Prötzel | Widespread bone-based uorescence in chameleons[END_REF][START_REF] Taboada | Naturally occurring uorescence in frogs[END_REF] , of light blue and green from skin and hairs 3,[START_REF] Anich | Bio uorescence in the platypus (Ornithorhynchus anatinus)[END_REF]6,8,[START_REF] Taboada | Naturally occurring uorescence in frogs[END_REF] , of yellow from birds' feathers [START_REF] Arnold | Fluorescent signaling in parrots[END_REF][START_REF] Mcgraw | A description of unique uorescent yellow pigments in penguin feathers[END_REF][16] , and shades of pink to red from feathers as well as from the pelage of most of the photoluminescent mammals [START_REF] Meisner | Psychedelic opossums: uorescence of the skin and fur of Didelphis virginiana[END_REF][6][START_REF] Reinhold | Mammals with uorescent fur : Observations from the Wet Tropics[END_REF][8][START_REF] Olson | Vivid bio uorescence discovered in the nocturnal Springhare (Pedetidae)[END_REF][START_REF] Weidensaul | Use of ultraviolet light as an aid in age classi cation of owls[END_REF] . This high variability implies that it is not the same UV-reactive molecular or structural compounds that are at the origin of this phenomenon depending on the species, suggesting probable different mechanisms at play. For instance, biological uids like urine are known to be photoluminescent 18,[START_REF] Leiner | The total uorescence of human urine[END_REF] , resulting in blue to yellow UV-PL observed. Recent studies found iridophores as responsible for greenish UV-PL in the dermis of a gecko 3 . The reddish UV-PL observed on the plumage of some birds was identi ed as caused by coproporphyrin III and protoporphyrin IX [START_REF] Church | Researches on Turacin, an animal pigment containing copper[END_REF][START_REF] Rimington | A reinvestigation of turacin, the copper porphyrin pigment of certain birds belonging to the Musophagidae[END_REF][START_REF] Negro | Porphyrins and pheomelanins contribute to the reddish juvenal plumage of black-shouldered kites[END_REF][START_REF] Okazaki | Distribution of protoporphyrin in bird feathers[END_REF][START_REF] Camacho | Correlates of individual variation in the porphyrin-based uorescence of red-necked nightjars (Caprimulgus ru collis)[END_REF][START_REF] Galván | Porphyrins produce uniquely ephemeral animal colouration: A possible signal of virginity[END_REF] , which are common molecules playing a crucial role in various biological processes and notably in heme biosynthesis 26 . A recent analysis on springhare pelage [START_REF] Olson | Vivid bio uorescence discovered in the nocturnal Springhare (Pedetidae)[END_REF] identi ed a mixture of other porphyrins, usually found in urine or feces of mammals [START_REF] Rimington | Porphyria in animals[END_REF] , along with an unidenti ed molecule. However, the question remains whether these compounds are also responsible for the reddish UV-PL observed in other mammals. Moreover, the precise spatial and spectral repartitions of these compounds within the pelage are still unknown, as well as their inter-and intraspeci c variability. Answering these questions would greatly help improving our knowledge on the biological origin and potential function of such photoluminescent mechanism in animals.

In addition to molecular identi cation, it remains unclear whether PL is actually seen and used by the organisms in their natural environment 1,28 . So far, two cases of sexual selection using PL have been experimentally demonstrated in budgerigars [START_REF] Arnold | Fluorescent signaling in parrots[END_REF] and in jumping spiders [START_REF] Lim | Sex-speci c UV and uorescence signals in jumping spiders[END_REF] . In mammals, based on the fact that UV-PL was predominantly observed in crepuscular or nocturnal animals, it was recently hypothesized that this may serve ecological functions in light-deprived or snow-covered environments such as intraspeci c communication or antipredator behavior [START_REF] Anich | Bio uorescence in the platypus (Ornithorhynchus anatinus)[END_REF]8,[START_REF] Olson | Vivid bio uorescence discovered in the nocturnal Springhare (Pedetidae)[END_REF] . These hypotheses have not been tested experimentally. Moreover, as the associated species are adapted to different ecosystems and lifestyles, from the semi-aquatic platypus to the terrestrial springhares and the arboreal ying squirrels, it remains unclear whether UV-PL has indeed a speci c ecological signi cance in nocturnal animals, or is simply a byproduct of a physiological condition such as pigmentation 1,28 .

Here we document a new selection of mammals exhibiting luminescence under UV, including hedgehogs (Erinaceus spp.) and ermine (Mustela erminea), substantiating the widespread occurrence of this phenomenon across this group. We focused on the reddish UV-PL observed in most of the species' pelage and used customized spectroscopy techniques to precisely identify in-situ the organic compounds at play as metal-free porphyrins. Furthermore, we used multispectral luminescence imaging to describe the spatial and spectral distributions of these porphyrinic compounds, demonstrating their speci c localization within hairs and spines along with their variable concentration across species. These results suggest a more pragmatic scenario in which this erratic accumulation of porphyrins into mammalian pelage, resulting in an observable photoluminescence under UV illumination, may not result from a speci c selection for nocturnal communication as previously argued, but from the non-pathological degradation process of porphyrins.

Terminological clari cations

Photoluminescence (PL) is a physical phenomenon in which a material reemits photons after a photonic excitation. It gathers two distinct phenomena, namely uorescence, or phosphorescence that can be differentiated by the difference in the electronic states involved in the radiative de-excitation pathway. Fluorescence and phosphorescence occur when the molecule in an excited state returns to the electronic ground state (essentially S 0 for most organic molecules), from the excited singlet or triplet state, respectively, by emission of a photon. The uorescence process occurs within a few tens of picoseconds to a few hundreds of nanoseconds, whereas phosphorescence, which corresponds to the forbidden transition between the excited and ground states, takes place on a timescale up to seconds, even minutes or more. UV-induced photoluminescence (UV-PL) is the process whereby absorbed UV photons trigger a radiative deexcitation at longer wavelengths. In contrast, bioluminescence (BL) corresponds to an emission of light generated by a living organism via chemical reactions.

Multiple cases of UV-PL and BL have been observed in various organisms either in their natural habitat or from preserved specimens. However, UV-PL is often referred to as "bio uorescence" in the literature, which might confuse either the scienti c community and the general public. Therefore, UV-PL is the appropriate technical term to use in this context, because it is neither proven whether it is uorescence or phosphorescence, and nor does it relate to bioluminescence. Moreover, although both phenomena result in spectacular luminous or glowing organisms, they consist in quite different mechanisms and are not observable under the same conditions. In practice, the UV-PL phenomenon is only observable to the human eye by using an arti cial source of ultraviolet light, associated with an optical lter to ensure error-free visual interpretation. Importantly, another noticeable difference is that the energetically demanding BL phenomenon can be correlated to identi able functions, while PL and UV-PL phenomena are more di cult to decipher 28 .

Results And Discussion

UV-induced photoluminescence is widespread in mammals Our investigation reveals that multiple species of mammals, representing the three major mammalian clades (monotremes, marsupials, and placentals), indeed show conspicuous photoluminescence when observed under long-wave UV illumination (365-395 nm; Fig. 1, Table 1). Also, we hereby document cases of UV-PL observed in two eutherian genera hitherto overlooked: the hedgehog (Erinaceus spp., Erinaceomorpha) which appears reddish, and the common ermine (Mustela erminea, Carnivora) which appears lavender to the human eye. Emission spectroscopy reveals that all analyzed mammal specimens emit broadly in the blue region with varying intensities (see the broad band at 450-480 nm in Fig. 1b). This blue UV-PL is particularly visible to the human eye on the poorly pigmented pelage in hedgehogs, ermines, and ying squirrels (Fig. 1a). We also con rm a visible blueish to greenish UV-PL on the pelage of the platypus, as previously reported [START_REF] Anich | Bio uorescence in the platypus (Ornithorhynchus anatinus)[END_REF] . As low pigmented human hair is also known to be visibly photoluminescent under UV in these speci c ranges due to their keratin bers [START_REF] Millington | Anomalous Fluorescence of White Hair Compared to Other Unpigmented Keratin Fibres[END_REF] , this phenomenon appears broadly shared in mammals.

Interestingly, a pink to red UV-PL is exhibited on the skin appendages (here hairs and spines) of phylogenetically distant species, such as marsupials (opossums) and placental mammals, the latter including ying squirrels (Euarchontoglires) and hedgehogs (Laurasiatheria) (Table 1, Fig. 1). Emission spectroscopy reveals in these mammals a series of peaks (often three) in the red region, between 600 and 690 nm, with variable intensity and position depending on the specimens (Fig. 1b), suggesting potential differences in the compounds at play or differences of the binding sites of the same compound [START_REF] Sandberg | Light-induced release of protoporphyrin, but not of zinc protoporphyrin, from erythrocytes in a patient with greatly elevated erythrocyte protoporphyrin[END_REF] . We additionally observed that the associated specimens exhibited variable intensity of observable reddish UV-PL. Notably, the older preserved specimens investigated appeared less intense, suggesting that the responsible compounds are degradable probably by light exposure and might be sensitive to storage condition. Furthermore, we observed that the localization of this reddish UV-PL on the pelage seems to vary within species. In our specimens, the belly was the most luminescent body region of opossum marsupials and ying squirrels, while it was the dorsal spines in hedgehogs (Fig. 1a). Lastly, the red UV-PL is not homogeneously distributed within individuals, as also reported in ying squirrels and springhares 8,9 , suggesting potential biological differences within individuals.

Porphyrin accumulation mammalian pelage is ubiquitous

We used excitation spectroscopy at 700 nm to probe, directly on solid samples, the absorption properties of the compounds responsible for the pink to red UV-PL observed in mammals' pelages. Spectra collected on hairs or spines show an intense band between 390 and 430 nm (so-called Soret band), followed by a series of bands, ten to a hundred times less intense, between 480 and 660 nm (so-called Q-bands) characteristic of free-base (nonmetalated) porphyrins (Fig. 2a). Porphyrins are tetrapyrroles, macrocyclic organic compounds including heme and chlorophyll (metalated) that are essential for life on Earth. Q-bands of decreasing intensity at 504.5, 539.6, 578.4 and 630.1 nm are undoubtedly indicative of protoporphyrin IX (PPIX) in the hedgehog's spines (Fig. 2a). However, the position and number of bands is different for the hairs of opossums and ying squirrels, indicating the presence of other porphyrins. Following ref. [START_REF] Dinello | Isolation and modi cation of natural porphyrins[END_REF] 's database, the position of Q-bands at 501.4, 535.4, 570.5 and 619.6 nm in the opossums' hairs (Fig. 2a) is consistent with uroporphyrin I or III, and to a lesser extent with porphyrin c.

The fth band observed at 638.4 nm most likely indicates that the uroporphyrin is protonated, as also suggested by the shoulder on the Soret band, two modi cations illustrated by our protonated protoporphyrin IX (PPIX2H) reference spectrum. For the ying rodents' hairs, the position of Q-bands at ~ 506, ~550, 578 and 640-646 nm only matches that of porphyrin S-411 [START_REF] Dinello | Isolation and modi cation of natural porphyrins[END_REF] , an analogue of coproporphyrin [START_REF] French | Identi cation of the acrylate porphyrin S-411 from meconium[END_REF] .

As part of the heme biosynthesis pathway, porphyrins are associated with all aerobic and anaerobic metabolisms and are ubiquitous in the cells of many organisms 26,[START_REF] De Oliveira Neves | Models for human porphyrias: Have animals in the wild been overlooked?: Some birds and mammals accumulate signi cant amounts of porphyrins in the body without showing the injurious symptoms observed in human porphyrias[END_REF] . Protoporphyrins and their natural derivatives, such as uroporphyrins and protoporphyrin S-411, are known to be photoluminescent and also involved in coloration, especially in birds feathers [START_REF] Church | Researches on Turacin, an animal pigment containing copper[END_REF][START_REF] Rimington | A reinvestigation of turacin, the copper porphyrin pigment of certain birds belonging to the Musophagidae[END_REF][START_REF] Negro | Porphyrins and pheomelanins contribute to the reddish juvenal plumage of black-shouldered kites[END_REF][START_REF] Okazaki | Distribution of protoporphyrin in bird feathers[END_REF][START_REF] Camacho | Correlates of individual variation in the porphyrin-based uorescence of red-necked nightjars (Caprimulgus ru collis)[END_REF][START_REF] Galván | Porphyrins produce uniquely ephemeral animal colouration: A possible signal of virginity[END_REF] . Interestingly, although protoporphyrins are normally excreted through feces in mammals, their overproduction and tissue accumulation are known but often associated with diseases called erythropoietic protoporphyrias. Other inherited metabolic porphyria disorders -described in humans with accumulation of δ-aminolevulinic acid and porphobilinogen -cause neurovisceral attacks (acute hepatic porphyrias), or/and skin lesions (cutaneous porphyrias with accumulation of porphyrin ring structures) depending on the affected step of the heme biosynthetic pathway [START_REF] Karim | Porphyrias: A 2015 update[END_REF]36 . In addition to humans, this condition has been described in a few other mammals such as ruminants, horses, and cats [START_REF] Rimington | Porphyria in animals[END_REF][START_REF] Tennant | Lessons from the porphyrias of animals[END_REF] . Interestingly, several cases of protoporphyrin accumulation not causing any disease were also reported in mammals. Notably, all members of the fox squirrel Sciurus niger are known to accumulate the free-base uroporphyrin I in their internal organs and skin without showing detrimental symptoms, as a result of the low activity of the enzyme uroporphyrinogen III synthase [START_REF] De Oliveira Neves | Models for human porphyrias: Have animals in the wild been overlooked?: Some birds and mammals accumulate signi cant amounts of porphyrins in the body without showing the injurious symptoms observed in human porphyrias[END_REF] . Other free-base porphyrins were recently identi ed as causing a non-pathological conspicuous reddish UV-PL on the pelage of several springhare species [START_REF] Olson | Vivid bio uorescence discovered in the nocturnal Springhare (Pedetidae)[END_REF] . Non-pathological porphyrin accumulation has also been described in a pet hedgehog 38 .

As our analyses were conducted on preserved collection specimens, we could not determine whether they exhibited porphyria symptoms before their death, and the causes of porphyrin accumulation in the mammals that we have sampled remain to be identi ed. Nonetheless, previous observations on living individuals suggest that this condition is predominantly not harmful for most mammal species. Also, as dermal lesions in humans are found in the skin where hairs are sparse [START_REF] Karim | Porphyrias: A 2015 update[END_REF] , it is possible that porphyrin accumulation occurring in the inert hair or spine tissues is naturally less problematic than the one occurring in living tissues such as those of the skin. Therefore, we suppose that most species may exhibit physiological solutions to adapt to the potential toxicity of porphyrin overproduction [START_REF] De Oliveira Neves | Models for human porphyrias: Have animals in the wild been overlooked?: Some birds and mammals accumulate signi cant amounts of porphyrins in the body without showing the injurious symptoms observed in human porphyrias[END_REF] . This suggests that porphyrin accumulation in the pelage of mammals, resulting in a reddish UV-PL phenomenon, is likely a much more common trait than previously assumed.

The platypus and ermine likely have other photoluminescent compounds in their pelage that remain to be identi ed. The analysis for these speci c UV-PL colors (blue-green and lavender) would require further investigation, because contrary to porphyrins they are not associated with characteristic spectra.

Does pelage photoluminescence have ecological function?

Our multispectral imaging reveals that the porphyrinic compounds responsible for the reddish UV-PL are located inside the spine and hair bers of mammals (Fig. 2b), as previously seen in springhares [START_REF] Olson | Vivid bio uorescence discovered in the nocturnal Springhare (Pedetidae)[END_REF] . This is particularly visible in the hedgehog' spines, where the PPIX is located within the inner lumen (Fig. 2b). This suggests that these porphyrinic compounds are excreted within the skin appendages, and throughout the natural process of continuous pelage growth. Also, we found that the spatial and spectral repartition of these compounds is not homogeneously distributed along the hairs and spines, and neither between individuals nor species. Indeed, in the hedgehog's spines, PPIX is coating the walls of the inner lumen from base to apex (Fig. 2b). In the hairs of marsupials and rodents, the porphyrins are present either throughout the total hair length (marsupials) or restricted to a more basal region ( ying squirrels). Additionally, we found that the intensity of UV-PL is also lower in areas that are otherwise less pigmented and in hairs that are thinner (e.g., ying squirrels) compared to the thicker and more pigmented spines of hedgehogs (Fig. 2b). Additionally, we found that alcohol-preserved specimens (e.g., marsupial from JAGUARS collections) show higher intensity of UV-PL, compared to the older and dry-preserved specimens of hedgehog or ying squirrels that we analyzed. This variation in degrees of reddish UV-PL intensity were also previously noted in museum specimens 6,8,9 . Because porphyrins are photodegradable compounds [START_REF] Poh-Fitzpatrick | Rates of plasma porphyrin disappearance in uorescent vs. red incandescent light exposure[END_REF] , it appears thus likely that the preservation of the reddish UV-PL on specimens' pelage may vary depending on both the preservation method employed and the amount of light they have been exposed to through time. This entails that one cannot draw the conclusion that a given species is not accumulating porphyrins in its pelage based on the apparent lack of UV-PL in museum specimens.

As this reddish UV-PL is predominantly found in crepuscular and nocturnal species, this phenomenon has been interpreted as potentially related to visual functions for intraspeci c communication or antipredator behavior in light-deprived environments [START_REF] Anich | Bio uorescence in the platypus (Ornithorhynchus anatinus)[END_REF]8,[START_REF] Olson | Vivid bio uorescence discovered in the nocturnal Springhare (Pedetidae)[END_REF][START_REF] Camacho | Correlates of individual variation in the porphyrin-based uorescence of red-necked nightjars (Caprimulgus ru collis)[END_REF] . However, in showing that the reddish UV-PL of mammals' pelage is induced by the accumulation of photodegradable protoporphyrins, it appears more likely that this phenomenon might simply be overrepresented in crepuscular and nocturnal specimens mainly because in their case the porphyrinic compounds are less degraded than in diurnal species. Furthermore, it should be kept in mind that it is far from evident that natural UV illuminating sources, even at dusk or dawn, are su cient for this UV-PL phenomenon to be perceived and contrasted from the re ected visible light by co-speci cs or predators [START_REF] Goutte | Intense bone uorescence reveals hidden patterns in pumpkin toadlets[END_REF] . Indeed, for the UV-PL to have a visual function, it would require that the animals are exposed, voluntarily or not, to su cient UV illumination so that the photoluminescence reemitted is seen by other individuals or species. Additionally, one should keep in mind that when it comes to UV-PL, the reemitted light is in the "visible spectrum" (400-700 nm), so whether the animals have an improved UV light vision (200-400 nm) does not provide them with any advantage to perceive such photoluminescence.

Given these results, we thus suggest an alternative hypothesis to interpret the accumulation of PPIX derivatives in the pelage of mammals, resulting in this observable UV-PL. We suggest that this phenomenon is a byproduct of the heme biosynthesis with no other function than to be excreted from the organism by being naturally degraded through the pelage 1,28 . Storing these residual molecules of the heme pathway -which might become toxic when produced in too high quantities 26,[START_REF] Karim | Porphyrias: A 2015 update[END_REF]36 -in skin appendages that are composed of inert tissues such as hair or spines and that are likely to be exposed to light, could hence help excreting these compounds without further energetically demanding metabolic processes. Given its distribution in the phylogeny, this perhaps represents a mechanism that was acquired early in the evolution of mammals. Finally, further inquisition on the functional signi cance of UV-induced photoluminescence in animals and plants should include a more thorough and comprehensive approach, incorporating both the ecology and physiology of the organisms exhibiting these phenotypes 1. To proceed with photoluminescent spectroscopy and multispectral imaging, we collected several samples of complete hairs and spines of known orientation and spatial localization on the pelage of several selected specimens (Table 1, Fig. 1a., Fig. 2b.). Spectroscopy and multispectral imaging measurements were also carried out on two complete specimens preserved in alcohol (JAGUARS-M535 and JAGUARS-M2838).

UV-induced photoluminescence macro-analysis and photography. Specimens were observed and photographed rst under natural or standard white light conditions. Then they were placed in the dark and illuminated using speci c UV light setups emitting at a distance of about 20 cm from the specimens. We initially used a 395 nm LED torch (100 LED ashlight, Youthink) following ref. 8 and associated with a yellow lter (K&F Concept) to annul the purple light emitted by this torch. Nevertheless, this setup may leave out some wavelengths that are not captured by the yellow lter and which can be misinterpreted as UV-PL. We therefore used a professional 365 nm compact lamp (4 W, UVL-21, UVP) associated with a strict UV black lter (Filter Band U-360 2IN SQ, Edmund Optics Ltd).

Photographs presented in Fig. 1.a. were obtained using the second setup, when the lter was placed directly on the UV emitting surface of the lamp. Photographs were taken using a Canon EOS 5D Mark III equipped with a 50mm macro lens and a UV blocking lter (UV390 Protect Filter, hama). White balance was systematically readjusted for each specimen.

Photoluminescence spectroscopy. Emission and excitation spectroscopy were performed using a spectro uorometer Fluorolog 3-22 from HORIBA Jobin Yvon. Spectra were collected using a bundle of optical ber equipped with a focusing optics allowing to perform measurements out of analysis compartment. Entrance and exit slits widths of the monochromators were set at 10 nm in order to ensure a balance between the collection of spectra with good signal-to-noise and a spectral resolution compatible with the detection of Q bands. The emission spectra were collected using an excitation at 400 nm. To record the excitation spectra, a 732 ± 34 nm bandpass lters (from Semrock) was placed at the entrance of the emission monochromator in order to collect the signal at 700 nm without any interference due to the second order diffraction of excitation light or due to stray light.

Photoluminescence multispectral imaging. False color luminescence images were assembled using black and white images collected with a home-made setup which consists of a low-noise 4-megapixel Si C-MOS camera (ORCA ash V4.0 V2 -Hamamatsu) with a sensitivity ranging from 200 to 1100 nm. The camera is tted with a UV-VIS-IR 60 mm 1:4 Apo Macro lens (CoastalOptics) in front of which is positioned a lter wheel holding 8 Interference band-pass lters (Semrock) to collect images in speci c spectral ranges. Illumination was provided by 16 LED lights ranging from 365 up to 700 nm (CoolLED pE-4000), coupled to a liquid light-guide ber tted with a ber-optic ring light-guide, allowing homogeneous illumination of the region of interest. False color images were generated by assigning red, green and blue color to speci c emission ranges using ImageJ 40 software.

Declarations Figures

Table 1 .

 1 Specimens analyzed. Collection abbreviation: ZMB= Museum für Naturkunde of Berlin; JAGUARS= Cayenne collection; MNHN= Muséum national d'Histoire naturelle of Paris; NRM= Swedish Museum of Natural History of Stockholm; AH= Anthony Herrel personal collection; NKMP= Naturkundemuseum of Potsdam. Type of analysis conducted: UVL= macro-observation under UV lamp, EmS= emission spectroscopy, Ex-MI= excitation spectroscopy & multispectral imaging. Visible color of their fur observed under UV lamp: NF= no luminescent color detected with the eye. Porphyrin detection obtained with spectroscopy analysis: 0= no, 1= yes.

	Mammal	Order	Species	Specimen number	Type of	Fur under	Porph.
	class				analysis	UV
	Prototheria	Monotremata	Ornithorhynchus	ZMB_Mam_35991	UVL,	green/cyan	0
			anatinus		EmS
	(egg-laying		(Platypus)
	mammals)
	Metatheria	Didelphimorphia	Caluromys	ZMB_Mam_47995	UVL	pink	-
	(marsupials)		lanatus (Brown-
			eared Woolly
			Opossum)
			Marmosa	JAGUARS-M535	UVL,	pink/red	1
			murina		EmS,
			(Linnaeus's		Ex-MI
			Mouse
			Opossum)
			Marmosa	MNHN 2001-1966	UVL	pink	-
			murina
			(Linnaeus's
			Mouse
			Opossum)
			Metachirus	MNHN 1988-68	UVL	pink	-
			nudicaudatus
			(Brown Four-
			eyed Opossum)
			Monodelphis	JAGUARS-M2962	UVL,	pink/red	1
			brevicaudata		EmS,
			(Guyanan short-		Ex-MI
			tailed Opossum)
			Monodelphis	JAGUARS-M2838	UVL,	pink/red	1
			brevicaudata		EmS,
			(Guyanan short-		Ex-MI
			tailed Opossum)
			Monodelphis	MNHN 1995-3216	UVL	pink	-
			brevicaudata
			(Guyanan short-
			tailed Opossum)
	Eutheria	Erinaceomorpha	Erinaceus	NRM_594242	UVL,	pink/red	-
	(placentals)		europaeus		EmS,
			(West European		Ex-MI
			Hedgehog)
			Erinaceus	ZMB_Mam_48212	UVL	pink/red	1
			roumanicus
			(Northern White-
			breasted
			Hedgehog)
		Carnivora	Mustela	NKMP-	UVL	lavender	-
			erminea	unnumbered
			(Beringian
			ermine)
			Mustela	NRM_20175124	UVL,	lavender	0
			erminea		EmS

  1,28 .

Methods

Specimens studied. examined a variety of specimens from various species of monotreme, marsupial, and placental mammals with preserved furs in either alcohol or as dry preparations, from museum collections of the Museum für Naturkunde of Berlin, the Swedish Museum of Natural History of Stockholm, the Muséum national d'Histoire naturelle of Paris, the JAGUARS collection of Cayenne, personal collection from Dr. Anthony Herrel, and the Naturkundemuseum of Potsdam. A selection of some specimens and associated methods of analysis are presented in Table

Acknowledgments

We thank the Museum für Naturkunde of Berlin, especially C. Funk, A. Rosemann, F. Mayer and D. Willborn; and the Naturkundemuseum of Potsdam, especially I. Pokorny, for lending us various specimens of mammals (Germany).

We are grateful to B. de Thoisy and A. Herrel for lending us fresh marsupial specimens from the JAGUARS collection (French Guyana). We thank the Dortmund Zoo, especially M. Stawinoga and I. Schappert for their support with data acquisition. Finally, we thank M. Jansen for helpful advice in data interpretation. E.A. was supported by the German Research Council (Deutsche Forschungsgemeinschaft; grant number AM 517/1-1). J.P. was supported by the Elsa-Neumann-Stipendium (Humboldt-Universität zu Berlin). S.L.D.T. was supported by the Fyssen Foundation and the Alexander Von Humboldt Foundation.

Data availability statement

The dataset generated for this study, i.e., the raw multispectral images and emission and excitation spectra, are available on Figshare (https://doi.org/10.6084/m9. gshare.XXX).

Code availability

The code used in this study can be accessed on Figshare (https://doi.org/10.6084/m9. gshare.XXX).

Author contributions E.A., J.P., S.L.D.T., P.G., and M.T. conceived this study. S.L.D.T., J.P., and E.A. conducted the macro-analysis and photography of collection specimens. D.C.K. helped in macro-analysis. P.G., M.T., B.H., R.G., and R.M. conducted the multispectral imaging and spectroscopy analyses and processing and identi ed the porphyrins. D.C.K., S.B., P.M. and S.S. equally helped with data interpretation. S.L.D.T, J.P. and E.A. drafted the manuscript with inputs from M.T. and P.G. All authors reviewed the nal version of the manuscript.

Competing interests

We have no competing interest to declare.

Materials & Correspondence

Material and correspondence requests should be addressed to J.P., E.

Supplementary Files

This is a list of supplementary les associated with this preprint. Click to download.

Toussaintetalspectra.xls

ToussaintetalRscript.txt RawMultispectralImages.zip