# NANOSCIENCE AND NANOMATERIALS FOR THE KNOWLEDGE AND CONSERVATION OF CULTURAL HERITAGE

EDITED BY : Luca Tortora, Gabriella Di Carlo, Maria Jesus Mosquera and Gabriel Maria Ingo PUBLISHED IN : Frontiers in Materials and Frontiers in Chemistry

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ISSN 1664-8714 ISBN 978-2-88966-297-5 DOI 10.3389/978-2-88966-297-5

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# NANOSCIENCE AND NANOMATERIALS FOR THE KNOWLEDGE AND CONSERVATION OF CULTURAL HERITAGE

Topic Editors:

Luca Tortora, Roma Tre University, Italy Gabriella Di Carlo, Consiglio Nazionale delle Ricerche Italiano (CNR), Italy Maria Jesus Mosquera, University of Cádiz, Spain Gabriel Maria Ingo, National Research Council (CNR), Italy

Citation: Tortora, L., Di Carlo, G., Mosquera, M. J., Ingo, G. M., eds. (2020). Nanoscience and Nanomaterials for the Knowledge and Conservation of Cultural Heritage. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88966-297-5

# Table of Contents

*05 Editorial: Nanoscience and Nanomaterials for the Knowledge and Conservation of Cultural HeritageNanoscience and Nanomaterials for the Knowledge and Conservation of Cultural Heritage*

Luca Tortora, Gabriella Di Carlo, Maria Jesus Mosquera and Gabriel Maria Ingo

*08 Raman Spectroscopy and Surface Enhanced Raman Scattering (SERS) for the Analysis of Blue and Black Writing Inks: Identification of Dye Content and Degradation Processes*

Daniela Saviello, Maddalena Trabace, Abeer Alyami, Antonio Mirabile, Piero Baglioni, Rodorico Giorgi and Daniela Iacopino

Federica Antonelli, Giulia Galotta, Giancarlo Sidoti, Florian Zikeli, Rossella Nisi, Barbara Davidde Petriaggi and Manuela Romagnoli

Monia Vadrucci, Giovanni De Bellis, Claudia Mazzuca, Fulvio Mercuri, Fabio Borgognoni, Emily Schifano, Daniela Uccelletti and Cristina Cicero

Alessandro Grottoli, Marzia Beccaccioli, Emma Zoppis, Rosita Silvana Fratini, Emily Schifano, Maria Laura Santarelli, Daniela Uccelletti and Massimo Reverberi

*89 Differential X-Ray Attenuation in MA-XRF Analysis for a Non-invasive Determination of Gilding Thickness*

Sergio Augusto Barcellos Lins, Stefano Ridolfi, Giovanni Ettore Gigante, Roberto Cesareo, Monica Albini, Cristina Riccucci, Gabriella di Carlo, Andrea Fabbri, Paolo Branchini and Luca Tortora

*98 Model Protective Films on Cu-Zn Alloys Simulating the Inner Surfaces of Historical Brass Wind Instruments by EIS and XPS*

Marzia Fantauzzi, Bernhard Elsener, Federica Cocco, Cristiana Passiu and Antonella Rossi

#### *112 Multi-Spectroscopic Approach for the Non-invasive Characterization of Paintings on Metal Surfaces*

Monica Albini, Stefano Ridolfi, Chiara Giuliani, Marianna Pascucci, Maria Paola Staccioli and Cristina Riccucci

*121 On-Demand Release of Protective Agents Triggered by Environmental Stimuli*

Chiara Giuliani, Elena Messina, Maria Paola Staccioli, Marianna Pascucci, Cristina Riccucci, Leonarda Francesca Liotta, Luca Tortora, Gabriel Maria Ingo and Gabriella Di Carlo

*131 Synthesis and Characterization of TEOS Coating Added With Innovative Antifouling Silica Nanocontainers and TiO*2 *Nanoparticles*

Ludovica Ruggiero, Maria Rosaria Fidanza, Morena Iorio, Luca Tortora, Giulia Caneva, Maria Antonietta Ricci and Armida Sodo

*142 Microchemical Investigation of Long-Term Buried Gilded and Silvered Artifacts From Ancient Peru*

Gabriel M. Ingo, Monica Albini, Angel D. Bustamante, Sandra del Pilar Zambrano Alva, Arabel Fernandez, Chiara Giuliani, Elena Messina, Marianna Pascucci, Cristina Riccucci, Paola Staccioli, Gabriella Di Carlo and Luca Tortora

# Editorial: Nanoscience and Nanomaterials for the Knowledge and Conservation of Cultural Heritage

Luca Tortora1,2 \*, Gabriella Di Carlo3 , Maria Jesus Mosquera4 and Gabriel Maria Ingo3

1 LASR3 Surface Analysis Laboratory, National Institute for Nuclear Physics (INFN) Roma Tre Division, Rome, Italy, 2 Department of Sciences, Roma Tre University, Rome, Italy, 3 Institute for the Study of Nanostructured Materials (ISMN), National Research Council (CNR), Rome, Italy, 4 Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Cadiz, Cadiz, Spain

Keywords: conservation, protection, cleaning, diagnostics, consolidants, corrosion, coatings, paintings

#### Editorial on the Research Topic

#### Nanoscience and Nanomaterials for the Knowledge and Conservation of Cultural Heritage

#### Edited by:

Saulius Juodkazis, Swinburne University of Technology, Australia

#### Reviewed by:

Soon Hock Ng, Swinburne University of Technology, Australia

> \*Correspondence: Luca Tortora<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Colloidal Materials and Interfaces, a section of the journal Frontiers in Materials

> Received: 14 September 2020 Accepted: 06 October 2020 Published: 29 October 2020

#### Citation:

Tortora L, Di Carlo G, Mosquera MJ and Ingo GM (2020) Editorial: Nanoscience and Nanomaterials for the Knowledge and Conservation of Cultural Heritage. Front. Mater. 7:606076. doi: 10.3389/fmats.2020.606076 "To protect and to serve" is the motto of the Los Angeles Police Department, adopted also by other law enforcement agencies; it underlines the necessity of protecting citizens from criminal acts and at the same time serving the community. Well, considering that works of art constitute a priceless good and they should be protected as such, this motto could be associated even to scientists working in the cultural heritage field. In fact, it is possible to safeguard the cultural heritage working for example on the development of innovative materials as protective agents or explaining degradation mechanisms occurring in artworks by using innovative materials and methods like those based on the nanotechnologies science.

In the last decade, emerging organic and inorganic nanomaterials have been designed, synthesized, and characterized for the really non-invasive and selective cleaning, the reliable consolidation, and the long-lasting reliable conservation also under unsuitable climate conditions. At the same time, sophisticated analytical techniques are more frequently combined to understand the nano and micro-chemical and structural features of artworks for the comprehensive knowledge of their "material" nature and the deciphering of manufacturing processes.

The structure of this special issue reflects these two aspects. It is composed of six original research articles dealing with the application of innovative nanomaterials for the protection of different substrates (archeological wood, steel, and stone material) and cleaning techniques for the disinfection of historical leather and selective removal of polymeric material. In addition, this special topic collection presents seven research articles describing the application of combined advanced analytical techniques to unveil different degradation mechanisms.

For what concerns the nanomaterials for the conservation of cultural heritage, Antonelli et al. report an article where they tested three different nanoscale consolidants to protect waterlogged archaeological wood samples. Lignin nanoparticles, bacterial nanocellulose, and cellulose nanocrystals were used on wood samples coming from two Italian archaeological sites. Problems related to color changing and differences in penetration of the nano-consolidants were extensively discussed. The paper proposed by Giuliani et al. shows a new generation of smart materials able to release the loaded inhibitor depending on the chemical environmental

conditions. Three inhibitors containing a carboxylic moiety, such as p-aminobenzoic, succinic, and caffeic acids were studied as protective agents of corrosion in simulated concrete pore solutions containing chloride ions; p-aminobenzoic acid was then used for the functionalization of silica nanoparticles and LDHs, adopted as nanocarriers for the inhibitors.

Seems to be a great deal of interest for the consolidant products used for stone. In fact, Ruggiero et al. propose a coating composed of a tetraethoxyorthosilicate matrix (TEOS), an elasticiser [hydroxyl-terminated polydimethylsiloxane (PDMS-OH)] and a non-ionic surfactant (n-octylamine). This TEOS-based composite coating was then functionalized with two different silica nanofillers with antifouling properties: commercial TiO2 nanoparticles and two different silica nanocontainers, loaded with the commercial biocide 2 mercaptobenzothiazole. In the same collection, Kapetanaki et al. reported a procedure for eco-friendly and one-pot synthesis of a consolidant incorporating in TEOS, PDMS, and nanoparticles of synthesized calcium oxalate. Cement mortars treated with this colloidal solution exhibited wellpreserved aesthetic surface parameters and significant maintenance of the treatment.

Particular attention was also payed paid to cleaning methods. X-ray beams were used by Vadrucci et al. for the sterilization of leather wallpaper fragments dating back to the 1700s. The disinfection treatment permitted to eliminate the biofilms present on the leather and produced by the following microorganisms: B. cereus and M. timonae. On the other hand, a chemical approach for the removal of aged or detrimental polymeric coatings from artworks was investigated by Baglioni et al. The interaction between nanostructured fluids (NSFs), usually adopted by restorers for cleaning procedures, and four different kinds of acrylic and vinyl polymer films deposited onto glass, marble, and polystyrene substrates was studied.

Integrated methodological approaches based on the combined use of complementary surface, interfaces, and bulk analytical techniques were applied on different heritage objects and materials as like inks, gilded copperbased buckles, historical metal plates, gilded diadem fragments, gilded plaques, and ancient brass wind instruments. Saviello et al. analyzed blue and black writing inks. Results coming from Raman spectroscopy and surface enhanced Raman scattering (SERS) revealed that all analyzed writing inks are composed of Crystal Violet and other triarylmethane dye mixtures, with, in some cases, the addition of phthalocyanine dyes. Raman and SERS were used even to investigate related aging processes due to the presence of light and high temperatures. Degradation processes were also studied by Graziani et al. In this case, time of flight secondary ion mass spectrometry (ToF-SIMS) depth profiling experiments and SEM-energy dispersive X-ray spectroscopy (EDS) were combined to spatially locate corrosion products present in a fire gilded brooch dating back to 16th−17th century AD and found during the dredging of the Tiber river (Rome, Italy). On the contrary, based on a non-destructive alternative, Lins et al. proposed an analytical approach to determine the gilding thickness for a similar gilded copper-based buckle. An innovative algorithm was developed to measure thin metallic layers deposited on a sample surface taking advantage of differential X-ray attenuation in MA-XRF analysis. Even in the article of Albini et al., the protagonists are copper-based objects. Painted metal surfaces were fully characterized by a plethora of complementary spectroscopic techniques such as Fourier transform infrared spectroscopy (FTIR), energydispersive X-Ray fluorescence spectroscopy (EDXRF), high spatial resolution field emission scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (EDS), and optical microscopy (OM). In particular, FTIR analysis revealed degradation products generated from the interaction between the organic binding media and metal substrates. A detailed study aimed to understand the manufacturing techniques used to give the gold or silver appearance in metal artifacts from ancient Peru was afforded by Ingo et al.. In the article, different metal objects, some of them dating back around 300–400 AD, found in the tombs of the Lords of Sipán (Lambayeque, Peru) and of the Lady of Cao (El Brujo, Peru) were characterized using complementary techniques as OM, SEM-EDS, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The abovementioned combined analytical approach permitted to the authors to have a detailed picture of the degradation process resulting from interactions between soil species and Ag-Cu-Au ternary alloys.

What happens when the surface of a brass comes in contact with saliva? Can be the saliva considered a potential corrosion agent? The problem was addressed by Fantauzzi et al. In the article, artificial saliva was placed in contact with brass surfaces, and the composition and morphology of the film formed after the chemical interaction were was investigated by XPS, SEM, and electrochemical impedance spectroscopy (EIS). Finally, the paper from Grottoli et al. shows the potentialities of the metagenomics applied to the cultural heritage conservation. A micro-invasive sampling method that involves the use of a simple adhesive tape followed by DNA extraction and PCR amplification permitted to identify the microbial communities colonizing the surface of the hypogeum of the Basilica di San Nicola in Carcere Church in Rome.

In conclusion, as you will appreciate, this special issue contains several hints and suggestions that fall in different fields of application strictly related to the cultural heritage. We hope that it can be a useful tool for conservation scientists, restorers, curators, or simply art lovers.

## AUTHOR CONTRIBUTIONS

All authors contributed equally to this article and have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

## ACKNOWLEDGMENTS

Topic Editors would like to thank all the authors for their contribution to this Special Issue. And, a great thank you also goes to reviewers and editorial staff from Frontiers in Materials and Frontiers in Chemistry for the successful completion of this special collection.

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Tortora, Di Carlo, Mosquera and Ingo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Raman Spectroscopy and Surface Enhanced Raman Scattering (SERS) for the Analysis of Blue and Black Writing Inks: Identification of Dye Content and Degradation Processes

Daniela Saviello1 , Maddalena Trabace2 , Abeer Alyami 1 , Antonio Mirabile3 , Piero Baglioni 2 , Rodorico Giorgi 2 and Daniela Iacopino1 \*

*1 Nanotechnology Group, Tyndall National Institute, University College Cork, Cork, Ireland, 2 Department of Chemistry & CSGI, University of Florence, Florence, Italy, 3 Mirabile, Paris, France*

#### Edited by:

*Gabriel Maria Ingo, Italian National Research Council (CNR), Italy*

#### Reviewed by:

*Armida Sodo, Roma Tre University, Italy Philippe Colomban, Centre National de la Recherche Scientifique (CNRS), France*

> \*Correspondence: *Daniela Iacopino<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Nanoscience, a section of the journal Frontiers in Chemistry*

Received: *12 September 2019* Accepted: *10 October 2019* Published: *25 October 2019*

#### Citation:

*Saviello D, Trabace M, Alyami A, Mirabile A, Baglioni P, Giorgi R and Iacopino D (2019) Raman Spectroscopy and Surface Enhanced Raman Scattering (SERS) for the Analysis of Blue and Black Writing Inks: Identification of Dye Content and Degradation Processes. Front. Chem. 7:727. doi: 10.3389/fchem.2019.00727* Raman spectroscopy and Surface Enhanced Raman Scattering (SERS) were applied to the analysis of blue and black writing inks. SERS was performed by application of plasmonic nanopastes constituted by Ag nanoparticles and Au nanorods directly on inks deposited on paper substrates under laser irradiation of 514 nm. It was found that SERS spectra were largely enhanced compared to Raman spectra and that Ag nanopastes produced much larger enhancements than Au nanopastes, due to a combination of plasmonic resonance, charge transfer, and molecular resonance effects. All analyzed writing inks resulted constituted by Crystal Violet and other triarylmethane dye mixtures, to which sometimes phthalocyanine dyes were also added (for example in Bic pens). SERS was also used for the identification of degradation processes occurring in artificially aged blue pens deposited on paper substrates. It was found that color alteration changed from ink to ink and varied from darkening to discoloration to slight fading, depending on the initial chemical composition. For inks containing Crystal Violet, two mechanisms associated to de-methylation and photo-reduction of excited dye to colorless leuco forms were identified.

Keywords: ballpoint pens, dyes, Raman, plasmonic nanopastes, SERS, degradation

## INTRODUCTION

Modern commercial inks are a complex mixture of several dyes and/or pigments constituting up to the 50% of the total ink formulation, carried either in a glycol-based solvent or benzyl alcohol (Bell, 2008; Houck and Siegel, 2010; Houck et al., 2013). Analysis of commercial writing inks is usually carried out in the context of forensic examination in order to assess dating and originality of documents and to correctly assess cases of crimes like falsification, questioned signatures and threatening letters. Accordingly, a vast number of publications in the last 20 years have focused on characterization of writing pens (i.e., identification of dyes and pigment contents in ballpoint and gel pens) and also on discrimination between inks. Therefore a number of techniques such as Thin Layer Chromatography (TLC) and mass spectrometry (MS) often coupled with optical analysis (ink absorption and luminescence) or image analysis have been routinely applied to

**8**

Saviello et al. Analysis of Writing Inks

the analysis, differentiation, and classification of writing inks (Weyermann et al., 2007; Djozan et al., 2008; Gallidabino et al., 2011; Sauzier et al., 2015). Recently, visible, Raman, and infrared spectroscopies have also gained popularity as techniques for providing rapid information on the chemical content of the ink mixtures and are increasingly pursued for their rapidity of analysis, little sample preparation and non-destructive analysis. Such approaches are also particularly amenable to the investigation of ink-based paper works, whereby the identification of dyes and pigment ink constituents is important to assess dating, originality and to implement correct conservation procedures thus ensuring long-term preservation. Among spectroscopic methods, Raman and Surface Enhanced Raman Scattering (SERS) spectroscopies have been widely used for identification of colorants in artworks and have shown as promising techniques for the identification of dye components in commercial inks (Mazzella and Buzzini, 2005; Bráz et al., 2014; Calcerrada and García-Ruiz, 2015). In particular, SERS revealed very effective in quenching the interference strong fluorescence emission sometimes accompanying the direct Raman illumination of writing inks thus enabling generation of high intensity spectra even under visible laser irradiation (Jeanmaire and Van Duyne, 1977; Geiman et al., 2009; Luo et al., 2013). However, the majority of SERS results suffer from poor reproducibility arising from the use of randomly aggregated Ag colloidal solutions, usually dropped on extracted samples (Seifar et al., 2001; White, 2003). In contrast, in order to guarantee reproducibility of analysis, SERS requires the use of reliable and robust plasmonic probes ideally applicable directly on the paper substrate. Recently, our group has developed plasmonic probes comprising Ag and Au nanopastes and nanoinks and has used SERS in combination with UV-vis spectroscopy for the analysis of blue and colored Bic pens, increasingly used for artistic purposes (Alyami et al., 2016, 2017, 2018).

Another important consideration in the field of artwork preservation is the aging of the ink deposited on a substrate (usually, but not necessary, paper). As soon as an ink line is deposited on a substrate, compositional changes occur rapidly due to loss of volatile components, photodegradation of dyes or polymerization of resins. All these processes happen concomitantly and they all contribute to significantly alter the aesthetic of paper works exposed to environmental conditions. Photofading of ballpoint pens has been traditionally performed by laser desorption/ionization (LDI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (Weyermann et al., 2006). However, in light of the need to preserve the integrity of analyzed objects, non-destructive techniques are nowadays preferred for ink-based artworks. Accordingly, Raman spectroscopy has been successfully used for the investigation of dye aging dynamics in writing inks and SERS was successfully applied by Cesaratto et al. to the tracking of triarylmethane dyes photodegradation on nineteenth century woodblock Japanese prints (Gorshova et al., 2016; Cesaratto et al., 2017).

In this work we propose the use of Raman and SERS spectroscopy for the analysis of a number of blue and black writing inks on paper substrates. The aim of the present study is to evaluate the ability and limitations of Raman spectroscopy for the analysis of writing inks and to assess the improvements brought about by the use of SERS. This includes routine chemical make-up identification as well as more complex analysis such as degradation assessments. The ability of SERS in overcoming fluorescence background limitations and increasing the sensitivity of Raman spectroscopy have been investigated already. However, results are contradictory as robust SERS probes for such type of analysis are missing. The novelty of the presented manuscript relies on the development of SERS metal nanopastes providing a reproducible method for the investigation of signal enhancement effects associated with the use of SERS compared to Raman spectroscopy. This is relevant for analysis of artworks whereby only small size samples are available for analysis. Moreover, the ability to synthesize SERS nanopastes of different plasmonic properties (Ag, Au) widens the application of SERS spectroscopy to Raman instrumentations of different excitation wavelengths and also to portable/handheld instrumentation.

### MATERIALS AND METHODS

#### Materials

Tetrachloroauric acid (99.5%), silver nitrate (ACS, 99%), sodium citrate (99 %), sodium borohydride (96%), ascorbic acid (CAS: 50817), cetyltrimethylammoniumbromide (CTAB, 99%), MeOH (CAS: 67561) were purchased from Sigma-Aldrich. All glassware was cleaned with aqua regia prior to nanopaste synthesis. Milli-Q water (resistivity > 18 M cm−1 ) was used throughout the experiments. Reference dye Crystal Violet (CAS: 548629) was also purchased from Sigma and used without further purification.

#### Synthesis of Plasmonic Nanopastes

Silver nanoparticle nanopastes were synthesized following the modification of the Lee and Meisel method reported by Polavarapu et al. (2014). Briefly, trisodium citrate solution (4.5 mL, 1.00 wt%) was added to an aqueous boiling solution containing AgNO3 (200 mL, 42 mg) under vigorous stirring. The reaction was boiled for another 1 h and then cooled to room temperature. The obtained Ag nanoparticles in water (200 mL) were centrifuged at 7,000 rpm for 20 min and then redispersed in water (2 mL) to obtain Ag nanopaste (3 mg/mL). Au nanorod nanopastes were synthesized by a modification of the seed mediated growth reported by Polavarapu et al. (2014). Specifically, a seed solution was prepared by adding 0.3 mL of an ice-cold aqueous sodium borohydride (NaBH4, 0.01 M) solution to an aqueous solution of 4.7 mL hexadecyltrimethylammonium bromide (CTAB, 0.1 M) and 25 µL of gold(III) chloride trihydrate (HAuCl4, 0.05 M) at 30◦C. An aliquot of 0.36 mL of the seed solution was added to a growth solution prepared by mixing 150 mL of CTAB (0.05 M), 1.5 mL HAuCl4 (0.05 M), 0.225 mL of silver nitrate (AgNO3, 0.01 M), and 5.0 mL of ascorbic acid (0.1 M) at 30◦C. The solution color changed from colorless to brownish-bluish after the addition of the seed solution to the grown solution. The obtained aqueous solution of gold nanorods was centrifuged twice and re-dispersed in water (1 mL) to get the nanorod paste.

### Commercial Writing Pens

Commercial writing pens were purchased from local stores. Real pen artworks were made by French artist Anne-Flore Cabanis and were kindly donated by paper conservator Antonio Mirabile. Artworks were made by Bic blue and black pens on commercial paper.

#### Scanning Electron Microscopy

Scanning electron microscopy (SEM) images of nanoinks deposited on SiO2 substrates were acquired using a field emission SEM (JSM-6700F, JEOL UK Ltd.) operating at beam voltages of 2 kV.

#### Thin Layer Chromatography

Thin layer chromatography was performed with silica plates 10 × 20 cm (Sigma-Aldrich). The writing inks and reference dye Crystal Violet were deposited as concentrated MeOH solutions. The TLC was developed in ethylacetate-ethanol-water (70:35:30 v/v) for 60 min.

#### Optical Characterization

UV-Vis spectra were acquired with an Agilent/HP 8453 UV-Vis Spectrophotometer (200 nm < λ < 1,100 nm). Raman spectra at 514 nm were obtained from a Renishaw inVia Raman system. An argon ion laser (1,800 l/mm grating) was employed as an excitation source. The laser beam was focused onto the sample through a Leica 20X objective with 0.4 N.A. Measured power at the sampling level was controlled at about 3 mW. Acquisition time was usually 10 s. Raman spectra at 785 nm were obtained from a Pelkin Elmer Raman station. The laser beam was focused onto the sample through a 50X objective (M Plan Achromat) with 0.75 N.A. The laser power was around 3.5 mW and typical acquisition time was 10 s. To obtain SERS spectra, 5 µL of plasmonic nanopastes were deposited on pen written lines on paper and left to evaporate for 30 min prior analysis.

### Artificial Aging

Paper samples with written blue pen features were subjected to three artificial aging cycles as follows: thermo-hygrometric and photo-oxidative for a total duration of 88 days. Every cycle comprised three phases: an initial phase in humidity chamber with thermo-hygrometric control of temperature at 25.5◦C and 80% relative humidity; a second phase of thermal degradation in oven at 80◦C and a third phase of neon light exposure to the front and back of the paper sheet.

TABLE 1 | List of band positions found in Raman spectra of analyzed inks.

\*ν*, stretching (s, symmetric; as, asymmetric);* δ*, bending.*

illumination wavelength.

## RESULTS AND DISCUSSION

#### Metal Nanopastes SERS Probes

Writing inks from 10 commercial pens (eight blue and two black) were analyzed. Details of used pens and optical microscopy images of deposited inks on paper are reported in **Figure S1**.

In order to perform SERS analysis directly on the pen lines, and therefore to avoid lengthy extraction processes, it is necessary to use colloidal plasmonic solutions able to homogeneously cover the colored paper fibers, in order to ensure reproducibility of results and high density of hot spots necessary for generation of high intensity spectral features. Toward this end, Ag nanoparticle and Au nanorod nanopastes were synthesized by slight modification of the "pen-on-paper" method proposed by Polavarapu et al. (2014). **Figures 1A,B** show SEM images of the synthesized nanopastes deposited on n-doped conductive SiO2 substrates. Ag nanopastes were mostly spherical in shape and had an average size of 65 ± 3 nm; the Au nanopastes displayed an elongated (rod-like) shape with average size of 13 ± 2 × 41 ± 4 nm. The absorbance spectra of synthesized nanopastes are reported as insets: the Ag nanopaste solution showed a relatively large plasmonic peak centered at 426 nm whereas the Au nanopaste solution was characterized by two peaks at 520 and 787 nm, associated to transversal and longitudinal plasmon resonances occurring in elongated nanoparticles (Johnson et al., 2002). **Figure 2B** shows a representative optical microscopy image of deposited Ag nanopaste on blue Staedler pen, showing formation of a well-defined SERS-active area on the analytical surface without smudging nor dissolution of the pen ink underneath. The representative SEM image showed in **Figure 1C** shows highly homogeneous distribution of plasmonic nanoparticles over large areas (>0.6 mm2 ) of the colored paper fibers. Such high density of adsorbed Ag nanoparticles results in a high density of hot spots suitable for generation of high intensity SERS spectra.

### Raman Spectroscopy and SERS of Writing Inks

Prior measurements of SERS spectra Raman spectra were recorded for five pens, in order to establish limitations of Raman spectroscopy and assess enhancement generated by subsequent application of SERS. All Raman spectra were recorded with 514 nm excitation wavelength and 3 mW laser power and are shown in **Figure 2** without background subtraction. Raman spectra of blue and black Bic and blue Staedler pens (**Figures 2A,B**) showed very similar bands (see **Table 1** for details) at 1,618, 1,587, 1,540 cm−1 , which were associated to the stretching of the benzene rings in triarylmethane compounds and 439, 420 cm−1 bands associated to bending of CNC bonds (Canamares et al., 2008). Based on previous findings by our group and others, the presence of crystal violet (CV, basic violet 3, C. I. 42555) was hypothesized for these pen inks (Alyami et al., 2016, 2017). It should be noted that previous analysis carried by our group on blue Bic pen ink whose components were separated by thin-layer chromatography (TLC) and subsequently identified by Raman/SERS spectroscopies showed also presence of a blue phthalocyanine component in the ink formulation. However, the phthalocyanine component did not give any Raman response at 514 nm excitation, confirming the results obtained in this work on the overall Bic pen ink showing only contribution from the CV component (Kunicki et al., 2013; Alyami et al., 2017). The Raman spectrum of blue Pilot pen (**Figure 2A**) showed good intensity bands centered at 1,614, 1,530, 1,359, 1,168, 438, and 419 cm−1 , which could also be attributed to the presence of a

triarylmethane mixture but not specifically to CV. The Raman spectrum of black Staedler (**Figure 2B**) also indicated possible presence of CV, although the intensity of obtained Raman band was too low and affected by significant fluorescence interference to allow unequivocal peak assignment.

**Figure 3** show SERS spectra recorded for each analyzed pen with both Ag and Au nanopastes. SERS spectra of additional blue writing inks are reported in **Figure S3**. The SERS spectra of each pen were plotted together with the corresponding Raman spectrum in order to give a straight forward visualization of the degree of enhancement obtained with both SERS probes compared to Raman conditions. In addition the blank Raman and SERS spectra of commercial Fabriano paper were also taken and are shown in **Figure S2**. The blank Raman spectrum was featureless; low intensity and broad peaks at 1,595, 1,519, and 1,407 cm−1 where observed with Ag nanopastes, one peak at 1,619 cm−1 peak was observed with Au nanopastes. The application of Ag nanopastes resulted in generation of high intensity SERS spectra for blue and black Bic and Staedler pens (**Figures 3A–E**). Specifically, spectra displayed intensities ca. 1 order of magnitude higher than intensities of corresponding Raman spectra (measured using the intensity of the 1,180 cm−1 band as reference) and showed all diagnostic peaks of CV. This enhancement was mainly attributed to an electromagnetic effect (EM), due to the use of an illumination wavelength in plasmonic resonance with the Ag nanopastes (**Figure 1C**). Details of the position of the bands found in all analyzed inks are reported in the SI (**Table S1**). The high intensity and clear formation of diagnostic bands allowed unequivocal identification of CV in both Bic and Staedler blue and black pen inks. Closer observation of the Bic and Staedler blue and black SERS spectra showed a red shift of frequencies compared to the Raman spectra. This in turn was associated to a chemical effect (CE) due to the formation of a chemical bond, and consequent charge transfer (CT), between CV and Ag via the central C atom whereby CV acted as an electron donor and Ag as electron acceptor (Canamares et al., 2008). Specifically, the C=C ring frequency was shifted from 1,618 to 1,620 cm−1 for the above ink pens. Further proof of occurrence of a CE was found in the following observed shifts: for Bic blue the N-phenyl frequencies appearing at 1,367 and 1,387 cm−1 in the Raman spectrum were shifted to 1,371 and 1,392 cm−1 , respectively in the SERS spectrum; for blue Staedler shifts from 1,369 and 1,386 cm−1 to 1,371 and 1,389 cm−1 were measured; for Bic/Staedler black inks bands were located at 1,370/1,373 and 1,388/1,388 cm−1 , but resolution of the Raman spectrum was too low to measure bands positions, respectively. Further proof of the occurrence of a CT process was given by the predominant enhancement of the non-totally symmetric (E) modes compared to the totally symmetric (A1) modes (Canamares et al., 2008; Selvakannan et al., 2013). As well as a CE a molecular resonance (MR) effect occurred for these pen inks, being the excitation wavelength in resonance with the absorption maximum of CV (588 nm with shoulder at 548 nm). Therefore, the large enhancement observed was attributed to the joint play of three effects: MR, EM, and CE.

In contrast, much less pronounced enhancement was obtained with Au nanopastes. This was expected as the major longitudinal plasmonic band of Au nanopastes was centered at 787 nm, away

from the SERS excitation wavelength. The small enhancement observed could be attributed to a modest electromagnetic effect associated to the transversal band at 520 nm and to the ability of CV to form a CT complex with Au via the N groups (Selvakannan et al., 2013).

Presence of phthalocyanine in blue Bic pen was confirmed by SERS spectrum taken at 785 illumination wavelength with Au nanopastes (**Figure S4a**). In contrast SERS spectrum of blue Staedler taken at 785 nm with Au nanopastes (**Figure S4b**) showed bands in same positions as SERS bands recorded at 514 nm illumination, therefore proving no presence of additional dye components. Surprisingly, the application of both Ag and Au nanopastes resulted in no enhancement generation for the blue Pilot pen ink (**Figure 3C**), suggesting a marked different chemical composition compared to the Bic and Staedler ink mixtures. The presence of Victoria blue R component was hypothesized for this pen ink based on resemblance between the recorded spectrum at 785 nm (see **Figure S4c**) with the spectra of Pilot pens and reference dyes reported in literature (Ho et al., 2016). Specifically, bands at 1,611, 1,360, 1,178, 760, 732, 460, and 421 cm−1 overlapped the spectrum of Victoria R reference taken at 785 nm reported in literature.

### Raman Spectroscopy of Real Artworks

In order to show the importance of database spectral collection for identification of inks used in artworks **Figure 4** shows Raman analysis performed on two artistic contemporary drawings made by writing blue and black pens. Specifically, **Figures 4a,b** shows optical microscopy images of the pen lines on the paper with areas of dense inks spots dis homogeneously distributed on the paper substrate, due to the dis homogeneous and fibrous morphology of the paper itself. **Figure 4b** shows Raman spectra of the blue and black drawings. Both spectra show bands associated to CV at 1,618, 1,586, 1,532, 1,445, 1,172, 916, 441, and 403 cm−1 . Careful comparison with Raman spectra of Bic pen (**Figure 2**) strongly suggested that this pen was used for the realization of the artistic drawing. As well as overlapping peak positions, the ratio of peaks 1,620/1,588 = 1.75 is equal to the ratio found in blue and black BIC pen Raman spectra.

## SERS for Investigation of Writing Inks Aging Processes

As writing inks are increasingly used for the realization of drawings and mixed media artistic creations it is of paramount importance to carry out investigations on color changes brought upon exposure of ink-based objects to light as such exposure is known to induce severe color changes compromising the aesthetic, informational, and value of such objects. Moreover, the elucidation of mechanisms leading to color changes in ink dyes is important for characterization and identification of documents and objects, identification of ink-dye constituents and informative for the application of tailored conservation and exposure protocols.

**Figures 5a,c,e** show optical images of blue Bic, Staedler, and Pilot inks on paper taken after three cycles of artificial aging. Compared to unaged conditions minor color changes were observed for the Pilot pen whereas Bic and Staedler pens showed substantial color changes resulting in darkening of the Bic pen ink and a dramatic discoloration of the Staedler pen ink. Corresponding SERS spectra of aged inks are reported in **Figures 5b,d,f**. Spectra were obtained with Ag nanopaste and measured at 514 nm laser excitation wavelength. For easiness of comparison aged pen SERS spectra were plotted together as the equivalent SERS spectra (i.e., same laser power and exposure) of unaged pen inks. Spectra were background subtracted in order to allow more direct comparison of peak intensity changes. The SERS spectrum of aged Bic pen showed diagnostic bands of CV.

spectra for blue Bic, Staedler, and Pilot inks on paper.

Compared to the unaged SERS spectrum, few key changes were observed. The ratio I1620/I1587 decreased from the 2.21 value in the unaged sample to the 1.19 value in the aged samples, the CV peak at 723 cm−1 shifted to 732 cm−1 ; the peaks at 744 and 771 cm−1 disappeared and the shoulders at 1,189 and 1,390 cm−1 disappeared. All these changes are in agreement with a mechanism of photo-induced N-demethylation already observed by Cesaratto et al. during their artificial aging studies on triarylmethylene dyes (Cesaratto et al., 2017). Specifically, the spectral changes observed in the Bic pen corresponded to what Cesaratto et al. observed at step 2 illumination, corresponding to a light dose of 0.12 MIx·h and associated to the occurrence of a N-demethylation process. Interestingly, faster degradation was observed for Staedler pen, as represented by the strong discoloration of exposed paper. Broad peaks were observed in the SERS spectrum of the aged pen centered at 1,613, 1,555, 1,536, 1,178, and 909 cm−1 .

Comparison of spectra showed strong resemblance with step 4 illumination (2.10 MIx·h) described by Cesaratto et al. and indicative of a chemical conversion from CV to fully Ndemethylated pararosaniline. These results are in agreement with data reported by Weyermann et al. who found that degradation kinetics of inks deposited on paper differed substantially from pen to pen (Weyermann et al., 2006). A few spectral changes also occurred for Pilot pen, although the uncertain dye mixture identification did not allow correlation with any specific degradation process. Specifically, peaks at 533, 937, and 1,073 cm−1 disappeared in the aged pen; also and the ratio I1616/I1587 decreased from 1.97 to 1.29 following the artificial aging process.

The striking color difference between aged Bic and Staedler pens was ascribed to the chemical composition of their constituting ink. It is known that Bic ink formulations contain a mixture of blue phthalocyanine and triarylmethane dyes and that while the latter are extremely sensitive to photo- and thermal irradiation, phthalocyanine dyes degrade at a much lower rate compared to triarylmethane dyes. Interestingly and in contrast with common perception that aging induces color fading, our optical images showed a darker blue coloration in aged Bic pen inks, probably due to residual presence of phthalocyanine dye. In contrast, strong discoloration was observed in the Staedler pen, which further proves lack of phthalocyanine in the ink mixture. The discoloration could be ascribed to a degradation path involving photoreduction of the excited dye cation to a colorless leuco form, as reported by several authors, which may occur in parallel with the described N-demethylation process (Gorshova et al., 2016).

#### CONCLUSION

Black and blue writing inks deposited on paper were investigated by Raman and SERS spectroscopies in order to identify major dye components. SERS measurements were performed in situ by deposition of a small droplet of plasmonic nanopaste on the analytical surface. Blue and black Bic and Staedler inks treated with Ag nanopastes gave SERS spectra of highly enhanced intensity compared to Raman spectra and SERS spectra taken at 514 nm illumination wavelength with Au nanopastes. The large enhancement was ascribed to a combination of EM, CT, and MR effects. SERS was also used to investigate the degradation process of blue writing inks through application of artificial aging processes involving light and high temperature exposure. Interestingly, in spite of the similar initial blue coloration, it was found that aged colored paper displayed very different colorations from each other. Specifically, the Bic paper displayed a darker blue color, the Staedler-colored paper displayed almost complete discoloration and the Pilot paper showed very small color changes. This was ascribed to the different chemical composition of the constituting inks, characterized by presence of phthalocyanine and triarylmethane dye in the case of Bic pen and only triarylmethane in the case of the Staedler pen. A degradation process involving N-demethylation of

#### REFERENCES

triarylmethane components was proposed based on spectral evidence. However, the intense discoloration observed for Staedler pen also suggested the concomitant formation of colorless leuco forms, due to photoreduction processes.

#### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/**Supplementary Material**.

#### AUTHOR CONTRIBUTIONS

DS and AA performed the Raman, SERS measurements, SEMs, and synthesis of Ag nanopastes. MT and RG performed the aging of samples. AM provided the pens and set up the aging protocols. DI and PB wrote the manuscript.

#### FUNDING

This work was funded by the European Union H2020 projects Nanorestart (646063) and Apache (814496) and the Irish Research Council (IRC) project GOIPD/2015/716.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2019.00727/full#supplementary-material

chromatography and image analysis. Forens. Sci Intern. 179, 199–205. doi: 10.1016/j.forsciint.2008.05.013

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Saviello, Trabace, Alyami, Mirabile, Baglioni, Giorgi and Iacopino. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Removing Polymeric Coatings With Nanostructured Fluids: Influence of Substrate, Nature of the Film, and Application Methodology

Michele Baglioni, Margherita Alterini, David Chelazzi, Rodorico Giorgi\* and Piero Baglioni\*

*Department of Chemistry and CSGI, University of Florence, Sesto Fiorentino, Italy*

#### Edited by:

*Luca Tortora, Roma Tre University, Italy*

#### Reviewed by:

*Claudia Mazzuca, University of Rome Tor Vergata, Italy Nunzio Tuccitto, University of Catania, Italy*

#### \*Correspondence:

*Rodorico Giorgi<EMAIL_ADDRESS>Piero Baglioni<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Colloidal Materials and Interfaces, a section of the journal Frontiers in Materials*

Received: *25 September 2019* Accepted: *18 November 2019* Published: *03 December 2019*

#### Citation:

*Baglioni M, Alterini M, Chelazzi D, Giorgi R and Baglioni P (2019) Removing Polymeric Coatings With Nanostructured Fluids: Influence of Substrate, Nature of the Film, and Application Methodology. Front. Mater. 6:311. doi: 10.3389/fmats.2019.00311* Cleaning is one of the most important and delicate operations in the conservation of cultural heritage, and, if not correctly performed, may irreversibly damage works of art. The removal of aged or detrimental polymeric coatings from works of art is a common operation in conservation, and nanostructured fluids (NSFs), such as aqueous swollen micelles and oil-in-water (o/w) microemulsions, are used as an alternative to non-confined organic solvents, which pose a series of non-negligible drawbacks. NSFs effectiveness in removing polymeric coatings has been thoroughly demonstrated in the last decades, while their cleaning mechanism is still under investigation. The present work deepens the knowledge on the removal mechanisms of NSFs, studying the interaction of a four-component NSF with four different types of acrylic and vinyl polymer films cast from solutions or aqueous polymer latexes on three substrates (glass, marble, and polystyrene) with different hydrophilicity and wettability. NSFs were applied either as non-confined or confined in cellulose poultices (traditionally employed by conservators), or in highly retentive chemical gels, observing the influence of the confining matrix on the removal process. It was found that the NSF/polymer film interaction is greatly dependent on the film structure and composition. Films formed from solvent solutions can be swollen by water/organic solvents mixtures or dewetted when a surfactant is added to the cleaning fluid; films formed from polymer latexes, on the other hand, are generally swollen even just by water alone, but poorly dewet. The substrate also plays an important role in the removal of polymer films formed from solutions, for instance the removal of an acrylic polymer from polystyrene could be achieved only through highly selective cleaning using NSF-loaded chemical hydrogels. These results can be key for conservators, providing innovative solutions to face new challenges in art preservation.

Keywords: polymer coatings, cleaning, nanostructured fluids, acrylics, vinyls, non-ionic surfactants, hydrogels, dewetting

## INTRODUCTION

Cleaning of works of art generally consists in the selective removal of materials that promote the degradation of the artifacts or alter their readability and appearance. Among these materials, aged or detrimental polymeric coatings are often found on works of art, and their removal is a common operation in art conservation. Synthetic polymers have been largely employed in the traditional restoration practice as varnishes, adhesives, protectives and consolidating agents. The presence of polymeric coatings on the surface of porous inorganic substrates (wall paintings, stone, mortars) drastically reduces water permeability and enhances the degradation induced by salts, up to consistent loss of the artifacts' surface layers (Carretti and Dei, 2004; Giorgi et al., 2010). Graffiti and vandalism are other well-known examples where selective removal of polymeric coatings must be carried out on artistic surfaces (Apostol et al., 2011; Sanmartín et al., 2014; Giorgi et al., 2017; Baglioni et al., 2018d). Other cases include the removal of aged pressure sensitive tapes from paper artworks (Bonelli et al., 2018), or of discolored and cracked varnishes from paintings (Burnstock and Kieslich, 1996; Baglioni et al., 2018a).

The vast amount of different solvent-sensitive materials found in classic and contemporary art poses continuous challenges to the safe removal of detrimental coatings (Kavda et al., 2017). Traditionally, restorers and conservators rely on the use of organic solvents to dissolve or swell unwanted materials. Solvents are typically applied either as non-confined, using cotton swabs, or thickened in viscous polymeric solutions and solvent gels (Burnstock and Kieslich, 1996; Burnstock and White, 2000; Baglioni et al., 2012c). However, these methods exhibit poor control and scarce selectivity, or involve the presence of residues from the cleaning system. In addition, health concerns arise from the toxicity of most solvents used in restoration.

Alternatively, nanostructured fluids (NSFs) such as aqueous swollen micelles and oil-in-water (o/w) microemulsions were proposed in the late 1980s for the removal of hydrophobic matter from porous inorganic substrates (Borgioli et al., 1995). Their effectiveness in removing polymeric coatings from different types of surfaces has been thoroughly demonstrated in the last decades (Carretti et al., 2003, 2007; Baglioni et al., 2010, 2012b, 2015a,b, 2016, 2018c). These NSFs are water-based systems where limited amounts of solvents are found either in the dispersed (as nano-sized droplets stabilized by surfactants) or in the continuous phase of the fluids. The large surface area developed by the nano-sized droplets, and the synergistic action of solvents and surfactants are responsible for enhanced cleaning power, while the presence of the continuous aqueous phase limits the re-deposition of detached hydrophobic matter on hydrophilic surfaces. Besides, being water-based, aqueous NSFs have reduced toxicity as opposed to traditional solvents. A further improvement was represented by the confinement of NSFs into retentive physical or chemical gels able to gradually release the fluids at the gel-artifact interface, maximizing control over the cleaning action (Baglioni et al., 2015c; Chelazzi et al., 2018).

While these systems represent the most advanced cleaning tools currently available to conservators, the interaction mechanism of NSFs with polymeric coatings is still being investigated.

The removal of high molecular weight macromolecules does not follow the rules of classical detergency. Studies performed on model samples, i.e., glass slides coated with an acrylate copolymer, have shown that dewetting takes place when waterbased NSFs are put in contact with the polymeric coating (Raudino et al., 2015, 2017; Baglioni et al., 2017, 2018b). The film swells and detaches from the surface. This behavior was also observed on mortar samples coated with the same polymer (Baglioni et al., 2018b). The nature of both the organic solvents and the surfactant(s) included in the cleaning formulation was found to be of fundamental importance in the whole process. The solvents swell the polymer and lower its glass transition temperature, increasing the mobility of the polymer chains; the surfactant reduces the interfacial tension, kinetically promoting the detachment of the polymer film from the surface, and initiating the dewetting process that eventually breaks down the coating into separate polymer droplets.

Despite providing fundamental insights, previous studies on this subject depicted only a partial picture of the interaction mechanisms between NSFs and polymeric coatings. Namely, all the studies were conducted only on a single type of polymer, i.e., Paraloid B72 R (poly(ethyl methacrylate/methyl acrylate) 70:30) cast from solution. Only glass or mortars were considered as substrates. Besides, simplified NSFs formulations were used to isolate the contribution of individual components in the system.

The present work aims to deepen the knowledge on the removal mechanisms of NSFs, studying the interaction of a four-component NSF with four different types of polymer films cast either from solutions or aqueous emulsions. In fact, it is well known that films of polymers cast from solutions have different physical structures than those cast from emulsions (Chevalier et al., 1992; Winnik, 1997; Steward et al., 2000), and polymers emulsions contain several additives; these can be crucial factors in determining the interaction process with the NSFs. For instance, hydrophilic additives in the polymer film might affect the interfacial tensions between the polymer, the substrate, and the NSF, which are key factors in wetting and dewetting processes. Three substrates were considered (glass, marble and polystyrene) with different hydrophilicity and wettability. At this stage, only laboratory samples and non-aged polymers were considered, so as to investigate simpler systems with controlled and known composition. Finally, NSFs were applied either as non-confined or confined in cellulose poultices (traditionally employed by conservators), or in highly retentive chemical gels, observing the influence of the confining matrix on the removal process in terms of controlled release and cleaning effectiveness. Our goal was to use a phenomenological approach to describe qualitatively the interaction between polymer films and NSFs, rather than providing a quantitative evaluation of polymer removal.

The NSF is composed of water, an alcohol ethoxylate nonionic surfactant, 2-butanol (BuOH), and 2-butanone (methyl ethyl ketone, MEK), and is representative of formulations actually employed in the conservation practice for the cleaning of real works of art.

The three selected substrates were coated with the four different polymers, obtaining a set of 12 samples, which were then exposed to the NSF, investigating the interaction mechanism for each combination. The effects of the NSF on the films were studied by means of optical microscopy and micro-reflectance infrared Fourier Transform spectroscopy 2D mapping of the areas of interest, which provides spatial resolution down to the micron-scale.

#### MATERIALS AND METHODS

#### Chemicals

C9−11E5,5 alcohol ethoxylate (Berol 266, AkzoNobel), sodium dodecylsulfate (SDS, Sigma-Aldrich, purity ≥ 99%), butanone (MEK, Sigma-Aldrich, purity 99%), 2-butanol (BuOH, Sigma-Aldrich, purity 99%), ethanol (EtOH, Fisher Chemical, purity ≥ 99%), white spirit (White Spirit, Fidea), D2O (EurisoTop, 98%), and deuterated butanone (d-MEK, C4H3D5O, Sigma-Aldrich, purity 98%) were used as received, without further purification. Water was purified with a Millipore Milli-Q gradient system (resistance > 18 MΩ cm).

#### Polymers Formulations

The four polymeric materials selected are widely used in the traditional restoration practice. They include: poly(vinyl acetate) (PVAc), commercially known as PVA K40 R , dissolved in ethanol; PVAc as an aqueous emulsion, commercially known as Vinavil NPC R ; poly(butyl methacrylate) (PBMA) dissolved in white spirit, commercially known as Plexisol P550 R ; poly(ethyl acrylate/methyl methacrylate) (PEA/PMMA) 40:60 as an aqueous emulsion, commercially known as Plextol B500 R . **Table 1** reports the main properties of the formulations. All the products were applied on the substrates as a 10% (w/w) solution/emulsion.

#### Differential Scanning Calorimetry (DSC) for the Glass Temperature (Tg) Determination

DSC measurements were performed on a DSC Q1000 from TA Instruments on small samples (2–5 mg) of the four dry films, according to the following procedure: equilibration at −50◦C; heating ramp from −50 to 60◦C at 10◦C/min; cooling ramp from 60 to −50◦C at 10◦C/min; heating ramp from −50 to 60◦C at 10◦C/min. The first heating/cooling ramp was used to equilibrate the sample, while the second heating ramp is the one used for the Tg determination. Each sample was run at least twice, in order to check for reproducibility of the measurement.

#### Substrates

The three different substrates selected for this work, i.e., glass, marble and polystyrene, were selected as they exhibit different wettability and are representative of artistic substrates frequently found in classic or contemporary art production. Glass slides of 5 × 5 × 0.3 cm3 , marble tiles of 5 × 5 × 1 cm3 , and polystyrene slides of 5 × 5 × 0.2 cm3 were used. The four different filming materials (polymers solutions or emulsions) were then laid manually on the top surfaces of each series of samples. 150 µl of each polymer formulation (VS, VE, AS, AE) was carefully spread using a pipette on a 4 × 5 cm2 area on the surface of glass, marble and polystyrene samples. A reference area was thus left untreated. The specimens were left drying for 30 days, and then the dry mass of the films was evaluated. The density of the dry films was then measured, by drying for 4 days in a ventilated oven at 60◦C weighed amounts of polymer solutions/emulsions that were poured into graduated vials. The results, reported in **Table 1**, made possible to calculate that the obtained films had an average thickness of ∼5–7µm, assuming that they are homogeneously spread all over the treated surface.

### Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy

ATR measurements were carried out with a Thermo Nicolet Nexus 870 spectrometer equipped with a liquid nitrogen-cooled MCT detector and a single reflection diamond crystal ATR unit. Spectra were recorded in the 4,000–650 cm−1 range (128 scans, 4 cm−1 resolution). ATR measurements were performed on small samples of spray-can paints' dried films, removed from the glass support.

#### NSF Preparation

The NSF selected for this work is composed as follows (w/w): H2O, 65.9%; C9−11E5,5, 3.5%; BuOH, 9.7%; MEK, 20.9%. In order to better understand the role of each component in the interaction with the polymer coatings, other liquid systems were also used, obtained removing some of the components from the complete NSF formulation. Namely, a water/C9−11E5,5 surfactant solution, and a water/BuOH/MEK solvents mixture were used. In both systems, the ratio between each component was the same as in the complete NSF formulation. It is worth noting that both BuOH and MEK are partly water-miscible [12.5 and 24% at 20◦C, respectively (Verschueren, 2001)] and this made possible to obtain a single-phase stable mixture of water and solvents even in the absence of surfactants.

#### Small-Angle Neutron Scattering

Small-angle neutron scattering (SANS) experiments were performed on the spectrometer V4 (Bensc-Helmholtz Zentrum Berlin). Two different configurations were employed (i.e., sample-to-detector distances, SD = 2 or 8 m) to cover a range of wave vectors q [q = (4π/λ)sin(θ/2), where λ is the wavelength of the incident neutron beam and θ the scattering angle] from 0.007 to 0.28 Å−1 . For each configuration a 6 Å neutron wavelength was used and the wavelength resolution, 1λ/λ, was <10%. Samples were contained in 1 mm thick quartz cells and kept at 20 ± 2 ◦C during the measurements. The scattering intensity was corrected for the empty cell contribution, transmission, and detector efficiency and was normalized to the absolute scale by direct measurement of the intensity of the incident neutron beam. The integration of the normalized 2D intensity distribution with respect to the azimuthal angle yielded the 1D scattering intensity distribution, I(q), in cm−1 . The reduction of the data was performed using standard BENSC procedures for small-angle isotropic scattering. The background from the incoherent scattering coming from each sample was determined from analysis of the Porod asymptotic limit and subtracted from the normalized spectra. Experimental data normalized to absolute scale were fitted using Igor routines (NCNR\_SANS\_

*aV, vinyl; A, acrylic; S, polymer applied as a solution; E, polymer applied as an emulsion.*

*bThe density was calculated as described in section Substrates.*

*cThe glass temperature (Tg) was measured by means of DSC analyses according to the procedure described in section Differential Scanning Calorimetry (DSC) for the Glass Temperature (Tg) Determination.*

package\_6.011) (Kline, 2006) available from NIST, National Institute for Standard and Technology, Gaithersburg, MD, running on Igor Pro© (Wavemetric Inc., Lake Oswego, Oregon; Version 6.22).

#### NSF/Polymer Interaction Experiments Immersion Tests

The aim of these tests was to investigate the direct interaction between cleaning fluids and polymer films, without the influence of a medium (i.e., cellulose pulp poultice or gel). To this purpose, the coated specimens were partly immersed in about 30 ml of the selected fluids, i.e., the minimum amount of fluid needed to treat the samples. The area of the polymer film exposed to the fluid action was kept constant and fixed to 1 × 5 cm2 . At selected time intervals, up to 10 min, the specimens were extracted from the fluid and let dry, and then visual inspection of possible changes in the film was carried out. At t = 0 min and t =10 min the superficial micromorphology of the films (after drying) was investigated by optical microscopy and FTIR 2D micro-reflectance mapping. Finally, after 10 min of incubation with each liquid system, a removal test was performed on all the samples, using a cotton swab soaked with water, in order to check the removability of the polymeric coatings.

#### Confocal Laser Scanning Microscopy (CLSM)

Confocal Microscopy experiments were performed on a Leica TCS SP8 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a 63× water immersion objective. The four polymers were stained with Coumarin 6, which was dissolved/dispersed in the liquid solutions or latexes. Then, 2–4 µm-thick polymer films were obtained by spin coating coverglasses for 60 s at 1,000 rpm. Coumarin 6 was excited with the 488 nm laser line of an argon laser. The emission of the dye was acquired with a PMT in the range 498–530 nm. CLSM experiments were performed to monitor the interaction of the different polymer films with the NSF. Briefly, 50 µl of the unlabeled liquid phase were put in contact with the coumarin 6-labeled polymer coated coverglass, and the morphological variations of the polymeric film were monitored over time.

#### Cleaning Tests

Two sets of cleaning tests were performed, in order to evaluate the influence on the application method of the NSF, and the effect of the supporting material (i.e., cellulose pulp poultice or chemical hydrogel) on the interaction between the NSF and the polymeric coating. Traditional cellulose pulp poultices were prepared by mixing 35 mg of cellulose pulp (Arbocel R , Zecchi, Firenze) and 160 µl of NSF. The obtained poultices were applied, interposing a Japanese paper sheet (Zecchi, Firenze) between the compress and the specimen surface, on 1 cm2 areas of each sample for 15 min. After removing the poultices, a gentle mechanical action was performed on the treated surface, by means of wet cotton swabs. Poly(hydroxyethyl methacrylate/poly vinylpyrrolydone) semi-interpenetrated networks (pHEMA/PVP SIPNs) are highly retentive hydrogels, previously proposed in combination with NSFs for the cleaning of works of art (Baglioni et al., 2018a), and prepared as described elsewhere (Domingues et al., 2013a,b). Gels having a size of 1 × 1 × 0.2 cm3 were used, able to upload ca. 160 µl of NSF, i.e., the same amount of cleaning fluid loaded in the poultices. The gels were loaded with the NSFs by immersion for 24 h, so as to exchange water within the gel with the NSFs. The gel was applied for 15 min, and at the end of the treatment a gentle mechanical action was performed on the coating residues.

#### Contact Angle Measurements

The contact angle of 5 µL sessile droplets of Milli-Q water on the selected material surface was measured with a Rame-Hart Model 190 CA Goniometer. The three substrates and the four polymer films were investigated. The equilibrium contact angle was measured in at least eight different areas, and the average value and standard deviation was evaluated.

#### Optical Microscopy

A Reichert Zatopan 353–890 microscope was used to collect micrographs. The instrument was coupled with a Nikon Digital Sight DS-Fi2 camera. The NIS-ELEMENTS software was used to capture and edit images.

### Fourier-Transform Infrared Spectroscopy−2D Micro-Reflectance Mapping

The Fourier transform infrared (FTIR) 2D imaging of the treated surfaces was carried out using a Cary 620–670 FTIR microscope, equipped with an FPA 128 × 128 detector (Agilent Technologies). The spectra were recorded directly on the surface of the samples in reflectance mode, with open aperture and

TABLE 2 | Contact angle of water droplets on the surface of the four selected polymeric coatings, and of the three types of substrates.

a spectral resolution of 8 cm−1 , acquiring 128 scans for each spectrum. Each analysis produces an IR map of 700 × 700 µm2 (128 × 128 pixels), with a spatial resolution of 5.5µm (i.e., each pixel has dimensions of 5.5 × 5.5 µm2 and is associated to an independent spectrum). In each map, the intensity of a characteristic peak of each polymer, e.g., the C=O ester stretching typical of both acrylics and vinyls, at about 1,730 cm−1 , was shown with a chromatic scale, following the order red > yellow > green > blue.

### RESULTS AND DISCUSSION

#### Polymer Films Characterization

The four polymeric coatings were characterized by means of FTIR-ATR and contact angle measurements. **Table 2** reports the values for the contact angle of water droplets laid on the surface of the polymer films, or of the selected substrates (glass, marble, polystyrene). The substrates exhibit a range of contact angles passing from hydrophilic (glass) to hydrophobic surfaces (polystyrene). Regarding the polymers, it can be noticed that the acrylics are more hydrophobic than the vinyls, and the films deriving from polymer latexes are more hydrophilic than those coming from solutions. This is likely due to the presence of surfactants, stabilizers, and other polar additives in the latexes emulsions, which affect the wettability of the film.

**Figure 1** shows the FTIR-ATR spectra of the four polymer films. The spectra of the two vinyl films match closely, confirming that the same type of polymers is present in both films. The main bands were assigned as follows (Doménech-Carbó et al., 2001; Learner and Institute, 2004): C-H stretching (2,928 cm−1 and a shoulder at 2,972 cm−1 ); C=O stretching (intense and narrow peak at 1,727 cm−1 ); asymmetric stretching of the C–O group (1,222 cm−1 , distinctive of PVAc); C–H in-plane bending (1,428 cm−1 and 1,371 cm−1 ); C–H out-of-plane bending (1,117 cm−1 ); C–O symmetric stretching (1,018 cm−1 ); C–C stretching (944 cm−1 ); C–H rocking (792 cm−1 ). Besides, the spectrum of VE shows a broad band at 3,335 cm−1 , which is likely due to the OH stretching of poly(vinyl alcohol) (PVA), i.e., one of the main additives of the Vinavil NPC R aqueous latex.

The spectra of AS and AE show slightly different features in the fingerprint and CH stretching regions, as expected given the different type of acrylate polymers found in those films.

The main bands of the acrylic films were assigned as follows (Doménech-Carbó et al., 2001; Learner and Institute, 2004; Pintus and Schreiner, 2011): C–H stretching (AS: peaks at 2,958, 2,933, 2,873 cm−1 ; AE: peaks at 2,887 cm−1 and 2,860 cm−1 ); C=O stretching (1,720 cm−1 ); C–H in-plane bending (1,465 and 1,380 cm−1 ; AE shows an additional peak is visible at 1,343

FIGURE 1 | ATR spectra of the four polymeric films selected.

cm−1 ); C–O stretching (AS: peaks at 1,240 and 1,065 cm−1 ; AE: peaks at 1,279, 1,241, 1,109, and 1,022 cm−1 ); C–C stretching (965 and 946 cm−1 ); C–H rocking (840 and 750 cm−1 ).

#### NSF Characterization

The selected NSF for this work is composed of water, a nonionic surfactant and two solvents, i.e., BuOH and MEK, which are partly miscible with water. The structure of this NSF was never studied before, thus acquiring information on the micelles' size and shape, and on the location of each component in the NSF, was deemed a preliminary step to help understanding the interaction of the fluid with the polymeric films. To this aim, SANS measurements were performed on four D2O based samples: (1) the D2O/C9−11E5.5 binary mixture; (2) the D2O/C9−11E5.5/BuOH ternary system; (3) the complete NSF formulation, i.e., D2O/C9−11E5.5/BuOH/MEK; (4) the complete NSF formulation with deuterated d-MEK fully replacing the regular MEK. The analysis of samples 3 and 4 represents a contrast variation experiment, in which everything is kept constant from one sample to the other, except for its contrast, by changing the scattering length density (SLD) of one or more chemicals (in this case, exchanging MEK with d-MEK).

**Figure 2**-top shows the SANS profiles for the four samples analyzed. The data were fitted according to two different models. The supramolecular aggregates, in the case of the binary surfactant/water and the water/surfactant/BuOH systems, were modeled as non-interacting polydisperse core-shell spheres, defined by two contrasts, i.e., bulk/shell and shell/core. On the other hand, the best fitting for the complete NSF was obtained by modeling the micelles as non-interacting prolate core-shell

ellipsoidal particles, again defined by a double contrast. The SLD of bulk, shell and core, i.e., respectively, ρbulk, ρshell, and ρcore, were calculated according to the SLD for neutrons of each chemical included in the formulations, as reported in **Table 3**. For globular micelles of homogeneous scattering length density, the total scattered intensity I(q) (cm−1 ) is given by (Sheu and Chen, 1988; Liu et al., 1995):

$$I\left(q\right) = \,^\text{N}\_{\text{P}} V\_{\text{P}}^2 \Delta \rho^2 P\left(q\right) \,\text{S}\left(q\right) + bk g\_{inc} \tag{1}$$

TABLE 3 | SLD for neutrons scattering of the chemicals included in the NSF.

*aFor the SLD calculation a C10 aliphatic chain was considered.*

where Np is the number density of the scattering particles (cm−3 ), Vp is the volume (cm3 ), 1ρ is the contrast term (cm−2 ), P(q) is the form factor, and S(q) is the structure factor. In this case S(q) = 1, as the particles were considered to be non-interacting. In the case of spherical core-shell aggregates, the particle scattering intensity is expressed as follows (Kline, 2006):

$$\begin{aligned} I \left( q \right) \\ \delta = \frac{\phi}{V\_P} \left[ \left( \rho\_{core} - \rho\_{shell} \right) \frac{3V\_c j\left( qr\_c \right)}{qr\_c} + \left( \rho\_{shell} - \rho\_{bulk} \right) \frac{3V\_P j\left( qr\_s \right)}{qr\_s} \right]^2 \end{aligned} \tag{2}$$

where j(x) is a spherical Bessel function and is expressed as:

$$j'(\mathbf{x}) = \frac{(\sin \mathbf{x} - \mathbf{x} \cos \mathbf{x})}{\mathbf{x}^2} \tag{3}$$

and where ϕ is the volume fraction of the micellar phase, Vc is the core volume, rc is the core radius, rs = rc + t (t is the shell thickness). Since this model takes into account a polydisperse core, which follows the Schultz distribution, the form factor calculated in Equation (2) is normalized by the average particle volume:

$$
\left< V \right> = \frac{4\pi}{3} \left< r\_c^3 \right> \tag{4}
$$

where:

$$
\left\langle r\_{\epsilon}^{3} \right\rangle = \frac{\left\langle z+2 \right\rangle \langle z+3 \rangle}{\left\langle z+1 \right\rangle^{2}} \left\langle r\_{\epsilon} \right\rangle \tag{5}
$$

and z is the width parameter of the Schultz distribution (Degiorgio et al., 1985):

$$z = \frac{1}{\left(\frac{\sigma}{\langle r\_{\ell} \rangle}\right)^2} - 1 \tag{6}$$

being σ 2 the variance of the distribution. The polydispersity index (PDI), reported in **Table 4** is defined as σ/<rc> [see equation (6)] and its value is comprised between 0 and 1.

In the case of monodisperse non-interacting prolate ellipsoids, on the other hand, when modeling asymmetric micelles with a

TABLE 4 | Fitting results of the SANS data acquired on the NSF.

core-shell scattering length profile, P(q) included in Equation (1) is usually calculated as an orientationally-averaged normalized form factor, P(q). First, the orientation-dependent form factor F(q, µ), is defined as follows (where µ is the cosine between the direction of the symmetry axis of the ellipsoid and the Q vector):

$$F\left(q,\mu\right) = f\left(\rho\right)\frac{\mathfrak{H}\left(u\right)}{u} + \left(1 - f\left(\rho\right)\right)\frac{\mathfrak{H}\left(\nu\right)}{\nu} \tag{7}$$

where j(x) is the same spherical Bessel function defined in Equation (3) and u and ν are expressed as:

$$u = q[\mu^2 a^2 + (1 - \mu^2)b^2]^{1/2} \tag{8}$$

$$\nu = q[\mu^2(a+t)^2 + (1-\mu^2)(b+t)^2]^{1/2} \tag{9}$$

which define the geometrical shape of the micelles, and where f(ρ) contains the contrast calculation, and a, b, t are the geometrical parameters of the ellipsoid, i.e., the major semi-axis, the minor semi-axis, and the shell thickness, respectively.

P(q) is then calculated as follows (Kotlarchyk and Chen, 1983):

$$\overline{P}\left(q\right) = \int\_0^1 d\mu \left| F(q,\mu) \right|^2 \tag{10}$$

Even though the measured samples included D2O instead of H2O, the structural picture emerging from the analysis of SANS data can be safely transferred to the H2O-based NSF, apart from possible slight changes (Baglioni et al., 2012a).

**Table 4** shows the main fitting results, while **Figure 2**-bottom contains a cartoon depicting the size and shape evolution of micelles following the addition of each component to the formulation. The size of C9−11E5.5 micelles in D2O was perfectly consistent with the length of the surfactant molecule and with the results of previous SAXS measurements performed on similar systems (Baglioni et al., 2017), where a r + t total micelle radius of about 35 Å had been obtained. A polydispersity index of 0.2 for the size of the hydrophobic core fits such systems, which usually have a PDI in the 0.1–0.5 range. According to fitting results, the structure and size of the micelles do not change significantly after the addition of BuOH (see **Figure 2**-bottom**)**. In fact, BuOH was found to be partitioned between the micellar and the aqueous bulk phase in a 30:70 ratio, meaning that most of the solvent is mixed with water. The fraction solubilized in the micelles is preferentially located in the shell, replacing D2O hydration molecules, and thus affects the micellar size and shape only slightly. The main effect of the inclusion of BuOH is to double the polydispersity of the core radius with respect to the binary water/surfactant system, even though the value is perfectly suitable for a micellar solution.

The results of the contrast variation experiments performed on the complete NSF formulation showed that the inclusion of a significant amount of MEK in the system completely alters both the size and shape of the aggregates (see **Figure 2**-bottom), as the result of a sphere-to-rod transition. This behavior was observed for similar systems containing the same surfactant (Baglioni et al., 2014) and is likely due to the system getting closer to its cloud point. In fact, micelles are known to grow and assume elongated shapes close to the clouding temperature, as observed in the present case. C9−11E5.5 has a cloud point of about 55–60◦C, which is known to be lowered by the interaction of this surfactant with MEK (Baglioni et al., 2014). The inclusion of MEK in the system does not alter significantly the partition coefficient of BuOH (PBuOH) between the micelles and the water phase, while MEK is mainly dissolved in the bulk phase (PMEK = 0.17).

Overall, SANS analysis clarified that the structure of the NSF is that of rod-like micelles, mainly composed of the sole surfactant, dispersed in a water/solvents mixture, indicating that the system is close to its cloud point. This structural information has relevant implications in terms of cleaning power of the NSF, as detergency is known to be maximized when these systems are close to the cloud point (Holmberg, 2002; Holmberg et al., 2002; Stubenrauch, 2008; Baglioni et al., 2014). Therefore, high cleaning effectiveness was expected from this NSF formulation.

#### NSF/Polymer Interaction

The interaction between the NSF and the four polymer films was initially investigated simply by immersing the coated specimens into four different liquid systems, i.e., water, water/surfactant, water/solvents, and the complete NSF. The micromorphology of the film was observed by optical microscopy at different times during the total 10 min of incubation of the specimens in the liquids, while 2D micro-FTIR mapping was performed on the polymer surface before exposure to the fluids and after 10 min of incubation. Finally, a removal test was performed on the polymers using wet cotton swabs, in order to check their removability.

Looking at the results of the immersion tests, illustrated in **Figures 3**–**6** and summarized in **Table 5**, a clear difference in the behavior of samples emerges, i.e., the polymer films deriving from latexes (VE and AE) are preferentially swollen and only in few cases partly dewetted, whereas the films deriving from solutions are unaffected by just water, are swollen by the surfactant solution, and are partly or completely dewetted when solvents are included in the liquid systems. For instance, **Figures 3**, **5** show how for chemically identical polymers (both PVAc) the type of the film plays a key role in determining the behavior when the film is exposed to the same NSF. 2D microFTIR mapping was crucial to confirm the location and distribution of the polymers on the micron-scale. In particular, when a film is

dewetted (see **Figures 3**, **4**) it was shown that no polymer residues are present (above the instrumental detection limit) outside the droplets on the substrate surface. In fact, it has been shown that the detection limit of an FPA detector is significantly lower than that of conventional mercury cadmium telluride (MCT) detectors for the FTIR detection of trace amounts of materials. The heterogeneous distribution of analytes can result in small areas of localized high concentration, which can be detected thanks to the high spatial resolution of the FPA detector (Chan and Kazarian, 2006). For instance, for polyvinyl alcohol and

polyvinyl acetate we verified that quantities < 1 pg/pixel (1 pixel = 5.5 × 5.5µm) can be detected.

Polymer coatings formed from aqueous emulsions are significantly more sensitive to the action of water [a well-known issue when emulsion-based acrylic paint layers are exposed to aqueous cleaning fluids (Murray et al., 2002; Ormsby and Learner, 2009; Willneff et al., 2014)], and tend to be swollen. This is justified by the presence of a non-negligible amount of hydrophilic additives and surfactants in the polymer emulsions, which remain in the film after drying and are able to interact with

cm−1 is shown with a chromatic scale, following the order red > yellow > green > blue. Visible light micrographs are approximately 1 cm2 large; the FTIR mapping areas have a size of 400µm × 550µm. The blue boxes highlight areas of the samples, which were investigated by 2D FTIR mapping; the position of the blue frame in the lower magnification images does not correspond to the actual position of the investigated areas.

water molecules, favoring the swelling of the film. The presence of hydrophilic additives can also explain why the swollen films tend to dewet less easily. The energetic balance of dewetting can be described by the spreading coefficient S, which for a polymer film on a glass surface, immersed in a liquid, is defined as (Baglioni and Chelazzi, 2013):

$$\text{J} = \begin{array}{c} \text{J} \\ \text{J} \end{array} - \begin{array}{c} \text{J} \\ \text{J} \end{array} - \begin{array}{c} \text{J} \\ \text{J} \end{array} \tag{11}$$

where γLG is the interfacial tension between glass and the liquid, γPG is the interfacial tension between glass and the polymer, and γLP is the interfacial tension between the liquid and the polymer. When S is negative, dewetting is energetically favored and occurs spontaneously unless an activation energy barrier hinders the process kinetically. Hydrophilic additives in the polymer film might lower the values of both γPG and γLP, making S less negative for a given liquid/polymer/glass set. On the other hand, the presence of surfactant additives may lower the glass transition temperature (Tg ) of the polymer films (as in the case of VE–see **Table 1**), making polymer chains more mobile, thus possibly decreasing the energy costs related to the formation of new interfacial regions during the detachment of the film, and lowering the activation energy necessary to initiate dewetting (Baglioni et al., 2017, 2018b; Montis et al., 2019).

Overall, in the case of the polymer films from emulsions investigated here, the films' thermodynamic stability seems to prevail on the kinetic drive of the dewetting process. However, the swollen films are softened and easily removable from glass and marble even with water and water/C9−11E5.5, meaning that the adhesion to the solid surface was sensibly reduced,

micro-reflectance FTIR map is reported, together with its relative micrograph in visible light. In each map, the intensity of the C=O ester stretching at about 1,730 cm−1 is shown with a chromatic scale, following the order red > yellow > green > blue. Visible light micrographs are approximately 1 cm2 large; the FTIR mapping areas have a size of 400µm × 550µm. The blue boxes highlight areas of the samples, which were investigated by 2D FTIR mapping; the position of the blue frame in the lower magnification images does not correspond to the actual position of the investigated areas.

while in the case of polystyrene the removal is slightly more difficult. When a polymer film formed from a solvent solution is exposed to a cleaning fluid, a more diversified behavior is observed. Water and water/C9−11E5.5 are partly or completely ineffective in removing the films. Instead, dewetting processes are induced by water/solvents mixtures or, more efficiently, by the synergistic action of solvents and surfactants (Gentili et al., 2012; Xu et al., 2012; Baglioni et al., 2017, 2018b). This is clearly exemplified by AS on marble, where water alone produces no visible effects on the polymer. The nonionic surfactant micellar solution is able to induce some swelling of the film, but not sufficient to grant its easy removal. A water/solvents mixture, on the other hand, produces a partial dewetting pattern, and the film can be partly removed using some mechanical action. This means that solvents are able to swell the polymer to such an extent that the Tg is lowered below room temperature, starting the dewetting process. However, it is only with the synergistic action of a surfactant, which lowers the interfacial tension at the liquid/substrate and liquid/polymer interfaces, that dewetting proceeds further, leading to easy and complete polymer removal. The substrate's chemical nature is a key factor. Increasing the hydrophobicity of the substrate, the affinity of the polymer film with the substrate increases, leading to less efficient polymer removal, culminating with the limit case of AS on polystyrene, which resulted completely irremovable.

The interaction between the complete NSF and the four polymer films was also monitored through CLSM imaging, as described in section Confocal Laser Scanning Microscopy

TABLE 5 | Results of immersion tests performed on polymer-coated glass, marble and polystyrene specimens.

*Both the film appearance and its removability after 10 min of incubation in the four different liquid systems are summarized in the table, following analysis with optical microscopy and 2D micro-FTIR mapping.*

*aAfter 10 min of incubation in the liquid systems, the removability of the coatings was checked performing a gentle mechanical action with a cotton swab soaked with water; x, no removal;* ±*, partial removal; o, complete removal.*

(CLSM). Homogeneous and reproducible 2–4µm thick films on coverglasses were obtained by spin coating. **Figure 7** summarizes the results of the investigation, which were in perfect agreement with what was observed during the immersion tests reported above. AS and VS, i.e., the polymer films cast from solutions, were completely and quickly dewetted by the NSF, while AE and VE, i.e., the films cast from polymer latexes, were just swollen. **Figure 7** shows that some small cracks and/or holes (of few microns) are visible in the VE and AE swollen films, which nonetheless maintain their overall coherence on a macro-scale. These holes possibly form in correspondence of previous film defects, which can be present on such thin films. Overall, the behavior of polymer films cast from aqueous latexes can be seen as if the physical process stopped at the very early stages of dewetting. This is in agreement with what observed during previous experiments, and seems to enforce the hypothesis that the amphiphilic additives in these films play a key role in inhibiting the dewetting process, which otherwise would likely occur.

The inhomogeneous aspect of the VE and AE films at t = 0 is due to the fact that the hydrophobic fluorescent dye was not evenly distributed in the aqueous polymer latex. As the films is swollen by the penetration of the organic solvents, the dye evenly spread through the film.

CLSM investigations, overall, allowed to confirm the results of the immersion tests on macroscopic samples, where the polymer thickness could not be directly measured and not accurately controllable.

Finally, the influence of the application methodology on the outcome of removal tests was evaluated. To this aim, the same amount of NSF was uploaded, respectively, in traditional cellulose pulp poultices and in pHEMA/PVP chemical hydrogels. The latter have been characterized and assessed in the last years, and their use for the removal of unwanted materials from water-sensitive substrates has been thoroughly reported (Domingues et al., 2013a; Baglioni et al., 2018a; Bonelli et al., 2018). Recently, SAXS and rheology studies showed that these gels act as "sponges," able to load different NSFs without being altered or dramatically alter the properties of the fluids (Baglioni et al., 2018a).

The two systems were applied for 15 min on the surface of the films, and then micrographs of the treated area were taken before checking the coating removability via gentle mechanical action with a wet cotton swab. As visible in **Figure 8**, the areas treated with the NSF-loaded poultice are generally more inhomogeneous than the ones treated with the NSF-loaded hydrogel. Moreover, several cellulose fibers were spotted on the samples that were in contact with the poultice, indicating the permanence of paper or cellulose pulp residues on the treated areas. Dewetting patterns could be clearly highlighted in the areas of AS on glass and marble treated with the NSF-loaded hydrogel, indicating a more controlled and reliable cleaning action. In any case, after the removal of either the compress or the gel, complete and easy removal could be obtained via a gentle mechanical action using wet cotton swabs, for all the specimens except AS on polystyrene. In this case, the acrylic polymer could not be removed after 15 min of application of either the NSF-loaded poultice or hydrogel (**Figures 9A,B**). We hypothesized that in this case the application time was too long, causing the migration of solvents through the AS coating up to the polymer/substrate interface,

while VE and AE films were still intact after 10 minutes of incubation with the NSF. In the bottom gray box, the 3D reconstruction of the four polymers' morphology after 10 min is reported. The different behavior between polymer films coming from solvent solutions or aqueous emulsions is clearly evidenced. Each side of the CLSM micrographs is 150µm long. *x* and *y* axes in the 3D reconstructions have the same length.

where they interacted with polystyrene creating a sort of joint or adhesion layer between the polymer and the substrate. This issue is easily overcome by using multiple shorter applications, assuming that the confining matrix of the NSF is enough retentive to release gradually the uploaded fluid on short time scales, which is the case of the pHEMA/PVP gels. In fact, repeated shorter applications (2 +2 +2 min) of the NSF loaded in the hydrogel allowed complete removal of the coating (as confirmed by the 2D microFTIR mapping), while uneven polymer residues were left from the same application using cellulose poultices (see **Figures 9C–F)**. The cartoon in **Figure 9** summarizes the removal process in the two cases: using the highly retentive hydrogel it is possible to perform a gradual action, which proceeds layer by layer, controlling the interaction of the NSF with the substrate. This approach was recently used to perform highly selective removal of overpaintings and vandalism (Giorgi et al., 2017). It is worth noting that the pHEMA/PVP gels used for cleaning interventions do not leave detectable residues on the treated surfaces (as checked with FTIR), while the only non-volatile residues in the NSFs (i.e., the surfactants) can be feasibly removed with a rinsing steps using the gels simply loaded with water (Baglioni et al., 2016).

FIGURE 9 | (A) The result of 15 min of application of the NSF-loaded poultice on AS laid on polystyrene; (B) The result of 15 min of application of the NSF-loaded gel on AS laid on polystyrene; (C) The result of 2 min + 2 min + 2 min of application of the NSF-loaded poultice on AS laid on polystyrene; (D) The result of 2 min + 2 min + 2 min of application of the NSF-loaded gel on AS laid on polystyrene. This is the only case that resulted in the complete removal of the polymer coating, as confirmed by the comparison between the micro-reflectance FTIR maps collected before (E) and after (F) the cleaning with the NSF-loaded hydrogel (the micro-picture is taken at the border between of the cleaned area, seen in blue). The cartoon on the right illustrates the mechanism proposed to explain the observed results. With the poultice (left), the solvents migrate through the polymer (1) and attack the polystyrene substrate, creating a junction or an enhanced adherence between the polymer film and the substrate (2). When a mechanical action is performed (3), an uneven layer of unremoved polymer layer is left stuck to the surface of polystyrene. Using the gel (right), the solvents migration is limited by the retention properties of the scaffold (1). The outer layers of the polymer coating are, then, slightly swollen (2) and easily removed by means of a gentle mechanical action (3). The gel is applied again (4) and the removal proceeds layer by layer, in a selective way, which allows for the complete and safe removal of the polymer film (5). Micrographs A, B, C, and D are 1.2 × 1.6 mm2 large; micrographs E and F (and relative FTIR maps) have a size of 400 × 550µm.

### CONCLUSIONS

This study focused on unveiling some key aspects of the NSFs/polymer coatings interaction depending on several different factors: (i) the chemical nature and, thus, hydrophilicity of the substrate; (ii) the chemical nature and physical structure of the polymeric films to be removed; (iii) the influence of the application methodology on the cleaning outcome. A series of systematic tests was performed and a coherent and clear picture emerged. A water/C9−11E5.5/BuOH/MEK NSF was selected for this study and was firstly characterized by means of SANS measurements, which showed that rod-like nonionic micelles are dispersed in a water/BuOH/MEK mixture close to the system cloud point. These features possibly make this NSF particularly effective in polymer removal. Then, glass, marble, and polystyrene specimens were coated with four different polymers, including two vinyls and two acrylics, each applied either as a solvent solution or as an aqueous emulsion. It was found that the NSF/polymer film interaction is greatly dependent on the film structure and composition. Films formed from solvent solutions can be swollen by water/organic solvents mixtures or dewetted when a surfactant is added to the cleaning fluid; films formed from polymer latexes, on the other hand, are generally swollen even just by water, but they tend not to dewet. This happens independently from the chemical nature of the polymer, and is a direct consequence of its structure and composition, which includes a significant amount of amphiphilic additives. These substances alter the energetic balance of the liquid/polymer/solid system and stabilize the film, which does not dewet. However, the films are easily removable from the substrates, meaning that the action of the cleaning fluid induces loss of adhesion, similarly to what happens during the first stages of the dewetting process that occurs for films cast from polymer solutions. The substrate also plays an important role in the removal of polymer films formed from solutions. In particular, the more the film is affine to the substrate, the harder its removal. In the limit case, the removal of an acrylic polymer from

### REFERENCES

polystyrene could be achieved only through selective cleaning action using a NSF-loaded highly retentive chemical hydrogel, which grants significantly more controlled performances than traditional cellulose pulp poultices. These results have 2-fold relevance: they deepen the knowledge of the physico-chemical processes that underpin phenomena of daily conservation practice, and provide conservators with innovative solutions to face new challenges in art preservation.

### DATA AVAILABILITY STATEMENT

The datasets generated for this study will not be made publicly available. The relevant data are already completely and clearly reported in the paper.

### AUTHOR CONTRIBUTIONS

MB, MA, DC, and RG contributed to the conception and design of the work, to the acquisition, the analysis, and the interpretation of experimental data. PB and RG revised the work critically giving a substantial intellectual contribution, and ultimately provided the final approval for its publication.

### FUNDING

This work was supported by the European Union (CORDIS)– Project NANORESTART (H2020-NMP-21-2014/646063) and PRIN 2017–MIUR. The access to SANS facilities has been supported by the European Commission under the 7th Framework Programme through the Key Action: Strengthening the European Research Area, Research Infrastructures, contract no. 226507 (NMI3).

#### ACKNOWLEDGMENTS

Dr. Uwe Keiderling was kindly acknowledged for his assistance during SANS experiments performed at the HZB.

of cultural heritage. Angew. Chem. Int. Ed. Engl. 57, 7355–7359. doi: 10.1002/anie.201710930

paintings. Appl. Phys. A 114, 705–710. doi: 10.1007/s00339-013- 8150-0

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Baglioni, Alterini, Chelazzi, Giorgi and Baglioni. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cellulose and Lignin Nano-Scale Consolidants for Waterlogged Archaeological Wood

Federica Antonelli 1 \*, Giulia Galotta2 , Giancarlo Sidoti 3 , Florian Zikeli 1 , Rossella Nisi 4 , Barbara Davidde Petriaggi 5 and Manuela Romagnoli 1

*1 Department for Innovation in Biological, Agro-Food and Forestry Systems (DIBAF), Tuscia University, Viterbo, Italy, 2 Biology Laboratory, Istituto Superiore per la Conservazione e il Restauro (ISCR), Rome, Italy, 3 Testing Materials Laboratory, Istituto Superiore per la Conservazione e il Restauro (ISCR), Rome, Italy, 4 BioFaber srl, Mesagne, Italy, 5 Underwater Archaeological Operations Unit, Istituto Superiore per la Conservazione ed il Restauro (ISCR), Rome, Italy*

#### Edited by:

*Gabriel Maria Ingo, Italian National Research Council, Italy*

#### Reviewed by:

*Rodorico Giorgi, Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Italy Maria Laura Santarelli, Sapienza University of Rome, Italy*

\*Correspondence:

*Federica Antonelli<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Nanoscience, a section of the journal Frontiers in Chemistry*

Received: *24 October 2019* Accepted: *09 January 2020* Published: *29 January 2020*

#### Citation:

*Antonelli F, Galotta G, Sidoti G, Zikeli F, Nisi R, Davidde Petriaggi B and Romagnoli M (2020) Cellulose and Lignin Nano-Scale Consolidants for Waterlogged Archaeological Wood. Front. Chem. 8:32. doi: 10.3389/fchem.2020.00032* Waterlogged archaeological wood comes from submerged archaeological sites (in lake, sea, river, or wetland) or from land waterlogged sites. Even if the wooden object seems to have maintained the original size and shape, the wood is more or less severely decayed because of chemical and biological factors which modify the normal ratio of cellulose and lignin in the cell wall. Drying procedures are necessary for the musealization but potentially cause severe shrinkages and collapses. The conservation practices focus not only on removing water from wood but also on substituting it with materials able to consolidate the degraded wood cell walls like polymers (e.g., PEG), sugars (e.g., lactitol), or resins (e.g., Kauramin). In the present work three different nano-scale consolidants were tested: lignin nanoparticles (LNPs) obtained form beech wood *via* a non-solvent method involving dialysis; bacterial nanocellulose (BC) obtained from cultures fed with agro-alimentary waste; cellulose nanocrystals (CNC) chemically extracted from native cellulose. Waterlogged archaeological wood samples of different species (oak, elm, stone pine, and silver fir) characterized by different levels of degradation were impregnated with the consolidants. The treatments efficiency was evaluated in terms of macroscopic observation of treated samples, anti-shrink efficiency (ASE) and equilibrium moisture content (EMC). The results obtained for the three consolidants showed substantial differences: LNPs and CNCs penetrated only about a millimeter inside the treated wood, while BC formed a compact layer on the surface of the cell walls throughout the thickness of the samples. In spite of successful BC penetration, physical evaluation of treatment efficiency showed that BC nanoparticles did not obtain a satisfying consolidation of the material. Based on the reported results more focused test protocols are optimized for future consolidation experiments.

Keywords: lignin nanoparticles (LNP), bacterial cellulose (BC), cellulose nanocrystals (CNC), equilibrium moisture content (EMC), anti-shrink efficiency (ASE), SEM, cultural heritage

## INTRODUCTION

The European standard EN335 (2013) (Conservation of cultural heritage—Guidelines for management of waterlogged wood on terrestrial sites of archaeological significance) defines waterlogged archaeological wood as: "wood whose structure has been filled with water through the sustained inclusion in a water saturated environment." This wood comes from submerged archaeological sites (in lake, sea, river, or wetland) or from land waterlogged sites. Generally, waterlogged wooden artifacts preserve their original size and shape but often they undergo severe cell wall decay due to chemical and biological factors. The pH and the salinity of water as well as the chemical nature of sediments (Hedges, 1990; Unger et al., 2001; Pearson, 2014) together with the action of biological degraders (e.g., erosion and tunneling bacteria and soft rot fungi) (Blanchette et al., 1989) affect the wood causing a more or less severe mass loss and increased porosity and permeability which lead to a spongy and weakened material.

Drying waterlogged archaeological wooden artifacts is necessary for the musealization but it is always a high risk procedure because it could cause severe shrinkages and collapses. Thus, conservation practices focus not only on removing water from wood, but also on consolidating the degraded cell walls aiming for stabilized shape and size of the artifact as well as to enable it to withstand the conditions of the future preservation environment (Grattan and Clarke, 1987). An effective consolidant confers on wood a good stabilization by using the minimal amount of product while being stable to variations in relative humidity. However, these are not the only features required from an effective consolidant, conservation history highlighted no treatment exists which is able to preserve an artifact forever. Thus, all the materials used in restoration practices must be removable or, at least, leave the object retreatable. Several materials have been tested and used for the consolidation of waterlogged archaeological wood (for a review see Christensen et al., 2012). By now, the most used consolidants are polymers (e.g., polyethilene glicol, PEG), sugars (e.g., lactitol), or resins (e.g., Kauramin) (Unger et al., 2001; Christensen et al., 2012).

The use of nanomaterials in the conservation of Cultural Heritage expanded during the last few decades (Baglioni et al., 2015). Different kinds of nanoparticles were mainly used to clean and consolidate wall paintings. Tests with nanosols of silica and alkaline nanoparticles were conducted to consolidate wood artifacts and to deacidify waterlogged archaeological wood (Chelazzi et al., 2006; Mahltig et al., 2008). Even if Cipriani et al. (2010) tested cellulose derived consolidants and Christensen et al. (2012) report of consolidation attempts made with cellulose whiskers, no additional literature is available about the consolidation of waterlogged archaeological wood with nanomaterials obtained from renewable sources.

The present work is aimed to find bio-inspired consolidants that could substitute the chemicals currently in use. In particular, focusing on the concepts of bioeconomy and circular-economy, rarely applied until now to the filed of Cultural Heritage, the study tested three cellulose and lignin nano-scale consolidants: lignin nanoparticles (LNPs), bacterial nanocellulose (BC), and cellulose nanocrystals (CNCs).

LNPs can be synthesized from technical lignin obtained as a by-product of paper industry or second generation ethanol biorefineries and have been proposed for a wide range of applications as e.g., active components or fillers in polymer blends introducing lignin properties like anti-oxidant activity or UV protection on a nano-scale level (Beisl et al., 2017).

Nanocellulose can be obtained by processing plant cellulose or as a product of primary metabolic processes of certain bacterial strains (Kargarzadeh et al., 2017). Bacterial cellulose (BC), is produced by the fermentation of Gluconoacetobacter species, which can produce high aspect ratio (length to diameter) nanofibers, with three-dimensional porous networks, retaining the highest purity. Because of its hydrophilic nature (99% of the constituents is water), flexibility, non-toxicity, good biocompatibility, and wide availability BC has been extensively used in diverse fields from food and paper industry to biomedical applications (Kunjalukkal Padmanabhan et al., 2017).

CNCs, also known as nanocrystalline cellulose, nanowhiskers, nanorods, and rod-like cellulose crystals, are obtained by hydrolysis with highly concentrated acids (6–8 M) and highpower mechanical or ultrasonic treatments of the crystalline domains of cellulose nanofibrils. Their dimensions and the degree of crystallinity depend on the cellulose source and extraction conditions (Abdul Khalil et al., 2014; Kargarzadeh et al., 2017).

### MATERIALS AND METHODS

#### Nanoparticles

Three different nanoparticles were tested as consolidants of waterlogged archaeological wood: lignin nanoparticles (LNPs), bacterial nanocellulose (BC), cellulose nanocrystals (CNCs).

LNPs were obtained from beech wood (Fagus sylvatica L.) from the Cimini Mountains in Lazio region, Italy (Piangoli Legno SNC, Soriano nel Cimino, VT). They were produced applying the "anti-solvent procedure" on acidolysis lignin using DMSO as lignin solvent, as reported in Zikeli et al. (2018). The obtained LNPs are hollow, sphere-shaped particles with diameters ranging between 40 and 120 nm.

BC was provided by Biofaber srl (Italy). It was obtained by fermentation of agro-alimentary waste, in accordance with Pal et al. (2017), in the form of hydrogel pellicles that were washed with distilled water and boiled in 0.5 M sodium hydroxide solution in order to purify the samples. Pellicles were dried at 60◦C to get dried BC membranes and then were grinded with a rotary mill in order to obtain cellulose nanospheres.

The BC prepared for the test had never been studied before, so morphological characterization of BC spay-dried on a stub was performed on a Zeiss (Sigma VP; Carl Zeiss, Germany) FESEM.

CNCs were supplied by CelluForce (Montreal, Canada). CelluForce NCC R is a cellulose hydrogen sulfate sodium salt, provided as a spray dried powder, white in color. The CNCs are spindle shaped crystals, 2.3–4.5 nm in diameter and 44–108 nm in length (from CelluForce product specifications).

### Wood Samples

Wood samples were obtained from two archaeological sites: Isola Sacra (Fiumicino, Italy) and the ancient port of Neapolis (Naples, Italy). The wood coming from Fiumicino belongs to the shipwreck named Isola Sacra 1 (sample P2550), an horeiatype vessel dated before the first half of the 3rd century AD and excavated in 2011 (Boetto et al., 2017). The wood from Naples pertains the shipwreck F (samples FTdis 4, Ftdis 9, and FTdis 48) (Di Donato et al., 2018), and a wooden pier (2514.28, 2514.30, and 2514.34) both dated to the roman age. The remains were recovered from the excavation area of Piazza Municipio in 2015 and 2018, respectively. After the recovery, the wood was stored in the biology laboratory of the Istituto Superiore per la Conservazione e il Restauro (IsCR). During all the storage period the fragments were left completely soaked to maintain the state of maximum water content.

From the wood samples 47 blocks were obtained with dimensions 2.5 × 2.5 × 1.5 cm and 3.0 × 1.5 × 1.5 cm, depending on the availability of the material. Ten blocks were used for each consolidant to be tested, the remaining were used as freeze- (7) and air-dried (10) not-consolidated controls.

Each sample was marked with an acronym indicating the consolidant (L, lignin nanoparticles; BC, bacterial cellulose; CNC, cellulose nanocrystals) or the drying method for controls (F, freeze-drying; A, air-drying), and the wood type (S, softwood; H, hardwood) followed by sequential numbers (**Table 1**).

#### Wood Characterization

Before the beginning of consolidation procedures the wood species were identified and the state of preservation of the material was characterized by micro-morphological and physical analyses according to the Italian standard and to the most used protocols (Jensen and Gregory, 2006; Schwarze, 2007; UNI11205, 2007; Capretti et al., 2008; Romagnoli et al., 2018).

The identification of wood species and micro-morphological examinations were carried out on thin sections (10–20µm) cut in the three anatomical planes (cross, longitudinal-radial and longitudinal-tangential) by mean of transmission light microscopy (DMRB, Leitz) as prescribed by UNI11118 (2004). Samples were cut by hand with a razor blade or using a cryo-microtome (Cryostat CM 1900, Leica). To identify the wood species the observed anatomical features were compared to literature works (Jacquiot, 1955; Jacquiot et al., 1973; Schweingruber and Baas, 1990).

To highlight the presence of micro-organisms the sections were stained with an aqueous solution of 1% w/v methylene blue in 50% lactic acid. Microbial decay was evaluated using both bright-field and polarized light microscopy in order to identify decay patterns and highlight the loss of crystalline cellulose.

To assess the decay by means of physical tests wood blocks were weighted and their volume was defined by the water displacement method. Weight was measured again after the samples were dried in an oven at 103 ± 2 ◦C up to a constant weight. The physical parameters used for the characterization are reported in **Table 2**. Reference values for basic density of nondecayed wood were taken from the literature (Giordano, 1986; Capretti et al., 2008; Macchioni et al., 2012).

TABLE 1 | The utilized samples, type of wood, archaeological sample and consolidant, or drying method.

### Methods Development of Impregnation and Wood Drying

Impregnation baths containing the nano-scale consolidants were prepared. LNPs' bath was obtained adding the nanoparticles to deionised water up to a concentration of 5 mg/ml. BC suspended in deionised water tended to precipitate, so to obtain a stable suspension several attempts were made. Following the suggestions of the Biofaber team, Tween 20, sucrose, polyethylene glycol 400 (PEG400), oil or a mix of them were added to the BC (concentration 5%), the suspensions were homogenized with the help of a magnetic shaker or a sonicator. The details of the performed tests are reported in **Table S1**. At the end of every test a more or less thick layer of BC had precipitated TABLE 2 | Physical parameters used to asses the wood decay.

*M0, anhydrous mass; Mf , mass at maximum water content; Vf , volume at maximum water content; Db, basic density of non-decayed wood.*

at the bottom of the container, except for the suspension obtained with procedure reported as T2 in **Table S1** (0.5% Tween 20 + 1% Cinnamon oil + deionised water, sonication 50% amplitude−1 h, 5% BC, magnetic shaking 500/600 rpm−1 h), which in consequence was selected as the procedure to prepare the impregnation baths.

To obtain a liquid suspension of CNCs, 3% of powdered crystals were added to deionised water and shaken on a magnetic shaker for 1 h. By adding a greater amount of CNCs the suspension became a gel and thus not suitable for the consolidation treatment.

To perform the consolidation tests, the waterlogged archaeological wood blocks were completely soaked in the baths for 1 month at room temperature. After the treatment, the blocks were frozen (−20◦C) and then freeze-dried following the procedure settled in the IsCR restoration laboratory Excavated Organic Materials. The lyophilizer (LIO 2000PNS) settings were: chamber temperature −30◦C; condenser temperature −50◦C; chamber vacuum 2 Pa. The wood temperature was monitored through the use of thermocouples positioned on the samples' surface. The pressure was raised up to room pressure and the blocks were removed from the lyophilizer when reaching a temperature of 18–20◦C.

F control blocks were kept completely soaked in deionised water and were successively frozen and freeze-dried under the same condition as treated samples. A controls samples were air-dried at room temperature.

During the consolidation treatments different problems arose for the three tested consolidants. Soon after the beginning of consolidation procedure LNPs and BC precipitated on the bottom of the container. Therefore, to keep the nanoparticles suspended the impregnation baths were gently shaken on a magnetic stirrer for the rest of the test. Additionally, the wood blocks were periodically rotated in order to avoid nanoparticles deposition on their upper surface. In spite of all the precautions taken, at the end of the treatments a more or less thick layer of LNPs and BC was present on the wood (**Figures S1A,B**). Those nanoparticles were removed with a soft brush before freezing the blocks.

After 1 week of consolidation test, the CNCs impregnation bath turned into a gel (**Figure S1C**). A test carried out on a small aliquot of the gel showed that to regain a liquid bath the addition of the double volume water was necessary. This solution was not considered in order to avoid an excessive dilution of the consolidant. At the end of the treatment, the wood blocks were extracted from the gel and the residual CNCs on the wood surface were removed with a soft brush.

### Evaluation of the Efficacy of the Treatments

A first evaluation of the efficacy of the treatments was obtained by macroscopic observation of the samples. Color, shape, presence of openings, and consistency were observed.

The penetration of the nanoparticles inside the wood tissues was evaluated by examining gold sputter-coated sections of treated blocks (LH5, BCH5, and CNCH5) and freeze-dried control (FH2) with a Scanning Electron Microscope (Zeiss EVO 60) in secondary electrons mode. For each sample three sections were observed: external cross section, internal cross section, and internal radial section. The internal sections were obtained splitting the block by hand, without the use of blades.

#### Anti-Shrink Efficiency

In order to evaluate the dimensional variations of treated wood, all the six sides of treated blocks, freeze- and air-dried controls were scanned on high definition scanner (HP scanjet G3010), together with a measuring ruler, before the consolidation treatment, and after the drying procedure (wood equilibrated at 20◦C, 50% RH). Using an image processing software (ImageJ 1.52n) the cross section surface and the average height of each block were measured. The obtained values were used to calculate the blocks' volume (VW, volume of waterlogged blocks; VD, volume of treated blocks and controls after drying).

For each block, the percentage shrinkage, S, was calculated following the equation:

$$\mathcal{S}(\%) = \frac{V\_W - V\_D}{V\_W} \ast 100$$

The S values obtained for each treated sample were used to determine the anti-shrink efficiency (ASE) with respect to freeze-dried (ASEF) and air-dried (ASEA) controls following the equations:

$$\begin{aligned} ASE\_F(\%) &= \frac{S\_F - S\_T}{S\_F} \* 100 \text{ and } \\ ASE\_A(\%) &= \frac{S\_A - S\_T}{S\_A} \* 100 \end{aligned}$$

Where SF and SA are the shrinkages of freeze- and air-dried controls, respectively, and ST is the shrinkage of treated blocks.

The ASEC, expressed as the percentage of shrinkage that has been suppressed by freeze-drying treatment as compared to the shrinkage of air-dried controls, was calculated as follows:

$$ASE\_C\left(\%\right) = \frac{S\_A - S\_F}{S\_A} \ast 100$$

#### Equilibrium Moisture Content

To evaluate the effect of the different consolidation treatments with respect to moisture absorption the equilibrium moisture content (EMC) was determined. The test was performed on the treated wood blocks and freeze-dried controls not used for SEM observations. The obtained results were compared to sound wood EMC from literature (Giordano, 1981) and to values obtained for sound wood controls. The latter, four blocks of silver

fir and six blocks of holm oak, 2.0 × 2.0 × 0.5 cm in dimensions, were marked with a progressive number.

The samples were put in a climatic chamber at 20◦C and the RH was varied according to the sequence 10, 35, 45, 55, 65, 85% reaching in each step the EMC (Giordano, 1981). The EMC for the RH value of 100% was obtained by putting the wood blocks in a closed container in presence of liquid water. At the end of the RH cycle, the samples were completely dried in a oven at 103 ± 2 ◦C up to a constant weight.

#### RESULTS AND DISCUSSION

#### Bacterial Cellulose Nanoparticles

SEM observations of bacterial nanocellulose allowed for characterizing the consolidant. Most part of the observed nanoparticles was more or less spherical in shape (**Figure 1**), in some cases small, irregularly shaped particles were observed. The size distribution (**Figure 2**) showed that the nanoparticles could be divided into two dimensional classes with diameter ranges of 0.04–0.50µm and 3– 50µm. Most part of the BC particles had a diameter of ca 10 µm.

#### Wood Characterization

Results regarding wood species identification and physical analyses are reported in **Table 3**. The softwood samples were referred to stone pine (Pinus pinea L.), silver fir (Abies alba Mill.) and cypress (Cupressus sempervirens L.). Regarding the hardwood samples, the sample FTdis 9 was identified as elm (Ulmus sp.) while the blocks obtained from the wooden pier belonged to the evergreen oak group and can be probably attributed to holm oak (Quercus ilex L).

Micro-morphological analyses of wood thin sections allowed for observing the microbial decay of the tissue which was

mainly ascribed to erosion bacteria (**Figure S2A**), even though fungal hyphae and spores were observed in several sections (**Figure S2B**). The observations carried out in polarized light showed a severe loss of the birefringence typical of cellulose (**Figures S2C,D**), a sign that the crystalline structure of the polymer was almost completely lost due to microbial attack.

The physical parameters used to assess the decay showed different levels of degradation for the selected woods. The highest MWC values were recorded for the holm oak samples (616, 741, and 648% respectively). For the three poles the residual density ranged between 17 and 20%. According to the degradation classes established by De Jong (Grattan and Clarke, 1987) and by McConnachie et al. (2008) these samples can be considered as highly degraded.

The stone pine samples were the best preserved of the analyzed woods with MWC value of 254% and RDb of 61%. Cypress showed an intermediate level of degradation with an MWC of 331% and an RDb of 41%. According to De Jong these woods belong to the intermediate class of degradation.

Silver fir and elm samples deserve a special mention. Based on the MWC values (193 and 250%) these samples resulted as the best preserved of all analyzed wood. However, the fir RDb value (108%) and the fact that elm was much more degraded (spongy tissue, not resisting to the cut) with respect to wood with similar MWC indicated that the results could not be considered as reliable. The wood was characterized by the presence of several shipworm tunnels filled with sediments. During the cleaning operations carried out before the tests it was not possible to remove all the sediments from the samples, especially from the innermost part. It is known that the presence of sediments and/or mineral depositions inside archaeological waterlogged wood influences the RDb values (Schniewind, 1990), so the unreliable results obtained for the physical analyses must be attributed to this cause.

### Evaluation of Efficacy of Consolidation Treatments

Macroscopic observation of treated samples and comparison between consolidated and un-consolidated wood allowed for a first evaluation of the effect of the respective consolidation treatments.

In contrast to air-dried controls, all treated samples and freeze-dried controls apparently maintained the original shape and no dimensional differences were perceivable by naked eye between differently consolidated blocks and freeze-dried controls (**Figure 3** and **Figures S3**, **S4**). In few cases, of both consolidated and freeze-dried blocks, small pieces of wood had detached during consolidation or drying procedure and small openings were present on the samples' surfaces.

Wood color and aspect were widely affected by consolidation treatments. LNPs treated samples resulted in dark brown color and a fine brown powder was present on the sample surface, even after freeze-drying. BC consolidated blocks were whitish or yellowish in color. When broken for SEM observation, it was evident that the color change had occurred throughout the block thickness. Finally, CNCs treated samples showed a color similar to the controls, but a thin, colorless film was present on almost all the surface of the blocks. That film was easily removable by touching the wood.

SEM observations of the treated wood samples allowed for observing how the nanoparticles had penetrated inside the wood tissues. **Figure 4** reports the images of external and internal cross sections and radial sections of all the observed blocks. The analysis of a control un-consolidated block showed that in the cross sections, both external and internal, cell lumens were empty and in several cases the secondary cell walls appeared mostly detached from the middle lamella (ML) with only thin filaments connecting the cell wall to the ML. In radial section the vessel and fiber walls appeared smooth and pits were well visible.