7232019 MampM-2Analytical and Microbiological Characterization of Paper Samples Exhibiting Foxing Stains1-2015-63

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Microsc Microanal 21 63ndash77 2015doi101017S143192761500001X

copy MICROSCOPY SOCIETYOF AMERICA 2015

Analytical and Microbiological Characterizationof Paper Samples Exhibiting Foxing Stains

Margarida Nunes1

Caacutetia Relvas1

Francisca Figueira2

Joana Campelo2

Antoacutenio Candeias12

Ana T Caldeira1 and Teresa Ferreira1

1Departamento de Quiacutemica Escola de Ciecircncia e Tecnologia Centro de Quiacutemica de Eacutevora amp Laboratoacuterio HERCULESUniversidade de Eacutevora Largo Marquecircs de Marialva 8 7000-809 Eacutevora Portugal 2Laboratoacuterio Joseacute de Figueiredo Direccedilatildeo Geral do Patrimoacutenio Cultural Rua das Janelas Verdes 1249-018 Lisboa Portugal

Abstract This work comprises the use of a multi-analytical approach combined with microbiological studies tocharacterize six paper samples containing foxing stains from the 20th century regarding their cellulose matrix1047297llers and sizing materials and to evaluate possible paper degradation that might have occurred during the foxing stains Photography under different illuminations and optical microscopy were used for morphologicalcharacterization of the paper samples and foxing stains Scanning electron microscopy coupled energy dispersivespectroscopy (SEM-EDS) was of particular importance for de1047297ning the presence of 1047297ber disorder and disruptionon the surface of some of the stains and localized accumulations of mineral-like particles on the surface of others

SEM-EDS attenuated total re1047298ection Fourier transform infrared spectroscopy (ATR-FT-IR) and energy dispersive X-ray 1047298uorescence (EDXRF) were used for the identi1047297cation of mineral 1047297llers whereas sizing agentswere analyzed using ATR-FT-IR EDXRF results showed that no differences within the standard deviation werefound in iron and copper contents between the foxed and unfoxed areas Fungi belonging to the genus Penicilliumspp were found in all the paper samples Unfoxed areas presented lower contamination than the foxed areas

Key words foxing stains unfoxed area paper degradation SEM-EDS ATR-FT-IR

INTRODUCTION

Paper has been used for centuries to record importanthistorical and cultural heritage events all over the world and

is present in thousands of private and public librariesmuseums and archives (Goltz et al 2010) Historical paperdiffers in many ways from contemporary paper (Manso amp Carvalho 2009) Before 1840 the raw materials used toproduce paper were cellulose 1047297bers obtained from rags andropes (Proniewicz et al 2002) With the increasing scarcity of rag 1047297bers wood gradually became the main source of cellulose In the hand papermaking process gelatin a very stable and impermeable 1047297lm-forming external sizing agentwas used As a consequence of industrialized machinepapermaking gelatin was replaced by the acidic rosinaluminum internal sizing which induced the formation of sulfuric acid promoting acid hydrolysis in paper (Erhardt amp

Tumosa 2005) In the 1980s this acidic sizing process wasreplaced by alkaline neutral sizing process using alkyl ketenedimer (AKD) (Espy 1990 Pinzari et al 2006 2010 Area amp Cheradame 2011 Song et al 2011)

Fillers were used by the end of the 18th century andchina clay (kaolin) was the most common 1047297ller used (Beazley1991) because of its good compatibility with the acid sizing process It was used until the second half of the 20th centurybeing replaced by alkaline calcium carbonate (CaCO3)

which is compatible with the neutral sizing of AKD being therefore denominated as an alkaline sizing process

Staining of documents may occur naturally as a result of paper aging and its constituents but it can be accelerated by

poor storage conditions or chemical effects (Castro et al 2008Goltz et al 2010 Abdel-Maksoud 2011) Foxing stains occurin the form of small isolated patches of discoloration scatteredon a leaf of paper which are typically rust brown or yellowishtoned they are usually limited in size with sharp or jaggededges often punctiform and sometimes circular (Derow amp Owen 1992 Montemartini Corte et al 2003 Manente et al2012) The study and reasoning behind the development of thistype of discoloration have been a subject of research for severalauthors (Ligterink et al 1991 Brandt et al 2009 Figueira et al2009 Manente et al 2012) Some have associated its occur-rence with biological attack others consider that foxing has achemical origin whereas others admit that both factors areresponsible for this type of degradation (Bicchieri et al 2002Buzio et al 2004 Choi 2007 Manso et al 2009 Zotti et al2011 Manente et al 2012) A contribution to distinguish theldquochemicalrdquo origin from the ldquobiologicalrdquo origin of foxing wasgiven by Piantanida et al (2005) and Coluzza et al (2008) whoused atomic force microscopy and showed that the topography of the two types of foxing is different

Choi (2007) in his literature review on foxing mentionsresearch activity since 1930s however as Bicchieri et al(2002) refer there are still no conclusive results on foxing formation mechanisms in spite of more intensive researchafter 1980 Although Bicchieri et al (2002) state that specialCorresponding author tasfuevorapt

Received April 13 2014 accepted January 6 2015

7232019 MampM-2Analytical and Microbiological Characterization of Paper Samples Exhibiting Foxing Stains1-2015-63

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reference must be made to iron and copper ions which areable to initiate free-radical chain reactions Florian (1996)and Florian amp Manning (2000) suggest that the autoxidationof lipids from conidia may be a possible cause for dis-coloration of the fungal material on paper 1047297bers

Ligterink et al (1991) brought a new perspective to thefoxing phenomenon by attributing its occurrence to a differ-

ence of humidity sorbency in the paper substrate which canoriginate from paper permeability de1047297cient paper sizing andirregularities in paper including folds tears and impurities Asa consequence of this 1047297nding Graaff (1994) and Eusman(1995) separately described the phenomenon of browning produced at the wetndashdry interface Independent from the various causes for discoloration development oxidation hasbeen cited as a common mechanism in all types of browning (Eusman 1995 Manente et al 2012 Jeong et al 2014)

This resulted in greater permeability of paper that may beresponsible for the large amount of foxing spots (Grijn et al2002 Figueira et al 2009) due to the introduction of mineralloadings to the 1047297ber mesh at the end of the 18th century (Beazley 1991) and to the alteration of the sizing processGelatin sizing was replaced by rosin and aluminum sulfate(papermakerrsquos alum) in the 19th century (Bruumlckle 1993 Can-non 2011) The presence and mobility of iron ions provenantfrom the humid section of the drying cylinders is also seen as afont of contaminants (Daniels amp Meeks 1994 Graaff 1994)

To the best of our knowledge only few studies (Mansoet al 2008 2011 Zotti et al 2011) were carried out to char-acterize papers of the 20th century The purpose of this study was to deepen our knowledge on foxing stains its chemical andbiological nature and morphological aspects associated with itparticularly in papers of the 20th century In order to achieve

this a multi-analytical nondestructive approach for the mor-phological and chemical characterization of foxed and unfoxedareas of unimportant paper samples was used in conjunctionwith a microbiological study We report the analysis of foxedand unfoxed areas by scanning electron microscopy coupledwith energy dispersive X-ray spectroscopy (SEM-EDS)attenuated total re1047298ection Fourier transform infrared spectro-scopy (ATR-FT-IR) and energy dispersive X-ray 1047298uorescence(EDXRF) Photography under standard raking and UVillumination and optical microscopy (OM) were also valuabletools for the characterization of foxing stains Microbiologicalstudies were carried out in order to evaluate the existence of

biotic attack in foxing stains

MATERIALS AND METHODS

Paper Sample Description

A set of six different foxed paper samples from the 20thcentury were obtained from the paper conservation studio of the Joseacute de Figueiredo Conservation Laboratory in LisbonThe paper samples used in this study had no artistic or his-torical value and the set was composed mainly of differenttypes of cardboard wood paper and inexpensive heavy drawing paper labeled P1 to P3 and P6 to P8 (Table 1)

Papers were selected by visual observation of the dif-ferent colors and morphologies of the foxing stains Based onthese parameters papers presenting only one typology of foxing stain were selected A minimum of ten points on eachpaper both on the stains and on the neighboring unfoxedpaper surface was subjected to chemical and morphologicalanalysis Samples from each paper containing foxing stainswere used in the microbiology studies Each sample wasproperly identi1047297ed and stored protected from light at con-stant room temperature

Photographic Imaging and OM

Photographic imaging and OM are in general the 1047297rst stepsused in evaluating color and morphology of the foxing stainsVisual observation and photographic register were obtainedusing a Nikon Coolpix 8700 (Tokyo Japan) camera placedon a column stand Images were captured in macro modeusing the same focal distance and 1047297 lm sensitivity (ISO 100)

under different illuminations (standard raking and trans-mitted light) UV light images were captured under the sameconditions as above except for 1047297lm sensitivity (ISO 400) using an UV Waldmann W portable lamp with two TL4 W08 F4T5BLB Philips lamps (Eindhoven The Netherlands)

Stains were also observed and registered with an OMusing a Leica MZ6 microscope lens (maximum numericalaperture of 0075 with 10times achromatic objective) (LeicaMicrosystems Wetzlar Germany) with raking light from aLeica CLS times 100 light spot (Leica Microsystems) For com-paring the stain dimensions and in order to include most of the stains encountered a magni1047297cation of 63times was chosenA magni1047297cation of 40times was used to evaluate the stains and

paper surface topographies The images were captured using an incorporated Leica DC200 camera (Leica Microsystems)

SEM-EDS

Morphological and topographical aspects of the papersamples on the foxed and unfoxed areas were evaluated by SEM SEM-EDS analysis enabled major element point ana-lysis and provided elemental composition mapping andsemi-quantitative evaluations This technique was particularly important for paper characterizationmdashnamely the identi1047297ca-tion of the 1047297llers used in the paper production

Table 1 Description of 20th Century Paper Samples Used inThis Study

Sample Description

P1 Window matboard of a painting by J Glamma StorbelleP2 Matboard backing of ldquoD Ana Mayorrdquo portraitP3 Inexpensive heavy drawing paper

P6 Matboard backing of the watercolor ldquoJoatildeo de DeusrdquoP7 Matboard with three windowsP8 Mechanical wood matboard backing

64 Margarida Nunes et al

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Small pieces of all the samples containing foxed andunfoxed areas were cut and placed face-up on two-sided sticky tapes on aluminum SEM specimen holders The study wascarried out on a variable pressure Hitachi S-3700N SEM(Hitachi High-Technologies Europe Krefeld North Rhine-Westphalia Germany) coupled with a Bruumlker XFlash 5010SDD EDX spectrometer (Billerica Massachusetts) A pressure

of 20 Pa in the chamber was used for the analyses Accelerating voltages for chemical analyses (20 kV) and imaging in thebackscattered mode (15 kV or 10 kV) were used

ATR-FT-IR

The ATR mode allowed a nondestructive analysis of the papersamples Infrared analysis was carried out at room temperatureand ambient humidity with a Bruumlker Alpha spectrometer(Billerica Massachusetts) coupled with a single-re1047298ection dia-mond ATR module All spectra were acquired in the absor-bance mode in the range from 4000 to 375 cmminus1 from a totalof 128 scans at 4 cmminus 1 resolution Spectra were recorded and

analyzed using OPUSMentor software (version 65)Before sample analysis background measurement was

performed in order to reduce the effect of atmospheric car-bon dioxide and water vapor Paper samples were positionedon the surface of the sample holder in direct contact with thediamond crystal and were subjected to some pressure Theidenti1047297cation of paper components and detection of microbialpresence were carried out by comparing the main features of the obtained spectra with published data (Derrick et al 1999Proniewicz et al 2001 2002 Zotti et al 2008 2011 Brandtet al 2009 Manente et al 2012) or with the ATR-FT-IR database (ATR-FTIR Library COMPLETE 2009) The absor-

bance spectra were normalized for comparison purposes

EDXRF

X-ray 1047298uorescence spectrometry is a nondestructive techni-que which makes it completely appropriate for the elementalanalysis of items of cultural heritage paper in particularSamples were analyzed directly without any preparationQualitative data from paper artifacts such as XRF peak height or intensity can be compared and used to identify differences in relative concentrations of media between artifactsor in areas in the same artifact for samples with similar com-position and thickness XRF analysis of paper is dif 1047297cult due to

its minimal thickness limited density and low concentrationsof the elements of interest (Barrett et al 2012)The analyses were carried out with a Bruumlker TRACER III-

SD (Billerica Massachusetts) handheld portable spectrometerwith a rhodium tube The S1 TRACER was used in a stationarybench top con1047297guration which is more adequate for paperanalysis with a yellow 1047297lter (3048 μm Al+254 μm Ti) thatremoves low-energy X-rays coming from the tube Thisreduced background levels and therefore improved the detec-tion limits (Barrett et al 2012) Use of the yellow 1047297lter wasadequate for analysis of metals (Ti to Ag and W to Bi) but therewas little sensitivity to elements below Ca Operating conditionsof 40 kV 125 μA and an acquisition time of 300 s were used for

all the analyses The characteristic radiation emitted by ele-ments present in the samples was collected by a silicon driftdetector that permitted an energy resolution of 14968 eV forMn Kα radiation at a count rate of 100 kcps Three analyses inthe foxed areas and in the unfoxed areas were carried out foreach sample An XRF cup containing water and covered with aProleenereg XRF 1047297lm was positioned behind the paper samples

being analyzed and together these were positioned against thenose of the instrument in order to supply a consistent lightpressure that kept samples in their 1047297nal position to be analyzedand diminished the distance between the sample and thedetector A beam spot of (3times 4) mm2 was used The equipmentalso had a camera that allowed visualization of the analyzedarea and captured the image and spot of analysis S1 PXRFsoftware (v 3830) was used to record the spectra and ARTAX software (v 5300) was used for spectra evaluation Net countsof the elements were also normalized to the Compton scatteredpeak by dividing the net counts for each analyzed elementobtained from spectra deconvolution by the net counts of theCompton scattered peak from the same spectrum (obtained by evaluation of the region of interest)

Microbiological Isolation and Characterization

Biological assays were carried out under aseptic conditionsSamples from foxed and unfoxed areas (~1 times 1 cm2) were col-lected and placed in sterilized test tubes in a suspension of transport MRD medium (Maximum Recovery Diluent Merck)(Darmstadt Bundesland Germany) Samples were preserved at4ordmC before the analytical procedure Samples were diluted in1 mL of solution and shaken mechanically for 1 h The sus-pensions were inoculated in selective media such as nutrientagar for bacterial isolation yeast extract peptone dextrose agar

for yeast development and malt extract agar and Cooke RoseBengal to isolate 1047297lamentous fungi Cultures were incubated at30degC for 24ndash48 h for the development of bacterial cultures andat 28degCfor 4ndash5 days for fungal growth To detect slow microbialgrowth plates were incubated at the same temperature for alonger time period All different colonies were picked to obtainpure cultures that were stored at 4ordmC and were periodically pinched to maintain active cultures The microbial isolatesobtained were characterized based on the observation of mac-roscopic features of the colonies (texture and color) and on themicro-morphology of the hyphae and reproductive structures(in the case of conidiaspore isolates) Preparations made from

fungal isolates were stained with methylene blue observed with20times (numerical aperture 050) and 50times (numerical aperture080) objectives with a Leica DM 2500P (Leica Microsystems)OM and were digitally recorded with a Leica DFC290HDcamera (Leica Microsystems) Fungal strains were identi1047297edonly at the genus level

R ESULTS AND D ISCUSSION

Contemporary paper is a complex matrix that may containcellulose signi1047297cant amounts of hemicellulose and ligninand non1047297brous components such as coloring agents 1047297llerscoating sizings among others

Analytical and Microbiological Characterization of Paper Samples 65

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Photographic Imaging and OM ObservationsPhotographic images (Table 2) permitted recording of thenatural tones of the paper samples and their respectivestains

The use of raking light enhanced imaging of the papertopography and revealed details of surface texture and planardistortions Photography obtained under these conditionsshowed that samples P1 and P7 had a mat and roughersurface than P2 and P3 whereas sample P6 exhibited a

smooth glossy surface with oxidized brown margins and P8exhibited a mat rough surface

Observation of photographic images under UV illumi-nation is a common method used in conservation practice toassess changes in the 1047298uorescence of paper (Manso et al2009 Michaelsen et al 2009) Florian amp Manning (2000)reported that fungal spots usually have a yellow 1047298uorescenceunder UV light which can be due to the presence of aromaticamino acids tyrosine tryptophan or phenylalanine in the

Table 2 Photographic Images of Paper Samples Under Different Illuminations

Sample Standard Light Raking Light UV Radiation

P1

P2

P3

P6

P7

P8

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proteins of the fungal structures however in the foxing spots it could also be due to degradation products fromcellulose oxidation According to Junior amp Ligterink (2001)1047298uorescence appears before discoloration and at a later stageand when discoloration has developed into dark-brownstains 1047298uorescence can no longer be observed Manso et al(2009) reported that 1047298uorescence can occur on nonstained

areas that are usually larger than the visibly stained areaswhich seems to be the case on sample P3 This was the only paper that showed 1047298uorescence

For a more detailed and an ampli1047297ed view of thedegraded areas by foxing OM was used (Table 3)

Manso et al (2009) analyzed 14 Portuguese drawingsfrom the 19th century and reported that from a

Table 3 Optical Microscopy Observations with Raking Light

Magni1047297cation

Sample 63times 40times Tone Foxing stains description

Cream toned

paper

Off-white

toned paper

Cream toned

paper

Cream toned

paper with

brown margins

Off-white

paper

Tanned cream

paper with

oxidized brown

surface middle

Not very intensive tonedfoxing stains of irregular

size and shape Diffuse

outer limits

Very small dark-brown

foxing stains of uniform

size and shape Sharp outer

limits

Not very intensive toned

foxing stains of irregular

size and shape Diffuse

outer limits

Very small dark-brown

nuclei surrounded by less-

intensive stain of irregular

size and shape Diffuse

outer limits

Intense sharp edged

mediumlarge brown

foxing Sharp outer

limits

Minute foxing

P1

P2

P3

P6

P7

P8

Analytical and Microbiological Characterization of Paper Samples 67

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topographical point of view there were no differencesbetween the foxed and unfoxed areas We also did notobserve any signi1047297cant alteration in the 1047297ber surface dis-position at these magni1047297cations

SEM-EDS Analyses

Analysis by SEM-EDS permitted examination of the mor-phology of the different paper samples and foxing stains and

evaluation of the elemental composition of the inorganicmaterials used as 1047297llers in paper production Investigation of the paper samples in the unfoxed areas showed no sub-stantial degradation of the cellulose 1047297bers The surfaces werestructurally organized with no broken 1047297bers Wood cellulose1047297bers of various dimensions were observed in detail inbackscattered electron images (Table 4) Moreover crystalsand aggregates (1047297ller materials and impurities) of heavierelements (higher atomic number) were observed as indicated

Table 4 Scanning Electron Microscopy Micrographs of Unfoxed and Foxed Areas in Paper Samples P1ndashP8

Sample Unfoxed Area Foxed Area

P 1

P 2

P 3

P6

P7

P8

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by particles of higher intensity in the backscattered electronimages of all samples These mineral-like deposits were quitenumerous in papers P6 P7 and P8

Images obtained from the foxed areas (Table 4) con-trasted with those from the unfoxed areas In fact twotypologies of stains were detected In samples P1 P2 and P31047297ber disruption and structural disorganization were

observed in the foxed areas whereas samples P6 P7 and P8only exhibited localized accumulations of particlesContrary to the results presented in other works (Flor-

ian amp Manning 2000 Rakotonirainy et al 2007 Manenteet al 2012) SEM analyses revealed minimal evidence of microbiological contamination in the foxed areas althoughfungal species were present in the foxed areas (see Micro-biological Presence On Paper Samples section) Similar1047297ndings were reported by Peters (2000)

EDS analysis was used to obtain information on theelemental composition of the papers In general modernpapers (20th century) contained the widest variety of 1047297llersin their composition (Manso et al 2011) In fact several1047297llers were detected for each paper although one or two weremore extensively used

The most frequently used calcium-based 1047297ller was calcite(CaCO3) being gypsum (CaSO4) also often used (Beazley 1991Manso et al 2011 Manente et al 2012) The detection of par-ticles rich in Ca in papers P1 and P7 suggests the use of a CaCO3

1047297ller EDS mapping of a foxed area in sample P7 is presented inFigure 1 Accumulation of CaCO3 particles probably due to poordistribution of the 1047297ller during paper production was observedin the foxed areas of this sample (see Table 4)

Particles rich in Al and Si were also detected in thissample Kaolin 1047297ller a hydrated aluminum silicate (Al2O3

SiO22H2O) may be found in most grades of 19th-century printing papers (Beazley 1991) The presence of Al and Siwas detected in all the paper samples suggesting the use of aluminum silicates as 1047297llers

EDS analysis (Fig 2a) of unfoxed areas in paper P6revealed the presence of particles where the molar ratio CaSwas ca 11 which is in accordance with the use of calciumsulfate (CaSO4)

EDS mapping of Ca and S insample P8(Fig 3) showed thedistribution of these two elements Accumulation of CaSO4

particles was detected in the foxed areas of samples P6 and P8probably like in sample P7 due to poor distribution of the 1047297ller

during paper production CaSO4

was also present in papers P1and P2 but no accumulation was present in the foxed areasTalc is hydrated magnesium silicate that is used to

enhance opacity and improve durability of paper (Wilson2006) Particles rich in magnesium and silicon were detectedin large amounts in samples P2 P3 and P6 An EDS spectrumof sample P2 is shown in Figure 2b

Particles rich in Ti were observed in sample P1 Tita-nium dioxide (TiO2) has a very good opacifying power and itcan also be used as a pigment Titanium was detected in largeamounts in this sample revealing that TiO2 was used as a1047297ller (Fig 2c) High levels of Ti were also discovered inpapers from 1919 and 1941 by Manso et al (2008)

Figure 1 Elemental (Al Si and Ca) mapping of sample P7 (foxedarea) BSE backscattered electron

Analytical and Microbiological Characterization of Paper Samples 69

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by particles of higher intensity in the backscattered electronimages of all samples These mineral-like deposits were quitenumerous in papers P6 P7 and P8

Images obtained from the foxed areas (Table 4) con-trasted with those from the unfoxed areas In fact twotypologies of stains were detected In samples P1 P2 and P31047297ber disruption and structural disorganization were

observed in the foxed areas whereas samples P6 P7 and P8only exhibited localized accumulations of particlesContrary to the results presented in other works (Flor-

ian amp Manning 2000 Rakotonirainy et al 2007 Manenteet al 2012) SEM analyses revealed minimal evidence of microbiological contamination in the foxed areas althoughfungal species were present in the foxed areas (see Micro-biological Presence On Paper Samples section) Similar1047297ndings were reported by Peters (2000)

EDS analysis was used to obtain information on theelemental composition of the papers In general modernpapers (20th century) contained the widest variety of 1047297llersin their composition (Manso et al 2011) In fact several1047297llers were detected for each paper although one or two weremore extensively used

The most frequently used calcium-based 1047297ller was calcite(CaCO3) being gypsum (CaSO4) also often used (Beazley 1991Manso et al 2011 Manente et al 2012) The detection of par-ticles rich in Ca in papers P1 and P7 suggests the use of a CaCO3

1047297ller EDS mapping of a foxed area in sample P7 is presented inFigure 1 Accumulation of CaCO3 particles probably due to poordistribution of the 1047297ller during paper production was observedin the foxed areas of this sample (see Table 4)

Particles rich in Al and Si were also detected in thissample Kaolin 1047297ller a hydrated aluminum silicate (Al2O3

SiO22H2O) may be found in most grades of 19th-century printing papers (Beazley 1991) The presence of Al and Siwas detected in all the paper samples suggesting the use of aluminum silicates as 1047297llers

EDS analysis (Fig 2a) of unfoxed areas in paper P6revealed the presence of particles where the molar ratio CaSwas ca 11 which is in accordance with the use of calciumsulfate (CaSO4)

EDS mapping of Ca and S insample P8(Fig 3) showed thedistribution of these two elements Accumulation of CaSO4

particles was detected in the foxed areas of samples P6 and P8probably like in sample P7 due to poor distribution of the 1047297ller

during paper production CaSO4

was also present in papers P1and P2 but no accumulation was present in the foxed areasTalc is hydrated magnesium silicate that is used to

enhance opacity and improve durability of paper (Wilson2006) Particles rich in magnesium and silicon were detectedin large amounts in samples P2 P3 and P6 An EDS spectrumof sample P2 is shown in Figure 2b

Particles rich in Ti were observed in sample P1 Tita-nium dioxide (TiO2) has a very good opacifying power and itcan also be used as a pigment Titanium was detected in largeamounts in this sample revealing that TiO2 was used as a1047297ller (Fig 2c) High levels of Ti were also discovered inpapers from 1919 and 1941 by Manso et al (2008)

Figure 1 Elemental (Al Si and Ca) mapping of sample P7 (foxedarea) BSE backscattered electron

Analytical and Microbiological Characterization of Paper Samples 69

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In sample P3 barium and sulfur were detected in molar ratioca BaS 11 Their presence reveals that barium sulfate wasused in the production of this paper (Fig 2d) Barium sulfateis usually added to paper as a coating in order to turn itglossy and was already detected in a magazine sample fromthe 20th century and in papers from 1919 and 1941 (Mansoet al 2008 2011)

Iron was present in samples P2 P3 and P6 Its presencecan be associated with clay 1047297llers or the papermaking

equipment Zinc was also found in sample P1 and itsoccurrence could be associated either with contaminationfrom the papermaking process or with white zinc oxide a1047297llerpigment more frequently used in the early 20th century (Manso et al 2011)

ATR-FT-IR Study

ATR-FT-IR analysis was carried out for the evaluation of 1047297llers and sizing materials In addition it was used to com-pare unfoxed paper surfaces with foxed areas It was also asuitable technique for evaluating the presence of fungi

Composition of Paper Figure 4 shows the spectra corresponding to paper samplesP1 to P8 obtained for unfoxed and foxed areas The mostimportant peak wave numbers and their interpretation areshown in Table 5

Cellulose peaks in all spectra were well recognized espe-cially in the 1047297ngerprint region at the range of 850ndash1500 cmminus 1

(Manente et al 2012) The vibrational pattern of cellulose is very complex in this range where stretching and deformationmodes of various groups couple The range between 900 and1200 cmminus 1 covers the CndashO a n d CndashC stretching anti-symmetric in-phase ring stretching anti-symmetric bridge

CndashOndashC as well as CndashCndashH a n d OndashCndashH deformation vibrations CndashOndashH in-plane bendings CndashCndashH OndashCndashH andCndashCndashH deformation stretching as well as HndashCndashH bending and wagging are observed in the 1200ndash1500 cmminus 1 range Inthe 1650 cmminus 1 region HndashOndashH bending vibrations of adsor-bed water molecules is expected (Proniewicz et al 2002) Abroad band peak at about 3300 cmminus 1 corresponds to the OndashHstretching mode of cellulosewater molecules whereas a peak in the range 2800ndash3000 cmminus 1 corresponds to the CndashH

stretching vibrations of cellulose (Brandt et al 2009 Manenteet al 2012)

Characteristic peaks of lignin (1669 1508 and 808cm minus 1) and broad absorbance in the 1800ndash1550 cm minus 1

region (Derkacheva amp Sukhov 2008 Manente et al 2012)were observed only in paper P8 (Fig 4f unfoxed area) Thissuggests that this paper is composed of mechanical woodpulp Mechanical pulping results in 1047297ber degradation andleaves a high level of residual lignin The ldquoyellowing rdquo thatoccurs upon exposure of these products to light arises fromphotochemical changes in the lignin component of the paper(Weinstock et al 1993) The relatively high band of lignin at

1508 cm

minus 1

in spectra of sample P8 is in accordance with thefact that the analysis was carried out on the margins of thepaper which were not exposed to daylight as can beobserved in Table 2 (Zotti et al 2008)

It also has to be emphasized that in addition to cellu-lose some hemicellulose bands are expectedmdashnamely thecharacteristic band at ca 815 cm minus 1 (Proniewicz et al 2002)In fact small amounts of hemicellulose were present insamples P2 P3 and P6 (Figs 4bndash4d unfoxed area)

The bands at 1425 and 870 cmminus 1 (Zotti et al 2008 2011Manente et al 2012) were due to CaCO3 The smaller bands atca 2500 and 1800 cmminus 1 (Boumlke et al 2004 ATR-FTIR Library COMPLETE 2009) also contributed to the detection of this

Figure 2 Energy dispersive X-ray spectra of samples (a ) P6 (b) P2 (c) P1 and (d) P3 (unfoxed areas)

70 Margarida Nunes et al

7232019 MampM-2Analytical and Microbiological Characterization of Paper Samples Exhibiting Foxing Stains1-2015-63

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compound These bands were noticed in samples P1 P2 P6P7 and P8 although in P7 (Fig 4e unfoxed area) the bandsincreased drastically in intensity in accordance with theextended use of this 1047297ller already detected by SEM-EDS

The shoulder at 3550ndash3400 cmminus1 and the increased smallpeaks at 1620 661 and 595 cmminus 1 were due to the presence of CaSO4 (Derrick et al 1999 Zotti et al 2008 2011 Manenteet al 2012) The band at 1140ndash1080 cmminus 1 (Derrick et al1999) due to asymmetric SO4

2minus stretching is superimposed

with the 1047297ngerprint region of cellulose although variations inintensity may provide some information about the presence of CaSO4 Paper P7 (Fig 4e unfoxed area) presents a less-intenseband ca 1110 cmminus 1 suggesting that CaSO4 is not present inthis paper whereas it is present in the rest of the samples Smallbands ca 524 and 470 cmminus1 due to kaolin (Proniewicz et al2002) were observed in all the samples with the most intense

bands detected in P7 The presence of aluminum silicate wasalready observed by EDS analysis (Fig 3)Spectra of samples P1 and P7 obtained from unfoxed areas

present a shoulder where two peaks can be assigned at around1640 and 1560 cmminus 1 These peaks are attributable to amide Iand amide II of a proteinaceous material (Derrick et al 1999Manente et al 2012) used in sizing On the other hand sampleP3 presents a shoulder at ca 1720 cmminus 1 (Derrick et al 1999Manente et al 2012) characteristic of resinaceous materials asrosin The shoulder at ca 1720 cmminus1 is not so easily detected insample P2 nevertheless a resinaceous sizing was also probably used for this sample For sample P8 due to the broad absor-bance in the 1800ndash1550 cmminus 1 region the FT-IR spectrumdoes not allow clear-cut identi1047297cation of the sizing materialIdenti1047297cation of the sizing material was also not possible forsample P6 The amide I band of a protein sizing (about 1650cmminus1) is visible whereas that of amide II (about 1545 cmminus1) ismasked by a linear decrease of absorbance that makes it dif 1047297cultto obtain a reliable deconvolution of spectral data

Characterization of Foxing StainsAccording to Zotti et al (2011) fungi show characteristicinfrared absorbance of OndashH groups and absorbed water(3700ndash3000 cmminus 1 and about 1635 cmminus 1) CndashH groups(about 2900 cmminus 1) polypeptide bonds (amide I at about1635 cmminus

1 and amide II at about 1540 cmminus

1) and poly-saccharide groups (about 1035 cm minus 1) The broad plateaubetween 1500 and 1200 cm minus 1 composed of several over-lapping bands is particularly interesting as it is typical of fungal agents and helps in differentiating the FT-IR spectraof paper subjected to biotic attack

Analyses of foxing stains on the six paper samplesrevealed the presence of fungi in all the foxed areas ( Table 5gray rows) con1047297rming biotic attack Band alterations in theregions assigned by Zotti et al (2011) were observed forpapers P3 and P7 (Figs 4c 4e foxed area) and minutemodi1047297cations of band shape and intensity were detected for

sample P2 (Fig 4b foxed area) In addition 1047297ber disorderand disruption were observed by SEM analyses for samplesP1 to P3 on foxing stains Being so modi1047297cations in intensity and shape of bands assigned to the vibration of OndashH andCndashH groups and to the 1047297ngerprint region of cellulose werealso due to morphological alteration of the surface

EDXRF

EDXRF analysis is a nondestructive technique sensitive toa wide range of elements It was therefore used to obtaininformation on elemental composition of the papersand foxing stains (Bicchieri et al 2002 Manso et al 2011)

Figure 3 Elemental (Ca and S) mapping of sample P8 (foxedarea) BSE backscattered electron

Analytical and Microbiological Characterization of Paper Samples 71

7232019 MampM-2Analytical and Microbiological Characterization of Paper Samples Exhibiting Foxing Stains1-2015-63

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By pointing the beam to different areas of the samples it waspossible to compare EDXRF spectra obtained from the fox-ing stains and from the unfoxed areas

Elements measured were Ti Cr Mn Fe Ni Cu Zn andPb Calcium was not considered because under the experi-mental conditions used there was little sensitivity to thiselement

Fluorescence counts for the detected elements dividedby the net counts of the Compton scattered peak obtained

from the same spectrum and multiplied by 10000 withassociated standard deviation are reported in Table 6 foreach analyzed sample

Iron and copper were found in all samples and could beassociated with the papermaking process Nevertheless theamounts in sample P1 are considerably higher comparedwith the rest of the samples The same observations werecarried out for lead zinc and chromium These results sug-gest that P1 was submitted to considerable contaminationduring the papermaking process The occurrence of zinc andiron can also be related to the presence of white zinc oxide apigment that was frequently used in the beginning of the 20thcentury (Manso et al 2011) and hematite an iron pigment

Titanium was present in samples P1 and P7 in accordance withthe results obtained by SEM-EDS for sample P1

When comparing the iron and copper contents for theunfoxed and the foxed areas no differences were observedwithin the standard deviation for all the samples A slightincrease in the zinc level within the standard deviation wasobserved in the foxed areas of samples P6 and P8 Spectra forsample P8 are presented in Figure 5

Manso et al (2009) analyzed foxing stains in drawings

from the 18th and 19th centuries and obtained similar resultsand found no differences between foxed and unfoxed areasof their samples for Al Si P S Mn Fe and Cu In factconsidering the reasoning behind the chemical nature of foxing and the expected differences in Fe and Cu contents inthe foxing stains it is surprising that for both studies nodifferences were observed

Microbiological Presence on Paper Samples

Viable fungi and bacteria were isolated from the six papersamples from foxed and unfoxed areas as described in theMaterials and Methods section The growth of bacterial

Figure 4 Attenuated total re1047298ection Fourier transform infrared spectra of unfoxed (black line) and foxed areas (gray

line) of paper samples (a ) P1 (b) P2 (c) P3 (d) P6 (e) P7 and (f ) P8

72 Margarida Nunes et al

7232019 MampM-2Analytical and Microbiological Characterization of Paper Samples Exhibiting Foxing Stains1-2015-63

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T a b l e 5

P e a k W a v e N u m b e r s ( c m minus

1

)

T e n t a t i v e A s s i g n m e n t a n d I n t e r p r e t a t i o

n o f A T R - F T - I R S p e c t r a o f S a m p l e s P 1 ndash P 8

W a v e N u m b

e r ( c m minus

1 )

P 1

P 2

P 3

P 6

P 7

P 8

T e n t a t i v e A s s i g n m e n t

I n t e r p r e t a t i o n

3 3 3 5

3 3 3 3

3 3 3 4

3 3 3 3

3 3 2 1

3 3 3 0

O ndash H s t r e t c h i n g

H y d r o x y l g r o u p o f c e

l l u l o s e w a t e r i n c e l l u l o s e

2 8 9 9

2 8 9 7

2 8 9 7

2 8 9 5

2 8 9 7

2 8 9 6

C ndash H s t r e t c h i n g

A l i p h a t i c h y d r o c a r b o n s

1 7 1 6

1 7 2 0

C = O s t r e t c h i n g

R e s i n a c e o u s s i z i n g

1 6 6 9

C = O s t r e t c h i n g

L i g n i n

1 6 3 6

1 6 3 6

1 6 1 8

1 6 3 6

1 6 4 7

O ndash H b e n d i n g

C a l c i u m s u l f a t e

1 6 4 6

1 6 4 7

C = O s t r e t c h i n g

A m i d e I ( p r o t e i n a c e o

u s s i z i n g )

1 6 5 1

1 6 5 3

1 6 5 3

1 6 4 7

1 6 5 1

1 6 2 4

C = O s t r e t c h i n g

A m i d e I ( p r e s e n c e o f

f u n g i )

1 5 5 8

1 5 5 8

1 5 5 8

1 5 5 9

1 5 5 8

1 5 5 9

C ndash N s t r e t c h i n g

A m i d e I I ( p r e s e n c e o f f u n g i )

1 5 5 8

1 5 6 0

C ndash N ndash H b e n d i n g

A m i d e I I ( p r o t e i n a c e o u s s i z i n g )

1 5 0 8

A r y l r i n g a s y m m e t r i c s t r e t c h i n g

L i g n i n

1 4 5 6

1 4 4 7

1 4 5 6

1 4 4 7

1 4 5 4

1 4 4 7

N ndash H b e n d i n g C ndash O ndash H b e n d i n g

A m i d e I I I ( p r e s e n c e o f f u n g i )

1 4 2 7

1 4 2 7

1 4 2 7

1 4 2 0

1 4 2 4

C O 3

2

minus

s t r e t c h i n g

C a l c i u m c a r b o n a t e

1 1 0 5

1 1 0 5

1 1 0 5

1 1 0 5

1 1 0 4

1 1 0 6

S O 4

2

minus

s t r e t c h i n g C ndash O ndash C s y m m e t r i c

s t r e t c h i n g

C a l c i u m s u l f a t e C e l l u

l o s e

1 0 2 9

1 0 3 2

1 0 2 9

1 0 2 9

1 0 2 7

1 0 2 9

C ndash O ndash C a s y m m e t r i c s t r e t c h i n g

C e l l u l o s e

8 7 5

8 7 6

8 7 7

8 7 9

8 7 4

8 7 6

O ndash C ndash O b e n d i n g s k e l e t a l v i b r a t i o n

C a l c i u m c a r b o n a t e c e l l u l o s e

8 1 4

8 1 3

8 1 3

C ndash O s t r e t c h i n g

H e m i c e l l u l o s e

8 0 8

C ndash H d e f o r m a t i o n o u t o f p l a n e a r o m

a t i c r i n g

L i g n i n

6 6 1

6 6 1

6 6 3

6 6 3

6 6 5

S O 4

2

minus

b e n d i n g

C a l c i u m s u l f a t e

5 9 2

5 9 2

5 9 5

5 9 2

5 9 5

C a l c i u m s u l f a t e

5 2 3

5 3 5

5 3 5

5 2 0

5 3 6

5 2 2

S i ndash O ndash A l s t r e t c h i n g

K a o l i n

4 6 7

4 7 2

4 6 8

4 5 9

4 6 9

4 6 9

S i ndash O ndash S i b e n d i n g

K a o l i n

R o w s i n g r a y r e f e r t o t h e f o x e d a r e a s p e c t r a O t h e r r e s u l t s w e r e o b t a i n e d f r o m t h e u n f o x e d a r e a s p e c t r a ( D e r r i c k e t a l 1 9 9 9 B ouml k e e t a l 2 0 0 4 S a i k i a amp P a r t h a s a r a t h y 2 0 1 0 )

A T R - F T - I R a t t e n u a t e d t o t a l r e 1047298 e c t i o n F o u r i e r t r a n s f o r m i n f r a r e d s p e c t r o s c o p y

Analytical and Microbiological Characterization of Paper Samples 73

7232019 MampM-2Analytical and Microbiological Characterization of Paper Samples Exhibiting Foxing Stains1-2015-63

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strains was not signi1047297cant and their study was thereforediscontinued

The count of fungi colony-forming units (cfu) was per-formed to determine the number of cells capable of forming colonies in a given environment Unfoxed areas presented alower degree of contamination compared with the foxedareas as shown by a small number of cfu rsquos (lt10 cfucm2)(data not shown) The results of cfucm2 obtained from thefoxed areas for each paper are presented in Figure 6

The P1 sample showed the highest number of cfucm2 inthe foxed areas indicating that this paper had the greatestdegree of microbial contamination All the fungal strainsisolated from the paper samples belonged to genera Penicilliumspp a strain that has been isolated from other paper materials

(Zyska 1997 Zotti et al 2008 Mesquita et al 2009 Michael-sen et al 2009) Identi1047297cation was subsequently con1047297rmed by OM examination of the isolates Microscopic features of thefungi isolated from the samples are reported in Table 7

The genera mostly associated with foxing are Peni-cillium Cladosporium and Aspergillus (Zotti et al 2008Mesquita et al 2009 Manente et al 2012) According toManente et al (2012) the strains isolated and identi1047297ed intheir work (Penicillium and Aspergillus) have a tendency toward xerophilia osmophylia and osmotolerance being

capable of surviving with low concentrations of water Forthis reason these ldquofoxing-causing fungirdquo are able to causedeterioration of paper (Montemartini Corte et al 2003)Abdel-Maksoud (2011) reported that biodeterioration of cellulose materials by fungi depends mainly on the chemicalcomposition of the support pH moisture content andrelative humidity of the environment temperature andillumination Besides the development of fungal structuresseveral metabolic compounds produced by fungi can alsoaccumulate in the paper support According to Sequeira et al(2012) most of these metabolic products continue theirdeleterious effects even after the fungus is dead

Cellulolytic tests performed with the isolated fungi fromthe six paper samples have shown that all the strains were

able to produce extracellular cellulolytic enzymes (data notshown) that have a degrading action on paper MontemartiniCorte et al (2003) veri1047297ed that many of the fungi examineddisplay cellulolytic activity to differing degrees with a pre-ference for more or less complex substrata

Contamination by Penicillium spp was the only onefound in this work nevertheless some microorganisms arenot able to grow under in vitro conditions The approachused here does not enable the complete characterization of the microbial community but allows identi1047297cation of isolated

Table 6 Net Counts Obtained from the Deconvolution of Experimental Spectra of the Foxed and Unfoxed Areas Divided by the NetCounts of the Region of Interest (ROI) of the Compton Scattered Peak Multiplied by 10000

Sample Name Ti Cr Mn Fe Ni Cu Zn Pb

P1 Unfoxed 6778plusmn 56 664plusmn 22 8170plusmn 106 84plusmn 04 4104plusmn 56 2802plusmn 21 26826plusmn 322Foxed 6764plusmn 22 679plusmn 16 8093plusmn 184 57plusmn 05 3929plusmn 58 2715plusmn 63 26236plusmn 347

P2 Unfoxed 347plusmn 07 75plusmn 08 130plusmn 07 2072plusmn 20 104plusmn 04 601plusmn 05 186plusmn 06

Foxed 336plusmn 07 70plusmn 04 148plusmn 54 2532plusmn 982 133plusmn 44 592plusmn 22 191plusmn 36P3 Unfoxed 345plusmn 06 54plusmn 01 395plusmn 11 14plusmn 11 447plusmn 02 89plusmn 01

Foxed 352plusmn 09 43plusmn 13 417plusmn 10 25plusmn 08 454plusmn 05 91plusmn 07P6 Unfoxed 344plusmn 23 111plusmn 16 75plusmn 05 1806plusmn 43 229plusmn 11 548plusmn 05 130plusmn 11

Foxed 375plusmn 14 103plusmn 06 97plusmn 07 1847plusmn 44 247plusmn 08 561plusmn 19 179plusmn 23P7 Unfoxed 978plusmn 04 44plusmn 03 1324plusmn 19 17plusmn 01 681plusmn 04 194plusmn 01

Foxed 966plusmn 12 55plusmn 08 1286plusmn 42 24plusmn 05 676plusmn 15 198plusmn 16P8 Unfoxed 369plusmn 07 60plusmn 06 62plusmn 03 1243plusmn 19 24plusmn 02 658plusmn 18 176plusmn 23

Foxed 387plusmn 05 52plusmn 06 52plusmn 08 1184plusmn 44 31plusmn 04 677plusmn 26 273plusmn 34

Figure 5 Energy dispersive X-ray 1047298uorescence spectra of sampleP8 in unfoxed and foxed areas

Figure 6 Colony forming units (cfu)cm2 from the foxed areas of paper samples P1ndashP8

74 Margarida Nunes et al

7232019 MampM-2Analytical and Microbiological Characterization of Paper Samples Exhibiting Foxing Stains1-2015-63

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microorganisms required for other assays namely thesimulation of foxing process with high density of cells

CONCLUSIONS

This work consisted of the study of six papers from the 20thcentury containing foxing stains The materials used in theproduction of papers and evaluation of a possible degradationprocess of the substrate as a result of the foxing stains wereevaluated Inorganic components used as 1047297llers were studiedusing SEM-EDS ATR-FT-IR and EDXRF whereas sizing

materials were analyzed by ATR-FT-IR SEM-EDS was used toevaluate the typologies of the foxing stains whereas EDXRFmicrobiological studies and ATR-FT-IR techniques were usedto assess the chemical nature and the biotic origin of foxingDescriptions of size color and shape of the foxing stains aswell as paper tonality and surface texture were done based onphotography under different illuminations and OM

Only foxing stains in the P3 sample 1047298uoresced under UVradiation suggesting that the degradation process was lessadvanced in this paper than in others No substantial differ-ences for the elements evaluated by EDXRFmdashnamely Fe and

Table 7 Microscopic Features of Isolated Fungal Strains from Foxed Areas of Samples P1ndashP8 and their Identi1047297cation

Microscopic features Identi1047297cation Samples

Penicillium spp 1

P1 P2

P3 P6

P7 P8

Penicillium spp 2

Penicillium spp 3

P1 P2

P3 P8

Penicillium spp 4

Analytical and Microbiological Characterization of Paper Samples 75

7232019 MampM-2Analytical and Microbiological Characterization of Paper Samples Exhibiting Foxing Stains1-2015-63

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Cumdashwere observed within the standard deviation between thefoxed and unfoxed areas for all the samples These resultssuggest that Fe and Cu are not the most important factors forthe foxing development process in these samples

ATR-FT-IR showed that fungi were present in the stainsof all the samples although the SEM-EDS technique did notdetected fungal contamination in them Band alterations in

the obtained spectra were observed for samples P3 and P7although paper P1 exhibited the greatest degree of microbialcontamination (highest number of cfucm2) Only fungibelonging to the genus Penicillium were observed in thepaper samples Unfoxed areas presented a lower degree of contamination when compared with foxed areas

Particularly important was the role of SEM-EDS forde1047297ning the typology of the foxing stains Two differenttypologies were found Fiber disorder and disruption werefound in samples P1 P2 and P3 and localized accumulationsof CaCO3 and CaSO4 particles were found in sample P7 andin samples P6 and P8 respectively

Several inorganic materials were found in each paperThe results revealed high amounts of 1047297llers in all samplesparticularly in papers P6 P7 and P8 TiO2 was used inpapers P1 and P7 whereas CaCO3 was the principal 1047297llerused in this last sample Apart from P7 CaCO3 was alsodetected in samples P1 P2 P6 and P8 Kaolin was detectedin all the samples CaSO4 does not seem to be present in P7Talc was observed in samples P2 P3 and P6 Barium sulfateusually added to paper for coating was present in sample P3

Sizing with a proteinaceous material in samples P1 andP7 suggests that they are conservation boards whereas P8where lignin was not removed is a mechanical wood two-ply paperboard with no applied coating Samples P2 and P3

seemed to be sized with a resin materialThe combination of several nondestructive techniques

allowed the characterization of paper composition (cellulosematrix 1047297llers and sizing materials) the evaluation of mor-phological aspects and the chemical and biotic nature of thefoxing stains

A CKNOWLEDGMENTS

The authors acknowledge Vanda Amaral and Nuno Carriccedilofor image processing They also acknowledge Ana MargaridaCardoso Catarina Miguel Joseacute Miratildeo and MassimoBeltrame for the suggestions and fruitful discussions

R EFERENCES

ABDEL-MAKSOUD G (2011) Analytical techniques used for theevaluation of a 19th century Quranic manuscript conditions

Measurement 44 1606ndash1617AREA MC amp CHERADAME H (2011) Paper aging and degradation

Recent 1047297ndingsandresearch methods Bioresources 6(4)5307ndash5337ATR-FTIR LIBRARY COMPLETE (2009) Vol 2copy 2009 ST Japan IncBARRETT T R OBERT S amp WADE J (2012) XRF analysis of historical

paper in open books In Studies in Archaeological SciencesShugar AN amp Mass JL (Eds) pp 191ndash214 Leuven BrusselsLeuven University Press

BEAZLEY K (1991) Mineral 1047297llers in paper Pap Conservator 15 17ndash27BICCHIERI M R ONCONI S R OMANO FP PAPPALARDO L CORSI M

CRISTOFERETTI G LEGNAIOLI S PALLESCHI V SALVETTI A amp TOGNONI E (2002) Study of foxing stains on paper by chemicalmethods infrared spectroscopy micro-X-ray 1047298uorescencespectrometry and laser induced breakdown spectroscopySpectrochim Acta B 57 1233ndash1249

BOumlKE H AKKURT S OumlZDEMIR S GOumlKTURK EH amp SALTIK EN

(2004) Quanti1047297cation of CaCO3ndashCaSO305H2OndashCaSO42H2Omixtures by FTIR analysis and its ANN model Mater Lett 58 723ndash726

BRANDT NN CHIKISHEV AY ITOH K amp R EBRIKOVA NL (2009)ATR-FTIR and FT-Raman spectroscopy and laser cleaning of old paper samples with foxing Laser Phys 19(3) 483ndash492

BRUumlCKLE I (1993) The role of alum in historical papermaking Abbey Newslett 17(4) 53ndash57

BUZIO R CALVINI P FERRONI A amp VALBUSA U (2004) Surfaceanalysis of paper documents damaged by foxing App Phys A 79383ndash387

CANNON A (2011) Interactions between adhesives from naturalsources and paper substrates Proceedings of Symposium 2011mdash

Adhesives and Consolidants for Conservation Research and Applications Ottawa pp 1ndash16 Available at httpwwwcci-iccgccasymposium2011 (retrieved March 3 2014)

CASTRO K PROETTI N PRINCI E PESSANHA S CARVALHO MLVICINI S CAPITANI D amp MADARIAGA JM (2008) Analysis of acoloured Dutch map from the eighteenth century The need fora multi-analytical spectroscopic approach using portableinstrumentation Anal Chim Acta 623 187ndash194

CHOI S (2007) Foxing on paper A literature review J Am Inst Conserv 46 137ndash152

COLUZZA C BICCHIERI M MONTI M PIANTANIDA G amp SODO A(2008) Atomic force microscopy application for degrada-tion diagnostics in library heritage Surf Interface Anal 40(9)1248ndash1253

DANIELS V amp MEEKS ND (1994) Foxing caused by copper alloy inclusions in paper In Symposium 88 Conservation of Historic and Artistic Works on Paper HD (ed) pp 229ndash233 OttawaCanada Canadian Conservation Institute

DEROW J amp OWEN A (1992) Foxing In Paper Conservation Catalog Bertalam S (Ed) pp 1ndash39 Washington DC American Institutefor Conservation of Historic and Artistic Works

DERKACHEVA O amp SUKHOV D (2008) Investigation of lignins by FTIR spectroscopy Macromol Symph 265 61ndash68

DERRICK MR STULIK D amp LANDRY JM (1999) Infrared Spectroscopy in Conservation Science Scienti 1047297c Tools for Conservation LosAngeles USA The Getty Conservation Institute

ERHARDT D amp TUMOSA C (2005) Chemical degradation of cellulosein paper over 500 years Restaurator 26(3) 151ndash158

ESPY HH (1990) The genesis of alkaline sizing and alkaline-curing wet-strength resins Alkaline Pap Advocate 3(3) 28ndash29Available at httpwwwcoolconservation-usorg (retrievedMarch 3 2014)

EUSMAN E (1995) Tideline formation in paper objects Cellulosedegradation at the wet dry boundary In Conservation ResearchStudies in the History of Art Monograph Series II (vol 51pp 11ndash27) Washington USA National Gallery of Art

FIGUEIRA F AFONSO M R OCHA AC amp CARVALHO ML (2009)Levantamento de manchas em desenhos dos seacutec XVI-XIX no MNAA Museologia 3 19ndash29

FLORIAN M-L (1996) The role of the conidia of fungi in fox spotsStud Conserv 41 65ndash75

76 Margarida Nunes et al

7232019 MampM-2Analytical and Microbiological Characterization of Paper Samples Exhibiting Foxing Stains1-2015-63

httpslidepdfcomreaderfullmm-2analytical-and-microbiological-characterization-of-paper-samples-exhibiting 1415

Cumdashwere observed within the standard deviation between thefoxed and unfoxed areas for all the samples These resultssuggest that Fe and Cu are not the most important factors forthe foxing development process in these samples

ATR-FT-IR showed that fungi were present in the stainsof all the samples although the SEM-EDS technique did notdetected fungal contamination in them Band alterations in

the obtained spectra were observed for samples P3 and P7although paper P1 exhibited the greatest degree of microbialcontamination (highest number of cfucm2) Only fungibelonging to the genus Penicillium were observed in thepaper samples Unfoxed areas presented a lower degree of contamination when compared with foxed areas

Particularly important was the role of SEM-EDS forde1047297ning the typology of the foxing stains Two differenttypologies were found Fiber disorder and disruption werefound in samples P1 P2 and P3 and localized accumulationsof CaCO3 and CaSO4 particles were found in sample P7 andin samples P6 and P8 respectively

Several inorganic materials were found in each paperThe results revealed high amounts of 1047297llers in all samplesparticularly in papers P6 P7 and P8 TiO2 was used inpapers P1 and P7 whereas CaCO3 was the principal 1047297llerused in this last sample Apart from P7 CaCO3 was alsodetected in samples P1 P2 P6 and P8 Kaolin was detectedin all the samples CaSO4 does not seem to be present in P7Talc was observed in samples P2 P3 and P6 Barium sulfateusually added to paper for coating was present in sample P3

Sizing with a proteinaceous material in samples P1 andP7 suggests that they are conservation boards whereas P8where lignin was not removed is a mechanical wood two-ply paperboard with no applied coating Samples P2 and P3

seemed to be sized with a resin materialThe combination of several nondestructive techniques

allowed the characterization of paper composition (cellulosematrix 1047297llers and sizing materials) the evaluation of mor-phological aspects and the chemical and biotic nature of thefoxing stains

A CKNOWLEDGMENTS

The authors acknowledge Vanda Amaral and Nuno Carriccedilofor image processing They also acknowledge Ana MargaridaCardoso Catarina Miguel Joseacute Miratildeo and MassimoBeltrame for the suggestions and fruitful discussions

R EFERENCES

ABDEL-MAKSOUD G (2011) Analytical techniques used for theevaluation of a 19th century Quranic manuscript conditions

Measurement 44 1606ndash1617AREA MC amp CHERADAME H (2011) Paper aging and degradation

Recent 1047297ndingsandresearch methods Bioresources 6(4)5307ndash5337ATR-FTIR LIBRARY COMPLETE (2009) Vol 2copy 2009 ST Japan IncBARRETT T R OBERT S amp WADE J (2012) XRF analysis of historical

paper in open books In Studies in Archaeological SciencesShugar AN amp Mass JL (Eds) pp 191ndash214 Leuven BrusselsLeuven University Press

BEAZLEY K (1991) Mineral 1047297llers in paper Pap Conservator 15 17ndash27BICCHIERI M R ONCONI S R OMANO FP PAPPALARDO L CORSI M

CRISTOFERETTI G LEGNAIOLI S PALLESCHI V SALVETTI A amp TOGNONI E (2002) Study of foxing stains on paper by chemicalmethods infrared spectroscopy micro-X-ray 1047298uorescencespectrometry and laser induced breakdown spectroscopySpectrochim Acta B 57 1233ndash1249

BOumlKE H AKKURT S OumlZDEMIR S GOumlKTURK EH amp SALTIK EN

(2004) Quanti1047297cation of CaCO3ndashCaSO305H2OndashCaSO42H2Omixtures by FTIR analysis and its ANN model Mater Lett 58 723ndash726

BRANDT NN CHIKISHEV AY ITOH K amp R EBRIKOVA NL (2009)ATR-FTIR and FT-Raman spectroscopy and laser cleaning of old paper samples with foxing Laser Phys 19(3) 483ndash492

BRUumlCKLE I (1993) The role of alum in historical papermaking Abbey Newslett 17(4) 53ndash57

BUZIO R CALVINI P FERRONI A amp VALBUSA U (2004) Surfaceanalysis of paper documents damaged by foxing App Phys A 79383ndash387

CANNON A (2011) Interactions between adhesives from naturalsources and paper substrates Proceedings of Symposium 2011mdash

Adhesives and Consolidants for Conservation Research and Applications Ottawa pp 1ndash16 Available at httpwwwcci-iccgccasymposium2011 (retrieved March 3 2014)

CASTRO K PROETTI N PRINCI E PESSANHA S CARVALHO MLVICINI S CAPITANI D amp MADARIAGA JM (2008) Analysis of acoloured Dutch map from the eighteenth century The need fora multi-analytical spectroscopic approach using portableinstrumentation Anal Chim Acta 623 187ndash194

CHOI S (2007) Foxing on paper A literature review J Am Inst Conserv 46 137ndash152

COLUZZA C BICCHIERI M MONTI M PIANTANIDA G amp SODO A(2008) Atomic force microscopy application for degrada-tion diagnostics in library heritage Surf Interface Anal 40(9)1248ndash1253

DANIELS V amp MEEKS ND (1994) Foxing caused by copper alloy inclusions in paper In Symposium 88 Conservation of Historic and Artistic Works on Paper HD (ed) pp 229ndash233 OttawaCanada Canadian Conservation Institute

DEROW J amp OWEN A (1992) Foxing In Paper Conservation Catalog Bertalam S (Ed) pp 1ndash39 Washington DC American Institutefor Conservation of Historic and Artistic Works

DERKACHEVA O amp SUKHOV D (2008) Investigation of lignins by FTIR spectroscopy Macromol Symph 265 61ndash68

DERRICK MR STULIK D amp LANDRY JM (1999) Infrared Spectroscopy in Conservation Science Scienti 1047297c Tools for Conservation LosAngeles USA The Getty Conservation Institute

ERHARDT D amp TUMOSA C (2005) Chemical degradation of cellulosein paper over 500 years Restaurator 26(3) 151ndash158

ESPY HH (1990) The genesis of alkaline sizing and alkaline-curing wet-strength resins Alkaline Pap Advocate 3(3) 28ndash29Available at httpwwwcoolconservation-usorg (retrievedMarch 3 2014)

EUSMAN E (1995) Tideline formation in paper objects Cellulosedegradation at the wet dry boundary In Conservation ResearchStudies in the History of Art Monograph Series II (vol 51pp 11ndash27) Washington USA National Gallery of Art

FIGUEIRA F AFONSO M R OCHA AC amp CARVALHO ML (2009)Levantamento de manchas em desenhos dos seacutec XVI-XIX no MNAA Museologia 3 19ndash29

FLORIAN M-L (1996) The role of the conidia of fungi in fox spotsStud Conserv 41 65ndash75

76 Margarida Nunes et al

7232019 MampM-2Analytical and Microbiological Characterization of Paper Samples Exhibiting Foxing Stains1-2015-63

httpslidepdfcomreaderfullmm-2analytical-and-microbiological-characterization-of-paper-samples-exhibiting 1515

FLORIAN ML-E amp MANNING L (2000) SEM analysis of irregularfungal fox spots in an 1854 book Population dynamics andspecies identi1047297cation Int Biodeterior Biodegrad 46 205ndash220

GRAAFF JH (1994) Research into the cause of browning of papermounted on mats In Contributions of the Central ResearchLaboratory to the Field of Conservation and RestorationVerschoor H Mosk J (Eds) pp 21ndash42 Amsterdam TheNetherlands The Laboratorium

GRIJN E KARDINAL A amp PORK H (2002) Research into paperdegradation from an historical starting-point A case-study of discoloration of 19th-century paper Contributions toConservation Mosk J amp Tennent NH (Eds) pp 119ndash126The Netherlands Research in Conservation at NetherlandsInstitute for Cultural Heritage

GOLTZ D ATTAS M YOUNG G CLOUTIS E amp BEDYNSKI M (2010)Assessing stains on historical documents using hyperspectralimaging J Cult Herit 11 19ndash26

JEONG M DUPONT A amp R ENEacute DE LA R IE E (2014) Degradation of cellulose at the wetndashdry interface II Study of oxidation reactionsand effect of antioxidants Carbohydr Polym 101 671ndash683

JUNIOR JL amp LIGTERINK F (2001) Spectroscopic characterization

of the 1047298

uorescence of paper at the wet-dry interface Restaurator 22(3) 133ndash145LIGTERINK F PORK H amp SMIT W (1991) Foxing stains and

discoloration of leaf margins and paper surrounding printing ink Elements of a complex phenomenon in books PapConservator 15 45ndash52

MANENTE S MICHELUZ A GANZERLA R R AVAGNAN G amp GAMBAROA (2012) Chemical and biological characterization of paper Acase study using a proposed methodological approach Int Biodeterior Biodegrad 74 99ndash108

MANSO M amp CARVALHO ML (2009) Application of spectroscopictechniques for the study of paper documents A surveySpectrochim Acta B 64 482ndash490

MANSO M CARVALHO ML QUERALT I VICINI S amp PRINCI E

(2011) Investigation of the composition of historical andmodern Italian papers by energy dispersive X-ray 1047298uorescence(EDXRF) X-ray diffraction (XRD) and scanning electronmicroscopy energy dispersive spectrometry (SEM-EDS) Appl Spectrosc 65(1) 52ndash59

MANSO M COSTA M amp CARVALHO ML (2008) Comparison of elemental content on modern and ancient papers by EDXRF

App Phys A 90 43ndash48MANSO M PESSANHA S FIGUEIRA F VALADAS S GUILHERME A

AFONSO M R OCHA AC OLIVEIRA MJ R IBEIRO I amp CARVALHOML (2009) Characterisation of foxing stains in eighteenth tonineteenth century drawings using non-destructive techniques

Anal Bioanal Chem 395 2029ndash2036MESQUITA N PORTUGAL A VIDEIRA S R ODRIacuteGUEZ-ECHEVERRIacuteA S

BANDEIRA AML SANTOS MJA amp FREITAS H (2009) Fungaldiversity in ancient documents A case study on the Archive of the University of Coimbra Int Biodeterior Biodegrad 63626ndash629

MICHAELSEN A PINtildeAR G MONTENARI M amp PINZARI F (2009)Biodeterioration and restoration of a 16th century book using a

combination of conventional and molecular techniques Acase study Int Biodeterior Biodegrad 63 161ndash168

MONTEMARTINI CORTE A FERRONI A amp SALVO AS (2003) Isolationof fungal species from test samples and maps damaged by foxing and correlation between these species and theenvironment Int Biodeterior Biodegrad 51 167ndash173

PETERS D (2000) An alternative to foxing Oxidation degradation asa cause of cellulosic discolouration Pap Restaurierung 1 801ndash806

PIANTANIDA G BICCHIERI M PINZARI F amp COLUZZA C (2005)Atomic force microscopy imaging directly on paper A study of library materials degradation Proc SPIE Opt Methods Arts

Archaeol 5857 217ndash227PINZARI F PASQUARIELLO C amp MICO A (2006) Biodeterioration of

paper A SEM study of fungal spoilage reproduced undercontrolled conditions Macromol Symp 238 57ndash66

PINZARI F ZOTTI M MICO A amp CALVINI P (2010) Biodegradationof inorganic components in paper documents Formation of calcium oxalate crystals as a consequence of Aspergillus terreusThom growth Int Biodeterior Biodegrad 64 499ndash505

PRONIEWICZ LM PALUSZKIEWICZ C WESELUCHA-BIRCZYNSKA ABARANSKI A amp DUTKA D (2002) FT-IR and FT-Raman study oh

hydrothermally degraded ground wood containing paper J Mol Struct 614 345ndash353PRONIEWICZ LM PALUSZKIEWICZ C WESELUCHA-BIRCZYNSKA A

MARJCHERCZYK H BARANSKI A amp KONIECZNA A (2001) FT-IR and FT-Raman study of hydrothermally degraded cellulose

J Mol Struct 596 163ndash169R AKOTONIRAINY MS HEUDE E amp LAVEacuteDRINE B (2007) Isolation

and attempts of biomolecular characterization of fungalstrains associated to foxing on a 19th century book J Cult Herit 8 126ndash133

SAIKIA BJ amp PARTHASARATHY G (2010) Fourier transform infraredspectroscopic characterization of kaolinite from Assam andMeghalaya Northeastern India J Mod Phys 1 206ndash210

SEQUEIRA S CABRITA EJ amp MACEDO MF (2012) Antifungal on

paper conservation An overview Int Biodeterior Biodegrad 7467ndash86

SONGXCHEN F amp LIU F (2011) Study on the reaction of alkyl ketenedimer (AKD) and cellulose 1047297ber Bioresources 7(1) 652ndash662

WEINSTOCK IA ATALLA RH AGARWAL UP amp MINOR JL (1993)Fourier transform Raman spectroscopic studies of a novel woodpulp bleaching system Spectrochim Acta A 49(5ndash6) 819ndash829

WILSON I (2006) Filler and coating pigments of papermaking InIndustrial Minerals amp Rocks Commodities Markets and UsesKogel JE Trivedi NC Barker JM amp Krukowski ST (Eds)pp 1287ndash1300 Colorado USA Society for Mining Metallurgyand Exploration Inc

ZOTTI M FERRONI A amp CALVINI P (2008) Micro fungalbiodeterioration of historic paper Preliminary FTIR andmicrobiological analyses Int Biodeterior Biodegrad 62 186ndash194

ZOTTI M FERRONI A amp CALVINI P (2011) Mycological and FTIR analysis of biotic foxing on paper substrates Int Biodeterior Biodegrad 65 569ndash578

ZYSKA B (1997) Fungi isolated from library materials A review of the literature Int Biodeterior Biodegrad 40(1) 43ndash51

Analytical and Microbiological Characterization of Paper Samples 77