Microsc. Microanal.

21, 6377, 2015

doi:10.1017/S143192761500001X

MICROSCOPY SOCIETY OF AMERICA 2015

Analytical and Microbiological Characterization

of Paper Samples Exhibiting Foxing Stains
Margarida Nunes,1 Ctia Relvas,1 Francisca Figueira,2 Joana Campelo,2 Antnio Candeias,1,2
Ana T. Caldeira,1 and Teresa Ferreira1,*
1
Departamento de Qumica, Escola de Cincia e Tecnologia, Centro de Qumica de vora & Laboratrio HERCULES,
Universidade de vora, Largo Marqus de Marialva, 8, 7000-809 vora, Portugal
2
Laboratrio Jos de Figueiredo, Direo Geral do Patrimnio Cultural, Rua das Janelas Verdes, 1249-018 Lisboa, Portugal

Abstract: This work comprises the use of a multi-analytical approach combined with microbiological studies to
characterize six paper samples, containing foxing stains, from the 20th century, regarding their cellulose matrix,
llers, 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 morphological
characterization of the paper samples and foxing stains. Scanning electron microscopy coupled energy dispersive
spectroscopy (SEM-EDS) was of particular importance for dening the presence of ber disorder and disruption
on the surface of some of the stains, and localized accumulations of mineral-like particles on the surface of others.
SEM-EDS, attenuated total reection Fourier transform infrared spectroscopy (ATR-FT-IR), and energy
dispersive X-ray uorescence (EDXRF) were used for the identication of mineral llers, whereas sizing agents
were analyzed using ATR-FT-IR. EDXRF results showed that no differences, within the standard deviation, were
found in iron and copper contents between the foxed and unfoxed areas. Fungi belonging to the genus Penicillium
spp. 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

I NTRODUCTION
Paper has been used for centuries to record important
historical and cultural heritage events all over the world and
is present in thousands of private and public libraries,
museums, and archives (Goltz et al., 2010). Historical paper
differs in many ways from contemporary paper (Manso &
Carvalho, 2009). Before 1840, the raw materials used to
produce paper were cellulose bers obtained from rags and
ropes (Proniewicz et al., 2002). With the increasing scarcity
of rag bers, wood gradually became the main source of
cellulose. In the hand papermaking process, gelatin, a very
stable and impermeable lm-forming external sizing agent,
was used. As a consequence of industrialized machine
papermaking, gelatin was replaced by the acidic rosin/
aluminum internal sizing, which induced the formation of
sulfuric acid, promoting acid hydrolysis in paper (Erhardt &
Tumosa, 2005). In the 1980s this acidic sizing process was
replaced by alkaline neutral sizing process using alkyl ketene
dimer (AKD) (Espy, 1990; Pinzari et al., 2006, 2010; Area &
Cheradame, 2011; Song et al., 2011).
Fillers were used by the end of the 18th century, and
china clay (kaolin) was the most common ller used (Beazley,
1991) because of its good compatibility with the acid sizing
process. It was used until the second half of the 20th century,
being replaced by alkaline calcium carbonate (CaCO3),
Received April 13, 2014; accepted January 6, 2015
*Corresponding author. tasf@uevora.pt

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., 2008;
Goltz et al., 2010; Abdel-Maksoud, 2011). Foxing stains occur
in the form of small isolated patches of discoloration, scattered
on a leaf of paper, which are typically rust, brown, or yellowish
toned; they are usually limited in size, with sharp or jagged
edges, often punctiform and sometimes circular (Derow &
Owen 1992; Montemartini Corte et al., 2003; Manente et al.,
2012). The study and reasoning behind the development of this
type of discoloration have been a subject of research for several
authors (Ligterink et al., 1991; Brandt et al., 2009; Figueira et al.,
2009; Manente et al., 2012). Some have associated its occurrence with biological attack, others consider that foxing has a
chemical origin, whereas others admit that both factors are
responsible for this type of degradation (Bicchieri et al., 2002;
Buzio et al., 2004; Choi, 2007; Manso et al., 2009; Zotti et al.,
2011; Manente et al., 2012). A contribution to distinguish the
chemical origin from the biological origin of foxing was
given by Piantanida et al. (2005) and Coluzza et al. (2008), who
used atomic force microscopy and showed that the topography
of the two types of foxing is different.
Choi (2007), in his literature review on foxing, mentions
research 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 research
after 1980. Although Bicchieri et al. (2002) state that special

64

Margarida Nunes et al.

reference must be made to iron and copper ions, which are

able to initiate free-radical chain reactions, Florian (1996)
and Florian & Manning (2000) suggest that the autoxidation
of lipids from conidia may be a possible cause for discoloration of the fungal material on paper bers.
Ligterink et al. (1991) brought a new perspective to the
foxing phenomenon by attributing its occurrence to a difference of humidity sorbency in the paper substrate, which can
originate from paper permeability, decient paper sizing, and
irregularities in paper including folds, tears, and impurities. As
a consequence of this nding, Graaff (1994) and Eusman
(1995) separately described the phenomenon of browning
produced at the wetdry interface. Independent from the
various causes for discoloration development, oxidation has
been 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 be
responsible for the large amount of foxing spots (Grijn et al.,
2002; Figueira et al., 2009) due to the introduction of mineral
loadings to the ber mesh at the end of the 18th century
(Beazley, 1991) and to the alteration of the sizing process.
Gelatin sizing was replaced by rosin and aluminum sulfate
(papermakers alum) in the 19th century (Brckle, 1993; Cannon, 2011). The presence and mobility of iron ions, provenant
from the humid section of the drying cylinders, is also seen as a
font of contaminants (Daniels & Meeks, 1994; Graaff, 1994).
To the best of our knowledge, only few studies (Manso
et al., 2008, 2011; Zotti et al., 2011) were carried out to characterize papers of the 20th century. The purpose of this study
was to deepen our knowledge on foxing stains, its chemical and
biological nature, and morphological aspects associated with it,
particularly in papers of the 20th century. In order to achieve
this, a multi-analytical nondestructive approach for the morphological and chemical characterization of foxed and unfoxed
areas of unimportant paper samples was used in conjunction
with a microbiological study. We report the analysis of foxed
and unfoxed areas by scanning electron microscopy coupled
with energy dispersive X-ray spectroscopy (SEM-EDS),
attenuated total reection Fourier transform infrared spectroscopy (ATR-FT-IR), and energy dispersive X-ray uorescence
(EDXRF). Photography under standard, raking, and UV
illumination and optical microscopy (OM) were also valuable
tools for the characterization of foxing stains. Microbiological
studies were carried out in order to evaluate the existence of
biotic attack in foxing stains.

Table 1. Description of 20th Century Paper Samples Used in

This Study.
Sample

Description

P1
P2
P3
P6
P7
P8

Window matboard of a painting by J. Glamma Storbelle

Matboard backing of D. Ana Mayor portrait
Inexpensive heavy drawing paper
Matboard backing of the watercolor Joo de Deus
Matboard with three windows
Mechanical wood matboard backing

Papers were selected by visual observation of the different colors and morphologies of the foxing stains. Based on
these parameters, papers presenting only one typology of
foxing stain were selected. A minimum of ten points on each
paper, both on the stains and on the neighboring unfoxed
paper surface, was subjected to chemical and morphological
analysis. Samples from each paper containing foxing stains
were used in the microbiology studies. Each sample was
properly identied and stored, protected from light, at constant room temperature.

Photographic Imaging and OM

Photographic imaging and OM are, in general, the rst steps
used in evaluating color and morphology of the foxing stains.
Visual observation and photographic register were obtained
using a Nikon Coolpix 8700 (Tokyo, Japan) camera placed
on a column stand. Images were captured in macro mode
using the same focal distance and lm sensitivity (ISO 100)
under different illuminations (standard, raking, and transmitted light). UV light images were captured under the same
conditions as above, except for lm sensitivity (ISO 400), using
an UV Waldmann W portable lamp with two TL4 W/08 F4T5/
BLB Philips lamps (Eindhoven, The Netherlands).
Stains were also observed and registered with an OM
using a Leica MZ6 microscope lens (maximum numerical
aperture of 0.075 with 1.0 achromatic objective) (Leica
Microsystems, Wetzlar, Germany) with raking light from a
Leica CLS 100 light spot (Leica Microsystems). For comparing the stain dimensions, and in order to include most of
the stains encountered, a magnication of 6.3 was chosen.
A magnication of 40 was used to evaluate the stains and
paper surface topographies. The images were captured using
an incorporated Leica DC200 camera (Leica Microsystems).

M ATERIALS AND M ETHODS

Paper Sample Description

SEM-EDS

A set of six different foxed paper samples from the 20th

century were obtained from the paper conservation studio of
the Jos de Figueiredo Conservation Laboratory in Lisbon.
The paper samples used in this study had no artistic or historical value and the set was composed mainly of different
types of cardboard, wood paper, and inexpensive heavy
drawing paper, labeled P1 to P3 and P6 to P8 (Table 1).

Morphological and topographical aspects of the paper

samples on the foxed and unfoxed areas were evaluated by
SEM. SEM-EDS analysis enabled major element point analysis and provided elemental composition mapping and
semi-quantitative evaluations. This technique was particularly
important for paper characterizationnamely, the identication of the llers used in the paper production.

Analytical and Microbiological Characterization of Paper Samples

Small pieces of all the samples containing foxed and

unfoxed areas were cut and placed face-up on two-sided sticky
tapes on aluminum SEM specimen holders. The study was
carried out on a variable pressure Hitachi S-3700N SEM
(Hitachi High-Technologies Europe, Krefeld, North RhineWestphalia, Germany) coupled with a Brker XFlash 5010
SDD 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 the
backscattered mode (15 kV or 10 kV) were used.

ATR-FT-IR
The ATR mode allowed a nondestructive analysis of the paper
samples. Infrared analysis was carried out at room temperature
and ambient humidity with a Brker Alpha spectrometer
(Billerica, Massachusetts) coupled with a single-reection diamond ATR module. All spectra were acquired in the absorbance mode, in the range from 4,000 to 375 cm 1, from a total
of 128 scans at 4 cm 1 resolution. Spectra were recorded and
analyzed using OPUS/Mentor software (version 6.5).
Before sample analysis, background measurement was
performed in order to reduce the effect of atmospheric carbon dioxide and water vapor. Paper samples were positioned
on the surface of the sample holder, in direct contact with the
diamond crystal, and were subjected to some pressure. The
identication of paper components and detection of microbial
presence were carried out by comparing the main features of
the obtained spectra with published data (Derrick et al., 1999;
Proniewicz et al., 2001, 2002; Zotti et al., 2008, 2011; Brandt
et al., 2009; Manente et al., 2012) or with the ATR-FT-IR
database (ATR-FTIR Library COMPLETE, 2009). The absorbance spectra were normalized for comparison purposes.

EDXRF
X-ray uorescence spectrometry is a nondestructive technique, which makes it completely appropriate for the elemental
analysis of items of cultural heritage, paper in particular.
Samples were analyzed directly without any preparation.
Qualitative 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 artifacts
or in areas in the same artifact for samples with similar composition and thickness. XRF analysis of paper is difcult due to
its minimal thickness, limited density, and low concentrations
of the elements of interest (Barrett et al., 2012).
The analyses were carried out with a Brker TRACER IIISD (Billerica, Massachusetts) handheld portable spectrometer
with a rhodium tube. The S1 TRACER was used in a stationary,
bench top conguration, which is more adequate for paper
analysis, with a yellow lter (304.8 m Al + 25.4 m Ti) that
removes low-energy X-rays coming from the tube. This
reduced background levels, and therefore improved the detection limits (Barrett et al., 2012). Use of the yellow lter was
adequate for analysis of metals (Ti to Ag and W to Bi), but there
was little sensitivity to elements below Ca. Operating conditions
of 40 kV, 12.5 A and an acquisition time of 300 s were used for

65

all the analyses. The characteristic radiation emitted by elements present in the samples was collected by a silicon drift
detector that permitted an energy resolution of 149.68 eV for
Mn K radiation at a count rate of 100 kcps. Three analyses in
the foxed areas and in the unfoxed areas were carried out for
each sample. An XRF cup containing water and covered with a
Proleene XRF lm was positioned behind the paper samples
being analyzed, and together these were positioned against the
nose of the instrument in order to supply a consistent light
pressure that kept samples in their nal position to be analyzed
and diminished the distance between the sample and the
detector. A beam spot of (3 4) mm2 was used. The equipment
also had a camera that allowed visualization of the analyzed
area, and captured the image and spot of analysis. S1 PXRF
software (v. 3.8.30) was used to record the spectra, and ARTAX
software (v. 5.3.0.0) was used for spectra evaluation. Net counts
of the elements were also normalized to the Compton scattered
peak by dividing the net counts for each analyzed element,
obtained from spectra deconvolution, by the net counts of the
Compton 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 conditions.
Samples from foxed and unfoxed areas (~1 1 cm2) were collected and placed in sterilized test tubes in a suspension of
transport MRD medium (Maximum Recovery Diluent, Merck)
(Darmstadt, Bundesland, Germany). Samples were preserved at
4C before the analytical procedure. Samples were diluted in
1 mL of solution and shaken mechanically for 1 h. The suspensions were inoculated in selective media such as nutrient
agar for bacterial isolation, yeast extract peptone dextrose agar
for yeast development, and malt extract agar and Cooke Rose
Bengal to isolate lamentous fungi. Cultures were incubated at
30C for 2448 h for the development of bacterial cultures and
at 28C for 45 days for fungal growth. To detect slow microbial
growth, plates were incubated at the same temperature for a
longer time period. All different colonies were picked to obtain
pure cultures that were stored at 4C and were periodically
pinched to maintain active cultures. The microbial isolates
obtained were characterized based on the observation of macroscopic features of the colonies (texture and color) and on the
micro-morphology of the hyphae and reproductive structures
(in the case of conidia/spore isolates). Preparations made from
fungal isolates were stained with methylene blue, observed with
20 (numerical aperture: 0.50) and 50 (numerical aperture:
0.80) objectives with a Leica DM 2500P (Leica Microsystems)
OM, and were digitally recorded with a Leica DFC290HD
camera (Leica Microsystems). Fungal strains were identied
only at the genus level.

RESULTS

AND

DISCUSSION

Contemporary paper is a complex matrix that may contain

cellulose, signicant amounts of hemicellulose and lignin,
and nonbrous components such as coloring agents, llers,
coating sizings, among others.

66

Margarida Nunes et al.

Table 2.

Photographic Images of Paper Samples Under Different Illuminations.

Sample

Standard Light

Raking Light

UV Radiation

P1

P2

P3

P6

P7

P8

Photographic Imaging and OM Observations

Photographic images (Table 2) permitted recording of the
natural tones of the paper samples and their respective
stains.
The use of raking light enhanced imaging of the paper
topography and revealed details of surface texture and planar
distortions. Photography obtained under these conditions
showed that samples P1 and P7 had a mat and rougher
surface than P2 and P3, whereas sample P6 exhibited a

smooth glossy surface with oxidized brown margins and P8

exhibited a mat rough surface.
Observation of photographic images under UV illumination is a common method used in conservation practice to
assess changes in the uorescence of paper (Manso et al.,
2009; Michaelsen et al., 2009). Florian & Manning (2000)
reported that fungal spots usually have a yellow uorescence
under UV light, which can be due to the presence of aromatic
amino acids tyrosine, tryptophan, or phenylalanine in the

Analytical and Microbiological Characterization of Paper Samples

Table 3.

67

Optical Microscopy Observations with Raking Light.

Magnication
Sample

6.3

40

Tone

Foxing stains description

P1

Off-white
toned paper

Not very intensive toned

foxing stains of irregular
size and shape. Diffuse
outer limits

P2

Cream toned
paper

Very small dark-brown

foxing stains of uniform
size and shape. Sharp outer
limits

P3

Cream toned
paper

Not very intensive toned

foxing stains of irregular
size and shape. Diffuse
outer limits

P6

Cream toned
paper with
brown margins

Very small dark-brown

nuclei surrounded by lessintensive stain of irregular
size and shape. Diffuse
outer limits

P7

Off-white
paper

Intense, sharp edged,

medium/large brown
foxing. Sharp outer
limits

P8

Tanned cream
paper with
oxidized brown
surface middle

Minute foxing

proteins of the fungal structures; however, in the foxing

spots, it could also be due to degradation products from
cellulose oxidation. According to Junior & Ligterink (2001),
uorescence appears before discoloration and at a later stage,
and when discoloration has developed into dark-brown
stains uorescence can no longer be observed. Manso et al.
(2009) reported that uorescence can occur on nonstained

areas that are usually larger than the visibly stained areas,
which seems to be the case on sample P3. This was the only
paper that showed uorescence.
For a more detailed and an amplied view of the
degraded areas by foxing, OM was used (Table 3).
Manso et al. (2009) analyzed 14 Portuguese drawings
from the 19th century and reported that from a

68

Margarida Nunes et al.

Table 4.

Scanning Electron Microscopy Micrographs of Unfoxed and Foxed Areas in Paper Samples P1P8.
Sample

Unfoxed Area

Foxed Area

P1

P2

P3

P6

P7

P8

topographical point of view there were no differences

between the foxed and unfoxed areas. We also did not
observe any signicant alteration in the ber surface disposition at these magnications.

SEM-EDS Analyses
Analysis by SEM-EDS permitted examination of the morphology of the different paper samples and foxing stains and

evaluation of the elemental composition of the inorganic

materials used as llers in paper production. Investigation of
the paper samples in the unfoxed areas showed no substantial degradation of the cellulose bers. The surfaces were
structurally organized with no broken bers. Wood cellulose
bers of various dimensions were observed in detail in
backscattered electron images (Table 4). Moreover, crystals
and aggregates (ller materials and impurities) of heavier
elements (higher atomic number) were observed as indicated

Analytical and Microbiological Characterization of Paper Samples

by particles of higher intensity in the backscattered electron

images of all samples. These mineral-like deposits were quite
numerous in papers P6, P7, and P8.
Images obtained from the foxed areas (Table 4) contrasted with those from the unfoxed areas. In fact, two
typologies of stains were detected. In samples P1, P2, and P3,
ber disruption and structural disorganization were
observed in the foxed areas, whereas samples P6, P7, and P8
only exhibited localized accumulations of particles.
Contrary to the results presented in other works (Florian & Manning, 2000; Rakotonirainy et al., 2007; Manente
et al., 2012), SEM analyses revealed minimal evidence of
microbiological contamination in the foxed areas, although
fungal species were present in the foxed areas (see Microbiological Presence On Paper Samples section). Similar
ndings were reported by Peters (2000).
EDS analysis was used to obtain information on the
elemental composition of the papers. In general, modern
papers (20th century) contained the widest variety of llers
in their composition (Manso et al., 2011). In fact, several
llers were detected for each paper, although one or two were
more extensively used.
The most frequently used calcium-based ller was calcite
(CaCO3), being gypsum (CaSO4) also often used (Beazley, 1991;
Manso et al., 2011; Manente et al., 2012). The detection of particles rich in Ca in papers P1 and P7 suggests the use of a CaCO3
ller. EDS mapping of a foxed area in sample P7 is presented in
Figure 1. Accumulation of CaCO3 particles, probably due to poor
distribution of the ller during paper production, was observed
in the foxed areas of this sample (see Table 4).
Particles rich in Al and Si were also detected in this
sample. Kaolin ller, a hydrated aluminum silicate (Al2O3.
SiO2.2H2O), may be found in most grades of 19th-century
printing papers (Beazley, 1991). The presence of Al and Si
was detected in all the paper samples, suggesting the use of
aluminum silicates as llers.
EDS analysis (Fig. 2a) of unfoxed areas in paper P6
revealed the presence of particles where the molar ratio Ca/S
was ca. 1:1, which is in accordance with the use of calcium
sulfate (CaSO4).
EDS mapping of Ca and S in sample P8 (Fig. 3) showed the
distribution of these two elements. Accumulation of CaSO4
particles was detected in the foxed areas of samples P6 and P8
probably, like in sample P7, due to poor distribution of the ller
during paper production. CaSO4 was also present in papers P1
and P2, but no accumulation was present in the foxed areas.
Talc is hydrated magnesium silicate that is used to
enhance opacity and improve durability of paper (Wilson,
2006). Particles rich in magnesium and silicon were detected
in large amounts in samples P2, P3, and P6. An EDS spectrum
of sample P2 is shown in Figure 2b.
Particles rich in Ti were observed in sample P1. Titanium dioxide (TiO2) has a very good opacifying power and it
can also be used as a pigment. Titanium was detected in large
amounts in this sample, revealing that TiO2 was used as a
ller (Fig. 2c). High levels of Ti were also discovered in
papers from 1919 and 1941 by Manso et al. (2008).

69

Figure 1. Elemental (Al, Si and Ca) mapping of sample P7 (foxed

area). BSE, backscattered electron.

70

Margarida Nunes et al.

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

In sample P3, barium and sulfur were detected in molar ratio

ca. Ba/S 1:1. Their presence reveals that barium sulfate was
used in the production of this paper (Fig. 2d). Barium sulfate
is usually added to paper as a coating in order to turn it
glossy and was already detected in a magazine sample from
the 20th century and in papers from 1919 and 1941 (Manso
et al., 2008, 2011).
Iron was present in samples P2, P3, and P6. Its presence
can be associated with clay llers or the papermaking
equipment. Zinc was also found in sample P1 and its
occurrence could be associated either with contamination
from the papermaking process or with white zinc oxide, a
ller/pigment 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
llers and sizing materials. In addition, it was used to compare unfoxed paper surfaces with foxed areas. It was also a
suitable technique for evaluating the presence of fungi.
Composition of Paper
Figure 4 shows the spectra corresponding to paper samples
P1 to P8 obtained for unfoxed and foxed areas. The most
important peak wave numbers and their interpretation are
shown in Table 5.
Cellulose peaks in all spectra were well recognized, especially in the ngerprint region at the range of 8501,500 cm 1
(Manente et al., 2012). The vibrational pattern of cellulose is
very complex in this range, where stretching and deformation
modes of various groups couple. The range between 900 and
1,200 cm 1 covers the CO and CC stretching, antisymmetric in-phase ring stretching, anti-symmetric bridge

COC, as well as CCH and OCH deformation

vibrations. COH in-plane bendings, CCH, OCH, and
CCH deformation stretching, as well as HCH bending
and wagging, are observed in the 1,2001,500 cm 1 range. In
the 1,650 cm 1 region, HOH bending vibrations of adsorbed water molecules is expected (Proniewicz et al., 2002). A
broad band peak at about 3,300 cm 1 corresponds to the OH
stretching mode of cellulose/water molecules, whereas a peak
in the range 2,8003,000 cm 1 corresponds to the CH
stretching vibrations of cellulose (Brandt et al., 2009; Manente
et al., 2012).
Characteristic peaks of lignin (1,669, 1,508, and 808
cm 1) and broad absorbance in the 1,8001,550 cm 1
region (Derkacheva & Sukhov, 2008; Manente et al., 2012)
were observed only in paper P8 (Fig. 4f, unfoxed area). This
suggests that this paper is composed of mechanical wood
pulp. Mechanical pulping results in ber degradation and
leaves a high level of residual lignin. The yellowing that
occurs upon exposure of these products to light arises from
photochemical changes in the lignin component of the paper
(Weinstock et al., 1993). The relatively high band of lignin at
1,508 cm 1 in spectra of sample P8 is in accordance with the
fact that the analysis was carried out on the margins of the
paper, which were not exposed to daylight, as can be
observed in Table 2 (Zotti et al., 2008).
It also has to be emphasized that, in addition to cellulose, some hemicellulose bands are expectednamely, the
characteristic band at ca. 815 cm 1 (Proniewicz et al., 2002).
In fact, small amounts of hemicellulose were present in
samples P2, P3, and P6 (Figs. 4b4d, unfoxed area).
The bands at 1,425 and 870 cm 1 (Zotti et al., 2008, 2011;
Manente et al., 2012) were due to CaCO3. The smaller bands at
ca. 2,500 and 1,800 cm 1 (Bke et al., 2004; ATR-FTIR Library
COMPLETE, 2009) also contributed to the detection of this

Analytical and Microbiological Characterization of Paper Samples

71

with the ngerprint region of cellulose, although variations in

intensity may provide some information about the presence of
CaSO4. Paper P7 (Fig. 4e, unfoxed area) presents a less-intense
band ca. 1,110 cm 1, suggesting that CaSO4 is not present in
this paper, whereas it is present in the rest of the samples. Small
bands ca. 524 and 470 cm 1 due to kaolin (Proniewicz et al.,
2002) were observed in all the samples with the most intense
bands detected in P7. The presence of aluminum silicate was
already 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 around
1,640 and 1,560 cm 1. These peaks are attributable to amide I
and amide II of a proteinaceous material (Derrick et al., 1999;
Manente et al., 2012) used in sizing. On the other hand, sample
P3 presents a shoulder at ca. 1,720 cm 1 (Derrick et al., 1999;
Manente et al., 2012) characteristic of resinaceous materials, as
rosin. The shoulder at ca. 1,720 cm 1 is not so easily detected in
sample P2; nevertheless, a resinaceous sizing was also probably
used for this sample. For sample P8, due to the broad absorbance in the 1,8001,550 cm 1 region, the FT-IR spectrum
does not allow clear-cut identication of the sizing material.
Identication of the sizing material was also not possible for
sample P6. The amide I band of a protein sizing (about 1,650
cm 1) is visible, whereas that of amide II (about 1,545 cm 1) is
masked by a linear decrease of absorbance that makes it difcult
to obtain a reliable deconvolution of spectral data.

Figure 3. Elemental (Ca and S) mapping of sample P8 (foxed

area). BSE, backscattered electron.

compound. These bands were noticed in samples P1, P2, P6,

P7, and P8, although in P7 (Fig. 4e, unfoxed area), the bands
increased drastically in intensity, in accordance with the
extended use of this ller already detected by SEM-EDS.
The shoulder at 3,5503,400 cm 1 and the increased small
peaks at 1,620, 661, and 595 cm 1 were due to the presence of
CaSO4 (Derrick et al., 1999; Zotti et al., 2008, 2011; Manente
et al., 2012). The band at 1,1401,080 cm 1 (Derrick et al.,
1999), due to asymmetric SO42 stretching, is superimposed

Characterization of Foxing Stains

According to Zotti et al. (2011), fungi show characteristic
infrared absorbance of OH groups and absorbed water
(3,7003,000 cm 1 and about 1,635 cm 1), CH groups
(about 2,900 cm 1), polypeptide bonds (amide I at about
1,635 cm 1 and amide II at about 1,540 cm 1), and polysaccharide groups (about 1,035 cm 1). The broad plateau
between 1,500 and 1,200 cm 1, composed of several overlapping bands, is particularly interesting, as it is typical of
fungal agents and helps in differentiating the FT-IR spectra
of paper subjected to biotic attack.
Analyses of foxing stains on the six paper samples
revealed the presence of fungi in all the foxed areas (Table 5,
gray rows), conrming biotic attack. Band alterations in the
regions assigned by Zotti et al. (2011) were observed for
papers P3 and P7 (Figs. 4c, 4e, foxed area), and minute
modications of band shape and intensity were detected for
sample P2 (Fig. 4b, foxed area). In addition, ber disorder
and disruption were observed by SEM analyses for samples
P1 to P3 on foxing stains. Being so, modications in intensity
and shape of bands assigned to the vibration of OH and
CH groups, and to the ngerprint region of cellulose, were
also due to morphological alteration of the surface.

EDXRF
EDXRF analysis is a nondestructive technique sensitive to
a wide range of elements. It was, therefore, used to obtain
information on elemental composition of the papers
and foxing stains (Bicchieri et al., 2002; Manso et al., 2011).

72

Margarida Nunes et al.

Figure 4. Attenuated total reection 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.

By pointing the beam to different areas of the samples, it was

possible to compare EDXRF spectra obtained from the foxing stains and from the unfoxed areas.
Elements measured were Ti, Cr, Mn, Fe, Ni, Cu, Zn, and
Pb. Calcium was not considered because under the experimental conditions used there was little sensitivity to this
element.
Fluorescence counts for the detected elements, divided
by the net counts of the Compton scattered peak, obtained
from the same spectrum and multiplied by 10,000, with
associated standard deviation, are reported in Table 6 for
each analyzed sample.
Iron and copper were found in all samples and could be
associated with the papermaking process. Nevertheless, the
amounts in sample P1 are considerably higher compared
with the rest of the samples. The same observations were
carried out for lead, zinc, and chromium. These results suggest that P1 was submitted to considerable contamination
during the papermaking process. The occurrence of zinc and
iron can also be related to the presence of white zinc oxide, a
pigment that was frequently used in the beginning of the 20th
century (Manso et al., 2011), and hematite, an iron pigment.

Titanium was present in samples P1 and P7, in accordance with

the results obtained by SEM-EDS for sample P1.
When comparing the iron and copper contents for the
unfoxed and the foxed areas, no differences were observed,
within the standard deviation, for all the samples. A slight
increase in the zinc level, within the standard deviation, was
observed in the foxed areas of samples P6 and P8. Spectra for
sample P8 are presented in Figure 5.
Manso et al. (2009) analyzed foxing stains in drawings
from the 18th and 19th centuries and obtained similar results
and found no differences between foxed and unfoxed areas
of their samples for Al, Si, P, S, Mn, Fe, and Cu. In fact,
considering the reasoning behind the chemical nature of
foxing, and the expected differences in Fe and Cu contents in
the foxing stains, it is surprising that for both studies no
differences were observed.

Microbiological Presence on Paper Samples

Viable fungi and bacteria were isolated from the six paper
samples, from foxed and unfoxed areas, as described in the
Materials and Methods section. The growth of bacterial

661
592
535
472

661
592
523
467

663
595
535
468

1,105
1,029
877
813

1,456

P6

663
592
520
459

1,447
1,427
1,105
1,029
879
813

1,647
1,559

1,636

3,333
2,895

P7

536
469

1,454
1,420
1,104
1,027
874

1,647
1,651
1,558
1,560

3,321
2,897

P8

808
665
595
522
469

1,508
1,447
1,424
1,106
1,029
876

1,624
1,559

1,669
1,647

3,330
2,896

Tentative Assignment

SiOAl stretching
SiOSi bending

OH stretching
CH stretching
C=O stretching
C=O stretching
OH bending
C=O stretching
C=O stretching
CN stretching
CNH bending
Aryl ring asymmetric stretching
NH bending; COH bending
CO32 stretching
SO42 stretching; COC symmetric stretching
COC asymmetric stretching
OCO bending; skeletal vibration
CO stretching
CH deformation out of plane, aromatic ring
SO42 bending

Hydroxyl group of cellulose; water in cellulose

Aliphatic hydrocarbons
Resinaceous sizing
Lignin
Calcium sulfate
Amide I (proteinaceous sizing)
Amide I (presence of fungi)
Amide II (presence of fungi)
Amide II (proteinaceous sizing)
Lignin
Amide III (presence of fungi)
Calcium carbonate
Calcium sulfate; Cellulose
Cellulose
Calcium carbonate; cellulose
Hemicellulose
Lignin
Calcium sulfate
Calcium sulfate
Kaolin
Kaolin

Interpretation

Rows in gray refer to the foxed area spectra. Other results were obtained from the unfoxed area spectra (Derrick et al., 1999; Bke et al., 2004; Saikia & Parthasarathy, 2010).
ATR-FT-IR, attenuated total reection Fourier transform infrared spectroscopy.

1,447
1,427
1,105
1,032
876
814

1,653
1,558

1,653
1,558

1,456
1,427
1,105
1,029
875

1,618

1,636

1,636
1,646
1,651
1,558
1,558

P3

3,334
2,897
1,720

3,333
2,897
1,716

P2

Wave Number (cm 1)

Peak Wave Numbers (cm 1), Tentative Assignment, and Interpretation of ATR-FT-IR Spectra of Samples P1P8.

3,335
2,899

P1

Table 5.

Analytical and Microbiological Characterization of Paper Samples

73

74

Margarida Nunes et al.

Table 6. Net Counts Obtained from the Deconvolution of Experimental Spectra of the Foxed and Unfoxed Areas, Divided by the Net
Counts of the Region of Interest (ROI) of the Compton Scattered Peak, Multiplied by 10,000.
Sample

Name

P1

Unfoxed
Foxed
Unfoxed
Foxed
Unfoxed
Foxed
Unfoxed
Foxed
Unfoxed
Foxed
Unfoxed
Foxed

P2
P3
P6
P7
P8

Ti

Cr

677.8 5.6
676.4 2.2
34.7 0.7
33.6 0.7
34.5 0.6
35.2 0.9
34.4 2.3
37.5 1.4
97.8 0.4
96.6 1.2
36.9 0.7
38.7 0.5

66.4 2.2
67.9 1.6
7.5 0.8
7.0 0.4
5.4 0.1
4.3 1.3
11.1 1.6
10.3 0.6
4.4 0.3
5.5 0.8
6.0 0.6
5.2 0.6

Mn

13.0 0.7
14.8 5.4
7.5 0.5
9.7 0.7
6.2 0.3
5.2 0.8

Fe

Ni

Cu

Zn

Pb

817.0 10.6
809.3 18.4
207.2 2.0
253.2 98.2
39.5 1.1
41.7 1.0
180.6 4.3
184.7 4.4
132.4 1.9
128.6 4.2
124.3 1.9
118.4 4.4

8.4 0.4
5.7 0.5
10.4 0.4
13.3 4.4
1.4 1.1
2.5 0.8
22.9 1.1
24.7 0.8
1.7 0.1
2.4 0.5
2.4 0.2
3.1 0.4

410.4 5.6
392.9 5.8
60.1 0.5
59.2 2.2
44.7 0.2
45.4 0.5
54.8 0.5
56.1 1.9
68.1 0.4
67.6 1.5
65.8 1.8
67.7 2.6

280.2 2.1
271.5 6.3
18.6 0.6
19.1 3.6
8.9 0.1
9.1 0.7
13.0 1.1
17.9 2.3
19.4 0.1
19.8 1.6
17.6 2.3
27.3 3.4

2,682.6 32.2
2,623.6 34.7

strains was not signicant and their study was, therefore,

discontinued.
The count of fungi colony-forming units (cfu) was performed to determine the number of cells capable of forming
colonies in a given environment. Unfoxed areas presented a
lower degree of contamination compared with the foxed
areas, as shown by a small number of cfus (<10 cfu/cm2)
(data not shown). The results of cfu/cm2, obtained from the
foxed areas for each paper, are presented in Figure 6.
The P1 sample showed the highest number of cfu/cm2 in
the foxed areas, indicating that this paper had the greatest
degree of microbial contamination. All the fungal strains
isolated from the paper samples belonged to genera Penicillium
spp., a strain that has been isolated from other paper materials
(Zyska, 1997; Zotti et al., 2008; Mesquita et al., 2009; Michaelsen et al., 2009). Identication was subsequently conrmed by
OM examination of the isolates. Microscopic features of the
fungi isolated from the samples are reported in Table 7.
The genera mostly associated with foxing are Penicillium, Cladosporium, and Aspergillus (Zotti et al., 2008;
Mesquita et al., 2009; Manente et al., 2012). According to
Manente et al. (2012), the strains isolated and identied in
their work (Penicillium and Aspergillus) have a tendency
toward xerophilia, osmophylia, and osmotolerance, being

capable of surviving with low concentrations of water. For

this reason, these foxing-causing fungi are able to cause
deterioration of paper (Montemartini Corte et al., 2003).
Abdel-Maksoud (2011) reported that biodeterioration of
cellulose materials by fungi depends mainly on the chemical
composition of the support, pH, moisture content, and
relative humidity of the environment, temperature, and
illumination. Besides the development of fungal structures,
several metabolic compounds produced by fungi can also
accumulate in the paper support. According to Sequeira et al.
(2012), most of these metabolic products continue their
deleterious effects even after the fungus is dead.
Cellulolytic tests performed with the isolated fungi from
the six paper samples have shown that all the strains were
able to produce extracellular cellulolytic enzymes (data not
shown) that have a degrading action on paper. Montemartini
Corte et al. (2003) veried that many of the fungi examined
display cellulolytic activity, to differing degrees, with a preference for more or less complex substrata.
Contamination by Penicillium spp. was the only one
found in this work; nevertheless, some microorganisms are
not able to grow under in vitro conditions. The approach
used here does not enable the complete characterization of
the microbial community but allows identication of isolated

Figure 5. Energy dispersive X-ray uorescence spectra of sample

P8 in unfoxed and foxed areas.

Figure 6. Colony forming units (cfu)/cm2 from the foxed areas of

paper samples P1P8.

Analytical and Microbiological Characterization of Paper Samples

Table 7.

75

Microscopic Features of Isolated Fungal Strains from Foxed Areas of Samples P1P8 and their Identication.
Microscopic features

Identication

Samples

Penicillium spp. 1

P1, P2,
P3, P6,
P7, P8

Penicillium spp. 2

Penicillium spp. 3

P1, P2,
P3, P8

Penicillium spp. 4

microorganisms required for other assays, namely the

simulation of foxing process with high density of cells.

CONCLUSIONS
This work consisted of the study of six papers from the 20th
century containing foxing stains. The materials used in the
production of papers and evaluation of a possible degradation
process of the substrate as a result of the foxing stains were
evaluated. Inorganic components used as llers were studied
using SEM-EDS, ATR-FT-IR, and EDXRF, whereas sizing

materials were analyzed by ATR-FT-IR. SEM-EDS was used to

evaluate the typologies of the foxing stains, whereas EDXRF,
microbiological studies, and ATR-FT-IR techniques were used
to assess the chemical nature and the biotic origin of foxing.
Descriptions of size, color, and shape of the foxing stains, as
well as paper tonality and surface texture, were done based on
photography under different illuminations and OM.
Only foxing stains in the P3 sample uoresced under UV
radiation, suggesting that the degradation process was less
advanced in this paper than in others. No substantial differences for the elements evaluated by EDXRFnamely, Fe and

76

Margarida Nunes et al.

Cuwere observed, within the standard deviation, between the

foxed and unfoxed areas for all the samples. These results
suggest that Fe and Cu are not the most important factors for
the foxing development process in these samples.
ATR-FT-IR showed that fungi were present in the stains
of all the samples although the SEM-EDS technique did not
detected fungal contamination in them. Band alterations in
the obtained spectra were observed for samples P3 and P7,
although paper P1 exhibited the greatest degree of microbial
contamination (highest number of cfu/cm2). Only fungi
belonging to the genus Penicillium were observed in the
paper samples. Unfoxed areas presented a lower degree of
contamination when compared with foxed areas.
Particularly important was the role of SEM-EDS for
dening the typology of the foxing stains. Two different
typologies were found. Fiber disorder and disruption were
found in samples P1, P2, and P3 and localized accumulations
of CaCO3 and CaSO4 particles were found in sample P7 and
in samples P6 and P8, respectively.
Several inorganic materials were found in each paper.
The results revealed high amounts of llers in all samples,
particularly in papers P6, P7, and P8. TiO2 was used in
papers P1 and P7, whereas CaCO3 was the principal ller
used in this last sample. Apart from P7, CaCO3 was also
detected in samples P1, P2, P6, and P8. Kaolin was detected
in all the samples. CaSO4 does not seem to be present in P7.
Talc was observed in samples P2, P3, and P6. Barium sulfate,
usually added to paper for coating, was present in sample P3.
Sizing with a proteinaceous material in samples P1 and
P7 suggests that they are conservation boards, whereas P8,
where 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 material.
The combination of several nondestructive techniques
allowed the characterization of paper composition (cellulose
matrix, llers, and sizing materials), the evaluation of morphological aspects, and the chemical and biotic nature of the
foxing stains.

ACKNOWLEDGMENTS
The authors acknowledge Vanda Amaral and Nuno Carrio
for image processing. They also acknowledge Ana Margarida
Cardoso, Catarina Miguel, Jos Miro, and Massimo
Beltrame for the suggestions and fruitful discussions.

REFERENCES
ABDEL-MAKSOUD, G. (2011). Analytical techniques used for the
evaluation of a 19th century Quranic manuscript conditions.
Measurement 44, 16061617.
AREA, M.C. & CHERADAME, H. (2011). Paper aging and degradation:
Recent ndings and research methods. Bioresources 6(4), 53075337.
ATR-FTIR LIBRARY COMPLETE (2009). Vol. 2 2009 ST Japan, Inc.
BARRETT, T., ROBERT, S. & WADE, J. (2012). XRF analysis of historical
paper in open books. In Studies in Archaeological Sciences,
Shugar, A.N. & Mass, J.L. (Eds.), pp. 191214. Leuven, Brussels:
Leuven University Press.

BEAZLEY, K. (1991). Mineral llers in paper. Pap Conservator 15, 1727.

BICCHIERI, M., RONCONI, S., ROMANO, F.P., PAPPALARDO, L., CORSI, M.,
CRISTOFERETTI, G., LEGNAIOLI, S., PALLESCHI, V., SALVETTI, A. &
TOGNONI, E. (2002). Study of foxing stains on paper by chemical
methods, infrared spectroscopy, micro-X-ray uorescence
spectrometry and laser induced breakdown spectroscopy.
Spectrochim Acta B 57, 12331249.
BKE, H., AKKURT, S., ZDEMIR, S., GKTURK, E.H. & SALTIK, E.N.
(2004). Quantication of CaCO3CaSO3.0.5H2OCaSO4.2H2O
mixtures by FTIR analysis and its ANN model. Mater Lett 58,
723726.
BRANDT, N.N., CHIKISHEV, A.Y. ITOH, K. & REBRIKOVA, N.L. (2009).
ATR-FTIR and FT-Raman spectroscopy and laser cleaning of
old paper samples with foxing. Laser Phys 19(3), 483492.
BRCKLE, I. (1993). The role of alum in historical papermaking.
Abbey Newslett. 17(4), 5357.
BUZIO, R., CALVINI, P., FERRONI, A. & VALBUSA, U. (2004). Surface
analysis of paper documents damaged by foxing. App Phys A 79,
383387.
CANNON, A. (2011). Interactions between adhesives from natural
sources and paper substrates. Proceedings of Symposium 2011
Adhesives and Consolidants for Conservation: Research and
Applications, Ottawa, pp. 116. Available at http://www.cci-icc.
gc.ca/symposium/2011 (retrieved March 3, 2014).
CASTRO, K., PROETTI, N., PRINCI, E., PESSANHA, S., CARVALHO, M.L.,
VICINI, S., CAPITANI, D. & MADARIAGA, J.M. (2008). Analysis of a
coloured Dutch map from the eighteenth century: The need for
a multi-analytical spectroscopic approach using portable
instrumentation. Anal Chim Acta 623, 187194.
CHOI, S. (2007). Foxing on paper: A literature review. J Am Inst
Conserv 46, 137152.
COLUZZA, C., BICCHIERI, M., MONTI, M., PIANTANIDA, G. & SODO, A.
(2008). Atomic force microscopy application for degradation diagnostics in library heritage. Surf Interface Anal 40(9),
12481253.
DANIELS, V. & MEEKS, N.D. (1994). Foxing caused by copper alloy
inclusions in paper. In Symposium 88: Conservation of Historic
and Artistic Works on Paper, H.D. (ed.), pp. 229233. Ottawa,
Canada: Canadian Conservation Institute.
DEROW, J. & OWEN, A. (1992). Foxing. In Paper Conservation Catalog,
Bertalam, S. (Ed.), pp. 139. Washington, DC: American Institute
for Conservation of Historic and Artistic Works.
DERKACHEVA, O. & SUKHOV, D. (2008). Investigation of lignins by
FTIR spectroscopy. Macromol Symph 265, 6168.
DERRICK, M.R., STULIK, D. & LANDRY, J.M. (1999). Infrared Spectroscopy
in Conservation Science. Scientic Tools for Conservation. Los
Angeles, USA: The Getty Conservation Institute.
ERHARDT, D. & TUMOSA, C. (2005). Chemical degradation of cellulose
in paper over 500 years. Restaurator 26(3), 151158.
ESPY, H.H. (1990). The genesis of alkaline sizing and alkalinecuring wet-strength resins. Alkaline Pap Advocate 3(3), 2829.
Available at http://www.cool.conservation-us.org (retrieved
March 3, 2014).
EUSMAN, E. (1995). Tideline formation in paper objects: Cellulose
degradation at the wet dry boundary. In Conservation Research,
Studies in the History of Art, Monograph Series II (vol. 51,
pp. 1127). Washington, USA: National Gallery of Art.
FIGUEIRA, F., AFONSO, M., ROCHA, A.C. & CARVALHO, M.L. (2009).
Levantamento de manchas em desenhos dos sc. XVI-XIX
no MNAA. Museologia 3, 1929.
FLORIAN, M.-L. (1996). The role of the conidia of fungi in fox spots.
Stud Conserv 41, 6575.

Analytical and Microbiological Characterization of Paper Samples

FLORIAN, M.L.-E. & MANNING, L. (2000). SEM analysis of irregular
fungal fox spots in an 1854 book: Population dynamics and
species identication. Int Biodeterior Biodegrad 46, 205220.
GRAAFF, J.H. (1994). Research into the cause of browning of paper
mounted on mats. In Contributions of the Central Research
Laboratory to the Field of Conservation and Restoration,
Verschoor, H., Mosk, J. (Eds.). pp. 2142. Amsterdam, The
Netherlands: The Laboratorium.
GRIJN, E., KARDINAL, A. & PORK, H. (2002). Research into paper
degradation from an historical starting-point: A case-study of
discoloration of 19th-century paper. Contributions to
Conservation, Mosk, J. & Tennent, N.H. (Eds.), pp. 119126.
The Netherlands: Research in Conservation at Netherlands
Institute for Cultural Heritage.
GOLTZ, D., ATTAS, M., YOUNG, G., CLOUTIS, E. & BEDYNSKI, M. (2010).
Assessing stains on historical documents using hyperspectral
imaging. J Cult Herit 11, 1926.
JEONG, M., DUPONT, A. & REN DE LA RIE, E. (2014). Degradation of
cellulose at the wetdry interface. II. Study of oxidation reactions
and effect of antioxidants. Carbohydr Polym 101, 671683.
JUNIOR, J.L. & LIGTERINK, F. (2001). Spectroscopic characterization
of the uorescence of paper at the wet-dry interface. Restaurator
22(3), 133145.
LIGTERINK, F., PORK, H. & SMIT, W. (1991). Foxing stains and
discoloration of leaf margins and paper surrounding printing
ink: Elements of a complex phenomenon in books. Pap
Conservator 15, 4552.
MANENTE, S., MICHELUZ, A., GANZERLA, R., RAVAGNAN, G. & GAMBARO,
A. (2012). Chemical and biological characterization of paper: A
case study using a proposed methodological approach. Int
Biodeterior Biodegrad 74, 99108.
MANSO, M. & CARVALHO, M.L. (2009). Application of spectroscopic
techniques for the study of paper documents: A survey.
Spectrochim Acta B 64, 482490.
MANSO, M., CARVALHO, M.L., QUERALT, I., VICINI, S. & PRINCI, E.
(2011). Investigation of the composition of historical and
modern Italian papers by energy dispersive X-ray uorescence
(EDXRF), X-ray diffraction (XRD), and scanning electron
microscopy energy dispersive spectrometry (SEM-EDS). Appl
Spectrosc 65(1), 5259.
MANSO, M., COSTA, M. & CARVALHO, M.L. (2008). Comparison of
elemental content on modern and ancient papers by EDXRF.
App Phys A 90, 4348.
MANSO, M., PESSANHA, S., FIGUEIRA, F., VALADAS, S., GUILHERME, A.,
AFONSO, M., ROCHA, A.C., OLIVEIRA, M.J., RIBEIRO, I. & CARVALHO,
M.L. (2009). Characterisation of foxing stains in eighteenth to
nineteenth century drawings using non-destructive techniques.
Anal Bioanal Chem 395, 20292036.
MESQUITA, N., PORTUGAL, A., VIDEIRA, S., RODRGUEZ-ECHEVERRA, S.,
BANDEIRA, A.M.L., SANTOS, M.J.A. & FREITAS, H. (2009). Fungal
diversity in ancient documents. A case study on the Archive of
the University of Coimbra. Int Biodeterior Biodegrad 63,
626629.
MICHAELSEN, A., PIAR, G., MONTENARI, M. & PINZARI, F. (2009).
Biodeterioration and restoration of a 16th century book using a

77

combination of conventional and molecular techniques: A

case study. Int Biodeterior Biodegrad 63, 161168.
MONTEMARTINI CORTE, A., FERRONI, A. & SALVO, A.S. (2003). Isolation
of fungal species from test samples and maps damaged by
foxing, and correlation between these species and the
environment. Int Biodeterior Biodegrad 51, 167173.
PETERS, D. (2000). An alternative to foxing? Oxidation degradation as
a cause of cellulosic discolouration. Pap Restaurierung 1, 801806.
PIANTANIDA, G., BICCHIERI, M., PINZARI, F. & COLUZZA, C. (2005).
Atomic force microscopy imaging directly on paper: A study of
library materials degradation. Proc SPIE Opt Methods Arts
Archaeol 5857, 217227.
PINZARI, F., PASQUARIELLO, C. & MICO, A. (2006). Biodeterioration of
paper: A SEM study of fungal spoilage reproduced under
controlled conditions. Macromol Symp 238, 5766.
PINZARI, F., ZOTTI, M., MICO, A. & CALVINI, P. (2010). Biodegradation
of inorganic components in paper documents: Formation of
calcium oxalate crystals as a consequence of Aspergillus terreus
Thom growth. Int Biodeterior Biodegrad 64, 499505.
PRONIEWICZ, L.M., PALUSZKIEWICZ, C., WESELUCHA-BIRCZYNSKA, A.,
BARANSKI, A. & DUTKA, D. (2002). FT-IR and FT-Raman study oh
hydrothermally degraded ground wood containing paper. J Mol
Struct 614, 345353.
PRONIEWICZ, L.M., PALUSZKIEWICZ, C., WESELUCHA-BIRCZYNSKA, A.,
MARJCHERCZYK, H., BARANSKI, A. & KONIECZNA, A. (2001). FT-IR
and FT-Raman study of hydrothermally degraded cellulose.
J Mol Struct 596, 163169.
RAKOTONIRAINY, M.S., HEUDE, E. & LAVDRINE, B. (2007). Isolation
and attempts of biomolecular characterization of fungal
strains associated to foxing on a 19th century book. J Cult
Herit 8, 126133.
SAIKIA, B.J. & PARTHASARATHY, G. (2010). Fourier transform infrared
spectroscopic characterization of kaolinite from Assam and
Meghalaya, Northeastern India. J Mod Phys 1, 206210.
SEQUEIRA, S., CABRITA, E.J. & MACEDO, M.F. (2012). Antifungal on
paper conservation: An overview. Int Biodeterior Biodegrad 74,
6786.
SONG, X., CHEN, F. & LIU, F. (2011). Study on the reaction of alkyl ketene
dimer (AKD) and cellulose ber. Bioresources 7(1), 652662.
WEINSTOCK, I.A., ATALLA, R.H., AGARWAL, U.P. & MINOR, J.L. (1993).
Fourier transform Raman spectroscopic studies of a novel wood
pulp bleaching system. Spectrochim Acta A 49(56), 819829.
WILSON, I. (2006). Filler and coating pigments of papermaking. In
Industrial Minerals & Rocks: Commodities, Markets, and Uses,
Kogel, J.E., Trivedi, N.C., Barker, J.M. & Krukowski, S.T. (Eds.),
pp. 12871300. Colorado, USA: Society for Mining, Metallurgy,
and Exploration, Inc.
ZOTTI, M., FERRONI, A. & CALVINI, P. (2008). Micro fungal
biodeterioration of historic paper: Preliminary FTIR and
microbiological analyses. Int Biodeterior Biodegrad 62, 186194.
ZOTTI, M., FERRONI, A. & CALVINI, P. (2011). Mycological and FTIR
analysis of biotic foxing on paper substrates. Int Biodeterior
Biodegrad 65, 569578.
ZYSKA, B. (1997). Fungi isolated from library materials: A review of
the literature. Int Biodeterior Biodegrad 40(1), 4351.