Journal of Cultural Heritage 61 (2023) 116–126

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Journal of Cultural Heritage

journal homepage: www.elsevier.com/locate/culher

Original article

Green biocides for the conservation of hypogeal mural paintings

raised from Western and Eastern traditions: Evaluation of interference
on pigments and substrata and multifactor parameters affecting their
activity
Daniela Isola a,b,∗, Flavia Bartoli a, Annalaura Casanova Municchia a, Hyun Ju Lee c,
Seon Hye Jeong d, Yong Jae Chung e, Giulia Caneva a
a
Department of Sciences, University Roma Tre, Viale Marconi 446, 00146, Rome, Italy
b
Department of Economics, Engineering, Society and Business Organization (DEIM), University of Tuscia, Largo dell’Università snc, 01100, Viterbo, Italy
c
Institute of Preventive Conservation for Cultural Property, Korea National University of Cultural Heritage, Buyeo, 33115, South Korea
d
International Heritage Education Center, Korea National University of Cultural Heritage, Buyeo, 33115, South Korea
e
Department of Heritage Conservation and Restoration, Graduate School of Cultural Heritage, Korea National University of Cultural Heritage, Buyeo, 33115,
South Korea

a r t i c l e i n f o a b s t r a c t

Article history: Hypogeal environments due to their limited air circulation, low constant temperatures and high humidity
Received 2 December 2022 could be threatened by microbial spreading. In cultural heritage preservation, natural bioactive molecules
Accepted 27 March 2023
have been proposed as a possible alternative to traditional biocides. Although several papers report the
in vitro effect of green biocides, scant information is available about their efficacy in direct application
Keywords: and interaction with historical materials. Then, we investigated five green biocidal formulations never
Biodeterioration control applied before to control the biodeterioration of subterranean mural painting raising from Italian and
Clove bud essential oil South Korean traditions. We tested three emulsified essential oil-based (oregano and clove bud) water
Etruscan/ Baekje wall paintings suspensions, and usnic acid-based water and 2-propanol/dimethyl sulfoxide (7:3) solutions for establish-
Oregano essential oil
ing: (i) possible interference with the reconstructed ancient wall paintings materials (colour and water
Pigments interference
adsorption changes), (ii) efficacy on recurrent biodeteriogens, trough optical/electronic microscopy, biolu-
Sustainable heritage conservation
minometric and culture-based methods. Results evidenced the different impact of such biocides on wall
paintings’ materials and pigments (e.g., higher on red and black Etruscan tiles). They also highlight the
importance of reconstruction experiments for customizing treatments, avoiding interference with histor-
ical materials (e.g., changes in colour) and biodeteriogens’ spreading. The active principles concentration
and stone porosity strongly affect the biocidal efficacy, but the solvent and emulsifier used also had a
relevant role.
© 2023 Consiglio Nazionale delle Ricerche (CNR). Published by Elsevier Masson SAS. All rights reserved.

1. Introduction organotrophic and phototrophic bacteria, algae and fungi [1,2,6,7].

Cultural heritage in underground conditions is typically sub-
jected to limited air circulation, low constant temperatures, pro-
longed darkness and high humidity throughout the year [1,2]. Sub-
terranean environments are therefore considered a challenge for
life, so phototrophs adapted to the scarcity of light and oligotrophs
adapted to long periods of starvation are their common inhabitants
[3–5]. These microorganisms are mainly organized in complex mu-
tualistic communities composed by chemolithoautotrophic, chemo-
∗
Corresponding author at: Department of Sciences, University Roma Tre, Viale
Marconi 446, 00146 Rome, Italy.
E-mail address: daniela.isola@uniroma3.it (D. Isola).
Biodeterioration processes cannot be attributed to a single micro-
bial group of these polymicrobial communities. Allochthonous in-
puts of microorganisms and organic matter can threaten the frag-
ile balance [8,9]. For example, beyond the mechanical action, root
penetration can also be a carrier for soil microorganisms, a drip-
ping line for water condensation, and their exudates, an organic
carbon source [10–12].
The alterations produced on the historical materials include
physical and chemical damages, as well as aesthetic alterations
[1,13–15], giving rise to exfoliation, salts (calcium sulphates/ ni-
trates/ carbonates, i.e. calcite) deposition [16–18], and discoloration
due to the microbial pigments. Much attention has been paid to
dark-pigmented fungi in painted tombs [5,19] or to whitish pati-

https://doi.org/10.1016/j.culher.2023.03.009
1296-2074/© 2023 Consiglio Nazionale delle Ricerche (CNR). Published by Elsevier Masson SAS. All rights reserved.
D. Isola, F. Bartoli, A. Casanova Municchia et al.
nas grown on dark walls [6,10,20,21]. In this context, eubacteria
and fungi deserve attention due to their ability to produce some
form of resistance (spores), the ability to spread, and the toler-
ance/resistance to some biocidal products [3,5,7,22].
When preventive measures fail in controlling microbial spread-
ing, direct control methods become necessary [4,22,23]. Tradi-
tional biocides use has become controversial because of the possi-
ble drawbacks to materials, microbial communities, operators and
the environment [24–30]. Then, the European Community issued
the Biocidal Products Regulation EU 528 [31], and similar actions
have been undertaken worldwide [32–34]. Beyond the toxicologi-
cal problem and the absence of interference with artworks [35–37],
biocides should be carefully evaluated considering the complexity
of the microbial community. The efficient use of biocides implies,
indeed, a deep knowledge of the product’s chemical features and
the role played by the resident bacterial community, sometimes
acting as a limiting factor for fungal spreading [3,24,28,38]. More-
over, the confinement and low ventilation characterizing the hy-
pogeal environment can favour the time of contact of the biocide,
increasing its effectiveness but also causing major health issues for
operators [36,37].
In the last decade, in contrast to traditional biocides, re-
searchers focused on substances that cause minimal or no en-
vironmental harm, defined as green or eco-friendly, mainly rep-
resented by natural or biologically derived substances standard-
izing new low-impact protocols [39–44]. Active molecules syn-
thetized by plants, marine organisms, and microorganisms have
been tested on different historical artefacts [30,41,45–50]. Living
microbial cells [51,52], physical methods and low impact chemi-
cals/ solvents were also applied with this purpose [14,17,53–55].
However, there is a general lack of information about the effect of
natural biocides on historical materials. In the frame of the inter-
national project between Italy and the Republic of Korea, we fo-
cused on hypogeal conservation and eco-friendly biocides able to
control biodeteriogens threatening our underground cultural her-
itage sites [4,5,21,45,56]. The biocides were selected considering
previous experiences and recent findings [41,43–45,53,57–59].
This research represents the first attempt to assess the interfer-
ence of green biocides with materials used in hypogeal wall paint-
ings raised from cultures developed far across time and space.
2. Research aim
This research wants to assess the efficiency and interference
of selected green biocides on reconstructed biological attacks and
materials composing Italian and Korean painted tombs (Etruscan
tombs of Tarquinia and Buyeo Royal Tomb No 1), paying atten-
tion to all components of the formulation (e.g., biocidal compound,
emulsifier and solvent).
3. Materials and methods
3.1. Reconstruction of heritage materials from study sites
3.1.1. The Etruscan necropolis of Monterozzi (Tarquinia, Italy) and its
wall paintings
The Monterozzi Necropolis represents one of the most relevant
Etruscan funerary complex due to the wall painted tombs of as-
tonishing beauty, spanning from 7th century B.C. to the 1st cen-
tury B.C. Since 2004 they were included along with the Etruscan
Necropolis of Cerveteri (Banditaccia) to the UNESCO World Her-
itage Sites [60]. The tombs were carved into “macco” rocky banks,
a highly porous (30–45%) bioclastic stone rich in Amphistegina mi-
crofossils [11,61].
The underground paintings, representing the Etruscans life and
afterlife (Fig. 1), were mainly prepared using a “secco” technique
117
Journal of Cultural Heritage 61 (2023) 116–126
where the colours (green, yellow, red, black, and blue) are super-
imposed to a whitish plaster without binders [61].
Thirteen samples 10 × 10 × 8 cm of macco stone were pre-
pared to faithfully reproduce the painted Etruscan tombs, apply-
ing as plaster layer a thin powdered “macco” and as preparatory
layer a mix of lime, thin powdered macco, and diatomaceous earth
(known as “fossil flour” or diatomite) rich in silicoaluminate [61].
Nine of them were left blank, the remaining ones were furtherly
coloured superimposing to the preparatory layer the most fre-
quently used mineral pigments (Table 1).
3.1.2. The Baekje Ancient Tomb No. 1 (Buyeo Royal Tombs, Republic
of Korea) and its wall paintings
Buyeo Royal Tombs consist of seven tombs built in the late 6th
to 7th centuries of the Baekje era (18 B.C.∼ 660 A.D.) and regis-
tered as a UNESCO World Heritage Site in 2015. Tomb No. 1, pre-
sumably the tomb of the king and the royal family, is the only
tomb with mural paintings (Fig. 1) and it consists of a square fu-
nerary chamber and a hollow road. Four types of rock were used
for the main chamber: augen gneiss (northern and eastern wall),
two mica-granite (western wall); hornblende schist (floor) and gra-
nodiorite (ceiling) [4,62]. These metamorphic (gneiss and schist)
and intrusive igneous rocks (granite and gronodiorite) are charac-
terized by medium hardness (5–7 Mohs scale) and very low poros-
ity (generally below 1%). A cover stone placed on top of the ver-
tically erected bricks walls completed the tomb [63]. The walls
of the main chamber are decorated with four guardian deities
(blue dragon, white tiger, red phoenix, and black tortoise) and lo-
tus flowers and cloud patterns are drawn on the ceiling, making
it a very important material for the study of Baekje painting. The
murals were prepared with the “Jobyeokji” ( in Chinese) tech-
nique where the draw is performed on a flat trimmed stone sur-
face without a mortar layer [56], and pigments (red, white, black,
and brown) are applied by mixing them with plant resins and veg-
etal or animal oils [64–67]. The murals, relatively clear at the time
of excavation (1915), gradually began to fade after opening leading
to a permanent closure of the tomb in 1971 [56,68].
A total of twenty-eight granite and gneiss 10 × 10 × 3 cm spec-
imens (fourteen each) were prepared; twenty-two of them were
left unpainted, and the remaining ones were prepared reproduc-
ing the Jobyeokji technique. The pigments (Table 1) were applied
by brush after mixing them with 3% animal skin glue (hide glue;
Nakagawa Gofun Enogu, Japan).
3.2. Reconstruction of biological attack: tiles inoculation/incubation
of different materials
3.2.1. The Etruscan tiles
A previous study on Etruscan tombs led to the isolation of
several dark-pigmented fungi responsible for discolorations [5].
Among them, Cladosporium sp. CCFEE 6625 and Acremonium-like
fungus CCFEE 6662 were chosen because recalcitrant to the tested
benzalkonium chloride-based biocides [5]. Additionally, a bacte-
rial strain identified as Rhodococcus sp. (BLASTn comparison of
SSU rDNA) was chosen because common and lacking any cross in-
hibitory activity. Small aliquots of fresh cultures were suspended
in sterile saline solution (NaCl 0.9%), counted, and then mixed in a
50 mL tube containing 20% Czapek-Dox broth (Oxoid). The inocu-
lum solution (cell nr/mL) consists of: Cladosporium sp. CCFEE 6625
1.56 × 10E4, Acremonium-like fungus CCFEE 6662 4.64 × 10E3,
Rhodococcus sp. 2.3 × 10E4.
Five 10 × 10 × 8 cm unpainted macco tiles (white preparatory
layer only) were used for efficacy tests; they weren’t preliminary
sterilized due to the size of the specimen and its frailty. Indeed, the
process has been considered more dangerous on the stone integrity
than effective in killing the resident community.
D. Isola, F. Bartoli, A. Casanova Municchia et al.
Fig. 1. Etruscan and Baekje ancient tombs. External view of A) the Etruscan and B) the Baekje tumuli. C, D) wall paintings details of Etruscan tombs. and E, F) burial chamber
and wall paintings of Baekje Royal Tomb No.1.
Table 1
Selection of pigments for Italian and Korean teams.
Italian pigments
CB Charcoal Black Carbonaceous material
Haem Haematite Fe2 O3
MG Malachite Green Cu2 (CO3 )(OH)2
EB Egyptian Blue CaOCuO(SiO2 )4
The Italian CB and Haem pigments were supplied by Winsor & Newton ltd, London, U.K; MG and EB
by Kremer Pigmente GmbH & Co.KG, Aichstetten, Germany. Korean pigments were supplied by Co.
Gail traditional pigment, Gyeonggi-do, Korea.
The inoculation was performed slowly, dropping (waiting for
the adsorption of each drop) 3 mL of the cell suspension to evenly
cover the upper surface. The tiles were dark incubated in a her-
metic ethanol-sterilized plastic box, raised from the bottom with a
metallic grid at 20 °C and relative humidity (RH) above or equal
to 90%. Thermo-hygrometric parameters were monitored daily to
maintain such value by spraying sterile distilled water (dH2 O) on
box walls. The incubation took three months to obtain a dense
growth.
3.2.2. The Baekje tiles
Microorganism distribution in Tomb No. 1 has been regularly
investigated since 2008, leading to the isolation of different bacte-
rial and fungal strains. Currently, a white patina is growing on the
painted walls in the main chamber composed mainly by different
species of the genus Streptomyces [21]. We selected Streptomyces
avidinii to represent the white patina-forming bacteria, Fusarium
oxysporum and Mortierella sp. for fungi. Previously tests showed
that none of these strains showed inhibitory activity against oth-
ers. Fresh cultures of each strain were suspended in sterile water,
then in 10% Czapek-dox broth (30 mL) and cultured at 30 °C and
150 rpm overnight. The inoculum solution (cell nr/ mL) consists of:
Fusarium oxysporum 1.3 × 10E4, Mortierella sp. 3. 0 × 10E4, Strep-
tomyces avidinii 4.6 × 10E5.
Ten granite and gneiss 10 × 10 × 3 cm specimens were used for
efficacy tests. Before inoculation five tiles for each substrate (gran-
ite and gneiss) were autoclaved (121 °C / 15 min) and slowly inoc-
ulated by dropping 1.5 mL of suspension allowing the uniform ab-
sorption. The specimens were placed in an ethanol-sterilized her-
metic box and incubated at 30 °C and RH above or equal to 95%
as occurring in Tomb No. 1. Since after 2 weeks of incubation
118
Journal of Cultural Heritage 61 (2023) 116–126
Korean pigments
Ci Cinnabar HgS
Haem Haematite Fe2 O3
OSW Oyster shell white CaCO3
there was no signs of growth, the inoculation was repeated using
potato dextrose broth (Difco, USA) as medium and incubated for
two months.
3.3. The biocidal products selection
According to the literature [41,43–45,53], five new biocidal for-
mulations were prepared from two plant essential oils and a lichen
allelopathic substance (Table 2): two from clove bud (Syzygium aro-
maticum (L.) Merr. & L. M. Perry), and one from oregano (Orig-
anum vulgare L.), and two from usnic acid, produced by lichens
like Usnea spp. [69]. The essential oils-based formulations were
water suspended and emulsifier-added. Due to the differences in
materials porosity and used pictorial techniques (Etruscan secco
and Jobyeokji, respectively), we tested two different clove bud es-
sential oil-based (CBEO-based) formulations differing in the con-
tent of the emulsifier. The formulation richer in emulsifier (Stone
Keeper, [45] has been tested on macco mock-ups. As the solvent
could affect the treatments outcomes, usnic acid was dissolved in
distilled water and in a mix of isopropyl alcohol (IPA, 2-propanol)
and dimethyl sulfoxide (DMSO), both solvents are already used in
cultural heritage conservation [53,70].
Samchun Chemical (Republic of Korea) for Tween 20 and
Span20; Sigma Aldrich for IPA, DMSO, aceton, and usnic acid were
the suppliers. Steam distilled clove bud essential oil (Co. What
Soap, Korea) and oregano essential oil (from dried flowering herb;
Code 7573, Now Foods, Bloomingdale, IL, USA) were used. The
composition, not precisely defined from the supplier, has been as-
sessed by GC–MS and reported in their major compounds in Table
S1.
D. Isola, F. Bartoli, A. Casanova Municchia et al. Journal of Cultural Heritage 61 (2023) 116–126

Table 2
The composition of the green biocides used.

Biocidal Product Abbreviation Composition for 100 ml

Oregano essential oil (OEO) Oregano essential oil 7.5 Ml

B1 Tween 20 1.0 mL
dH2 O 91.5 mL
Clove bud essential oil Clove bud essential oil: 7.5 mL
(CBEO)-“Stone keeper” (SK) [45] Tween 20: 7.0 mL
B2
Span 20 3.0 mL
dH2 O 82.5 mL
Clove bud essential oil (CBEO) Clove bud essential oil 7.5 mL
B2/b Tween 20 1.0 mL
dH2 O 91.5 mL
Usnic acid water based Usnic acid 0.07 g
B3
Acetone then 10 ml of acetone 100 mL
solution (0.2 mM) were brought
up to 100 mL with dH2 O
Usnic acid IPA+ DMSO Usnic acid (0.2 mM) 10 mL
B4 IPA 63 mL
DMSO 27 mL

Table 3
Experiment synopsis.

Italian Team Korean Team

Oregano oil-based (B1) B1 B1

Clove bud oil-based “SK” (B2) B2
Tested biocides Clove bud oil-based (B2/b) B2/b
Usnic acid water solution (B3) B3 B3
Usnic acid IPA/DMSO solution (B4) B4 B4

Stone substrates Macco Gneiss and Granite

Cladosporium sp. CCFEE 6625 Fusarium oxysporum

Inoculated biodeteriogens Acremonium-like CCFEE 6662 Mortierella sp.
Rhodococcus sp. Streptomyces avidinii

CIELAB colorimetry CIELAB colorimetry

Sponge test Sponge test
Performed interference tests
Portable microscope Stereo microscope
Raman Spectroscopy

Optical microscope Stereo microscope

Assessment tools for
SEM Bioluminometer
biocides efficiency tests
Viability test culture Viability test culture

3.4. Interference tests of biocides
Eight macco (four with white preparatory layers and four
painted), four granite, and four gneiss (one bare and three painted
each) tiles were used for colour and water adsorption measure-
ments. Some differences in application mode, observation times
and maintenance conditions arise from differences in materials and
study sites. All tests were performed before the biocide application,
and later after one and three months from treatment. Table 3 re-
ports the complete synopsis of the experiment.
3.4.1. Assessment of colour changes
Colour measurements were performed using the CIE-LAB colour
space according to the European Standard UNI-EN15886 [71].
An EOPTIS CLM-194 handheld colourimeter with a measure-
ment diameter of 8 mm was used for macco tiles; each reported
measure was the average value of 15 measurements (5 in tripli-
cate). Each coloured tile block was divided in four parts using pa-
per sheets (as the biocides to be tested), and control was obtained
from the central area (Figure S1). The biocidal application has been
performed by brushing on white tiles and spaying on coloured
ones, due to fragile cohesion of the non-bonded pigments.
For Baekje gneiss and granite painted and non-painted tiles, a
Minolta CR-400 (Osaka, JPN) was used for colour measurements,
taken as above and the biocidal products were all applied by brush.
The global colour variation E∗ was calculated following the
formula:

Eab
∗
= L∗2 + a∗2 + b∗2
where the colour coordinates are: L∗ (lightness; 0= absolute black,
100 = absolute white), a∗ (position between green (a∗ <0) and
red/magenta (a∗ >0)) and b∗ (position between blue (b∗ <0) and
yellow (b∗ >0)). Restorers normally accept E∗ < 4 as a limit value
for the visual impact of surface treatments [28].
3.4.2. Assessment of possible changes in water absorption
According to the European standard UNI-EN 17655 [72], the
contact sponge method was used to evidence possible changes
in water absorption before and after the treatment, on non-
pigmented tiles; pigments indeed could be removed by sponge.
A 1034 Rodac plate, containing a 5.5 cm ∅ natural sponge by
Spontex, was imbibed with 3 g of distilled water (5 g for gneiss
and granite trials) and manually pressed against a vertical stone
surface for 1 min. The total weight (sponge + plate) was measured
before and after contact, and their difference gave the water ab-
sorbed. The applied formula is:
 g  Pi − Pf
Wa ∗ min =
cm2 23.76 ∗ t
where Wa is water absorbed per surface unit per minute, Pi is the
initial weight of the sponge inside the plate, Pf is the weight of the

119
D. Isola, F. Bartoli, A. Casanova Municchia et al.
sponge inside the plate after the contact, 23.76 is the sponge area
and t is the time of contact.
3.4.3. Stereomicroscope examination of major surface changes
A possible increase of porosity and/or detachments of macco
stone (Etruscan) was assessed by a Dino-Lite stereomicroscope at
60 × and 120 × magnifications using white and UV light (5 repli-
cas/tile before and after applications) to detect any variation. Sim-
ilarly, the surface of the Jobyeokji specimens (Korean) was ob-
served, before and after the treatments, at 20 × and 40 × mag-
nifications using a stereo microscope (SMZ18, Nikon, JPN).
3.4.4. Raman spectroscopy
On reproduced Etruscan paintings was used a Renishaw Ra-
man In-Via Reflex μ-spectrometer coupled with a confocal micro-
scope equipped with a 785 nm diode laser (nominal output power
300 mW) to characterize possible new substances formed by bio-
cide interaction with materials. The laser power at the surface was
held to ≤5 mW to avoid sample degradation. The backscattered
light is dispersed by a 1200 line/mm grating, and the Raman signal
is detected by a Peltier cooled (−70 °C) 576 × 384 pixel CCD de-
tector. All the spectra were collected in the range 20 0–40 0 0 cm−1
at room temperature. Nominal spectral resolution, at these work-
ing conditions, was about 3 cm−1 .Raman spectra were collected by
using 20 × and 50 × objectives. WiRE software has been used for
spectra acquisition and Origin software for the spectra elaboration.
3.5. Efficacy assessments of biocidal formulations
3.5.1. Macco stone trials
Each colonized tile was divided into three areas (about
10 × 3.3 cm) and biocides (one for tile) applied by brush one, two,
and three successive times respectively. Between the subsequent
applications, the tile was left to dry reproducing the common re-
storer practice. Similarly, sterile distilled water was applied as con-
trol. The treated tiles were stored in a hermetic box, and conditions
were maintained under control (20 °C and RH ≥ 90%). After one
month, biomass was sampled using a sterile surgical blade, and the
treatments efficacy evaluated by the following described methods.
The optical microscopy observations were performed with
Olympus BX41 (Olympus, Tokyo, Japan), following the UNI 10923
guidelines [73]. For SEM (FE-SEM, SUPRATM 35, Carl Zeiss SMT,
Oberkochen, Germany), the fractured fragments were spurred with
gold in a vacuum using K550 unit (Emitech Technologies Ltd. Kent,
England) and then observed [74].
The biomass for viability trials was taken by scraping an area of
1 × 10 cm for each dose of each tested biocide, collected in sterile
Eppendorf tubes (1.5 mL, pre-weighted) and weighed before stor-
ing at −20 °C until processing. Each sample was divided in three
weighted aliquots. Scalar dilution (three) of controls and treated
samples were prepared in sterile saline solution (NaCl 0.9%), plated
in triplicate onto Potato Dextrose Agar (PDA; VWR, Chemicals, Bel-
gium), incubated at room temperature (20 ± 1 °C), read after 3–5
days, and. counts expressed as UFC/mg (of material scraped) ± SD.
After surface samplings the four treated tiles were maintained
in transparent box and inspected until the end of the experiment
(four months from treatment).
3.5.2. Gneiss and granite trials
The biocides application was performed as previously de-
scribed. The test tiles were stored in an airtight box at 30 °C, RH
≥95%., and specimens were observed under a stereo microscope
(Nikon SMZ18, 20 ×) from the first to the fourth week from the
treatment. The microbial activity was measured, and viability cul-
ture analysis performed.
120
Journal of Cultural Heritage 61 (2023) 116–126
ATP Bioluminometer (Clean-trace NG3, 3 M, USA) was used to
confirm the residual microbial activity on the tile surfaces. Biomass
was collected by swabbing in a straight line along the full length
(10 × 0.3 cm) of the specimens treated with natural biocides (after
one, two, and three applications). After reaction the relative light
units (RLU) measured, repeated 3–5 times, and converted into per-
centages (%) by comparison to their controls. Sterile cotton swabs
were used to collect biomass for viability tests, washed in sterile
dH2 O, and suspensions serially diluted. Each dilution was plated in
triplicate onto PDA (Difco, USA) and incubated at 30 °C for three
days and count expressed as. UFC/mL.
3.5.3. Statistical analysis
To assess outcomes significance after biocides application two-
way ANOVA with Tukey post-hoc test comparison (p < 0.05), re-
gardless of rows and columns, was performed using GraphPad
Prism version 9.3.1 (GraphPad Software, San Diego, CA, USA).
4. Results
4.1. Interference tests
4.1.1. Colorimetric measurements
Macco white tiles showed relevant chromatic changes (E∗ >4)
when treated with usnic acid in water (B3) (Table 4) due to a de-
crease in L∗ and an increase of the b∗ in the yellow position
(Table S2) also visible by the naked eye. The results from painted
macco showed that all biocides caused significant total chromatic
variation (E∗ >4) with notable variability depending on the pig-
ment. The (Haem) red and black (CB) colours were the most af-
fected: the stronger alteration on red tiles was an evident whiten-
ing in correspondence to B2 application (L∗ 11.7) (Table 4, Ta-
ble S2); while on black tiles whitening alteration was recorded af-
ter B1 treatment (OEO-based), B2 (CBEO-based - SK) and B3 (UA-
water solution) (L∗ > 4). (Table S2). The white superposed patinas
were interpreted as calcite through Raman spectra (Figure S2) (i.e.,
(CaCO3 ): 1086 cm−1 , 712 cm−1 and a shoulder at 281 cm−1 ). The
green (MG) tiles did not show evident chromatic variation, except
for the B1 where a darkening has been recorded (L∗ −3.9). Oth-
erwise, very high E∗ values were recorded in the blue (EB) sam-
ples (Table 4), mainly due to the uneven painting layer. Indeed,
high E∗ values were also recorded in the control areas and mi-
croscopic observation did not underline any superficial alteration.
The non-painted Korean tiles were not affected by the tested
biocidal formulations (Table 2) with E∗ comprised between 0.2
(B1 and B2/b on gneiss) and 0.9 (B4 on gneiss) on bare tiles. Slight
differences were also recorded on cinnabar (Ci) and haematite
(Haem) tiles (gneiss and granite). The gneiss cinnabar painted tile
treated with B2/b biocide was the only test recording a relevant
colour change with E∗ >4. Otherwise, the results achieved on
oyster shell white (OSW) should not be considered suitable be-
cause affected by fungal contamination.
4.1.2. Water absorption
Water absorption measurements before and after treatment
did not lead to significant changes on white layered macco, and
non-painted granite and gneiss. Indeed, the measurements on the
treated materials were always superimposable to the non-treated.
4.2. Biocides efficiency
4.2.1. Direct observation, optical and SEM microscopy
After one month from treatment, optical microscope observa-
tions of samples taken from macco tiles did not evidence visible
cell alterations. Instead, SEM evidenced several collapsed/deflated
cells, recorded specially after three applications. Deflated cells
D. Isola, F. Bartoli, A. Casanova Municchia et al. Journal of Cultural Heritage 61 (2023) 116–126

Table 4
The colour difference of treated and untreated areas of mural paintings Italian and Korean
mock-ups after four months from biocides applications. The differences are reported as E∗
value, in bold the differences ≥ 4. The complete data are reported in supplementary material
(Table S2).

Samples B1 B2- B2/b B3 B4

Etruscan Macco White 1.2 1.5 – 4.8 0.9

wall paintings preparatory layer
Charcoal black 4.6 4.6 – 7.1 3.0
Haematite 6.5 18.1 – 5.6 5.6
Malachite green 4.2 3.6 – 2.5 3.4
Egyptian blue 2.2 3.4 – 6.2 22.3∗
Baekje Ancient Gneiss Non-painted 0.2 – 0.2 0.2 0.9
wall paintings Cinnabar 3.1 – 4.1 1.1 1.7
Haematite 0.7 – 0.8 0.8 0.4
Oyster shell white 9.9∗∗ – 10.9∗∗ 10.4∗∗ 11.0∗∗
Granite Non-painted 0.5 – 0.3 0.3 0.7
Cinnabar 1.3 – 2.7 0.9 1.1
Haematite 0.4 – 0.6 1.0 0.4
Oyster shell white 9.6∗∗ – 10.6∗∗ 11.2∗∗ 8.7∗∗

B1: oregano oil-based water suspension; B2: clove bud oil-based water suspension (Stone
keeper); B2/b: clove bud oil-based water suspension; B3: usnic acid-based water- solution;
B4: usnic acid-based IPA/DMSO solution.
∗
indicates not suitable data for an uneven blue pigment.
∗∗
not suitable data due to fungal contamination.

Fig. 2. B2-treated macco tile, direct observation after two months from treatment. A-C) A: the blue squares evidenced the enlarged details in figures B and C; the white
arrows point the new, lush fluffy fungal growth.

were absent after B3 (UA-water solution) treated samples, and spo-
radic on those treated with B2. After two months from treatment,
the tile treated with B2 showed a diffuse and dense fluffy fungal
growth (Fig. 2). No relevant changes were observed using a stereo
microscope after treatments on granite and gneiss tiles.
recording the higher metabolic activity after one and four weeks
in both stone types. B2/b biocide, instead, showed a significative
increase in metabolic activity after four weeks (Fig. 3).
5. Discussion

4.2.2. Viability plate test and bioluminometric assay
Plate observation evidenced the presence of Cladosporium sp.
CCFEE 6625 (inoculated) and Penicillium sp. (macco’s resident my-
coflora) in all infected tiles (Fig. 3A).
None of the biocidal formulation gave significant changes in cell
viability after the first application on macco tiles (Table 5). Viabil-
ity progressively decreases for B1, B2 and B4 biocides; this last re-
sulted the most effective (Table 5, Fig. 3A). Otherwise, in gneiss
and granite colonized tiles has been recorded a striking reduction
of viable cells (up to 95.6% for gneiss tiles and 98.4% for granite)
since the first biocides application. Three applications accounted a
very low viability for all the biocides tested without significant dif-
ferences between them (Table 5).
The bioluminometric tests gave a two steps sight on residual
metabolic activity. The outcomes recorded with biocides B1 and B4
were similar (Fig. 3). Notable differences were recorded with the
other two biocides. In detail B3 resulted the less efficient biocide,
Cultural heritage needs more appropriate conservation strate-
gies in light of sustainability. Considering the risk of biocides in-
terference with materials and optimizing the treatments’ efficacy
is therefore of utmost importance.
5.1. Green biocides and materials interferences
The only formulation causing relevant changes on white macco
tiles was usnic acid in water (B3), which lead to yellowing. Usnic
acid in IPA/DMSO (B4) recorded the lowest colour change (0.5 vs
5.5 B3) suggesting an improved solving effect.
The whitish alterations recorded on charcoal black and
haematite could be due to water being calcite detected after treat-
ment with B1, B2 and B3 biocides. Indeed, water can penetrate
deeper than alcohol (this last fast evaporates and less solve salts)
and wash away the calcite particles present in the lime prepara-
tory layer and, during water evaporation, salts in solution could

121
D. Isola, F. Bartoli, A. Casanova Municchia et al. Journal of Cultural Heritage 61 (2023) 116–126

Fig. 3. Synopsis of the biocidal efficacy results on macco, gneiss and granite tiles. A) Results achieved after three applications on macco tiles are reported following an
increasing order of efficacy. In the upper part are indicated the methods used to assess the biocidal effect. On the left the biocides used. The bar is 10 μm. B) Bubble chart
of the gneiss and granite tiles result reported following an increasing order of efficacy. Bubbles were calculated as average percentage respect their relative controls. In the
upper part are indicated the biocides used, on the left are indicated the methods used to assess the biocidal effect namely viability (Cell n.) and metabolic activity (ATP); on
the right side, the stone types. The complete bioluminometric data and percentage values are reported in supplementary material Table S3 and Table S4 respectively.
Fig. 3 note. B1: oregano oil-based water suspension; B2: clove bud oil-based water suspension (Stone keeper); B2/b: clove bud oil-based water suspension; B3: usnic acid-
based water- solution; B4: usnic acid-based IPA/DMSO solution.

Table 5
Viability test results achieved in macco, gneiss, and granite trials. Results are expressed as UFC/mg ± SD for macco and UFC/mL± SD
for gneiss and granite trials. Different letters indicate significative differences within each experiment.

C B1 B2 B2/b B3 B4

Macco
1st application 812.23±106.3a 909.54±41.2a – 925.4 ± 63.5a 602.31±23.4b
2nd application 907.4 ± 89.4a 584.23±84.3b 618.07±56.5b – 949. 12±79.5a 186.25±13.3c
3rd application 272.33± 46.14c 573.33±51.7b – 1013.3 ± 96.4a 26.39±1.8d
Gneiss
1st application 43.3 ± 13.1b – 253.3 ± 68.5c 186.7 ± 45.0c 33.3 ± 12.5b
2nd application 753.3 ± 12.5a 20.3 ± 5.0b – 126.7 ± 12.5bc 53.7 ± 13.9b 15.3 ± 4.5b
3rd application 2.3 ± 1.2b – 63.3 ± 26.2b 28.3 ± 8.5b 2.7 ± 1.7b
Granite
1st application 42.7 ± 8.1b – 320.3 ± 92.0b 66. 7 ± 11.6b 137.3 ± 6.2b
2nd application 2620±545.2a 27.0 ± 4.5b – 173.7 ± 47.6b 192.0 ± 23.8b 67.7 ± 12.7b
3rd application 8.0 ± 4.3b – 113±7.9b 72.3 ± 16.0b 21.0 ± 1.6b

B1: oregano oil-based water suspension; B2: clove bud oil-based water suspension (Stone keeper); B2/b: clove bud oil-based water
suspension; B3: usnic acid-based water- solution; B4: usnic acid-based IPA/DMSO solution; C is the control.

122
D. Isola, F. Bartoli, A. Casanova Municchia et al.
migrate outwards and recrystallize calcite on the surface. The phe-
nomenon is closely related to the local microclimate parameters
such as aerial CO2 , prolonged high humidity conditions, and the
presence of other salts [75,76]. Calcium carbonate has indeed lim-
ited solubility (14 mg/L) in pure water. However, if carbon dioxide
is present, the solubility increases by more than a factor of 100,
and the carbonate ion goes into solution as a hydrogen carbon-
ate [77]. The hypogeal microenvironmental conditions favour cal-
cite formations, as frequently reported on paintings [75,78,79] and
it should not be underestimated when using water-based products.
Otherwise, B4 (IPA/DMSO solvent) did not lead to visible changes
on the black tile; and those recorded on the haematite tile was
due to an increase in the red/magenta component (a∗ 5.5). Mala-
chite green painted tile decreased in lightness after applying the
OEO-based product only (E 4.2, B1). Egyptian blue tile treated
with B1 and B2 did not lead to visible colour changes. The lack of
calcite formations on the green and the blue pigment could be ex-
plained by the different pigments’ permeability affecting the crys-
tallisation rate [75]; further investigations are needed for confir-
mation. Nebulizer and brush application modes could badly affect
the substrate, but knowledge advancements are needed to quanti-
tatively and qualitatively measure their outcomes.
Negligible colour changes have been recorded on bare granite
and gneiss tiles and those haematite-painted. Cinnabar gneiss tile
treated with B2/b formulate were the only affected by a visible
change (E 4.1). No reliable measures have been achieved on oys-
ter shell white.
The colorimetric readings on historical artefacts could be influ-
enced by many factors, such as the application mode. This last is
strongly constrained by the cohesion of materials. Indeed, even if
the Jobyeokji technique uses organic binders, the cohesion is not
strong enough as the brush use could be detrimental. Experiments
performed by Lee [21] spraying till wetting three different concen-
trations of OEO and CBEO (5%, 7.5%, and 10%) on three traditional
painting layers (pigment+ 3% hide glue layered on glass -pigments:
oyster shell white, cinnabar, and haematite) do not evidenced sig-
nificant colour changes (E∗ <4) after one month from the ap-
plication. Differently, it was reported that oyster shell white pig-
ment after brush application of the same CBEO (at 5%, 7.5%, 10%)
caused yellowing discouraging its application on murals [54]. This
evidence suggests as the application method (brush vs spray) can
change the quantity of biocide applied per surface unit and then
the outcomes.
The comparison between Italian and Korean test tiles under-
lined different interactions caused by biocides on substrates. The
whitish patina due to calcite recrystallization, frequently recorded
in the Italian tiles, was absent in the Korean ones because of the
lack of carbonates in stone and preparatory layer. These aspects
should be considered to define the application limit for a product
or a solvent that can correctly work on a substrate but induce al-
teration on another. The different outcomes recorded for the usnic
acid based-biocide (B3) evidenced as the amount of the product
used and the background colour (e.g., white for macco and shaded
grey for gneiss and granite) could affect the results.
5.2. Biocidal efficiency tests
These tests gave relevant evidence referred to the importance
of (i) the experiment comparability, (ii) the biocidal efficacy of the
molecules, (iii) emulsifiers in EOs-based formulates, (iv) the sol-
vent used.
5.2.1. Green biocides and experiment comparability
In recent years, the biocidal use of substances of natural
origin, particularly essential oils, is increasing. These are com-
plex mixtures whose antimicrobial features are associated with
123
Journal of Cultural Heritage 61 (2023) 116–126
a broad spectrum of secondary metabolites ranging from pheno-
lics, quinones, flavonoids, alkaloids, terpenoids, to polyacetylenes
[80]. The biocidal properties of CBEO and OEO have been asso-
ciated mainly to the pivotal presence of eugenol and carvacrol
respectively, and in less extent to the synergic action with mi-
nor molecules [81,82]. The comparison with bibliographic sources
evidenced huge qualitative and quantitative differences with the
products used. For example, eugenol in CBEO has been reported
6.3 times (v/v) more concentrated than in the EO we used [83].
This difference was even greater in OEO where carvacrol has been
reported to be up to 26 times (v/v) more concentrated than in the
EO used [84], and up to 7 times in the final biocidal formulations
(91.37 mM vs 13.17 mM). This fact could justify the contrasting re-
sults already recorded on EOs effectiveness [41,43], but also point
out the attention on the non-active fraction. Indeed, the balance
between the active and non-active compounds should be deeply
evaluated [85], as the advantages should exceed possible unwanted
effects on materials. In this light, information on the EO compo-
nents in addition to the product supplier and the product code,
are basic for protocols standardization.
5.2.2. Biocidal power
Three main factors contribute to the biocidal effect: the toxic-
ity of the active principle for the target organism, the dose, and
the time of contact [25]. The cytotoxic effect is directly or indi-
rectly tied to plasma membranes damages. Eugenol [CAS 97-53-
0] interacts with bacterial cell membranes affecting the transport
of ions and ATP, inhibiting the enzymatic activities and, reducing
fungal protein synthesis, DNA replication, and spore lysis [86]. Car-
vacrol [CAS 499-75-2] affects the structure and function of bacte-
rial and fungal cell membranes [83,85]. The lipophilic nature of us-
nic acid [CAS 7652-61-0] promotes its cytotoxic effect. Being highly
membrane-permeable, usnic acid easily diffuses through biological
membranes, resulting in the breakdown of proton gradients [87].
The B1 (OEO-based) biocidal activity, due the low content in car-
vacrol (1.28 g L-1; 13.17 mM), could be confidently related to the
other major components (e.g., o-cymene α -pinene, β -pinene). Fur-
ther investigations are necessary because their biocidal power and
mechanism of action are missing.
The efficacy trials performed on gneiss and granite lead to sim-
ilar outcomes with a drastic decrease of the viable cells since the
first application (up to 95%). This did not happen in macco tri-
als where not significative changes with controls were recorded
after the first application. This could be tied to the species in-
volved or, most confidently, to the contact time in the different
substrates. Being macco stone characterized by high porosity, the
biocidal product could penetrate in the substrate even far from
the colonized surface, and successive applications were confidently
necessary to record significant biocidal effects. The same could be
assessed for the usnic acid water-based formulation whose lead to
some effect on gneiss and granite (better on the former) and none
on macco trials.
5.2.3. The importance of emulsifier in EOs-based formulates
All commercial biocides contain minority chemicals not spec-
ified in their composition but included in the patent of the com-
mercial formulas [4]. These could contribute to the product efficacy
or not. A poor balance between active and non-active ingredients
can support a very active growth especially if the non-active in-
gredients work as a carbon and energy source [85]. This happened
on macco when the CBO formulation (B2, Stone Keeper) has been
applied leading to a tertiary bioreceptivity [88].
Stone Keeper formulation (B2), characterized by a higher con-
tent in emulsifiers (Tween-20 and Span 20), was used to ensure a
more homogeneous application of the product on a highly porous
surface. The surfactants were confidently used as nutrients by the
D. Isola, F. Bartoli, A. Casanova Municchia et al.
survived settlers since Tween 20, for example, is commonly used
to assess the fungal/bacterial esterase activity [24] in plate as-
says. A similar response has been recorded through biolumines-
cent assays. Indeed, gneiss treated with B2/b (having only Tween-
20 as emulsifier) evidenced after four weeks from treatment an
increased metabolic activity up to 42 times on gneiss and 4 times
on granite. The new biocidal products design should then consider
that eco-friendly chemicals could support microbial life; the non-
active chemicals should be reduced as what does not kill them
should not feed.
5.2.4. The importance of the solvent
Water content in cultural heritage materials and temperature
represents the most important factors leading to physical, chem-
ical and microbial deterioration [1]. The RH in underground sites
is generally high, but it could be subject to fluctuations depend-
ing on, for example, ground depth, rock substrate, and even their
orientation [11].
The sudden introduction of water in a so fragile confined envi-
ronment could be deleterious if organic material is present and not
accompanied by a strong biocidal action, where it could be used
for microbial metabolism. IPA [CAS 67-63-0] and DMSO [CAS 67-
68-5], instead, aroused great interest because can produce a bio-
cidal/sublethal effect on microorganisms acting on biomembranes.
Both are used in several fields, including medical applications on
humans [89,90] due to their relatively low acute toxicity (oral LD50
in rats is 5045 mg Kg-1 and 14,500 mg Kg-1 for IPA and DMSO re-
spectively), and DMSO ability to carry other substances in dermal
applications [91].
Considering the different biocidal outcomes produced by B3
(usnic acid water solution) and B4 (usnic acid in IPA/DMSO)
in macco trials, the contribution given by the solvent is sig-
nificant. Furthermore, considering water could promote life and
calcite crystallization in underground environments, the solvent
IPA/DMSO mixture (if not interfering with colours or materials) de-
serves consideration.
6. Conclusions
The tested green biocides gave variable outcomes for inter-
ference with pigments and substrata, and for efficiency. Recon-
struction experiments were fundamental in preventing side effects
on materials (e.g., colour changes), biodeteriogens spreanding and
allowing treatment customization. The biocidal power combined
with the effects of emulsifier and solvents needs to be considered
before any treatment. Full details on the EO composition are ba-
sic for protocol standardization (experiment comparability) and to
assess their properties.
Funding
This research was funded by a grant from the Italian Ministry
Foreign Affairs and International Cooperation Program “Research of
conservation environment and eco-friendly damage control of cul-
tural heritage Korea and Italy”, grant number PGR06686 and sup-
ported by the International Research & Development Program of
the National Research Foundation of Korea (NRF) funded by the
Ministry of Science and ICT, grant number 2019K1A3A1A250 0 0303.
Acknowledgments
The authors wish to thank Adele Cecchini and Massimo Legni
(Ass. Amici Tombe di Taquinia) and the Direzione Regionale Musei
Lazio (Daniela De Angelis, Direttrice del Sito UNESCO di Cerveteri
e Tarquinia) and the Soprintendenza Archeologia Belle Arti e Pae-
saggio per l’Area Metropolitana di Roma, la Provincia di Viterbo e
124
Journal of Cultural Heritage 61 (2023) 116–126
l’Etruria Meridionale (Daniele Federico Maras), Buyeogun, Baekje
World Heritage Center for sharing information and pictures of
restoration activities.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.culher.2023.03.009.
References
[1] G. Caneva, M.P. Nugari, O. Salvadori, Plant Biology for Cultural Heritage: Biode-
terioration and Conservation, The Getty Conservation Institute, Los Angeles,
CA, 2008.
[2] L. Bruno, D. Billi, S. Bellezza, P. Albertano, Cytomorphological and genetic char-
acterization of troglobitic Leptolyngbya strains isolated from Roman hypogea,
Appl. Environ. Microbiol. 75 (2009) 608–617, doi:10.1128/AEM.01183-08.
[3] F. Bastian, C. Alabouvette, C. Saiz-Jimenez, Bacteria and free-living amoeba in
the Lascaux Cave, Res. Microbiol. 160 (2009) 38–40, doi:10.1016/j.resmic.2008.
10.001.
[4] G. Caneva, D. Isola, H.J. Lee, Y.J. Chung, Biological risk for hypogea: shared data
from Etruscan tombs in Italy and ancient tombs of the Baekje dynasty in Re-
public of Korea, Appl. Sci. 10 (2020) 6104, doi:10.3390/app10176104.
[5] D. Isola, L. Zucconi, A. Cecchini, G. Caneva, Dark-pigmented biodeteriogenic
fungi in Etruscan hypogeal tombs: new data on their culture-dependent diver-
sity, favouring conditions, and resistance to biocidal treatments, Fungal Biol.
125 (2021) 609–620, doi:10.1016/j.funbio.2021.03.003.
[6] Y. Ma, H. Zhang, Y. Du, T. Tian, T. Xiang, X. Liu, F. Wu, L. An, W. Wang,
J.D. Gu, H. Feng, The community distribution of bacteria and fungi on an-
cient wall paintings of the Mogao Grottoes, Sci. Rep. 5 (2015) 7759 1-9,
doi:10.1038/srep07752.
[7] D. He, F. Wu, W. Ma, Y. Zhang, J.D. Gu, Y. Duan, R. Xu, H. Feng, W. Wang,
S.W. Li, Insights into the bacterial and fungal communities and microbiome
that causes a microbe outbreak on ancient wall paintings in the Maijishan
Grottoes, Int. Biodeterior. Biodegradation 163 (2021) 105250, doi:10.1016/j.
ibiod.2021.105250.
[8] I. Domínguez-Moñino, V. Jurado, M.A. Rogerio-Candelera, B. Hermosin, Human
impact on show caves: chewing gum stuck to the walls, in: M.A.R. Candel-
era (Ed.), The Conservation of Subterranean Cultural Heritage, CRC Press, 2014,
pp. 247–252, doi:10.1201/b17570-30.
[9] C. Saiz-Jimenez, The Conservation of Subterranean Cultural Heritage, CRC Press,
London, 2014.
[10] M. Diaz-Herraiz, V. Jurado, S. Cuezva, L. Laiz, P. Pallecchi, P. Tiano, S. Sanchez-
Moral, C. Saiz-Jimenez, Deterioration of an Etruscan tomb by bacteria from the
order Rhizobiales, Sci. Rep. 4 (2014) 3610, doi:10.1038/srep03610.
[11] G. Caneva, S. Langone, F. Bartoli, A. Cecchini, C. Meneghini, Vegetation cover
and tumuli’s shape as affecting factors of microclimate and biodeterioration
risk for the conservation of Etruscan tombs (Tarquinia, Italy), Sustainability 13
(2021), doi:10.3390/su13063393.
[12] D. Isola, F. Bartoli, S. Langone, S. Ceschin, L. Zucconi, G. Caneva, Plant DNA
barcode as a tool for root identification in hypogea: the case of the Etr-
uscan tombs of Tarquinia (Central Italy), Plants 10 (2021) 1138, doi:10.3390/
plants10061138.
[13] W. Wang, X. Ma, Y. Ma, L. Mao, F. Wu, X. Ma, L. An, H. Feng, Molecular charac-
terization of airborne fungi in caves of the Mogao Grottoes, Dunhuang, China,
Int. Biodeterior. Biodegradation 65 (2011) 726–731, doi:10.1016/j.ibiod.2011.04.
006.
[14] F. Cappitelli, C. Cattò, F. Villa, The control of cultural heritage microbial deteri-
oration, Microorganisms 8 (2020) 1–20, doi:10.3390/microorganisms8101542.
[15] S.E. Favero-Longo, H.A. Viles, A review of the nature, role and control of
lithobionts on stone cultural heritage: weighing-up and managing biodete-
rioration and bioprotection, World J. Microbiol. Biotechnol. 36 (2020) 1–18,
doi:10.1007/s11274- 020- 02878- 3.
[16] A. Cirigliano, M.C. Tomassetti, M. Di Pietro, F. Mura, M.L. Maneschi, M.D. Gen-
tili, B. Cardazzo, C. Arrighi, C. Mazzoni, R. Negri, T. Rinaldi, Calcite moonmilk
of microbial origin in the Etruscan Tomba degli Scudi in Tarquinia, Italy, Sci.
Rep. 8 (2018) 15839 1-10, doi:10.1038/s41598- 018- 34134-y.
[17] C. Lepinay, A. Mihajlovski, S. Touron, D. Seyer, F. Bousta, P. Di Martino, Bacterial
diversity associated with saline efflorescences damaging the walls of a French
decorated prehistoric cave registered as a World Cultural Heritage Site, Int.
Biodeterior. Biodegradation 130 (2018) 55–64, doi:10.1016/j.ibiod.2018.03.016.
[18] A. Cirigliano, F. Mura, A. Cecchini, M.C. Tomassetti, D.F. Maras, M. Di Paola,
N. Meriggi, D. Cavalieri, R. Negri, A. Quagliariello, J.E. Hallsworth, T. Rinaldi,
Active microbial ecosystem in Iron-Age tombs of the Etruscan civilization, En-
viron. Microbiol. 23 (2021) 3957–3969, doi:10.1111/1462-2920.15327.
[19] T. Kiyuna, K.D. An, R. Kigawa, C. Sano, J. Sugiyama, Two new Cladophialophora
species, C. tumbae sp. nov. and C. tumulicola sp. nov., and chaetothyrialean
fungi from biodeteriorated samples in the Takamatsuzuka and Kitora Tumuli,
Mycoscience 59 (2018) 75–84, doi:10.1016/j.myc.2017.08.008.
[20] W. Ma, F. Wu, T. Tian, D. He, Q. Zhang, J.D. Gu, Y. Duan, D. Ma, W. Wang,
H. Feng, Fungal diversity and its contribution to the biodeterioration of mural
paintings in two 1700-year-old tombs of China, Int. Biodeterior. Biodegradation
152 (2020) 104972, doi:10.1016/j.ibiod.2020.104972.
D. Isola, F. Bartoli, A. Casanova Municchia et al.
[21] H.J. Lee, in: Growth Characteristics and Control Methods of the Microorgan-
isms in Mural Tombs, Korea National University of Cultural Heritage, Buyeo,
2021, p. 221.
[22] M.A. Kakakhel, F. Wu, J.D. Gu, H. Feng, K. Shah, W. Wang, Controlling biodete-
rioration of cultural heritage objects with biocides: a review, Int. Biodeterior.
Biodegradation 143 (2019) 104721, doi:10.1016/j.ibiod.2019.104721.
[23] D. Isola, F. Bartoli, P. Meloni, G. Caneva, L. Zucconi, Black fungi and stone her-
itage conservation: ecological and metabolic assays for evaluating coloniza-
tion potential and responses to traditional biocides, Appl. Sci. 12 (2022) 2038,
doi:10.3390/app12042038.
[24] D. Isola, L. Selbmann, P. Meloni, E. Maracci, S. Onofri, L. Zucconi, Detrimen-
tal rock black fungi and biocides: a study on the Monumental Cemetery of
Cagliari, in: M.A. Rogerio-Candelera, M. Lazzari, E. Cano (Eds.), Science and
Technology for the Conservation of Cultural Heritage, CRC Press, London, 2013,
pp. 83–86.
[25] G. Caneva, M.P. Nugari, S. Pinna, O. Salvadori, Il Controllo Del Degrado Biologi-
co- I Biocidi Nel Restauro Dei Materiali Lapidei, Ed. Nardini, Firenze (Collana
Arte e Restauro), 1996 1-200. ISBN 88-404-4039-9.
[26] F. Cappitelli, F. Villa, in: Novel antibiofilm non-biocidal strategies. Microor-
ganisms in the Deterioration and Preservation of Cultural Heritage, Springer
Nature Switzerland AG, Cham, Switzerland, 2021, pp. 117–135, doi:10.1007/
978- 3- 030- 69411- 1_5. pagEdith Joseph (Ed).
[27] C. Zhang, F. Cui, G.M. Zeng, M. Jiang, Z.Z. Yang, Z.G. Yu, M. Khu, L.Q. Shen, Qua-
ternary ammonium compounds (QACs): a review on occurrence, fate and tox-
icity in the environment, Sci. Total Environ. 518 (2015) 352–362, doi:10.1016/j.
scitotenv.2015.03.007.
[28] D. Pinna, in: Coping with Biological Growth on Stone Heritage Objects: Meth-
ods, Products, Applications, and Perspectives, Apple Academic Press, New York,
2017, p. 382, doi:10.1201/9781315365510.
[29] R. Zarzuela, M. Luna, L.A. Carrascosa, M.J. Mosquera, Preserving cul-
tural heritage stone: innovative consolidant, superhydrophobic, self-clean-
ing, and biocidal products, in: M. Hosseini, I. Karapanagiotis (Eds.), Ad-
vanced Materials for the Conservation of Stone, Springer, Cham, 2018, pp.
259–275.
[30] S. Lo Schiavo, F. De Leo, C. Urzì, Present and future perspectives for biocides
and antifouling products for stone-built cultural heritage: ionic liquids as a
challenging alternative, Appl. Sci. 10 (2020) 6568, doi:10.3390/APP10186568.
[31] EU 528/2012 European Union, Regulation (EU) No 528/2012 of the European
Parliament and of the Council of 22 May 2012 concerning the making available
on the market and use of biocidal products, Off. J. Eur. Union Lett. 167 (2012)
1–123.
[32] UNESCO, APSARA. 2012. Angkor Charter -guidelines for Safeguarding the World
heritage site of Angkor, 5° Siem Reap, Cambodia (December 2012).
[33] Park, J.G., 2014. Enactment of New Biocides Regulation in Korea, Research Re-
port, 1037-1215.
[34] United States Environmental Protection Agency USA EPA, 2017 https://www.
epa.gov/pesticides.
[35] M. La Russa, A. Macchia, S.A. Ruffolo, F. De Leo, M. Barberio, P. Barone,
G.M. Crisci, C. Urzi`, Testing the antibacterial activity of doped TiO2 for pre-
venting biodeterioration of cultural heritage building materials, Int. Biodete-
rior. Biodegradation 96 (2014) 87–96, doi:10.1016/j.ibiod.2014.10.002.
[36] C. Urzì, F. De Leo, L. Krakova, D. Pangallo, L. Bruno, Effects of biocide treat-
ments on the biofilm community in Domitilla’s catacombs in Rome, Sci. Total
Environ. 572 (2016) 252–262, doi:10.1016/j.scitotenv.2016.07.195.
[37] L. Rugnini, G. Migliore, F. Tasso, N.T.W. Ellwood, A.R. Sprocati, L. Bruno, Biocidal
activity of phyto-derivative products used on phototrophic biofilms growing on
stone surfaces of the domus Aurea in Rome (Italy), Appl. Sci. 10 (2020) 6584,
doi:10.3390/app10186584.
[38] P.M. Martin-Sanchez, A. Nováková, F. Bastian, C. Alabouvette, C. Saiz-Jimenez,
Use of biocides for the control of fungal outbreaks in subterranean environ-
ments: the case of the Lascaux Cave in France, Environ. Sci. Technol. 46 (2012)
3762–3770, doi:10.1021/es2040625.
[39] S. Sasso, A.Z. Miller, M.A. Rogerio-Candelera, B. Cubero, M.L. Coutinho,
L. Scrano, S.A. Bufo, Potential of natural biocides for biocontrolling pho-
totrophic colonization on limestone, Int. Biodeterior. Biodegradation 107 (2016)
102–110, doi:10.1016/j.ibiod.2015.11.017.
[40] M. Veneranda, L. Blanco-Zubiaguirre, G. Roselli, G. Di Girolami, K. Castro,
J.M. Madariaga, Evaluating the exploitability of several essential oils con-
stituents as a novel biological treatment against cultural heritage biocoloniza-
tion, Microchem. J. 138 (2018) 1–6, doi:10.1016/j.microc.2017.12.019.
[41] M.R. Fidanza, G. Caneva, Natural biocides for the conservation of stone cultural
heritage: a review, J. Cult. Herit. 38 (2019) 271–286, doi:10.1016/j.culher.2019.
01.005.
[42] KNUCH, in: Research of Conservation Environment and Eco-Friendly Damage
Control of Cultural Heritage Korea and Italy, National Research Foundation of
Republic of Korea, 2020, p. 2020.
[43] A. Ebadollahi, M. Ziaee, F. Palla, Essential oils extracted from different species
of the Lamiaceae plant family as prospective bioagents against several detri-
mental pests, Molecules 25 (2020) 1556, doi:10.3390/molecules25071556.
[44] L. Gatti, F. Troiano, V. Vacchini, F. Cappitelli, A. Balloi, An in vitro evaluation
of the biocidal effect of Oregano and Cloves’ volatile compounds against mi-
croorganisms colonizing an oil painting—a pioneer study, Appl. Sci. 11 (2021)
78, doi:10.3390/app11010078.
[45] S.H. Jeong, H.J. Lee, D.W. Kim, Y.J. Chung, New biocide for eco-friendly biofilm
removal on outdoor stone monuments, Int. Biodeterior. Biodegradation 131
(2018) 19–28, doi:10.1016/j.ibiod.2017.03.004.
125
Journal of Cultural Heritage 61 (2023) 116–126
[46] C. Genova, E. Fuentes, P. Sanmartín, G. Favero, B. Prieto, Phytochemical com-
pounds as cleaning agents on granite colonized by phototrophic subaerial
biofilms, Coatings 10 (2020) 295, doi:10.1016/j.ibiod.2012.05.033.
[47] I. Romano, G. Granata, A. Poli, I. Finore, E. Napoli, C. Geraci, Inhibition of
bacterial growth on marble stone of 18th century by treatment of nanoen-
capsulated essential oils, Int. Biodeterior. Biodegradation 148 (2020) 104909,
doi:10.1016/j.ibiod.2020.104909.
[48] A.A. Argyri, A.I. Doulgeraki, E.G. Varla, V.C. Bikouli, P.I. Natskoulis,
S.A. Haroutounian, G.A. Moulas, C.C. Tassou, N.G. Chorianopoulos, Evalua-
tion of plant origin essential oils as herbal biocides for the protection of caves
belonging to natural and cultural heritage Sites, Microorganisms 9 (2021)
1836, doi:10.3390/microorganisms9091836.
[49] P. Cremonesi, A. Casoli, Enzymes as tools for conservation of works of art, J.
Cult. Herit. 50 (2021) 73–87, doi:10.1016/j.culher.2021.06.005.
[50] F. Gabriele, A. Vetrano, L. Bruno, C. Casieri, R. Germani, L. Rugnini, N. Spreti,
New oxidative alginate-biocide hydrogels against stone biodeterioration,
Int. Biodeterior. Biodegradation 163 (2021) 105281, doi:10.1016/j.ibiod.2021.
105281.
[51] E. Marin, C. Vaccaro, M. Leis, Biotechnology applied to historic stoneworks con-
servation: testing the potential harmfulness of two biological biocides, Int. J.
Conserv. Sci. 7 (2016) 227–238.
[52] G. Ranalli, E. Zanardini, Biocleaning on cultural heritage: new frontiers of mi-
crobial biotechnologies, J. Appl. Microbiol. (2021), doi:10.1111/jam.14993.
[53] G. Toreno, D. Isola, P. Meloni, G. Carcangiu, L. Selbmann, S. Onofri, G. Caneva,
L. Zucconi, Biological colonization on stone monuments: a new low impact
cleaning method, J. Cult. Herit. 30 (2018) 100–109, doi:10.1016/j.culher.2017.
09.004.
[54] M. Mascalchi, C. Orsini, D. Pinna, B. Salvadori, S. Siano, C. Riminesi, Assessment
of different methods for the removal of biofilms and lichens on gravestones of
the English Cemetery in Florence, Int. Biodeterior. Biodegradation 154 (2020)
105041, doi:10.1016/j.ibiod.2020.105041.
[55] S.E. Favero-Longo, E. Matteucci, D. Pinna, M.G. Ruggiero, C. Riminesi, Efficacy of
the environmentally sustainable microwave heating compared to biocide ap-
plications in the devitalization of phototrophic communities colonizing rock
engravings of Valle Camonica, UNESCO world heritage site, Italy, Int. Biodete-
rior. Biodegradation 165 (2021) 105327, doi:10.1016/j.ibiod.2021.105327.
[56] KNUCHResearch of Conservation Environment and Eco-Friendly Damage Con-
trol of Cultural Heritage Korea and Italy, National Research Foundation of Re-
public of Korea, Seoul, Korea, 2021.
[57] C. Gazzano, S.E. Favero-Longo, P. Iacomussi, R. Piervittori, Biocidal effect
of lichen secondary metabolites against rock-dwelling microcolonial fungi,
cyanobacteria and green algae, Int. Biodeterior. Biodegradation 84 (2013) 300–
306, doi:10.1016/j.ibiod.2012.05.033.
[58] J.M. Lee, S.S. Hur, Isolation and identification of antifungal compounds from
Eugenia caryophyllata extracts, J. Korean Appl. Sci. Technol. 31 (4) (2014) 740–
747, doi:10.12925/jkocs.2014.31.4.740.
[59] M. Stupar, M.L. Grbić, A. Džamić, N. Unković, M. Ristić, A. Jelikić, J. Vukojević,
Antifungal activity of selected essential oils and biocide benzalkonium chloride
against the fungi isolated from cultural heritage objects, S. Afr. J. Bot. 93 (2014)
118–124, doi:10.1016/j.sajb.2014.03.016.
[60] A. Cecchini, Le tombe dipinte di Tarquinia. Vicenda Conservativa, Restauri e
Tecnica di Esecuzione, Nardini Editore, Florence, Italy (2012) 15–102.
[61] D. Gadia, C. Bonanomi, M. Marzullo, A. Rizzi, Perceptual enhancement of de-
graded Etruscan wall paintings, J. Cult. Herit. 21 (2016) 904–909, doi:10.1016/
j.culher.2016.04.009.
[62] J.H. Park, G.H. Lee, C.H. Lee, Consideration for historical application of augen
gneiss and petrographic characteristics for rock properties of Donghachong
tomb from royal tombs of Neungsanri in Buyeo, Korea, Econ. Environ. Geol.
52 (1) (2019) 91–106, doi:10.9719/EEG.2019.52.1.91.
[63] Buyeo-gun, The Investigation on the Conservation Environment of the Ancient
Tomb No. 1 in Neungsan-ri, 2008, p. 334. Korea.
[64] H.S. Lee, K.S. Han, Y.J. Chung, Publication Institution: KNUCH, Korea National
University of Cultural Heritage Printing office name, JeongJin, 2015 Page:
1∼227.
[65] Buyeo-gun·Baekje World Heritage Center, 2016. Monitoring on conservation
environment of the Ancient Tomb No. 1 in Neungsan-ri, Research Report, p.
139.
[66] Buyeo-gun·Baekje World Heritage Center, 2017. Continuous monitoring of the
Ancient Tomb No. 1 in Neungsan-ri, Buyeo, Research Report, p. 166.
[67] Buyeo-gun·Baekje World Heritage Center, 2018. Continuous monitoring of the
Ancient Tomb No. 1 in Neungsan-ri, Buyeo, Research Report, p. 171.
[68] N.S. Lee, in: Baekje Kingdom in Neungsan-ri Tomb at Buyeo, The Research In-
stitute of Baekje Culture, Gongju City, Korea, 20 0 0, pp. 1–24.
[69] K. Ingolfsdottir, Usnic acid, Phytochemistry 61 (2002) 729–736, doi:10.1016/
S0 031-9422(02)0 0383-7.
[70] M.E.S. Osman, A.A.E.N. El-Shaphy, M.M. Ayid, Evaluation of the inhibitory effect
of dimethyl sulfoxide on fungal degradated archaeological wood, Int. J. Con-
serv. Sci. 8 (2017) 431–440.
[71] UNI EN 15886:2010. Conservation of cultural property - test methods - colour
measurement of surfaces ICS: 97.195.
[72] UNI EN 17655:2022 Conservation of cultural heritage - determination of water
absorption by contact sponge method.
[73] UNI 10923, 2001. Beni culturali: Materiali lapidei naturali ed artificiali. Allesti-
mento di Preparati Biologici per l’osservazione al Microscopio Ottico.
[74] Normal 8/81, 1981. Esame delle caratteristiche morfologiche al microscopio
elettronico a scansione (SEM).
D. Isola, F. Bartoli, A. Casanova Municchia et al.
[75] D. Tapete, B. Mazzei, F. Fratini, C. Riminesi, R.M. Del Fà, E. Cantisani, B. Sac-
chi, O.A. Cuzman, M.G. Patrizi, L. Scaletti, P. Tiano, Monitoring hypogeum sys-
tems affected by crystallisation processes among conservation needs, microcli-
mate factors and accessibility to heritage, Conservazione e Valorizzazione dei
siti archeologici: approcci scientifici e problemi di metodo (2013).
[76] D. Tapete, F. Fratini, B. Mazzei, E. Cantisani, E. Pecchioni, Petrographic study of
lime-based mortars and carbonate incrustation processes of mural paintings in
Roman catacombs, Periodico di Mineralogia 82 (2014) 503.
[77] N.A. Wojas, A. Swerin, V. Wallqvist, M. Jarn, J. Schoelkopf, P.A. Gane, P.M. Claes-
son, Surface-modified and unmodified calcite: effects of water and saturated
aqueous octanoic acid droplets on stability and saturated fatty acid layer or-
ganization, Langmuir 37 (48) (2021) 14135–14146, doi:10.1021/acs.langmuir.
1c02387.
[78] R. Mazzeo, E. Joseph, V. Minguzzi, G. Grillini, P. Baraldi, D. Prandstraller, Sci-
entific investigations of the Tokhung-Ri tomb mural paintings (408 AD) of the
Koguryo era, Democratic People’s Republic of Korea, J. Raman Spectrosc. 37
(2006) 1086–1097, doi:10.1002/jrs.1592.
[79] P. Pallecchi, G. Giachi, M.P. Colombini, F. Modugno, E. Ribechini, The painting
of the Etruscan “Tomba della Quadriga Infernale” (4th century BC), in Sarteano
(Siena, Italy): technical features, J. Archaeol. Sci. 36 (12) (2009) 2635–2642,
doi:10.1016/j.jas.2009.07.019.
[80] K. Myszka, M.T. Schmidt, M. Majcher, W. Juzwa, M. Olkowicz, K. Cza-
czyk, Inhibition of quorum sensing-related biofilm of Pseudomonas fluorescens
KM121 by Thymus vulgare essential oil and its major bioactive compounds,
Int. Biodeterior. Biodegradation 114 (2016) 252–259, doi:10.1016/j.ibiod.2016.
07.006.
[81] V. Vinciguerra, F. Rojas, V. Tedesco, G. Giusiano, L. Angiolella, Chemical char-
acterization and antifungal activity of Origanum vulgare, Thymus vulgaris es-
sential oils and carvacrol against Malassezia furfur, Nat. Prod. Res. 33 (2019)
3273–3277, doi:10.1080/14786419.2018.1468325.
[82] D. Pinna, Can we do without biocides to cope with biofilms and lichens on
stone heritage? Int. Biodeterior. Biodegradation 172 (2022) 105437, doi:10.
1016/j.ibiod.2022.
126
Journal of Cultural Heritage 61 (2023) 116–126
[83] F.C. De Medeiros, F.N. Gouveia, H.R. Bizzo, R.F. Vieira, C.H. Del Menezzi, Fungi-
cidal activity of essential oils from Brazilian Cerrado species against wood de-
cay fungi, Int. Biodeterior. Biodegradation 114 (2016) 87–93, doi:10.1016/j.ibiod.
2016.06.003.
[84] B. Prieto, P. Sanmartín, J. Cancelo-González, L. Torres, B. Silva, Impact of her-
bicide treatments on the construction materials in the Roman wall of Lugo,
Spain (UNESCO World Heritage Site), Appl. Sci. 11 (2021) 5276, doi:10.3390/
app11115276.
[85] G. Zhang, C. Gong, J. Gu, Y. Katayama, T. Someya, J.D. Gu, Biochemical reac-
tions and mechanisms involved in the biodeterioration of stone world cultural
heritage under the tropical climate conditions, Int. Biodeterior. Biodegradation
143 (2019) 104723, doi:10.1016/j.ibiod.2019.104723.
[86] F. Antonelli, M. Bartolini, M.L. Plissonnier, A. Esposito, G. Galotta, S. Ricci,
B. Davidde Petriaggi, C. Pedone, A. Di Giovanni, S. Piazza, F. Guerrieri, M. Ro-
magnoli, Essential oils as alternative biocides for the preservation of wa-
terlogged archaeological wood, Microorganisms 8 (2020) 2015, doi:10.3390/
microorganisms8122015.
[87] Y.N. Antonenko, L.S. Khailova, T.I. Rokitskaya, E.S. Nosikova, P.A. Nazarov,
O.A. Luzina, N.F. Salakhudinov, E.A. Kotova, Mechanism of action of an old
antibiotic revisited: role of calcium ions in protonophoric activity of usnic
acid, Biochimica et Biophysica Acta (BBA)-Bioenergetics 1860 (2019) 310–316,
doi:10.1016/j.bbabio.2019.01.005.
[88] P. Sanmartín, A.Z. Miller, B. Prieto, H.A. Viles, Revisiting and reanalysing the
concept of bioreceptivity 25 years on, Sci. Total Environ. 770 (2021) 145314,
doi:10.1016/j.scitotenv.2021.145314.
[89] N.C. Santos, J. Figueira-Coelho, J. Martins-Silva, C. Saldanha, Multidisciplinary
utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular as-
pects, Biochem. Pharmacol. 65 (2003) 1035–1041, doi:10.1016/S0006-2952(03)
0 0 0 02.
[90] J.M. Boyce, Alcohols as surface disinfectants in healthcare settings, Infect. Con-
trol Hosp. Epidemiol. 39 (2018) 323–328, doi:10.1017/ice.2017.301.
[91] S.W. Jacob, D.C. Wood, Dimethyl sulfoxide (DMSO) toxicology, pharmacology,
and clinical experience, Am. J. Surg. 114 (1967) 414–426.