Ci a ion: Cos an ini, I.; Cas o, K.;
Mada iaga, J.M.; A ana, G. Analy ical
Techniques Applied o he S udy o
Indus ial A chaeology He i age:
The Case o Plaiko Zubixe Foo b idge.
Molecules 2022,27, 3609. h ps://
doi.o g/10.3390/molecules27113609
Academic Edi o s: Ma ia Luisa
As ol i, Ma ia Pia Samma ino and
Emanuele Dell’Aglio
Recei ed: 8 May 2022
Accep ed: 1 June 2022
Published: 4 June 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
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4.0/).
molecules
A icle
Analy ical Techniques Applied o he S udy o Indus ial
A chaeology He i age: The Case o Plaiko Zubixe Foo b idge
Ila ia Cos an ini *, Kepa Cas o, Juan Manuel Mada iaga and Go ka A ana
Depa men o Analy ical Chemis y, Facul y o Science and Technology, Uni e si y o he Basque
Coun y UPV/EHU, P.O. Box 644, 48080 Bilbao, Spain; kepa.cas [email p o ec ed] (K.C.);
[email p o ec ed] (J.M.M.); [email p o ec ed] (G.A.)
*Co espondence: [email p o ec ed]
Abs ac :
In his wo k, mic o-Raman spec oscopy and mic o-ene gy-dispe si e X- ay luo escence
spec oscopy (
µ
-EDXRF) we e applied on mic osamples aken om he Plaiko Zubixe oo b idge
(1927) loca ed in Onda oa (Basque Coun y, Spain) in o de o in es iga e he o iginal pain coa ing
and make an e alua ion o he conse a ion s a e be o e i s es o a ion. Elemen al and molecula
images we e acqui ed o he s udy o he compounds dis ibu ion. Some mode n pigmen s such as
ph halocyanine blue and g een pigmen s, minium, calcium ca bona e, P ussian blue, and hema i e
we e iden i ied. Ba ium sul a e and i anium dioxide we e ecognized as opaci ie agen s. Thanks
o he s udy o he s a ig aphies, i has been possible o de e mine he o iginal pain laye , which
includes lead whi e, ul ama ine blue, ca bon black, and ba ium sul a e. In addi ion, colo ime ic
analyses made i possible o know he CIELab alues o he o iginal laye in o de o ep oduce he
o iginal colou du ing he planned es o a ion wo k. The massi e p esence o chlo ine de ec ed
by
µ
-EDXRF and he co osion p oduc s o he us laye , in pa icula akaganei e and hema i e,
highligh ed he a mosphe ic impac in he conse a ion o he b idge because hey we e due o he
e ec o bo h ma ine ae osol and o he p esence o acidic componen s in he en i onmen coming
om an h opogenic ac i i y. This wo k demons a ed he use ulness o a scien i ic app oach o he
s udy o indus ial a chaeology he i age wi h he aim o con ibu e o i s conse a ion and es o a ion.
Keywo ds: indus ial he i age; µ-Raman spec oscopy; µ-EDXRF; pigmen s; conse a ion s a e
1. In oduc ion
The a chaeological indus ial he i age is a ela i ely new concep , bo n in he 1970s,
when he need o p ese e he p oo s o indus ializa ion p ocess a e hey had been allen
in o disuse o abandoned was decla ed [
1
]. Indeed, he indus ial he i age conce ns a
pa icula ype o he i age ha includes objec s, in as uc u es, and wo ks c ea ed du ing
he indus ial e olu ion, mainly o p ac ical a he han deco a i e pu poses ha ha e
had a s ong impac on he e i o y and some o which ha e now been ecen ly decla ed
his o ical he i age.
On he one hand, he conse a ion o he indus ial he i age conce ns indi idual
objec s ha can ep esen a symbol o he ci y and i s inhabi an s. On he o he hand,
he ehabili a ion o he indus ial he i age can in ol e en i e u ban a eas, p oducing
an e iden inc ease in ou ism and p omo ing a social, en i onmen al, and economic
de elopmen o he ci ies [
2
,
3
]. The e o e, ega dless o he social impac , he indus ial
he i age conse a ion is unques ionably a opical issue.
I s es o a ion could be ca ied ou wi h scien i ic s anda ds, espec ing he o iginal
appea ance and he economic and echnological en i onmen o he ime, o by c ea ing a
new wo k, a eplica o he exis ing. In o he si ua ions, al hough he o iginal appea ance o
a wo k was di e en , he au ho i ies can decide o es o e he appea ance ha he wo k
had in he las ew yea s. These la e cases a e mainly linked o social o cul u al easons.
Molecules 2022,27, 3609. h ps://doi.o g/10.3390/molecules27113609 h ps://www.mdpi.com/jou nal/molecules
Molecules 2022,27, 3609 2 o 15
Howe e , a scien i ic diagnos ic app oach, h ough he use o diagnos ic echniques,
capable o gaining knowledge o he echnologies and ma e ials used, has no been widely
adop ed in his ield o esea ch as has been he case o o he ypes o wo ks o a [
4
–
6
].
Cu en ly, he e a e no many examples o scien i ic esea ch in he li e a u e conce ning
he s udy o indus ial he i age. One o he mos ecen is he wo k by Tisso e al. [
7
] on
he pain coa ings o h ee ene gy gene a o s om he ea ly 20 h-cen u y powe plan a
Le ada de Toma (Po ugal) ha shows he impo ance o applying a scien i ic-diagnos ic
me hod e en o he s udy o objec s belonging o he indus ial e olu ion [5].
The conse a ion o he indus ial cul u al he i age is s ongly in luenced by he
en i onmen in which he objec is loca ed. Bo h me al and s eel, o which he indus ial
he i age is mainly composed, a e conside ed among he mos esis an ma e ials, and
o his eason hey ha e been used o he cons uc ion o b idges and in as uc u es.
Despi e his, i hey a e loca ed nea sou ces o humidi y o en i onmen s wi h high
ela i e humidi y alues, hey can su e om as e oxida ion p ocesses o e he yea s.
In pa icula , a ma ine a mosphe e is one o he mos co osi e en i onmen s o me allic
s uc u es due o he in luence o ma ine ae osol. I is composed by o ganic and ino ganic
ma e dissol ed in wa e and includes p ima y (PMA) and seconda y ae osol (SMA)
pa icles. The p ima y ae osol is composed by suspended sea wa e d ops ich in chlo ide-
ions, gene a ed by he in e ac ion be ween wind and wa es on he su ace o he sea, which
a e deposi ed on he e es ial su aces acco ding o a d y o we deposi ion p ocess [
8
].
The high con en o ai bo ne chlo ides, mainly in he o m o NaCl o KCl, eac ex ensi ely
wi h i on ma e ials [9].
In addi ion, i is well known ha he p esence o SO
2
as well as he ac ion o o he acid
gases, such as NO
x
and CO
2
, can cause he inc ease o he co osion a e in me als h ough
we o d y deposi ion mechanisms [
10
,
11
]. In we deposi ion, he a mosphe ic acid gases e-
ac wi h he humidi y and/o ainwa e , gi ing ise o hei acidic ae osols (H
2
CO
3
, H
2
SO
4
,
and HNO
3
). The acidic na u e o he mois u e ilm deposi ed on he su ace gene a es i s
he oxida ion and hen he dissolu ion o he me al, accele a ing he co osion mechanisms
and he consequen o ma ion o ni a e, sul a e, and ca bona e sal s. In d y deposi ion,
he a mosphe ic gases can eac di ec ly wi h solid pa icles deposi ed on he su aces [
12
].
Mo eo e , in an u ban si e close o he coas , he ma ine ae osol is ich in ai bo ne pa ic-
ula e ma e including me als such as Pb, Cd, C , Mn, Cu, Mo, Rh, Ni, As, Ti, V, and Hg
coming om combus ion p ocesses, a ic, and indus ial ac i i y [
13
]. In addi ion, ac o s
such as u bulence o he ai , chemical a ini y be ween pollu an s, and he ma e ial and
eac i i y o he pollu an s can accele a e he deposi ion phenomena [
10
]. Al hough in some
cases he co osion p oduc s ha e a p o ec i e unc ion [
14
–
16
], in coas al a mosphe es he
p esence o ce ain co osion p oduc s, such as akaganei e (FeO
0.833
(OH)
1.167
Cl
0.167
) [
17
],
can accele a e he co osion a e in me al wo ks o his o ical in e es .
Thus, he ehabili a ion o he i on-building he i age has been necessa y because o he
de e io a ion p oduced by na u al and an h opogenic ac o s ha endange ed hei su i al
and hei use ulness o he socie y o which hey we e designed. This is he case wi h
he Onda oa oo b idge. The oo b idge belongs o he adi ion o mobile i on b idges
buil in nume ous na igable channels. I s pa icula i y o being one o he ew emaining
o a ing b idges p ese ed oday, he only one in Spain, makes i a unique a chi ec u al
elemen wo hy o being p ese ed and a symbol o he coun y. Due o i s p eca ious
s a e o conse a ion, a es o a ion in e en ion was planned, which also had he aim o
es o ing i s o iginal colou .
Thus, he p esen wo k aims a iden i ying he o iginal colou o he oo b idge o
Onda oa wi h a iew o i s u u e es o a ion so ha i would be possible o eco e i s
o iginal appea ance, since he me allic s uc u e has been subjec ed, as a whole, o a ious
ch oma ic changes om i s cons uc ion o he las in e en ions. In addi ion, he impac
o ma ine ae osol and he ha bou en i onmen in he b idge will be documen ed by he
cha ac e iza ion o di e en co osi e compound and bioma ke s.
Molecules 2022,27, 3609 3 o 15
Fo his pu pose, a scien i ic diagnos ic s udy was necessa y. The s udy was ca ied ou
on six mic o samples, i e o hem as c oss-sec ions, by means o elemen al (mic o-ene gy-
dispe si e X- ay luo escence spec oscopy) and molecula analysis (Raman spec oscopy)
a e a ca e ul obse a ion unde an op ical mic oscope. Colo ime ic analyses we e also
pe o med o know he colou alues o he di e en pigmen s used.
2. Resul s and Discussion
2.1. Cha ac e iza ion o he Pain Laye s
Two samples collec ed in di e en a eas in he la ges piece, which belongs o he
low pa o he ailing (Figu e S1a, Supplemen a y Ma e ials) ecei ed in he labo a o y,
p esen ed he same s a ig aphic composi ion; he e o e, only he esul s o one sample a e
shown below. Speci ically, h ee di e en homogeneous laye s, wo ou e mos g een ones
(Figu e S2a,b) and one inne ed/o ange (Figu e S2c), we e ecognized by obse ing he
samples wi h a s e eomic oscope.
Thanks o Raman analyses, i was possible o iden i y he compounds ha cha ac e ize
he di e en pain laye s. The ou e mos laye (laye a in Figu e S2) o da k g een colou
was composed o ph halocyanine g een (C
32
H
3
Cl
13
CuN
8
, Raman bands a 688, 740, 744,
815, 977, 1079, and 1208 cm
−1
, Figu e 1a) [
18
], while he in e media e laye (laye b in
Figu e S2) o ligh e g een colou was made mainly wi h he ph halocyanine blue pigmen
(C
32
H
16
N
8
Cu, Raman bands a 236, 257, 483, 594, 680, 747, 779, 832, 953, 1007, 1108, 1143,
and 1193 cm
−1
, Figu e 1b) [
18
]. In addi ion, in many Raman spec a a e y weak peak a
1038 cm
−1
was isible (Raman band ma ked in he ed ci cle in Figu e 1b). This band could
belong o he yellow azo pigmen PY100 used in small amoun s, mixed wi h ph halocyanine
blue, o ob ain a g een colou . Con a y o he g een colou ed laye s, he inne mos o ange
laye (laye c in Figu e S2) was made by mixing wo compounds, minium (Pb
3
O
4
Raman
bands: 122, 152, 224, 313, 390, and 548 cm
−1
) [
19
] and a smalle amoun o ba ium sul a e
(BaSO
4
, Raman bands: 453, 460, 618, and 987 cm
−1
, Figu e 1c) [
20
] since all he Raman
spec a eco ded in his a ea showed he main ea u es o bo h compounds.
Molecules 2022, 27, x FOR PEER REVIEW 3 o 15
Fo his pu pose, a scien i ic diagnos ic s udy was necessa y. The s udy was ca ied
ou on six mic o samples, i e o hem as c oss-sec ions, by means o elemen al (mic o-
ene gy-dispe si e X- ay luo escence spec oscopy) and molecula analysis (Raman spec-
oscopy) a e a ca e ul obse a ion unde an op ical mic oscope. Colo ime ic analyses
we e also pe o med o know he colou alues o he di e en pigmen s used.
2. Resul s and Discussion
2.1. Cha ac e iza ion o he Pain Laye s
Two samples collec ed in di e en a eas in he la ges piece, which belongs o he
low pa o he ailing (Figu e S1a, Supplemen a y Ma e ials) ecei ed in he labo a o y,
p esen ed he same s a ig aphic composi ion; he e o e, only he esul s o one sample
a e shown below. Speci ically, h ee di e en homogeneous laye s, wo ou e mos g een
ones (Figu e S2a,b) and one inne ed/o ange (Figu e S2c), we e ecognized by obse ing
he samples wi h a s e eomic oscope.
Thanks o Raman analyses, i was possible o iden i y he compounds ha cha ac e -
ize he di e en pain laye s. The ou e mos laye (laye a in Figu e S2) o da k g een col-
ou was composed o ph halocyanine g een (C32H3Cl13CuN8, Raman bands a 688, 740,
744, 815, 977, 1079, and 1208 cm−1, Figu e 1a) [18], while he in e media e laye (laye b in
Figu e S2) o ligh e g een colou was made mainly wi h he ph halocyanine blue pigmen
(C32H16N8Cu, Raman bands a 236, 257, 483, 594, 680, 747, 779, 832, 953, 1007, 1108, 1143,
and 1193 cm−1, Figu e 1b) [18]. In addi ion, in many Raman spec a a e y weak peak a
1038 cm−1 was isible (Raman band ma ked in he ed ci cle in Figu e 1b). This band could
belong o he yellow azo pigmen PY100 used in small amoun s, mixed wi h ph halocya-
nine blue, o ob ain a g een colou . Con a y o he g een colou ed laye s, he inne mos
o ange laye (laye c in Figu e S2) was made by mixing wo compounds, minium (Pb3O4
Raman bands: 122, 152, 224, 313, 390, and 548 cm−1) [19] and a smalle amoun o ba ium
sul a e (BaSO4, Raman bands: 453, 460, 618, and 987 cm−1, Figu e 1c) [20] since all he Ra-
man spec a eco ded in his a ea showed he main ea u es o bo h compounds.
Figu e 1. Raman spec a o he compounds iden i ied in he subsample (SUBS-1a) in c oss-sec ion:
ph halocyanine g een (a), ph halocyanine blue wi h aces o azo pigmen PY100 (in he ed ci cle)
(b), minium plus ba ium sul a e (*) (c).
As can be seen in he Raman spec oscopy images (Figu e 2) ca ied ou conside ing
he band wi h he highes in ensi y o each compound, he g een laye s consis o ph hal-
ocyanine g een (Figu e 2b) and ph halocyanine blue (Figu e 2c), espec i ely. On he o he
hand, he p esence o ba ium sul a e used in mix u e wi h minium was con i med due o
Figu e 1.
Raman spec a o he compounds iden i ied in he subsample (SUBS-1a) in c oss-sec ion:
ph halocyanine g een (
a
), ph halocyanine blue wi h aces o azo pigmen PY100 (in he ed ci cle) (
b
),
minium plus ba ium sul a e (*) (c).
As can be seen in he Raman spec oscopy images (Figu e 2) ca ied ou conside ing
he band wi h he highes in ensi y o each compound, he g een laye s consis o ph halo-
cyanine g een (Figu e 2b) and ph halocyanine blue (Figu e 2c), espec i ely. On he o he
hand, he p esence o ba ium sul a e used in mix u e wi h minium was con i med due
Molecules 2022,27, 3609 4 o 15
o he p esence o he wo compounds in he same pic o ial laye , as is e iden om he
o e lapping o he Raman images in Figu e 2e, . The p esence o he yellow azo pigmen
was iden i ied on wo pic o ial laye s in mix u e wi h ph halocyanine blue and e en wi h
minium and ba ium sul a e like in he Raman image shown in Figu e 2d. The Raman map
was ob ained conside ing he band a 1038 cm
−1
, assigned o SO
3−
symme ic s e ch [
21
],
in o de o a oid he o e lapping wi h ba ium sul a e since bo h compounds ha e a Raman
bands in he same posi ion (989 cm−1).
Molecules 2022, 27, x FOR PEER REVIEW 4 o 15
he p esence o he wo compounds in he same pic o ial laye , as is e iden om he
o e lapping o he Raman images in Figu e 2e, . The p esence o he yellow azo pigmen
was iden i ied on wo pic o ial laye s in mix u e wi h ph halocyanine blue and e en wi h
minium and ba ium sul a e like in he Raman image shown in Figu e 2d. The Raman map
was ob ained conside ing he band a 1038 cm−1, assigned o SO3− symme ic s e ch [21],
in o de o a oid he o e lapping wi h ba ium sul a e since bo h compounds ha e a Ra-
man bands in he same posi ion (989 cm−1).
Figu e 2. Raman image o a c oss-sec ional sample (sample SUBS-1a) (50×) collec ed om he ailing
piece (a) and i s molecula composi ion: ph halocyanine g een (b), ph halocyanine blue (c), yellow
azo pigmen (d), ba ium sul a e (e) and minium ( ).
Thus, in his pa o he b idge, a e applying ed lead, ba ium sul a e, plus a yellow
azo pigmen , a g een pain (ph halocyanine blue and yellow azo pigmen ) was applied,
and o e i a da k-g een one (ph halocyanine g een),which was he colou ha is isible
cu en ly. The use o ed lead, cu en ly banned due o i s oxici y, was p obably em-
ployed as an ioxidan pain , ollowing he ules in he second hal o 20 h cen u y[22].
Unlike he p e ious case, he subsamples ha we e collec ed om he piece ha pe mi
he mo emen o he b idge (Figu e S1b) showed a di e en s a ig aphic composi ion om
a i s obse a ion wi h he s e eoscopic mic oscope. The i s analysed subsample (SUBS-
1b), collec ed om a g een a ea, shows ou homogeneous and well-de ined laye s (Figu e
S3). The ou e mos pic o ial laye was en i ely composed o ph halocyanine g een (Figu e
S4a). In he second one, whi e in colou , which p obably ep esen s he p ime laye , u ile
(α-TiO2, Raman bands a: 442 and 608 cm−1) [23]and calcium ca bona e (CaCO3, Raman bands
a : 282 and 1086 cm−1) [23] we e de ec ed, as seen in Figu e S4b. Addi ionally, he inne mos
g een laye o a ligh e shade was composed o a mix u e o P ussian blue (Fe4 [Fe (CN)6]3,
Raman bands a: 277, 364, and 530 cm−1) [24] and ba ium sul a e (Figu e S4c).
Ano he pic o ial laye was ecognized, p e iously applied, hanks o he Raman
spec oscopy images showed in Figu e 3. A i s glance, his laye had he same hue as
he p e ious one and only by obse a ion wi h a s e eoscopic mic oscope i was no pos-
sible o ecognize he wo di e en laye s. Howe e , he Raman images made i possible
o dis inguish one mo e pic o ial laye , en i ely composed o ph halocyanine blue (Figu e
3g). Thus, in his sample ou laye s we e ound, espec i ely, om he oldes o he mos
ecen : ph halocyanine g een, u ile mixed wi h calcium ca bona e, P ussian blue wi h
ba ium sul a e, and ph halocyanine blue.
Figu e 2.
Raman image o a c oss-sec ional sample (sample SUBS-1a) (50
×
) collec ed om he ailing
piece (
a
) and i s molecula composi ion: ph halocyanine g een (
b
), ph halocyanine blue (
c
), yellow
azo pigmen (d), ba ium sul a e (e) and minium ( ).
Thus, in his pa o he b idge, a e applying ed lead, ba ium sul a e, plus a yellow
azo pigmen , a g een pain (ph halocyanine blue and yellow azo pigmen ) was applied,
and o e i a da k-g een one (ph halocyanine g een),which was he colou ha is isible
cu en ly. The use o ed lead, cu en ly banned due o i s oxici y, was p obably employed
as an ioxidan pain , ollowing he ules in he second hal o 20 h cen u y [22].
Unlike he p e ious case, he subsamples ha we e collec ed om he piece ha pe mi
he mo emen o he b idge (Figu e S1b) showed a di e en s a ig aphic composi ion
om a i s obse a ion wi h he s e eoscopic mic oscope. The i s analysed subsample
(SUBS-1b), collec ed om a g een a ea, shows ou homogeneous and well-de ined laye s
(
Figu e S3
). The ou e mos pic o ial laye was en i ely composed o ph halocyanine g een
(Figu e S4a). In he second one, whi e in colou , which p obably ep esen s he p ime
laye , u ile (
α
-TiO
2
, Raman bands a: 442 and 608 cm
−1
) [
23
] and calcium ca bona e
(CaCO
3
, Raman bands a : 282 and 1086 cm
−1
) [
23
] we e de ec ed, as seen in Figu e S4b.
Addi ionally, he inne mos g een laye o a ligh e shade was composed o a mix u e o
P ussian blue (Fe
4
[Fe (CN)
6
]
3
, Raman bands a: 277, 364, and 530 cm
−1
) [
24
] and ba ium
sul a e (Figu e S4c).
Ano he pic o ial laye was ecognized, p e iously applied, hanks o he Raman
spec oscopy images showed in Figu e 3. A i s glance, his laye had he same hue as he
p e ious one and only by obse a ion wi h a s e eoscopic mic oscope i was no possible
o ecognize he wo di e en laye s. Howe e , he Raman images made i possible o
dis inguish one mo e pic o ial laye , en i ely composed o ph halocyanine blue (Figu e 3g).
Thus, in his sample ou laye s we e ound, espec i ely, om he oldes o he mos ecen :
ph halocyanine g een, u ile mixed wi h calcium ca bona e, P ussian blue wi h ba ium
sul a e, and ph halocyanine blue.
Molecules 2022,27, 3609 5 o 15
Molecules 2022, 27, x FOR PEER REVIEW 5 o 15
Figu e 3. Op ical image (a) and Raman spec oscopy image o he sample in sec ion showing i s
molecula composi ion: ph halocyanine g een (b), u ile (c) and calcium ca bona e (d), ba ium sul-
a e (e), P ussian blue ( ), and ph halocyanine blue (g). The black line h ough he sample is a c ack
in he sample.
Consequen ly, he s a ig aphy o his subsample showed ha on a black laye (i s
s a ig aphy is desc ibed in he ollowing subsample), a ph halocyanine blue p ime was
applied (laye g in Figu e 3), on which ano he laye was composed o P ussian blue and
ba ium sul a e (laye e + in Figu e 3). Nex , ano he whi e p ime composed o u ile and
calcium ca bona e was applied (laye c + d in Figu e 3), and inally, a g een laye o ph hal-
ocyanine g een (laye b in Figu e 3), which was he one isible nowadays.
Raman analysis on he su aces o he subsample (SUBS-2b) aken om a black a ea
showed he p esence o ca bon black, homogeneously dis ibu ed h oughou he en i e
su ace. Unde nea h, he e was a he e ogeneous laye consis ing mainly o hema i e ound
in g ains o di e en sizes (Fe2O3, Raman bands a: 222, 240, 290, 405, 490, and 608 cm−1,
Figu e S5a)[25]. In addi ion o i on oxide, he analyses on his laye ha e made i possible
o ecognize a a ious poin s o he Raman spec um cha ac e is ic o a ma e ial com-
posed o silicon and ca bon (simila o silicon ca bide wi e, SiC, wi h Raman bands a : 150,
763, 786, 795, and 964 cm−1, Figu e S5b)[26]. Raman bands belonging o ba ium sul a e
we e also iden i ied in he same laye . The dis ibu ion o he h ee compounds is indi-
ca ed in he Raman images in Figu e S6.
The obse a ion o one o he subsamples (SUBS-3b) aken in he piece shown in Fig-
u e 1b, which is pa o he sys em ha allowed he mo emen o he b idge, allowed us
o ecognize a mo e complex s a ig aphy. This sample was collec ed in he lowe a ea o
he da k piece whe e p obably he o iginal pain ing could emain. The i s ou e laye
was da k in colou applied o e a he e ogeneous ed and whi e laye ha p esen ed g ains
o di e en sizes as in he piece p e iously desc ibed. Addi ionally, a hicke laye o black
colou and ano he o g een colou could be clea ly ecognized. Acco ding o he obse -
a ion wi h he s e eomic oscope, he i s laye applied in he sample was a he e ogene-
ous laye o g ey colou wi h da k and blue g ains as shown in Figu e 4. The de ail o he
blue-g eyish laye , whose hickness was a ound 1.5 mm, is showed in Figu e 4c. In he
lowe pa o he men ioned laye o blue-g ey pain , emains o i on oxide lakes ( eddish
colou ) de ached om he me allic su ace we e also obse ed.
Figu e 3.
Op ical image (
a
) and Raman spec oscopy image o he sample in sec ion showing i s
molecula composi ion: ph halocyanine g een (
b
), u ile (
c
) and calcium ca bona e (
d
), ba ium
sul a e (
e
), P ussian blue (
), and ph halocyanine blue (
g
). The black line h ough he sample is a
c ack in he sample.
Consequen ly, he s a ig aphy o his subsample showed ha on a black laye (i s
s a ig aphy is desc ibed in he ollowing subsample), a ph halocyanine blue p ime was
applied (laye g in Figu e 3), on which ano he laye was composed o P ussian blue and
ba ium sul a e (laye e + in Figu e 3). Nex , ano he whi e p ime composed o u ile
and calcium ca bona e was applied (laye c + d in Figu e 3), and inally, a g een laye o
ph halocyanine g een (laye b in Figu e 3), which was he one isible nowadays.
Raman analysis on he su aces o he subsample (SUBS-2b) aken om a black a ea
showed he p esence o ca bon black, homogeneously dis ibu ed h oughou he en i e
su ace. Unde nea h, he e was a he e ogeneous laye consis ing mainly o hema i e ound
in g ains o di e en sizes (Fe
2
O
3
, Raman bands a: 222, 240, 290, 405, 490, and 608 cm
−1
,
Figu e S5a) [
25
]. In addi ion o i on oxide, he analyses on his laye ha e made i possible
o ecognize a a ious poin s o he Raman spec um cha ac e is ic o a ma e ial composed
o silicon and ca bon (simila o silicon ca bide wi e, SiC, wi h Raman bands a : 150, 763,
786, 795, and 964 cm
−1
, Figu e S5b) [
26
]. Raman bands belonging o ba ium sul a e we e
also iden i ied in he same laye . The dis ibu ion o he h ee compounds is indica ed in
he Raman images in Figu e S6.
The obse a ion o one o he subsamples (SUBS-3b) aken in he piece shown in
Figu e 1b, which is pa o he sys em ha allowed he mo emen o he b idge, allowed us
o ecognize a mo e complex s a ig aphy. This sample was collec ed in he lowe a ea o he
da k piece whe e p obably he o iginal pain ing could emain. The i s ou e laye was da k
in colou applied o e a he e ogeneous ed and whi e laye ha p esen ed g ains o di e en
sizes as in he piece p e iously desc ibed. Addi ionally, a hicke laye o black colou and
ano he o g een colou could be clea ly ecognized. Acco ding o he obse a ion wi h
he s e eomic oscope, he i s laye applied in he sample was a he e ogeneous laye o
g ey colou wi h da k and blue g ains as shown in Figu e 4. The de ail o he blue-g eyish
laye , whose hickness was a ound 1.5 mm, is showed in Figu e 4c. In he lowe pa o he
men ioned laye o blue-g ey pain , emains o i on oxide lakes ( eddish colou ) de ached
om he me allic su ace we e also obse ed.
Molecules 2022,27, 3609 6 o 15
Molecules 2022, 27, x FOR PEER REVIEW 6 o 15
Figu e 4. C oss-sec ions a di e en magni ica ion (a–c) o he pain ing sample SUBS-3b om a black
a ea whe e he o iginal pain laye is isible.
A i s , he subsample was analysed by mic o-ene gy dispe si e X- ay luo escence
(μ-EDXRF) o s udy i s elemen al composi ion. The elemen al maps o each elemen on
he analysed sample a e shown in Figu e 5. The µ-XRF analysis indica ed he p esence o
chlo ine, i on, silicon, ba ium, and zinc, uni o mly dis ibu ed in he ou e pa o he sub-
sample. Su p isingly, chlo ine was no de ec ed in he g een laye indica ing he absence
o ph alocyanine g een in his case as he g een pigmen . I on and silicon belong o he
i s in e io laye whe e hema i e and he silicon ca bide compound we e de ec ed. On
he o he hand, some o hese elemen s such as i on, chlo ine, silicon, ba ium, and zinc
did no belong o he ex e io g een pain laye , bu a he hey we e elemen s apped by
he a mosphe ic pa icles o he ma ine ae osol ha a i ed a he b idge con inuously.
Al hough he ma ine ae osol is mainly composed o chlo ides, i can also ca y ine pa i-
cles in suspension composed o beach sand (in his si e, he e is a beach close o he b idge)
ha mainly con ibu es o inc ease Si and Fe concen a ion on he su ace. The e idence
o i on in he lowe a ea o he sample, on he o he hand, e e s o emains o he me al
suppo ha belongs o he b idge s uc u e and his con ibu es o he mass ac ion o Fe
in he sample. The p esence o ba ium inside he subsample was e iden occupying much
o i s su ace and i was he elemen wi h he highes concen a ion in he sample. Among
he main elemen s o his in e io , sul u , lead, calcium, and zinc s and ou . The semi-
quan i a i e alues o each elemen in he sample SUBS-3b a e shown in Table 1.
In addi ion, in u ban and ha bou a eas, o he ions a e also p esen in a suspended
way such as Ba2+, Zn2+, Ca2+, K+, Mg2+, Fe3+, Al3+, S 2+, NH4+, HCO3−, and B −. The sou ce o
hese anions and ca ions can eside in he in luence o ma i ime a ic, po ac i i ies, and
also indus y o oad a ic [27].
Figu e 4.
C oss-sec ions a di e en magni ica ion (
a
–
c
) o he pain ing sample SUBS-3b om a black
a ea whe e he o iginal pain laye is isible.
A i s , he subsample was analysed by mic o-ene gy dispe si e X- ay luo escence
(
µ
-EDXRF) o s udy i s elemen al composi ion. The elemen al maps o each elemen on
he analysed sample a e shown in Figu e 5. The
µ
-XRF analysis indica ed he p esence
o chlo ine, i on, silicon, ba ium, and zinc, uni o mly dis ibu ed in he ou e pa o
he subsample. Su p isingly, chlo ine was no de ec ed in he g een laye indica ing
he absence o ph alocyanine g een in his case as he g een pigmen . I on and silicon
belong o he i s in e io laye whe e hema i e and he silicon ca bide compound we e
de ec ed. On he o he hand, some o hese elemen s such as i on, chlo ine, silicon, ba ium,
and zinc did no belong o he ex e io g een pain laye , bu a he hey we e elemen s
apped by he a mosphe ic pa icles o he ma ine ae osol ha a i ed a he b idge
con inuously. Al hough he ma ine ae osol is mainly composed o chlo ides, i can also
ca y ine pa icles in suspension composed o beach sand (in his si e, he e is a beach close
o he b idge) ha mainly con ibu es o inc ease Si and Fe concen a ion on he su ace.
The e idence o i on in he lowe a ea o he sample, on he o he hand, e e s o emains
o he me al suppo ha belongs o he b idge s uc u e and his con ibu es o he mass
ac ion o Fe in he sample. The p esence o ba ium inside he subsample was e iden
occupying much o i s su ace and i was he elemen wi h he highes concen a ion in he
sample. Among he main elemen s o his in e io , sul u , lead, calcium, and zinc s and ou .
The semi-quan i a i e alues o each elemen in he sample SUBS-3b a e shown in Table 1.
Table 1. EDXRF elemen al da a (w .%) o sub sample SUBS-3b.
Sample ID Mg Al Si S Cl K Ca Fe Zn S Ba Pb C
SUBS-3b 1.63 0.78 3.4 7.2 16.4 0.3 10.8 12.8 3.44 0.98 28.2 13.9 0.2
In addi ion, in u ban and ha bou a eas, o he ions a e also p esen in a suspended way
such as Ba
2+
, Zn
2+
, Ca
2+
, K
+
, Mg
2+
, Fe
3+
, Al
3+
, S
2+
, NH
4+
, HCO
3−
, and B
−
. The sou ce
o hese anions and ca ions can eside in he in luence o ma i ime a ic, po ac i i ies,
and also indus y o oad a ic [27].
Molecules 2022,27, 3609 7 o 15
Molecules 2022, 27, x FOR PEER REVIEW 7 o 15
Figu e 5. Mic o-ene gy-dispe si e X- ay luo escence spec oscopy (µ-EDXRF) maps o he sample
SUBS-3b om a black a ea.
Table 1. EDXRF elemen al da a (w .%) o sub sample SUBS-3b.
Sample ID
Mg
Al
Si
S
Cl
K
Ca
Fe
Zn
S
Ba
Pb
C
SUBS-3b
1.63
0.78
3.4
7.2
16.4
0.3
10.8
12.8
3.44
0.98
28.2
13.9
0.2
The molecula composi ion o he mos laye s was he same as in he p e ious sub-
sample (SUBS-2b), since ca bon black was iden i ied on he ou side and, unde nea h, he e
was a he e ogeneous laye wi h hema i e, ba ium sul a e, and he silicon ca bide. This
pigmen composi ion accoun s o he p esence o he elemen s Si, S, Ba, and Fe iden i ied
by µ-EDXRF analysis. In his sample, he g een colou was no ela ed wi h he use o
ph halocyanine pigmen s. Indeed, P ussian blue, he yellow pigmen lead ch oma e (Ra-
man bands a 337, 360, 376, 402, and 840 cm−1 Figu e S7a) and ba ium sul a e we e he
majo componen s o he g een colou a ion iden i ied in he in e io o he subsample. In
addi ion, lead whi e (2PbCO3·Pb(OH)2) was ound in some poin s o analysis in his a ea.
Thus, he p esence o lead was e y i egula , and i ep esen s he second main elemen ,
o he o al sample a e Ba. Howe e , a majo con en o lead appea s in he lowe pa o
he sample ( he one in con ac wi h me allic i on) ha could be associa ed wi h he use o
lead whi e, iden i ied mos ly in he blue and g ey laye s (as will be seen la e as well) and
only in aces in he g een laye . The low alues o ch omium in he XRF semi-quan i ica-
ion, belonging o lead ch oma e, was jus i ied wi h he high abso p ion coe icien bo h
o lead o he same pigmen and o lead whi e used in he mix u e. Addi ionally, by Ra-
man spec oscopy o he mino compounds we e de ec ed in ha blue-g een laye , such
as gypsum (Raman bands a 412, 492, 617, 668 1008, and 1134 cm−1, Figu e S7b)[28] and
ana ase (β-TiO2, Raman bands a 140, 192, 393, 512, and 635 cm−1, Figu e S7c)[29].
In he oldes g ey pain laye , in mos o he analysed poin s, Raman spec a o ba ium
sul a e we e eco ded, wi h all he cha ac e is ic bands (Raman bands: 453, 460, 618, 648,
987, and 1140 cm−1, Figu e 6a) bo h in he ma ix and in loose g ains o di e en sizes. Ad-
di ionally, in he black and blue g ains, Raman spec a o ca bon black (Raman bands: 1347,
and 1602 cm−1, Figu e 6b) and ul ama ine blue (Al6Na8O24S3Si6, Raman bands: 260, 546, 583,
805, 1095, and 1644 cm−1, Figu e 6c)[30] we e eco ded, espec i ely. In addi ion, lead whi e
Figu e 5.
Mic o-ene gy-dispe si e X- ay luo escence spec oscopy (
µ
-EDXRF) maps o he sample
SUBS-3b om a black a ea.
The molecula composi ion o he mos laye s was he same as in he p e ious sub-
sample (SUBS-2b), since ca bon black was iden i ied on he ou side and, unde nea h, he e
was a he e ogeneous laye wi h hema i e, ba ium sul a e, and he silicon ca bide. This
pigmen composi ion accoun s o he p esence o he elemen s Si, S, Ba, and Fe iden i-
ied by
µ
-EDXRF analysis. In his sample, he g een colou was no ela ed wi h he use
o ph halocyanine pigmen s. Indeed, P ussian blue, he yellow pigmen lead ch oma e
(Raman bands a 337, 360, 376, 402, and 840 cm
−1
Figu e S7a) and ba ium sul a e we e he
majo componen s o he g een colou a ion iden i ied in he in e io o he subsample. In
addi ion, lead whi e (2PbCO
3·
Pb(OH)
2
) was ound in some poin s o analysis in his a ea.
Thus, he p esence o lead was e y i egula , and i ep esen s he second main elemen , o
he o al sample a e Ba. Howe e , a majo con en o lead appea s in he lowe pa o he
sample ( he one in con ac wi h me allic i on) ha could be associa ed wi h he use o lead
whi e, iden i ied mos ly in he blue and g ey laye s (as will be seen la e as well) and only
in aces in he g een laye . The low alues o ch omium in he XRF semi-quan i ica ion,
belonging o lead ch oma e, was jus i ied wi h he high abso p ion coe icien bo h o lead
o he same pigmen and o lead whi e used in he mix u e. Addi ionally, by Raman spec-
oscopy o he mino compounds we e de ec ed in ha blue-g een laye , such as gypsum
(Raman bands a 412, 492, 617, 668 1008, and 1134 cm
−1
, Figu e S7b) [
28
] and ana ase
(β-TiO2, Raman bands a 140, 192, 393, 512, and 635 cm−1, Figu e S7c) [29].
In he oldes g ey pain laye , in mos o he analysed poin s, Raman spec a o ba ium
sul a e we e eco ded, wi h all he cha ac e is ic bands (Raman bands: 453, 460, 618, 648,
987, and 1140 cm
−1
, Figu e 6a) bo h in he ma ix and in loose g ains o di e en sizes.
Addi ionally, in he black and blue g ains, Raman spec a o ca bon black (Raman bands:
1347, and 1602 cm
−1
, Figu e 6b) and ul ama ine blue (Al
6
Na
8
O
24
S
3
Si
6
, Raman bands: 260,
546, 583, 805, 1095, and 1644 cm
−1
, Figu e 6c) [
30
] we e eco ded, espec i ely. In addi ion,
lead whi e (Raman band a 1050 cm
−1
) was iden i ied in he o iginal pain laye , al hough
in lesse quan i y compa ed o he o he compounds (Figu e 6d). All he pigmen s ound
in he o iginal pain laye o he oo b idge a e shown in he Raman spec a collec ed in
Figu e 6.
Molecules 2022,27, 3609 8 o 15
Molecules 2022, 27, x FOR PEER REVIEW 8 o 15
(Raman band a 1050 cm−1) was iden i ied in he o iginal pain laye , al hough in lesse quan-
i y compa ed o he o he compounds (Figu e 6d). All he pigmen s ound in he o iginal
pain laye o he oo b idge a e shown in he Raman spec a collec ed in Figu e 6.
No chlo ine compounds we e ound by Raman spec oscopy in his sample. The
p esence o his elemen , iden i ied by µ-EDXRF only in he ou e mos pa o he sample,
is possibly due o he cha ac e is ics o he na u al ma ine en i onmen in which he
b idge is loca ed, as discussed la e in de ail.
Figu e 6. Raman spec a eco ded in he o iginal g ey laye (sample SUBS-3b): ba ium sul a e (a),
ca bon (b), ul ama ine blue (c), and lead whi e (d).
The esul s allow econs uc ing he execu ion o pain ing on he ailing s uc u e. I s
o iginal colou was g eyish blue (see blue laye o Figu e 4) composed o he mix u e o
ul ama ine blue and ca bon black. These pigmen s we e mixed wi h lead whi e and ana-
ase, wi h hese la e ones p obably being used as opaci ie s [31]. On op o his g eyish
blue laye , ano he pain o a mo e g eenish blue hue was applied (see g eenish laye
abo e he blue in Figu e 4) composed o P ussian blue mixed wi h lead ch oma e and
dispe sed in ba ium sul a e (wi h aces o gypsum) and ana ase. O e ime, a hema i e
an ioxidan p ime was applied and o e i , a black o co e possibly all us o ma ion
and colou lakes.
2.2. Colo ime ic S udies
Colou measu emen s in CIELab colou space we e collec ed om he pieces o he
b idge (Figu e S1) deli e ed o he labo a o y (Table 2) ha pe mi he eplica ion o col-
ou . The e o e, he alues o he g een colou and o he o iginal g eyish blue colou we e
eco ded, ob ained by ca e ully sc aping he su ace o he piece ha allows he o a ion
o he b idge.
Table 2. Lab alues o he colou s measu ed in he samples in Figu e S1.
Colou
L *
a *
b *
G een (Figu e S1a)
34.96
−11.22
1.68
G eyish blue (Figu e S1b)
44.47
−0.14
2.07
* The CIELAB colo space is e e ed wi h as e isks o p e en con usion wi h Hun e Lab.
2.3. E alua ion o he S a e o Conse a ion o he I on S uc u e o he B idge
Figu e 6.
Raman spec a eco ded in he o iginal g ey laye (sample SUBS-3b): ba ium sul a e (
a
),
ca bon (b), ul ama ine blue (c), and lead whi e (d).
No chlo ine compounds we e ound by Raman spec oscopy in his sample. The p es-
ence o his elemen , iden i ied by
µ
-EDXRF only in he ou e mos pa o he sample, is
possibly due o he cha ac e is ics o he na u al ma ine en i onmen in which he b idge is
loca ed, as discussed la e in de ail.
The esul s allow econs uc ing he execu ion o pain ing on he ailing s uc u e.
I s o iginal colou was g eyish blue (see blue laye o Figu e 4) composed o he mix u e
o ul ama ine blue and ca bon black. These pigmen s we e mixed wi h lead whi e and
ana ase, wi h hese la e ones p obably being used as opaci ie s [
31
]. On op o his g eyish
blue laye , ano he pain o a mo e g eenish blue hue was applied (see g eenish laye
abo e he blue in Figu e 4) composed o P ussian blue mixed wi h lead ch oma e and
dispe sed in ba ium sul a e (wi h aces o gypsum) and ana ase. O e ime, a hema i e
an ioxidan p ime was applied and o e i , a black o co e possibly all us o ma ion and
colou lakes.
2.2. Colo ime ic S udies
Colou measu emen s in CIELab colou space we e collec ed om he pieces o he
b idge (Figu e S1) deli e ed o he labo a o y (Table 2) ha pe mi he eplica ion o colou .
The e o e, he alues o he g een colou and o he o iginal g eyish blue colou we e
eco ded, ob ained by ca e ully sc aping he su ace o he piece ha allows he o a ion o
he b idge.
Table 2. Lab alues o he colou s measu ed in he samples in Figu e S1.
Colou L * a * b *
G een (Figu e S1a) 34.96 −11.22 1.68
G eyish blue (Figu e S1b) 44.47 −0.14 2.07
*The CIELAB colo space is e e ed wi h as e isks o p e en con usion wi h Hun e Lab.
2.3. E alua ion o he S a e o Conse a ion o he I on S uc u e o he B idge
The Raman analyses we e also applied o he s udy o co osion p oduc s in he i on
s uc u e gene a ed by he exposu e o he b idge o he ma ine en i onmen ha a ou ed
i s disin eg a ion. The s a e o conse a ion o he pieces deli e ed o he labo a o y was
di e en om each o he ; he e o e, an oxidized chip (SUBS-4b) wi hou a pain laye om
he piece in Figu e S1b and a sample (SUBS-2a) wi h pain laye om he base o he ailings
(Figu e S1a) we e selec ed.
Molecules 2022,27, 3609 9 o 15
The Raman maps (Figu e 7) ca ied ou in he oxidized sample SUBS-2a, ea ed as
c oss-sec ion, highligh ed he p esence o goe hi e (
α
-FeOOH, Raman bands: 250, 330,
390, 478, and 550 cm
−1
as shown in Figu e 8a) [
32
] as he main compound in he in e nal
a ea o he oxidized chip, as seen in he Raman image shown in Figu e 7a. The p esence
o magne i e (Fe
3
O
4
, Raman bands: 550 and 663 cm
−1
shown in Figu e 8c) [
33
] was also
iden i ied by poin -by-poin Raman spec oscopy, and i s p esence is impo an in he
us a ea as shown in he Raman image o Figu e 7c. On he o he hand, he p esence o
lepidoc oci e (
γ
-FeO(OH), (Raman bands: 215, 249, 305, 345, 376, 523, 645, and 1300 cm
−1
as
depic ed in Figu e 8b) in he edges o he sample and in co espondence o mic o ac u es
o he subsample was iden i ied as seen om he Raman image shown in Figu e 7b. This
obse a ion is in good ag eemen wi h o he in es iga ions ha documen ed he o ma ion
o lepidoc oci e associa ed o local mo e ae a ed condi ions [
34
,
35
]. This species o i on
oxyhyd oxide is known o be one o he mos uns able o ms o he co osion compounds,
which can ans o m in o he mo e s able goe hi e wi h he succession o we –d y cycles
du ing he passi a ion o he co osion p ocesses [
36
]. This means ha in he sample, he
decay p ocess has no been ye comple ed and is s ill going on.
Molecules 2022, 27, x FOR PEER REVIEW 9 o 15
The Raman analyses we e also applied o he s udy o co osion p oduc s in he i on
s uc u e gene a ed by he exposu e o he b idge o he ma ine en i onmen ha a-
ou ed i s disin eg a ion. The s a e o conse a ion o he pieces deli e ed o he labo a-
o y was di e en om each o he ; he e o e, an oxidized chip (SUBS-4b) wi hou a pain
laye om he piece in Figu e S1b and a sample (SUBS-2a) wi h pain laye om he base
o he ailings (Figu e S1a) we e selec ed.
The Raman maps (Figu e 7) ca ied ou in he oxidized sample SUBS-2a, ea ed as
c oss-sec ion, highligh ed he p esence o goe hi e (α-FeOOH, Raman bands: 250, 330, 390,
478, and 550 cm−1 as shown in Figu e 8a) [32] as he main compound in he in e nal a ea
o he oxidized chip, as seen in he Raman image shown in Figu e 7a. The p esence o
magne i e (Fe3O4, Raman bands: 550 and 663 cm−1 shown in Figu e 8c) [33] was also iden-
i ied by poin -by-poin Raman spec oscopy, and i s p esence is impo an in he us a ea
as shown in he Raman image o Figu e 7c. On he o he hand, he p esence o lepido-
c oci e (γ-FeO(OH), (Raman bands: 215, 249, 305, 345, 376, 523, 645, and 1300 cm−1 as de-
pic ed in Figu e 8b) in he edges o he sample and in co espondence o mic o ac u es o
he subsample was iden i ied as seen om he Raman image shown in Figu e 7b. This
obse a ion is in good ag eemen wi h o he in es iga ions ha documen ed he o -
ma ion o lepidoc oci e associa ed o local mo e ae a ed condi ions [34,35]. This species o
i on oxyhyd oxide is known o be one o he mos uns able o ms o he co osion com-
pounds, which can ans o m in o he mo e s able goe hi e wi h he succession o we –d y
cycles du ing he passi a ion o he co osion p ocesses [36]. This means ha in he sam-
ple, he decay p ocess has no been ye comple ed and is s ill going on.
Figu e 7. Raman images show he dis ibu ion o goe hi e (a), lepidoc oci e (b), and magne i e (c) in
he sample SUBS-2a.
Figu e 7.
Raman images show he dis ibu ion o goe hi e (
a
), lepidoc oci e (
b
), and magne i e (
c
) in
he sample SUBS-2a.
Molecules 2022, 27, x FOR PEER REVIEW 10 o 15
Figu e 8. Raman spec a o goe hi e (a), lepidoc oci e plus as axan hin (A) (b), magne i e (c), akaga-
nei e wi h aces o as axan hin (A) and ca bon (C) (d), and hema i e (e) om he sample SUBS-4b.
In ag eemen wi h ou esul s, p e ious s udies on us su aces on mild s eel demon-
s a ed ha in ma ine en i onmen s, lepidoc oci e de elops p e e en ially on he ou e -
mos su ace, i espec i e o he chlo ide ion deposi ion a e, while magne i e and akaga-
nei e (no ound in his sample), an oxyhyd oxide o med in chlo ine ich a mosphe es,
mainly o m nea he base s eel. In addi ion, acco ding o Diaz e al.[37] wi h he inc ease
o he exposi ion ime, us laye s become hicke , and he lepidoc oci e is pa ially ans-
o med in o goe hi e gene a ing a s a i ied bilaye s uc u e o us consis ing o a po ous
ou e laye o lepidoc oci e and an inne laye o compac goe hi e.
As men ioned, e en magne i e was iden i ied in he subsample as a co osion p od-
uc . I s o ma ion is commonly de ec ed as a decay compound in us de eloped in ma ine
a mosphe es, and i is usually de ec ed in he inne zone closes o he base s eel, whe e
he lowe oxygen a ailabili y a ou s i s de elopmen [38,39].
Raman esul s e ealed ha he s uc u e was also a ec ed by biological coloniza-
ion. The ca o enoid pigmen as axan hin, ecognized by he h ee main bands a 1509
cm−1 (ν1 C═C), 1152 cm−1 (ν2 C-C and 1001 cm−1 (ν3 C-H) and e en by he o e ones a 957,
1191, 1448, 2150, 2296, and 2650 cm−1, was de ec ed in ex ended a eas o he sample [40].
The spec um in Figu e 8b shows as axan hin and lepidoch oci e in he same spo a ea,
showing how he coloniza ion p ocess ex ends also o he oxidized us s. Among he ca-
o enoid pigmen s, as axan hin is he mos oxidized species and is known o be syn he-
sized by pho osyn he ic o ganisms such as cyanobac e ia, ungi, and algae as a de ense
mechanism agains a mosphe ic pollu ion. Fo his eason, i was p oposed as bioindica-
o s o high concen a ion o SO2 in he a mosphe e [41]. In ou s udy, he p esence o
as axan hin in co osion pa ina was p obably ela ed o he acidic en i onmen in which
he b idge was loca ed. The indus ial ac i i y p esen in he ou ski s o he ci y and he
close p oximi y o he po a e he esponsible o he high concen a ion o his compound
in he oxidized mic osample.
Fu he mo e, su ace analyses we e ca ied ou on a mic osample (SUBS-4b) col-
lec ed om an oxidized a ea o he sample ha allowed he mo emen o he b idge (Fig-
u e S1b). Only he p esence o lepidoc oci e, wi h a globula mo phology, was iden i ied
in he inne su ace o he me al agmen . On he o he hand, he ex e io side showed a
Figu e 8.
Raman spec a o goe hi e (
a
), lepidoc oci e plus as axan hin (A) (
b
), magne i e (
c
), aka-
ganei e wi h aces o as axan hin (A) and ca bon (C) (
d
), and hema i e (
e
) om he sample SUBS-4b.
