Talan a 251 (2023) 123757
A ailable online 4 Augus 2022
0039-9140/© 2022 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
Compa ison o Mg/Ca concen a ion se ies om Pa ella dep essa limpe
shells using CF-LIBS and LA-ICP-MS
Ma ina Ma ínez-Minche o
a
,
b
,
*
, Adol o Cobo
a
,
b
,
c
,
d
, Ana M´
endez-Vicen e
e
, Jo ge Pisone o
,
Ne ea Bo del
, Igo Gu i´
e ez-Zugas i
d
, Pa ick Robe s
g
,
h
, ´
Al a o A izabalaga
i
,
Jos´
e Valdiande
a
, Jesús Mi apeix
a
,
b
,
c
, Jos´
e Miguel L´
opez-Higue a
a
,
b
,
c
,
Asie Ga cía-Esc´
a zaga
g
,
i
,
j
,
**
a
Pho onics Enginee ing G oup, Uni e sidad de Can ab ia, Spain
b
Ins i u o de In es igaci´
on Sani a ia Valdecilla (IDIVAL), Spain
c
CIBER-BBN, Ins i u o de Salud Ca los III, 28029, Mad id, Spain
d
Ins i u o In e nacional de In es igaciones P ehis ´
o icas de Can ab ia (Uni e sidad de Can ab ia, Gobie no de Can ab ia, Banco San ande ), Uni . de Can ab ia, Spain
e
Se icios Cien í ico-T´
ecnicos, Unidad de Ensayos Medioambien ales. Uni e sidad de O iedo, Spain
Depa amen o de Física, Uni e sidad de O iedo, Spain
g
Depa men o A chaeology, Max Planck Ins i u e o he Science o Human His o y, Ge many
h
School o Social Sciences, Uni e si y o Queensland, Queensland, Aus alia
i
Depa amen o de Geog a ía, P ehis o ia y A queología, Uni e sidad Del País Vasco (UPV/EHU), Spain
j
Depa men o P ehis o y and Ins i u e o En i onmen al Science and Technology (ICTA), Uni e si a Au `
onoma de Ba celona, Bella e a, Spain
ARTICLE INFO
Keywo ds:
CFLIBS
LA-ICP-MS
LIBS
Mg/Ca p o iles
A chaeology
Mollusk shells
Paleoclima e
ABSTRACT
The elemen al composi ion o ma ine mollusk shells can o e aluable in o ma ion abou en i onmen al con-
di ions expe ienced by a mollusk du ing i s li espan. P e ious s udies ha e shown signi ican co ela ions be-
ween Mg/Ca concen a ion a ios measu ed on biogenic ca bona e o mollusk shells and sea su ace empe a u e
(SST). He e we p opose he use o Lase -Induced B eakdown Spec oscopy (LIBS) and he alida ion o he
Calib a ion-F ee LIBS (CF-LIBS) app oach o he apid measu emen and es ima ion o Mg/Ca mola concen-
a ion p o iles wi hin Pa ella dep essa Pennan , 1777 limpe shells. To achie e hese objec i es, esul s de i ed
om CF-LIBS me hodology a e compa ed wi h hose ob ained om an es ablished analy ical echnique o his
pu pose, such as Lase Abla ion Induc i ely Coupled Plasma Mass Spec ome y (LA-ICP-MS). Concen a ion
se ies ob ained wi h bo h me hodologies show de ined empo al pa e ns and e lec he season-o -cap u e in
each specimen. The esul s e idence a signi ican co ela ion (R
2
=0.63–0.81) be ween CF-LIBS and LA-ICP-MS
Mg/Ca mola concen a ion p o iles wi hin ou li e-collec ed P. dep essa shells. A e aged e o o he mola
concen a ion es ima ed wi h CF-LIBS was lowe han 10% in e e y specimen. The compa ison be ween he
esul s ob ained om wo echniques used in his s udy has allowed us o demons a e o he i s ime ha Mg/
Ca mola concen a ion measu ed in biogenic ca bona es we e accu a ely in e ed using CF-LIBS echnique. The
CF-LIBS app oach alida ion ep esen s g ea po en ial o he apid and la ge-scale paleoen i onmen al and
a chaeological analysis o his mollusk species, which is equen ly ound in a chaeological si es.
1. In oduc ion
Ma ine mollusks ha e been exploi ed as a ood esou ce by humans
in he pas ; hence, hei shells a e equen ly p ese ed and ound in
a chaeological si es all o e he wo ld, om low o high la i udes [1–4].
Shell chemical composi ion can ac as a powe ul eco de o seasonal
sea su ace empe a u e (SST) a ia ions expe ienced by a mollusk in he
pas , enabling esea che s o econs uc pas clima e condi ions [5–7],
as well as o accu a ely es ablish he pe iod o he yea when i died/was
collec ed by humans [3,8–10]. The co ela ion be ween s able oxygen
iso ope a ios de i ed om biogenic ca bona e (δ
18
O) and SST has been
* Co esponding au ho . Pho onics Enginee ing G oup, Uni e sidad de Can ab ia, Spain.
** Co esponding au ho . Depa men o P ehis o y and Ins i u e o En i onmen al Science and Technology (ICTA), Uni e si a Au `
onoma de Ba celona, Bella e a,
Spain
E-mail add esses: [email p o ec ed] (M. Ma ínez-Minche o), [email p o ec ed] (A. Ga cía-Esc´
a zaga).
Con en s lis s a ailable a ScienceDi ec
Talan a
jou nal homepage: www.else ie .com/loca e/ alan a
h ps://doi.o g/10.1016/j. alan a.2022.123757
Recei ed 23 Ma ch 2022; Recei ed in e ised o m 5 July 2022; Accep ed 18 July 2022
Talan a 251 (2023) 123757
2
ex ensi ely demons a ed in p e ious wo ks [11–13]. Al hough i is a
e y commonly used me hod in paleoclima ic and a chaeological
s udies, oxygen iso ope ac iona ion is in luenced by oxygen iso ope
composi ion o he seawa e (δ
18
O
sw
) [14,15]. This poses a no able
limi a ion o paleo econs uc ions whe e he alues o δ
18
O
sw
a e un-
known. This has mo i a ed he de elopmen o independen paleo-
empe a u e p oxies. A co ela ion be ween SST and he quan i y o
some ace elemen s (magnesium, s on ium, e c.) inco po a ed in o he
biogenic calcium ca bona e ma ix has also been ound o shells o
ce ain species [16–18]. Indeed, ace elemen a ios o biogenic ca -
bona es, mos commonly Mg/Ca in biogenic calci e and S /Ca in
a agoni e, ha e been widely in es iga ed as po en ial empe a u e
p oxies o se e as complemen s o al e na i es o δ
18
O ca bona e
analysis [19].
Lase -Induced B eakdown Spec oscopy (LIBS), an a omic spec os-
copy echnique based on he op ical analysis o lase -gene a ed plasmas
om he sample ma e ial, has p o en o be pa icula ly p omising in his
ega d hanks o i s high h u-pu . When a high-ene gy lase pulse is
ocused on he sample, a ma e ial abla ion is p oduced, gene a ing a
plasma whose composi ion is p opo ional o he elemen al concen a-
ion o he sample. The plasma emi s op ical adia ion depending on he
p esen elemen s, which enable hei iden i ica ion by hei unique
spec al signa u es [20]. LIBS has been widely employed on a a ie y o
ma e ials and has seen a di e si y o applica ions such as he analysis o
me allic alloys [21–23], biomedicines [24,25], mine als and ocks
[26–28], soils [29], as well as i s use in o ensic s udies [30] and
a chaeology and cul u al he i age [31–33]. LIBS o e s mul iple ad-
an ages o e o he analy ical echniques including e sa ili y, no
con ac equi emen s, mul i-elemen al analysis, i being p ac ically
non-des uc i e, and a educ ion in, o lack o , sample p epa a ion [34].
Measu ing solid samples, such as mollusk shells, wi h he commonly
used echniques o his pu pose (ICP-OES o ICP-MS) equi es
mic o-milled sampling p ocedu es and subsequen acid diges ion, which
can be highly ime-consuming compa ed o LIBS p ocedu e which o e s
a numbe o bene i s in he con ex o mo e limi ed sample p epa a ion
equi emen s, lowe cos s, and an inc eased apidi y in measu emen
and accumula ion o da a om mul iple indi iduals om a gi en con ex
[35–37]. Lase abla ion-induc i ely coupled plasma-mass spec ome y
(LA-ICP-MS) is a highly accep ed and widely used echnique o he
analysis o majo , mino and ace elemen s, as well as iso ope- a io
de e mina ion [38–43]. Due o he use o lase abla ion as a sampling
me hod, his analy ical echnique combines he ad an age o no
equi ing acid diges ion o he sample wi h he ad an ages o ICP
echniques ela ed o p ecision and obus ness. Howe e , despi e i s
good analy ical pe o mance, LA-ICP-MS su e s om e y high cos and
signi ican complexi y when compa ing bo h echnologies. The e o e,
LIBS o e s mo e e sa ili y, imp o ed speed, ease o ope a ion, a o d-
abili y and po abili y [44]. P e ious wo k has demons a ed ha
Mg/Ca a ios ob ained along he g ow h axis o limpe s using LIBS
showed e y well-de ined seasonal cycles ha co ec ly e lec ed he
annual a ia ions in SST [36,37,45–47]. Howe e , one o he main is-
sues wi h he LIBS echnique is ha he spec al line in ensi ies om a
gi en elemen a e s ongly dependen on he physical and chemical
p ope ies o he sample ( he so-called ma ix e ec ) [37]. Fo his
eason, a e e ence ma e ial wi h almos iden ical p ope ies o he
sample should be used o calib a ion plo s. Due o he absence o spe-
ci ic ma ix-ma ched calib a ion s anda ds o ma ine mollusk shell
biogenic ca bona es, p e ious s udies employed wo line emission in-
ensi ies om LIBS spec a o ob ain Mg/Ca alues gi en in a bi a y
uni s [36,46]. The use o line a ios can ne e heless lead o
LIBS-de i ed a iabili y due o changes in expe imen al condi ions such
as op ical alignmen , empe a u e o spec ome e con igu a ion and
a iabili y be ween specimens. Mola concen a ion a ios o elemen s
should p o ide signi ican ad an ages o e he line a io app oach.
The calib a ion- ee LIBS p ocedu e (CF-LIBS) is a quan i a i e
algo i hmic me hod ha allows he elemen al concen a ion o ma e ials
o be ob ained wi hou he a o emen ioned ma ix e ec [48]. This is
accomplished wi hou he necessi y o calib a ion cu es o e e ence
samples, and i is no a ec ed by possible a ia ions o expe imen al
pa ame e s such as lase ene gy, spo size, o sample oughness. This
me hod appea s o be sui able o analyzing samples wi h complex
ma ices o which ma ix-ma ched s anda ds a e no a ailable, e.g.,
ocks, mine als and me eo i es [49–52] o a chaeological a i ac s
[53–56]. This app oach allows us o ob ain ela i e concen a ions in
mmol/mol uni s. None heless, i equi es alida ion because CF-LIBS is
an algo i hm ha elies on some assump ions abou plasma cha ac e -
is ics, which may di e om eal condi ions. The e o e, he nume ical
concen a ion ha we ob ain mus be checked agains he ac ual alues.
P e ious wo ks applying he CF-LIBS app oach o ma ine mollusk
shell biogenic ca bona es highligh ed he paleoen i onmen al and
a chaeological po en ial o his no el me hodology [37]. Ne e heless,
addi ional s udy is s ill equi ed o imp o e he algo i hm en a i ely
used by Ga cía-Esc´
a zaga e al. [37] o es ima e Mg/Ca mola concen-
a ions in biogenic ca bona es. He e, he e o e, we p opose he ali-
da ion o he Calib a ion-F ee LIBS app oach o he es ima ion o he
mola a ios o bo h magnesium and calcium elemen s in Pa ella dep essa
Pennan , 1777 limpe shells. In o de o alida e he LIBS echnique o
his applica ion, he mola a io sequences ha e been compa ed o
LA-ICP-MS measu emen s o he same shells. Con i ma ion o his
echnique would open up signi ican a enues o he apid measu emen
o shells om a chaeological and o he con ex s and he expansion o
sample sizes used in he assessmen o season o collec ion o pas
paleoclima e.
2. Ma e ials and me hods
2.1. Sample p epa a ion
P. dep essa limpe shells we e li e-collec ed a Lang e Beach along
he coas o Can ab ia (No h o Spain) in he mon h o Sep embe ,
which co esponds o he end o he wa me pe iod o he yea , i.e., he
summe season. To o m he ma ine mollusk shells, he ca bona e p e-
cipi a es in he o m o g ow h inc emen s om he apex o he shell
edge. P epa a ion o he shells was ca ied ou a he Ins i u e o P e-
his o y (IIIPC) o he Uni e si y o Can ab ia (Spain). The shells we e
sec ioned along he g ow h axis ollowing he p ocedu es applied in
p e ious esea ch on he same limpe species [37]. Fou selec ed
Lis o abb e ia ions
A A gon
Ba Ba ium
Ca Calcium
CCD Cha ge-coupled de ice
CF-LIBS Calib a ion F ee Lase Induced B eakdown
Spec oscopy
ICP-MS Induc i ely Coupled Plasma Mass Spec ome y
ICP-OES Induc i ely Coupled Plasma Op ical Emission
Spec ome y
LA-ICP-MS as Lase Abla ion Induc i ely Coupled Plasma Mass
Spec ome y
LTE Local The modynamic Equilib ium
LIBS Lase -Induced B eakdown Spec oscopy
Mg Magnesium
mmol milimol
Nd-YAG Neodymium Doped Y ium–Aluminum-Ga ne
NIST Na ional Ins i u e o S anda ds and Technology
SST Sea Su ace Tempe a u e
S S on ium
M. Ma ínez-Minche o e al.
Talan a 251 (2023) 123757
3
samples we e pa ially coa ed wi h an epoxy esin along he maximum
g ow h axis o a oid he shell b eaking when cu . Sec ioning was pe -
o med using a Buehle Isome low-speed saw and a diamond wheel.
F om each limpe , a 3 mm sec ion was ob ained (Fig. 1a).
The sec ions we e ixed on o a glass mic oscope slide and g ound on
glass pla es using 600 and 800 SiC g i powde and polished wi h a 1
μ
m
diamond suspension g i un il he in e nal g ow h lines and inc emen s
we e clea ly isible. The sec ion was used o elemen al analyses using
LA-ICP-MS and LIBS on he calci e laye om he shell apex ( he i s
po ion o he shell g ow h) o he shell edge ( he las po ion o he shell
g ow h) (Fig. 1b). The measu emen s we e pe o med ollowing a pa h
in he calci e laye o he limpe , om he apex o he ou e edge,
c ossing he g ow h lines. (Fig. 1c). (Supplemen a y Figs. 1–4).
2.2. LA-ICP-MS se up and analyses
LA-ICP-MS me hodologies begin by abla ing he sample ma e ial
using a lase sou ce; hen, he abla ed sample is ionized wi hin he
induced-coupled plasma (ICP) sou ce. Once p oduced, he ions a e
de ec ed and sepa a ed acco ding o hei m/z a io. Quan i ica ion is
based on compa a i e measu emen using ex e nal e e ence ma e ials
and in e nal s anda diza ion. Sample analyses we e accomplished a he
Uni e si y o O iedo (Spain). The LA-ICP-MS se up (Fig. 2) consis s o a
lase abla ion uni (193 nm A F excime lase - Analy e G2, Teledyne -
Pho on Machines-) coupled o a quad upole mass spec ome e (model
7700x om Agilen Technologies).
The analyses we e pe o med as ansec s (line scans) ollowing
limpe g ow h lines om he apex o he shell o he edge. P io o
analysis, a p e-abla ion was ca ied ou o clean he su ace o he lim-
pe s. Fo his pu pose, a sligh ly la ge spo size and a highe abla ion
speed we e employed. Expe imen al condi ions a e shown in Table 1.
The iso opes se in he me hod we e
25
Mg,
43
Ca,
88
S and
137
Ba wi h
an in eg a ion ime (dwell ime) o 100 ms o each iso ope, esul ing in
a o al acquisi ion ime o 0.4 s. Unde hese condi ions, a he se
scanning speed, a la e al esolu ion o app oxima ely 20
μ
m was ob-
ained along he line ajec o y.
ICP-MS analysis condi ions we e op imized by he abla ion o he
in e na ional e e ence s anda d NIST612 glass ( luence 5 J/cm
2
, ci -
cula spo ∅ 85
μ
m, scan a e 5
μ
m/s, equency 10 Hz), moni o ing
oxide p oduc ion and plasma obus ness by he a ios
248
ThO/
232
Th
(0.5%) and
238
U/
232
Th (~1.15), espec i ely.
The in e na ional s anda d e e ence ma e ials used o calib a ion
we e he syn he ic glasses SRM 610 and 612 om The Na ional Ins i u e
o S anda ds and Technology (NIST), as well as he MACS3 calcium
ca bona e s anda d e e ence om he U.S. Geological Su ey (USGS).
All s anda ds we e measu ed in iplica e a he beginning and end o he
analy ical session as well as e e y ~30 min o moni o sensi i i y d i .
Da a ea men and p ocessing we e pe o med wi h he assis ance o
Ioli e4 so wa e [57] aking MACS3 and he wo NIST 610 and 612
s anda ds as calib a ion and seconda y s anda ds, espec i ely.
Elemen al concen a ions we e exp essed as mmol/mol in ela ion o
calcium, which was selec ed as he in e nal s anda d elemen wi h a
alue o 40.4% weigh .
2.3. LIBS se up and measu emen s
A selec ion o ou P. dep essa shell samples we e measu ed using a
LIBS se up a he Uni e si y o Can ab ia (Spain), a e he pe o mance
o he LA-ICP-MS measu emen s and ollowing he same pa h. The
expe imen al se up (Fig. 3) comp ises a pulsed lase sou ce ha p o-
duces he ma e ial abla ion o he sample and a spec ome e o collec
and analyze he ligh emi ed om he plasma. The lase sou ce is a Q-
Swi ched Nd:YAG double-pulsed lase (Lo is LS-2134D) ope a ing a a
wa eleng h o 1064 nm, wi h 16 ns pulse wid h and 10 Hz epe i ion
a e. The pulse ene gy was se o 35 mJ. The lase spo was ocused wi h
a lens o 75 mm ocal leng h, p oducing a spo o 200
μ
m diame e and
0.2
μ
m dep h (a e age) o each lase sho , esul ing in an i adiance
alue a he sample su ace app oxima ely equal o 7 GW/cm
2
. The ligh
emi ed om he plasma gene a ed on he sample su ace was cap u ed
by a collima ing lens and conduc ed o he spec ome e h ough wo
op ical ibe s.
One was a used silica, sola iza ion esis an , op ical ibe wi h 1 mm
co e diame e , coupled o ano he , which was made up o a bundle o
eigh op ical ibe s, each one wi h a 200
μ
m co e diame e and a o al
diame e o he bundle o a ound 800
μ
m. The spec ome e used in his
s udy is an eigh -channel A an es ULS2048-USB2-RM CCD spec ome e
wi h a o al wa eleng h ange om 178 o 889 nm and a esolu ion om
0.015 o 0.06 nm. The collec ion o he plasma-emi ed ligh was se o
s a 1
μ
s a e he second lase pulse, wi h an acquisi ion ga e o 1 ms
( he minimum cap u e ime o his non-ga ed spec ome e ). A ough-
lens ision sys em wi h a CCD came a allowed he inspec ion o he
sample su ace. The sample posi ion was con olled by a mo o ized XYZ
posi ione , p og ammed o ollow a measu emen pa h along he shell
su ace.
In his expe imen , he spec a acquisi ion was pe o med in he ai
a a mosphe ic p essu e and ambien empe a u e. Measu emen s we e
made e e y 25
μ
m, and 12 consecu i e lase pulses pe poin we e
eco ded o a e aging.
2.4. CF-LIBS applica ion
The algo i hm used is an implemen a ion o he me hod o iginally
p oposed by Ciucci e al. [48]. The CF-LIBS app oach is a quan i a i e
me hodology ha p oduces accu a e quan i a i e measu emen s o he
elemen al composi ion o a sample wi hou using calib a ion cu es and
allows o e coming he ma ix e ec . Fo his pu pose, some assump ions
abou he plasma condi ions should be made. Fi s , we assume ha he
abla ion is s oichiome ic, i.e., he plasma composi ion is ep esen a i e
o he ac ual composi ion o he sample be o e he abla ion. In addi ion,
we assume ha he plasma is in local he modynamic equilib ium (LTE)
in he eal- ime and space obse a ion window. Finally, he hi d
assump ion is ha he plasma lines a e op ically hin. Ano he expe i-
men al equi emen o he CF-LIBS is ha he spec al ange o mea-
su emen should include measu able lines om all he elemen s p esen
Fig. 1. Limpe sampling p ocess.
M. Ma ínez-Minche o e al.
Talan a 251 (2023) 123757
4
in he sample [58].
Based on LTE app oxima ion, he line in eg al in ensi y co e-
sponding o he ansi ion le els E
k
and E
i
o an a omic species s is
desc ibed in Eq. (1):
Iki =FCsAki
gke−(Ek/kBT)
Us(T)(1)
whe e I
ki
ep esen s he measu ed in eg al line in ensi y; F is an expe -
imen al ac o ; C
s
is he concen a ion o he emi ing a omic species s;
A
ki
is he ansi ion p obabili y o he gi en line; g
k
is he k le el de-
gene acy; K
B
is he Bol zmann cons an ; T is he plasma empe a u e,
and U
s
(T) is he pa i ion unc ion o he emi ing species a he plasma
empe a u e. Applying loga i hm o Eq. (1), we ob ain:
ln Iki
gkAki
= − Ek
kBT+ln CsF
Us(T)(2)
The equa ion can be w i en as a linea equa ion o he ollowing
o m
y=mx +qs
whe e y=ln Iki
gkAki
,x=Ek,m= − 1
kBT,qs=ln CsF
Us(T)(3)
Using he de ini ions below and he pa ame e s om he da abase,
e e y measu ed line can be ep esen ed as a poin in he 2D Bol zmann
plane, which is he space iden i ied by he x and y coo dina es o Eq. (2).
E e y a omic species can be ep esen ed as pa allel lines. The slope (m)
o he lines ob ained om he Bol zmann plo is ela ed o he plasma
empe a u e T, while he in e cep (q
s
) is ela ed loga i hmically o he
species concen a ion C
s
. F om he in e cep alue, he concen a ion o
he co esponding species can be calcula ed as ollows:
Cs=1
FUs(T)eqs(4)
The expe imen al ac o F can be ob ained by no malizing he sum o
he concen a ion o all he species o one:
∑
s
Cs=1(5)
The linea i ing o each elemen mus ha e e y simila slopes,
Fig. 2. LA-ICP-MS se up.
Table 1
LA-ICP-MS ope a ing condi ions.
P e-abla ion pa ame e s Abla ion pa ame e s ICP pa ame e s
Ene gy ( luence) =2 J/
cm
2
Pulse equency =10
Hz
Spo size, diame e =
65
μ
m
Scan speed =200
μ
m/
s
Ene gy ( luence) =5 J/cm
2
Pulse equency =10 Hz
Spo size, diame e =30
μ
m
Scan speed =50
μ
m/s
Cell gas (He) low a e =800
ml/min
RF Powe =1600 W
Plasma gas (A ) =15
L/min
Ca ie gas (A ) =0.8
L/min
Sample Dep h =4.3
mm
Fig. 3. LIBS se up used o spec a acquisi ion.
M. Ma ínez-Minche o e al.
Talan a 251 (2023) 123757
5
which means all he elemen s ha e he same empe a u e, and he
plasma is in LTE. To ob ain he alue o he plasma empe a u e mo e
accu a ely, a Saha–Bol zmann plo (combina ion o exci a ion Bol z-
mann dis ibu ion and Saha ioniza ion dis ibu ion) mus be used. In his
me hod, emission lines om a oms and ions o he same elemen a e
included in he linea eg ession. The Saha ioniza ion equa ion (Eq. (6))
shows he ela ion be ween he concen a ions o neu al and ionic
species o he same elemen .
Cs II F
Cs I F=2Us II (T)
Us I (T)
(2
π
mekBT)3
2
Neh3e
−(Eion
kBT)(6)
Saha-Bol zmann plo s can be explained as:
x={EI
k o neu al lines EII
k+Eion o ionic lines (7)
y={ln Iki
gkAki
o neu al lines ln Iki
gkAki
−ln(2(2
π
mekBT)3
2
Neh3) o ionic lines
(8)
In Eq. (6), E
ion
ep esen s he ioniza ion ene gy; m
e
is he mass o he
elec on, and N
e
is he elec on densi y. The elec on densi y N
e
can be
ob ained independen ly om measu emen s o S a k b oadening.
To ob ain accu a e esul s om he CF-LIBS algo i hm, he abo e-
men ioned c i e ia mus be ul illed, mainly, LTE and op ically hin
condi ions. Fo ou expe imen al se up, wi h a non-ga ed spec ome e ,
e i ica ion o LTE condi ions is a ele an issue, because he ime
in eg a ion window is always longe han he plasma li e ime, and only
he cap u e delay om he lase sho can be accu a ely con olled. LTE
condi ions in ol e, on one hand, dominan collision p ocesses o e
adia i e ones, which a e mo e easily ma ched a he beginning o he
plasma e olu ion (sho e delays). On he o he hand, he equi ed small
a e o a ia ion o he modynamic pa ame e s o he plasma is be e
ul illed a la e s ages (longe delays). As a comp omise, we ha e chosen
he epo ed alue o 1
μ
s delay, a widely used alue o CF-LIBS [58].
Wi h his delay, he con inuum backg ound emission is educed while
he o e all a omic emission is s ill high. In hese condi ions, he
con ibu ion o he op ical emission spec um o he much less in ense
plasma a la e s ages esul s in a “ga ed-like” ope a ion wi h a sho e
e ec i e cap u e window, which u he helps o achie e he LTE con-
di ions [59]. In ou expe imen s, we ha e e i ied ha he Ca I and Mg I
emission lines all o 10% o in ensi y (wi h espec o hei maximum
alue) a 6
μ
s a e he lase sho , while Ca II and Mg II lines almos
disappea a e 2
μ
s. This sugges s a “ga ed-like” ope a ion wi h an
equi alen empo al cap u ing window o 5
μ
s wi h 1
μ
s delay.
To assu e ha he emission lines en e ing he CF-LIBS algo i hm a e
op ically hin, a selec ion p ocedu e has been implemen ed, ollowing
ad ice in Fu e al. [60]. Fi s , an a e age spec um om all measu e-
men s in each sample is analyzed, and he emission lines a e iden i ied.
Selec ion o he sui able ones is based on line in ensi y, signal o back-
g ound a io, o e lapping wi h lines o he same o di e en species, and
isola ion om o he lines. Fo he su i ing lines, hei spec oscopic
pa ame e s ( om NIST [61] and Genie da abases [62]) ha e been
conside ed: lowe ansi ion ene gy, ansi ion p obabili y, ansi ion
p obabili y accu acy, a ailabili y o he S a k b oadening pa ame e ,
and a wide ange o uppe ene gy le els o he Saha-Bol zmann plo s.
3. Resul s and discussion
3.1. LA-ICP-MS concen a ion p o iles
LA-ICP-MS da a we e i s ly ob ained, gi ing Mg/Ca concen a ion
sequences along he g ow h axis. As he spo diame e o LA-ICP-MS
measu emen s is app oxima ely six imes smalle han he LIBS c a e
(30
μ
m s 200
μ
m), a olling a e age o six da a poin s was calcula ed o
enable compa ison o he concen a ion measu emen s o bo h
echniques. The esul ing sequences a e smoo he han he aw se ies, as
seen in Fig. 4.
3.2. CF-LIBS app oach esul s
A i s s ep in he applica ion o he CF-LIBS algo i hm is he selec ion
o sui able emission lines ha will be used in he Saha-Bol zmann plo s
o calcula e he plasma empe a u e, elec onic densi y, and elemen al
concen a ion. Wi h he c i e ia desc ibed in sec ion 2.4, wel e emis-
sion lines (six om Ca and six om Mg) ha e been selec ed. As an
impo an c i e ion in ou applica ion, selec ed lines should be a ailable
in all spa ial poin s o he shell, so he same numbe o lines a e used o
c ea e he Saha-Bol zmann plo s in each one. This helps o educe he
a iabili y o he a io sequences. The selec ed lines a e shown in
Table 2, wi h he las column showing he alue o he op ical dep h
pa ame e k
, which is ela ed o he deg ee o sel -abso p ion [58]. I
mus be no ed ha ou o hem a e esonan lines (i.e., ze o lowe en-
e gy le el) and he wo Mg II lines ha e a high alue o k
, usually
indica ing ha hey a e p one o sel -abso p ion. In ou expe imen s,
howe e , we ha e ound ha hese ou esonan lines ( wo o hem om
Ca I and wo om Mg II) a e o e y low in ensi y bu high signal o noise
a io, and do no show appa en sel -abso p ion, as he i ing e o o an
op ically hin Lo en zian p o ile is lowe han 7%. Thei inclusion in he
CF-LIBS algo i hm esul , in con as , in a mo e s able a io sequence and
does no a ec he concen a ion alues signi ican ly: emo ing he
esonan lines modi ies he concen a ion alues by less han 2% while
a iabili y is much la ge , due o he lowe numbe o lines used in he
Saha-Bol zmann plo s.
Once LIBS spec a we e eco ded and lines selec ed, CF-LIBS calcu-
la ions we e pe o med along he shell g ow h axis o he ou selec ed
limpe s. Tempe a u e and elec onic densi y we e necessa y o ob ain
Mg and Ca concen a ions. Elec onic densi y is ob ained h ough he
a e aged S a k b oadening o selec ed emission peaks. Saha-Bol zmann
plo s we e ep esen ed o e e y limpe specimen in o de o ob ain
plasma empe a u e (in Fig. 5 a Saha-Bol zmann plo o Mg and Ca
co esponding o sample LAN.565 is shown). To assess he LTE condi-
ions, he McWhi e c i e ion has been used [60]. Plasma empe a u es
ob ained om he Saha-Bol zmann plo s (see Table 3) a e a ound 7500
K; om his, minimum elec onic densi y o achie e LTE condi ions
should be 1.8E16 cm
−3
, while ou a e age alue is 5.7E16cm
−3
. As an
addi ional necessa y condi ion o LTE [58], we ha e e i ied ha he
di usion leng h o a non-homogeneous, non-s a iona y plasma (calcu-
la ed in ou case as 0.025 mm) is much sho e han he a ia ion leng h
(wi h ypical alues o a ew mm in ou expe imen al condi ions).
The calcula ions equi ed o ob ain he concen a ions a each spa ial
poin we e ca ied ou by a Ma lab implemen a ion o he CF-LIBS al-
go i hm desc ibed be o e in sec ion 2.4. The code is a ailable a h
ps://gi hub.com/acobo/CFLIBS.
3.3. CF-LIBS and LA-ICP-MS compa ison
The maximum and minimum alues o he mola Mg/Ca concen-
a ion a io (Table 4) we e easonably consis en be ween he ou
specimens and he echniques applied. Concen a ion a io alues ob-
ained he ein we e la gely simila o esul s p e iously measu ed on
calci e ca bona e on Pa ella ulga a Linnaeus, 1758 limpe species using
he ICP-OES echnique [36] (a e aged minimum and maximum we e
13.0 and 24.1 mmol Mg/mol Ca, espec i ely), and esul s om Pa ella
spp. Limpe s shells using ICP-MS [18] (a e aged minimum and
maximum Mg/Ca concen a ion a ios we e 14.4 and 23.1 mmol/mol).
Mo eo e , concen a ion se ies ob ained by bo h me hods showed
e y well-de ined empo al pa e ns co e ing wa me and colde pe-
iods. Mg/Ca concen a ion se ies calcula ed wi h CF-LIBS p esen s a
good isual i wi h he LA-ICP-MS p o iles, as can be seen in Fig. 6.
P e ious wo k, including esea ch conduc ed on he species s udied
he e [37], demons a ed ha highe alues in Mg/Ca a ios measu ed in
M. Ma ínez-Minche o e al.
Talan a 251 (2023) 123757
6
ma ine mollusk shells a e ela ed o highe SST, and lowe alues in
Mg/Ca a ios sugges lowe SST [36,47]. He e, o bo h analy ical
echniques, he ou specimens show a high Mg/Ca alue a he end o
he sequence ( a igh po ion o he Mg/Ca a io se ies) whe e he edge
o he shell is loca ed (i.e. he mos ecen ly o med pa o he limpe ).
As hese specimens we e collec ed a he end o he summe , his da a
co ec ly e lec s he season o cap u e (high SST). Limpe s LAN.565,
LAN.566 and LAN.567 clea ly show seasonal cycles in he concen a ion
se ies (Fig. 6b, c, and 6d). By con as , he sequence o he limpe
LAN.563 shows a less clea sinusoidal end in bo h, he LIBS and
LA-ICP-MS measu emen se ies (Fig. 6a). The di e en beha io on he
Fig. 4. Mg/Ca concen a ion alues ob ained wi h LA-ICP-MS p ocedu e (g ey line) compa ed o he same da a applying a olling a e age (black line).
Table 2
Spec oscopic pa ame e s o neu al and ionized a omic lines o Ca and Mg used
in CF-LIBS calcula ions. Da a om NIST [61].
Elemen Ioniza ion Wa eleng h
(nm)
Aki(s
−1
) ×
10
8
gk Ek
(eV)
k
Ca I 227.546 0.301 3 5.447 11.11
Ca I 239.856 0.167 3 5.167 7.61
Ca I 558.197 0.060 7 4.743 5.63
Ca II 315.887 3.100 4 7.047 7.45
Ca II 370.603 0.880 2 6.468 1.98
Ca II 373.690 1.700 2 6.468 3.86
Mg I 382.935 0.899 3 5.946 14.84
Mg I 516.732 0.113 3 5.108 5.99
Mg I 517.268 0.339 3 5.108 17.96
Mg I 518.360 0.561 3 5.108 24.04
Mg II 279.553 2.600 4 4.434 415.30
Mg II 280.270 2.570 2 4.422 208.03
Fig. 5. Saha-Bol zmann plo o limpe LAN.565 o he elemen s Ca and Mg.
Table 3
Plasma empe a u e o he limpe s analyzed.
Sample LAN.563 LAN.565 LAN.566 LAN.567
Te (K) 7445 ±111 7506 ±100 7374 ±110 7414 ±136
M. Ma ínez-Minche o e al.
Talan a 251 (2023) 123757
7
Mg/Ca se ies in his specimen is unknown, bu se e al s udies sugges
ha he magnesium inco po a ion o he calci e shell may depend on
ac o s o he han empe a u e and may e en be a iable wi hin he
same species [63]. Physiological p ocesses in ol ed in shell o ma ion
may change h oughou i s g ow h and in oduce impu i ies and he -
e ogenei ies in i s composi ion [64,65] due o ac o s such as s ess,
me abolic ac i i y o con ol o shell c ys al elonga ion [66]. In his case,
i is possible ha he limpe has s opped g owing las win e , and win e
empe a u es a e no e lec ed. G ow h s ops may ha e occu ed by
exceeding he he mal h eshold o by on ogeny, a ec ing he co ec
e lec ion o win e and/o summe seasons, as i was p e iously
obse ed o his same mollusk species a his la i ude [67].
Rega ding he LA-ICP-MS and CF-LIBS eco ds, hey show a signi i-
can co ela ion be ween hem. E en hough bo h se ies a e e y simila
in each sample, some pa s o he shell p esen no able di e ences in
hei concen a ion alues. The abla ed ma e ial, which is measu ed
wi h LIBS and LA-ICP-MS, is no s ic ly he same because he second
echnique applied (LIBS) was measu ed on ma e ial some mic ons
deepe han he ma e ial i s ly abla ed and measu ed by LA-ICP-MS
[68]. This ac may lead o di e ences in spa ial concen a ions using
bo h echniques. Reg ession coe icien s (Fig. 7) we e calcula ed in o de
o quan i y he simila i y be ween he concen a ions ob ained using
bo h echniques applied and in e e y specimen. Reg ession coe icien
alues measu ed a y om R
2
=0.63 o R
2
=0.81. In addi ion, he
a e aged e o s o calcula ed concen a ion by CF-LIBS in ela ion o he
LA-ICP-MS alues we e ob ained, esul ing in 7.96% o LAN.563,
9.65% o LAN.565, 7.75% o LAN.566 and 9.84% o LAN.567.
3.4. Implica ions o paleoclima ology and a chaeology
Du ing he las wo decades, se e al s udies ha e highligh ed he
sui abili y o ace elemen analyses on biogenic ma ine mollusk shells
as a high- esolu ion clima e eco de [16,18,69]. In his con ex , LIBS
echnique has been p esen ed as a apid and low-cos me hodology o
measu e elemen al composi ion along he ma ine shell g ow h. The e-
sul s ob ained so a on di e en species ha e epo ed a signi ican
co ela ion be ween Mg/Ca a io alue p o iles along he shell g ow h
and SST du ing he mollusks li espan [36,37,45–47]. This sugges s ha
his p oxy could ha e e y high implica ions o u u e paleoclima e and
a chaeological in es iga ions.
Ne e heless, o he s udies ha e also shown ha ace elemen a io
de i ed om mollusc shells as clima e eco ds also p esen a deep
weakness. Hausmann e al. [45] and Laza e h e al. [70] ha e epo ed
changes in elemen al composi ion along isoch onous g ow h line-
s/inc emen s (i.e., laye s deposi ed a he same ime by he mollusks).
Addi ional s udies ha e also epo ed g ea a iabili y in he elemen al
mola concen a ions be ween e y nea g ow h lines/inc emen s [65,
71], which is a e y ele an handicap o using ace elemen a ios as a
sui able p oxy o deciphe en i onmen al condi ions du ing he pas .
In es iga ions p e iously conduc ed by Hausmann e al. [35,45] ha e
shown ha measu ing all a ea o he shell sec ion is e y use ul o be e
unde s anding how ace elemen s a e inco po a ed wi hin shell ca -
bona e laye s deposi ed a he same ime. To conduc 2D measu emen
in he nea u u e could be a e y use ul app oach o accu a ely
econs uc ing SST du ing he pas , since his no el me hod could allow
us o ob ain an a e age alue o he same g ow h lines/inc emen s (as
o he ICP based echniques o e ). Addi ionally, o a be e unde -
s anding o how ca bona e is deposi ed by he mollusk will also enable
us o de e mine he adequa e measu emen pa h along he shell g ow h
o each species.
Table 4
Maximum and minimum Mg/Ca ela i e concen a ion esul s om CF-LIBS and
LA-ICP-MS o he ou mollusk shell samples.
Sample LAN.563 LAN.565 LAN.566 LAN.567
Maximum CF-LIBS (mmol Mg/
mol Ca)
25.6 27.4 26.1 27.7
Minimum CF-LIBS (mmol Mg/
mol Ca)
15.1 14.1 14.8 14.2
Maximum LA-ICP-MS (mmol
Mg/mol Ca)
29.5 26.4 27.2 26.3
Minimum LA-ICP-MS (mmol
Mg/mol Ca)
14.0 12.8 13.2 12.5
Fig. 6. CF-LIBS and LA-ICP-MS concen a ion p o iles o he ou limpe specimens.
M. Ma ínez-Minche o e al.
Talan a 251 (2023) 123757
8
4. Conclusions
This s udy p esen s, o he i s ime, a compa a i e analysis o Mg/
Ca concen a ion p o iles measu ed wi hin shells o he common limpe
P. dep essa using he LA-ICP-MS and CF-LIBS app oaches. The
Calib a ion-F ee LIBS app oach has been success ully applied o ou
Pa ella dep essa limpe specimens. This me hod es ima es mola con-
cen a ions o calcium and magnesium p esen in ma ine mollusk shells.
Bo h me hods iden i y expec ed ends in Mg/Ca concen a ion a ios in
he edge o he shell ( he mos ecen ly deposi ed ma e ial). Season o
cap u e (i.e., end o summe ) is co ec ly e lec ed in all 4 samples using
bo h LA-ICP-MS and he CF-LIBS app oach. Howe e , LAN.563 spec-
imen show a a he noisy sequence so ha he win e and summe
seasons p e ious o i s collec ion canno be clea ly deduced. This
beha io can be explained by he hypo hesis ha magnesium inco po-
a ion is in luenced no only by empe a u e bu also by physiological
ac o s as well as he exis ence o in a-species a iabili y o he exis-
ence o g ow h s ops [63,64,72]. The esul s de i ed om his s udy
show a ela i ely high co ela ion be ween he concen a ion da a ob-
ained wi h he LA-ICP-MS echnique and CF-LIBS me hodology, wi h
eg ession coe icien alues om 0.63 o 0.81 and a e age ela i e e o
alues lowe han 10% in e e y sample. The a o emen ioned me ics
make he CF-LIBS app oach a eliable way o de e mine Mg/Ca mola
concen a ion wi hin P. dep essa limpe shells. The signi ican co ela-
ion obse ed be ween Mg/Ca p o iles de i ed om bo h echniques
ha e e ealed, o he i s ime, ha he algo i hm used he ein enables
o co ec ly es ima e he mola concen a ions o he chemical elemen s
in biogenic ca bona es. CF-LIBS is a aluable app oach o paleoclima ic
and a chaeological s udies, yielding eco ds o empe a u e seasonali y
du ing he li e o an indi idual as well as he ime o yea when mollusks
om a chaeological si es we e collec ed as a ood esou ce by pas
human occupan s. Ne e heless, as his s udy uses jus ou specimens,
u he s udies a e s ill equi ed o u he alida e he app oach.
Ins i u ional Re iew Boa d s a emen
This in es iga ion does no in ol e esea ch on animal’s ansgenic
small labo a o y animals, animals ansgenic a m animals, non-human
p ima es o animals cloned a m animals. Du ing he de elopmen o
his s udy mode n mollusk specimens we e collec ed. The equi ed au-
ho iza ions we e ob ained om he Fishing Ac i i y Se ice o he
Can ab ian Go e nmen . The s udy was conduc ed acco ding o he
guidelines o he Decla a ion o Helsinki, and app o ed by he Ins i u-
ional Re iew Boa d (o E hics Commi ee) o Fishing Ac i i y Se ice o
he Can ab ian Go e nmen . In o med.
C edi au ho s a emen
Ma ina Ma ínez-Minche o: Concep ualiza ion, Me hodology,
In es iga ion, W i ing – O iginal D a , Valida ion. Adol o Cobo:
Concep ualiza ion, Me hodology, So wa e, W i ing - O iginal D a ,
Valida ion. Ana M´
endez-Vicen e: In es iga ion, W i ing - Re iew &
Edi ing, Valida ion. Jo ge Pisone o: Resou ces, W i ing - Re iew &
Edi ing, Valida ion. Ne ea Bo del: Resou ces, W i ing - Re iew & Edi -
ing, Valida ion. Igo Gu i´
e ez-Zugas i: Resou ces, W i ing - Re iew &
Edi ing, Valida ion. Pa ick Robe s: W i ing - Re iew & Edi ing, Vali-
da ion. ´
Al a o A izabalaga: W i ing - Re iew & Edi ing, Valida ion.
Jos´
e Valdiande: In es iga ion, Valida ion. Jesús Mi apeix: In es iga ion,
W i ing - Re iew & Edi ing. Jos´
e Miguel L´
opez-Higue a: Supe ision,
Valida ion. Asie Ga cía-Esc´
a zaga: Concep ualiza ion, Me hodology,
In es iga ion, Supe ision, W i ing – O iginal D a , Valida ion.
Fig. 7. Linea eg ession o CF-LIBS and LA-ICP-MS o he ou specimens.
M. Ma ínez-Minche o e al.
Talan a 251 (2023) 123757
9
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgmen s
This esea ch was pe o med as pa o he p ojec s HAR2016-75605-
R and HAR2017-86262-P unded by he Spanish Minis y o Economy
and Compe i i eness, MINECO. This wo k is also pa o he p ojec s
PID2019-107270RB-C21 and MCI-21-PID2020-113951-GB-I00 unded
by MCIN/AEI/10.13039/501100011033 and FEDER “A way o make
Eu ope”. This in es iga ion was also suppo ed by he Alexande on
Humbold Founda ion hough a Humbold Resea ch Fellowship (no
code a ailable). This s udy is also pa o he p ojec PID2021-
126937NB-I00 (PALEOCROSS) unded by MCIN/AEI/10.13039/
501100011033 and by ERDF "A way o making Eu ope”. MMM esea ch
was unded by p edoc o al g an BES-2017-080076 om MINECO.
Du ing he de elopmen o his esea ch AGE was unded by he Basque
Coun y Pos doc o al P og amme (g an numbe POS_2020_2_0032), by
he Uni e si y o La Rioja h ough a pos doc o al g an (no code a ail-
able) and he is cu en ly suppo ed by he Ca alonia Pos doc o al P o-
g amme h ough a Bea iu de Pin´
os ellowship (g an numbe 2020 BP
00240). This s udy has also been suppo ed by he P ehis o ic Resea ch
Consolida ed G oup o he Basque Coun y Uni e si y (IT-1435-22),
unded by he Basque Coun y Go e nmen . We would also like o hank
he Max Planck Socie y o unding, as well as he Uni e sidad de Can-
ab ia (UC), Ins i u o In e nacional de In es igaciones P ehis ´
o icas de
Can ab ia (IIIPC), G upo de Ingenie ía Fo ´
onica (GIF), Uni e si y o he
Basque Coun y (UPV/EHU) and Uni e si y o La Rioja (UR) o
p o iding suppo .
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j. alan a.2022.123757.
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