Jou nal o Haza dous Ma e ials 452 (2023) 131280
A ailable online 28 Ma ch 2023
0304-3894/© 2023 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-
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Resea ch A icle
Fa e and e ec s o g aphene oxide alone and wi h so bed benzo(a)py ene in
mussels My ilus gallop o incialis
Nago e Gonz´
alez-So o
a
, Nago e Blasco
a
, Mi eia I azola
b
, Eide Bilbao
a
, Lúcia Guilhe mino
c
,
Mi en P. Caja a ille
a
,
*
a
CBET Resea ch G oup, Dep . Zoology and Animal Cell Biology, Science and Technology Facul y and Plen zia Ma ine S a ion, Uni e si y o he Basque Coun y (UPV/
EHU), Basque Coun y, Spain
b
Dep . Analy ical Chemis y and Plen zia Ma ine S a ion, Uni e si y o he Basque Coun y (UPV/EHU), Basque Coun y, Spain
c
Eco oxicology Resea ch G oup, ICBAS, Ins i u e o Biomedical Sciences o Abel Salaza and Resea ch G oup o Eco oxicology, S ess Ecology and En i onmen al Heal h
(ECOTOX), CIIMAR, In e disciplina y Cen e o Ma ine and En i onmen al Resea ch, Uni e si y o Po o, Po ugal
HIGHLIGHTS GRAPHICAL ABSTRACT
•GO was ound in diges i e ac lumen
and eces o mussels exposed o GO o
GO+BaP.
•BaP bioaccumula ed in mussels exposed
o GO+BaP and especially o BaP.
•An en i onmen ally ele an concen a-
ion o GO was geno oxic o mussel
hemocy es.
•In lamma ion in diges i e gland/gonad
and oocy e a esia ound in exposed
mussels.
•E ec s o ca ied BaP and enhanced
oxici y o GO+BaP s GO o BaP we e
iden i ied.
ARTICLE INFO
Edi o : Ka ina S. B. Miglio anza
Keywo ds:
G aphene amily nanoma e ials
Polycyclic a oma ic hyd oca bons
T ojan Ho se e ec
Bi al e mollusks
Biological esponses
ABSTRACT
G aphene oxide (GO) has gained a g ea scien i ic and economic in e es due o i s unique p ope ies. As
inco po a ion o GO in consume p oduc s is ising, i is expec ed ha GO will end up in oceans. Due o i s high
su ace o olume a io, GO can adso b pe sis en o ganic pollu an s (POPs), such as benzo(a)py ene (BaP), and
ac as ca ie o POPs, inc easing hei bioa ailabili y o ma ine o ganisms. Thus, up ake and e ec s o GO in
ma ine bio a ep esen a majo conce n. This wo k aimed o assess he po en ial haza ds o GO, alone o wi h
so bed BaP (GO+BaP), and BaP alone in ma ine mussels a e 7 days o exposu e. GO was de ec ed h ough
Raman spec oscopy in he lumen o he diges i e ac and in eces o mussels exposed o GO and GO+BaP while
BaP was bioaccumula ed in mussels exposed o GO+BaP, bu especially in hose exposed o BaP. O e all, GO
ac ed as a ca ie o BaP o mussels bu GO appea ed o p o ec mussels owa ds BaP accumula ion. Some e ec s
Abb e ia ions: CDNB, 2,4-Dini ochlo obenzene; AChE, Ace ylcholines e ase; BaP, Benzo(a)py ene; Ca , Ca alase ac i i y; DMSO, Dime hyl sul oxide; GC/MS-
QqQ, Gas ch oma og aphy ollowed by iple quad upole mass spec ome y; GC–MS, Gas ch oma og aphy–mass spec ome y; GPx, Glu a hione pe oxidase; GST,
Glu a hione S- ans e ase; GO-PEI, GO unc ionalized wi h polye hyleneimine; GI, Gonad index; GFNs, G aphene amily nanoma e ials; GO, G aphene oxide;
GO+BaP, G aphene oxide wi h so bed BaP; IDH, Isoci a e dehyd ogenase; NMs, Nanoma e ials; NR, Neu al ed; NADPH, Nico inamide adenine dinucleo ide
phospha e; PFOS, Pe luo ooc ane sul ona e; POPs, Pe sis en o ganic pollu an s; PAHs, Polycyclic a oma ic hyd oca bons; KP, Po assium phospha e; ROS, Reac i e
oxygen species; SOD, Supe oxide dismu ase; T is, T is(hyd oxyme hyl)-aminome han.
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (M.P. Caja a ille).
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Recei ed 9 No embe 2022; Recei ed in e ised o m 13 Ma ch 2023; Accep ed 22 Ma ch 2023
Jou nal o Haza dous Ma e ials 452 (2023) 131280
2
obse ed in mussels exposed o GO+BaP we e due o BaP ca ied on o GO nanopla ele s. Enhanced oxici y o
GO+BaP wi h espec o GO and/o BaP o o con ols we e iden i ied o o he biological esponses, demon-
s a ing he complexi y o in e ac ions be ween GO and BaP.
1. In oduc ion
Nanoma e ials (NMs) a e commonly de ined as a di e se class o
ma e ials wi h a leas one dimension a he nanoscale (<100 nm) [1]. In
2011, he Eu opean Commission de ined a nanoma e ial as: “a na u al,
inciden al o manu ac u ed ma e ial con aining pa icles, in an unbound s a e
o as an agg ega e o as an agglome a e and whe e, o 50% o mo e o he
pa icles in he numbe size dis ibu ion, one o mo e ex e nal dimensions is in
he size ange 1–100 nm. In speci ic cases and whe e wa an ed by conce ns
o he en i onmen , heal h, sa e y o compe i i eness he numbe size dis-
ibu ion h eshold o 50% may be eplaced by a h eshold be ween 1% and
50% [2]. Among NMs, ca bon-based NMs ha e a ac ed g ea scien i ic
and echnological a en ion due o he physico-chemical p ope ies o
hei nanome ic s uc u es. Ca bon-based NMs include among o he s,
ulle enes, ca bon nano ubes, ca bon black o g aphene; bu among all
o hem, g aphene s ands ou o he unique p ope ies ha make i he
hinnes , s onges and ligh es known ma e ial [3].
G aphene amily nanoma e ials (GFNs) a e used in a wide ange o
applica ions including elec onic de ices, a new gene a ion o ba e ies,
senso s [4], biomedical applica ions [5], an ico osion coa ings [6,7],
ag icul u al p ocedu es [8] o en i onmen al applica ions such as was e
wa e ea men s [4], wa e desalina ion [9] and pollu an s emo al [4,
10]. The e a e mo e han 26,000 g aphene ela ed pa en s [11] and
mo e han 100 g aphene based p oduc s [12]. In ac , he p oduc ion o
GFNs is highe in compa ison o he es o NMs [13] and i is expec ed o
con inue g owing as he expensi e and low e icien me hods used
nowadays o g aphene p oduc ion a e imp o ed. The e o e, g aphene
p oduc ion is expec ed o each 3800 ones, wi h a wo h o 300 millions
by 2027 [14].
G aphene oxide (GO) is a p ecu so in g aphene syn hesis and one o
he mos s udied g aphene de i a es [15] whose eac i i y and capaci y
o chemical unc ionaliza ion [12] a e impo an cha ac e is ics ela ed
o he p esence o unc ional oxygen g oups bo h on he su ace (hy-
d oxyl and epoxy g oups) and in he edges (ca boxyl g oups) o he shee
[16]. Due o he gene al in e es on GO and i s inc eased p oduc ion in
he las yea s, GO is being eleased in o he en i onmen du ing i s li e
cycle [17] bo h h ough di ec elease (e.g., sewage e luen s, i e
in lux) o indi ec ly (e.g., ae ial deposi ion, dumping and un o ) [18].
The e o e, GO will de ini ely each coas al and ma ine ecosys ems [12]
and p obably will in e ac wi h di e en componen s o he na u al
sys em, which may al e beha iou , anspo , a e and oxici y o GO
[19]. T anspo and a e o GFNs a e go e ned mainly by he s abili y o
suspensions, which may be al e ed by en i onmen al ac o s such as
salini y, o ganic ma e concen a ion, oxida ion s a us and bio-
u ba ion. In aqua ic en i onmen s, GO can dispe se and o m ela i ely
s able suspensions ha endu e in he wa e column [18,20]. Such
beha io may acili a e he up ake o GO by a la ge numbe o o ganisms
h ough di e en ou es such as inges ion o espi a ion, as i has been
desc ibed o o he NMs [21]. In addi ion, he high pe sis ence o GO can
esul in hei bioaccumula ion and biomagni ica ion in ood webs [4,
22], inc easing hei po en ial impac in ma ine ecosys ems, e en i he
GO concen a ions eleased in o he en i onmen a e ela i ely low
[23]. The e o e, le els o GO in su ace wa e s should be a p ima y
conce n [24]. Howe e , he en i onmen al concen a ions o GO a e s ill
la gely unknown [12]. The p esence o GO was al eady de ec ed in he
biomass om was ewa e ea men plan s [25]. Recen s udies conside
ha he p edic ed en i onmen al concen a ion o GO could be simila
o ha desc ibed o o he NMs such as mul i-walled ca bon nano ubes,
which is in he ange 0.001–1000
μ
g/L o aqua ic en i onmen s [26].
Toxici y o NMs is s ongly ela ed o hei size, shape, su ace
p ope ies o chemical composi ion, which a e key cha ac e is ics o
isk assessmen [1,27]. O e all, a cellula le el, GFNs ha e been e-
po ed o dec ease in eg i y o he cell´s plasma memb ane, possibly as a
consequence o en y o nanoshee s in o cells by di ec pene a ion o
endocy osis [18]. Shee s can also dis up he plasma memb ane due o
induced in agina ions o pe o a ions [28,29], o e en by he des uc-
i e ex ac ion o lipids [30]. Dis up ion o he plasma memb ane and
in e naliza ion o GFNs can p o oke he o ma ion o eac i e oxygen
species (ROS) and lead o oxida i e s ess [29], which is conside ed one
o he main unde lying mechanisms o oxici y o NMs [27]. Oxida i e
damage caused by he inc eased in acellula p oduc ion o ROS can
lead o mi ochond ial and lysosomal dys unc ion and inally o a
dec ease in he iabili y o hemocy e cells o ma ine mussels [29]. In
addi ion, oxida i e s ess and/o physical cell damage can also cause
DNA damage esul ing in he agmen a ion and des uc ion o nucleic
acids [31]. These al e a ions a he cellula le el may lead o e ec s a
highe biological le els, such as educ ion o me abolic ac i i y [4,32],
his opa hological lesions [33,34], al e a ions in beha io and locomo o
unc ions [35] and ad e se impac on he ep oduc ion capaci y, g ow h
and su i al [36].
In aqua ic en i onmen s, gene ally NMs do no appea alone, bu a e
ound wi hin complex mix u es o chemical con aminan s o igina ed
bo h om na u al and an h opogenic sou ces. Se e al s udies ha e
demons a ed ha GFNs show a g ea adso p ion capaci y o pe sis en
o ganic pollu an s (POPs) such as polycyclic a oma ic hyd oca bons
(PAHs), mainly due o hei la ge su ace a ea and hyd ophobici y
[37–40]. This adso p ion p oduces accumula ion o o ganic pollu an s
on he su ace o he NM, which may inc ease hei up ake by aqua ic
o ganisms [41] and hei po en ial ad e se e ec s [39], a phenomenon
known as T ojan ho se e ec .
O iginally, Limbach e al. [42] in oduced he T ojan ho se concep
o e e o he ex ended oxici y caused by me al nanopa icles in com-
pa ison o hei soluble o ms, due o he con inuous elease o me al
ions in and ou o cells. La e , ca ie o T ojan ho se e ec was in o-
duced o designa e he inc eased up ake and accumula ion o en i on-
men al pollu an s acili a ed by NMs [43,44]. Thus, he T ojan ho se
e ec is gene ally de ined as he possible h ea o NMs due o hei
abili y o adso b and ca y adso bed compounds o o ganisms, a phe-
nomenon known o occu o many NMs and se e al me al o o ganic
pollu an s [45–48]. Mos wo ks a e based on co-exposu e o NMs and
en i onmen al pollu an s and he oxici y o he mix u e is usually
compa ed o ha o he dissol ed pollu an and no o ha o he NM
alone [46,48] which could lead o an unde es ima ion o he con ibu-
ion o he NM o he oxici y o he mix u e [46]. Di e en e iews
conside ha a T ojan ho se e ec occu s when he exposu e o he
mix u e causes a signi ican ly highe oxicological e ec han he
exposu e o he compounds sepa a ely [45–50].
Among PAHs, benzo(a)py ene (BaP) is a p io i y pollu an [51,52],
commonly used in eco oxicology s udies and known o cause e ec s a
di e en le els o biological o ganiza ion [53]. BaP is a geno oxic and
ca cinogenic agen capable o p oducing issue and DNA damage [54,
55], oxida i e s ess [56], pe oxisome p oli e a ion [57], lysosomal
dys unc ion [58], endoc ine dis up ion [59], among o he e ec s in
ma ine o ganisms, including bi al es. In addi ion, BaP can in e ac wi h
eme ging pollu an s o high conce n, such as mic oplas ics and NMs, in
ma ine mussels esul ing in oxicological in e ac ions [55,60]. Among
a ge o ganisms, mussels (My ilus sp.), a e conside ed model o ganisms
o he e alua ion o pollu an s including mic o and nanoscale pa icu-
la e ma e ials, due o hei highly de eloped mechanisms o cellula
in e naliza ion o pa icles h ough endocy osis and phagocy osis, o
N. Gonz´
alez-So o e al.
Jou nal o Haza dous Ma e ials 452 (2023) 131280
3
physiological unc ions such as in acellula diges ion and cellula im-
muni y [27,61,62].
To imp o e he basis o he isk assessmen o GFNs o he ma ine
en i onmen , i is e y impo an o s udy he oxici y o GFNs, alone
and combined wi h POPs in ma ine o ganisms, such as mussels. The e-
o e, he aim o his wo k was o in es iga e he a e and e ec s o GO,
alone o wi h so bed BaP, in adul ma ine mussels (M. gallop o incialis)
using Raman spec oscopy and a ba e y o biological esponses,
espec i ely. O e all, his wo k con ibu es o unde s and he T ojan
ho se e ec o GFNs owa ds BaP in mussels.
2. Ma e ials and me hods
2.1. Ob en ion o GO and p epa a ion o GO wi h so bed BaP
Comme cial nanopla ele s o g aphene oxide (GO) we e pu chased
om G aphenea (San Sebas ian, Spain) as s able suspensions. Acco ding
o he supplie , he concen a ion o GO in he dispe sion was 10 mg/mL.
This was expe imen ally con i med by measu ing GO in aqueous dis-
pe sions wi h a UV– is spec opho ome e (UV-2550, Shimadzu) a 660
nm, whe e g aphene ollows he Lambe -Bee law. Fo ha aim, a
calib a ion cu e (Fig. S1) was p epa ed om dispe sions wi h known
GO concen a ions. The concen a ion o GO expe imen ally de e mined
was 9.91 mg/mL.
Acco ding o he manu ac u e ’s in o ma ion, nanopla ele s showed
la e al dimensions anging om 500 nm o ew mic ons and hickness
was <2 nm. Oxygen con en was abou 40% w . Cha ac e iza ion o he
same ba ch o GO by ansmission elec on mic oscopy and a omic o ce
mic oscopy was epo ed p e iously by Ma ínez-´
Al a ez e al. [63].
The p o ocol o p epa e GO wi h so bed benzo(a)py ene (BaP) was
based on p e ious wo k (Ma ínez-´
Al a ez e al. [63]). B ie ly, a e
p epa ing he BaP solu ion o 100
μ
g/L con aining 0.01% dime hyl
sul oxide (DMSO, pu i y 99% Sigma, S . Louis, Missou i) in a glass
bo le, GO was added in a 0.5 mg:10 mL GO/BaP p opo ion (weigh /-
olume). So p ion p ocess was allowed by shaking samples in an o bi al
shake (300 pm) o 24 h in he da k a 21 ±1 ◦C. A e 24 h, samples
we e cen i uged in an Alleg a X30R cen i uge (9509 g, 30 min).
Supe na an was disca ded and he pelle was e-suspended in 50 mL o
MilliQ wa e . Samples we e o exed be o e dosing in o anks. Samples
con aining GO alone we e p ocessed in he same way, bu using only
MilliQ wa e .
Fo so p ion expe imen s [63], samples we e p epa ed in he same
way desc ibed abo e. A e cen i uga ion, absence o GO in supe na-
an s was assessed by spec opho ome y a 230 nm. Then, BaP was
quan i ied in supe na an s by gas ch oma og aphy/mass spec ome y
a e solid phase mic o ex ac ion. Based on he BaP concen a ion
measu ed in he aqueous phase, he amoun o BaP so bed o GO was
indi ec ly calcula ed. This p ocess was done in iplica e and ials con-
aining BaP solu ions wi hou GO we e p ocessed in pa allel o moni o
po en ial BaP loss due o e apo a ion, deg ada ion, so p ion on o he
ial walls o o he ac o s du ing he expe imen al p ocedu e. Acco ding
o Ma ínez-´
Al a ez e al. [63] he p opo ion o BaP so bed on o GO
was 96.7% ±0.5%.
2.2. Sampling and acclima ion o mussels
Roughly 460 mussels My ilus gallop o incialis (3.5–4.5 cm shell
leng h) we e collec ed in Feb ua y 2019 in Mundaka, Basque Coun y
(43◦24 ´04.9”N, 2◦41 ´41.6”W). Mussels we e main ained in aqua ia a-
cili ies a he Plen zia Ma ine S a ion (PiE) o he Uni e si y o he
Basque Coun y (UPV/EHU), o acclima ion du ing 21 days. Acclima-
ion was ca ied ou in a 300 L polyp opylene ank wi h a eci cula ing
seawa e sys em. Ma ine wa e was collec ed wi h a pump a 10 m dep h
in he mou h o he Bu oi es ua y (43◦24′21′′N, 2◦56′47′′W) and il e ed
(pa icles ≤3 µm) be o e eaching he ma ine s a ion. Mussels we e no
ed o wo days and hen hey we e ed once a day wi h he Isoch ysis
galbana mic oalgae (2 ×10
7
cells/mussel-day) o 19 days. I. galbana (T-
Iso clone) cul u es we e ob ained om he Animal Physiology Labo a-
o y a UPV/EHU. Du ing acclima ion, ligh egime was 12 L/12D and
oom empe a u e was kep a 18 ◦C. Wa e pa ame e s we e checked
daily wi h a mul ichannel p obe. The a ia ion o wa e pa ame e s
du ing he acclima ion pe iod was (mean ±s anda d de ia ion): salini y
o 32.40 ±0.27, pH o 7.25 ±0.35 and empe a u e o 16.02 ±0.08 ◦C.
The dissol ed O
2
was always abo e 80%.
Fig. 1. Summa y o he expe imen al design. A: Exposu e g oups, B: p epa a ion o g aphene oxide (GO) wi h adso bed benzo(a)py ene (BaP) and C: exposu e se up
o he GO+BaP g oup as an example.
N. Gonz´
alez-So o e al.
Jou nal o Haza dous Ma e ials 452 (2023) 131280
4
2.3. Mussel exposu e
In o de o keep a homogeneous suspension o GO, wi hou agg e-
ga ion o p ecipi a ion, a wa e eci cula ion sys em consis ing o wo
wa e pumps was ins alled in he aqua ia. Be o e mussel exposu e, anks
we e exposed o 24 h o GO a he same concen a ion es ed in o de o
sa u a e he sys em.
A e 21 days o acclima ion, mussels we e exposed o 7 days o
g aphene oxide (GO), g aphene oxide wi h so bed BaP (GO+BaP) o BaP
alone in wo 20 L eplica e anks pe ea men (GO R1 and GO R2,
GO+BaP R1 and GO+BaP R2, BaP R1 and BaP R2) wi h 57 mussels each
(Fig. 1). Two con ol anks, also wi h 57 mussels each, we e un in
pa allel (Con ol R1, Con ol R2). Mussel samples we e aken a e 7
days o exposu e. An exposu e concen a ion o 500
μ
g/L GO was
selec ed, based on en i onmen ally ele an concen a ions o mul i-
walled ca bon nano ubes, which ange om 1
μ
g/L o 1 mg/L [12].
Fo he GO+BaP exposu e g oups, he nominal concen a ion o BaP
incuba ed wi h GO was 100
μ
g/L. Finally, o he BaP exposu e g oups,
96.7
μ
g/L BaP was used as he equi alen BaP concen a ion so bed in
GO+BaP p epa a ions [63].
Du ing he expe imen , wa e was changed daily. Th oughou
exposu e, mussels we e ed once a day wi h I. galbana (2 ×10
7
cells
/mussel-day) wo hou s be o e changing wa e . While wa e o anks
was changed, 5 mussels pe ank we e selec ed andomly and placed in
indi idual glass con aine s wi h clean wa e o collec eces (Fig. 1). The
ligh egime and oom empe a u e we e kep a 12 L/12D and 18 ◦C,
espec i ely. Wa e pa ame e s we e checked daily wi h a mul ichannel
p obe: salini y (32.93 ±0.06 PSU), dissol ed O
2
(>77%), pH (7.48
±0.23) and empe a u e (16.62 ±0.25 ◦C).
2.4. Bioaccumula ion o BaP
The p esence o BaP in seawa e was checked by gas ch oma og a-
phy–mass spec ome y (GC–MS) analysis in wa e collec ed a e 20 h
o exposu e in all he anks acco ding o he p o ocol desc ibed in Ka -
sumi i e al. [64]. B ie ly, wis e s i ba s (20 mm leng h and 0.5 mm
ilm hick, Ge s el GmbH & Co. KG, Mülheim an de Ruh , Ge many)
we e employed o ex ac ion o BaP om he ma ine wa e . Twis e
ba s we e in oduced in samples (~20 mL) du ing 195 min. Then,
wis e ba s we e cleaned wi h Milli-Q wa e and d ied wi h pape is-
sue. BaP was deso bed om he wis e ba s using a comme cial he mal
deso p ion TDS-2 uni connec ed o a CIS-4 injec o (Ge s el GmbH &
Co. KG,Mülheiman de Ruh , Ge many). The deso p ion uni was hen
connec ed o an Agilen 6890 gas ch oma og aph coupled wi h an Agi-
len 5975 mass spec ome e sys em (Agilen Technologies, Palo Al o,
USA) o BaP de e mina ion.
Mussels o chemical analysis (14–18 mussels pe ank) we e s o ed
a −40 ◦C and analyzed a IPROMA (Cas ellon, Spain) o de e mine
bioaccumula ion o BaP in whole mussel issues. Mussels we e lyophi-
lized and homogenized. Ex ac ion was pe o med wi h ace one and
dichlo ome hane using he QuEChERS me hod [65]. Concen a ion o
BaP was de e mined using gas ch oma og aphy ollowed by iple
quad upole mass spec ome y (GC/MS-QqQ) [66], using BCR-682
mussel issue as e e ence ma e ial. The limi o quan i ica ion in he
analyses was 5 ng/g d y weigh .
2.5. De e mina ion o g aphene oxide in mussel issues and eces
Th ee mussels pe ank we e dissec ed, ozen in liquid ni ogen and
main ained a −40 ◦C un il u he analysis. Then 20 µm sec ions we e
ob ained in a c yos a (Leica CM 3050 S) and obse ed unde an inVia
Renishaw mic oscope in o de o ge Raman spec a using a 532 nm
lase . Condi ions we e se using he 100x objec i e as ollows: 1–5µm
s eps in he issue, 0.2–0.4 s, 10% lase in ensi y, 1 accumula ion and
ocused in 1200 nm. Fo each sample, se ial c yo ome sec ions we e
ixed in Bake ’s solu ion ( o maldehyde 4% ( / ), NaCl 2% (w/ ),
calcium ace a e 1% (w/ )) o 15 min, insed in dis illed wa e and
s ained 20 s in 0.1% oluidine blue o ge he opog aphic e e ence o
he issue sample.
Mussels´ eces we e also collec ed and analyzed by Raman spec os-
copy. Mussel eces we e comple ely d ied and placed in an aluminum
oil be o e ge ing he Raman spec um. Condi ions we e se using a
100x objec i e as ollows: 100% lase in ensi y, 1 s, 80 accumula ion
and ocused in 1200 nm. As e e ence, spec a o mic oalgae Isoch ysis
galbana, BaP s ock solu ion and DMSO we e ob ained in he same
condi ions.
2.6. Cellula bioma ke s in hemocy es
Hemolymph o 8 mussels pe ank was wi hd awn om he pos e io
adduc o muscle and cell iabili y, ca alase ac i i y and DNA damage in
e ms o mic onuclei o ma ion we e measu ed in hemocy es o indi-
idual mussels. Neu al ed (NR) up ake was assessed acco ding o
Bo en eund & Pue ne [67] wi h modi ica ions explained in
Gonz´
alez-So o e al. [60]. Ca alase ac i i y (Ca ) was assessed acco ding
o Aebi [68] as modi ied in Gonz´
alez-So o e al. [60]. P o ein concen-
a ion was measu ed ollowing he B ad o d me hod
69
o no malize
abso bance da a. Ca ac i i y was exp essed as he consump ion o mM
H
2
O
2
/min/mg p o ein.
The mic onucleus assay was pe o med acco ding o Du oudie e al.
[70]. Mic onuclea ed cells we e classi ied ollowing he accep ed
c i e ia o mussels: well-p ese ed cell cy oplasm, mic onuclei no
ouching he main nucleus, simila o weake s aining han he main
nucleus and size o mic onuclei ≤1/3 in compa ison o he main nu-
cleus. O he nuclea abno mali ies such as binuclea ed cells, occu ence
o nucleoplasmic b idges and nuclea buds we e sco ed acco ding o
Pin o-Sil a e al. [71] and Bolognesi & Fenech [72]. Resul s a e epo ed
in ‰ equencies.
2.7. Enzyme ac i i ies in mussel issues
Diges i e gland, gills and adduc o muscle o 10 mussels pe ank
we e dissec ed ou , ozen in liquid ni ogen and main ained a −80 ◦C
un il u he analysis. Adduc o muscle was homogenized in 0.1 M po-
assium phospha e (KP) bu e (pH 7.2) o ace ylcholines e ase (AChE)
de e mina ion. Gills we e cu in wo hal s; one hal was homogenized in
0.1 M KP bu e (pH 6.5) o glu a hione S- ans e ase (GST) de e mi-
na ion and he second hal in 0.1 M KP bu e (pH 7.4) o Ca , glu a-
hione pe oxidase (GPx) and supe oxide dismu ase (SOD)
de e mina ion. Diges i e glands we e di ided in h ee pa s. The i s
pa was used o GST de e mina ion, he second o Ca , GPx and SOD,
and he las piece was homogenized in 50 mM is bu e (T is(hyd ox-
yme hyl)-aminome han, pH 7.8) o isoci a e dehyd ogenase (IDH)
de e mina ion. Tissues we e homogenized in each bu e ollowing a
1:10 p opo ion, issue weigh : olume o bu e .
The ac i i y o he enzyme AChE and IDH, GST, GPx and SOD we e
de e mined as desc ibed in p e ious s udies whe e some modi ica ions
o he o iginal ecnhiques we e made [73,74]. B ie ly, AChE ac i i y was
de e mined acco ding o Ellman e al. [75] a 412 nm and exp essed as
he p oduc ion o 5,5’-di hiobis-(2-ni obenzoic acid) in nmol/min/mg
p o ein. IDH ac i i y was de e mined acco ding o Ellis & Goldbe g [76]
a 340 nm and exp essed as he p oduc ion o nico inamide adenine
dinucleo ide phospha e (NADPH) in nmol/min/mg p o ein. GST ac i i y
was de e mined acco ding o Habig e al. [77] a 340 nm and exp essed
as he p oduc ion o 2,4-Dini ochlo obenzene (CDNB) conjuga es wi h
he hiol g oup o glu a hione in nmol/min/mg p o ein. Ca ac i i y was
de e mined as p e iously explained. GPx ac i i y was de e mined ac-
co ding o Floh´
e& Günzle [78] a 340 nm and exp essed as he con-
sump ion o NADPH in nmol/min/mg p o ein. SOD ac i i y was
de e mined acco ding o McCo d & F ido ich [79] a 550 nm and gi en
in SOD uni s (1 SOD uni =50% inhibi ion o he educ ion o cy o-
ch ome C pe mg p o ein). Each enzyme ac i i y was no malized o
N. Gonz´
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Jou nal o Haza dous Ma e ials 452 (2023) 131280
5
p o ein concen a ion using B ad o d me hod [69].
2.8. His opa hology o he diges i e gland
Diges i e glands o 10 mussels pe ank we e dissec ed ou and
p ocessed ollowing a s anda d p o ocol o his ology [80]. B ie ly, is-
sues we e ixed in 4% o malin in indi idual casse es and dehyd a ed
h ough a g aded se ies o e hanol ha inished in xylene using an
au oma ic issue p ocesso (Leica ASP300; Leica Ins umen s, We zla ,
Ge many). Then, samples we e embedded in pa a in and 5 µm sec ions
we e cu in a Lei z 1512 mic o ome (Leica Ins umen s, We zla , Ge -
many). Slides we e d yed in an o en a 37 ◦C (24 h) and s ained wi h
hema oxylin/eosin [81] using an au os aine XL V2.02 (Leica). Slides
we e moun ed in DPX and analyzed unde a BX51 ligh mic oscope
(Olympus, Tokyo, Japan).
Vacuoliza ion, a ophy and nec osis o he diges i e ubule epi he-
lium, ib osis, hemocy ic in il a ion, agg ega ion o b own cells in he
connec i e issue and in diges i e ubules and p esence o pa asi es we e
assessed in he diges i e gland ollowing Villalba e al. [82], Ga mendia
e al. [83] and Bignell e al. [84]. The p e alence o each al e a ion
(numbe o indi iduals showing each pa hology di ided by he numbe
o indi iduals o each g oup) was calcula ed as pe cen age.
2.9. Game e de elopmen , gonad index and his opa hology o gonad
Man le o he same animals used o he his opa hological analysis o
he diges i e gland we e dissec ed ou and p ocessed ollowing he
s anda d p o ocol o his ology desc ibed be o e.
Sex a io, game e de elopmen al s ages and gonad index (GI) we e
de e mined. Six game e s ages we e dis inguished [85] and a gonad
index (GI) alue, anging om 0 ( es ing gonad) o 5 (ma u e gonad),
was assigned o each de elopmen al s age as in Gonz´
alez-So o e al.
[60], adap ed om Kim e al. [86].
Oocy e a esia and nec osis, ib osis, hemocy ic in il a ion, agg e-
ga ion o b own cells, and occu ence o pa asi es we e also assessed in
gonads ollowing O iz-Za agoi ia & Caja a ille [87]. P e alences we e
Table 1
Bioaccumula ion o BaP in mussel so issues (ng/g d y weigh ) in con ol mussels and in mussels exposed o GO, GO+BaP, and BaP o 7 days. N =numbe o mussels
is indica ed. LoQ =Limi o Quan i ica ion: 5 ng/g d y weigh .
Con ol GO GO+BaP BaP
R1
N=14
R2
N=18
R1
N=16
R2
N=14
R1
N=14
R2
N=17
R1
N=15
R2
N=14
<LoQ <LoQ <LoQ <LoQ 23 28 18,700 18,400
Fig. 2. A-C: Hea maps showing he de ec ion o g aphene oxide (GO) in c yos a sec ions o he diges i e gland o di e en mussels; A: Con ol mussel, B: mussel
exposed o GO o 7 days, C: mussel exposed o GO+BaP o 7 days. D: Raman spec a om he zones highligh ed in ed in map B (g een) and he spec um ob ained
o he GO s ock ( ed). E: Raman spec um om he map in A. Scale ba s: A and B: 100 µm and C: 500 µm.
N. Gonz´
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6
calcula ed as o he diges i e gland. In addi ion, in ensi y o oocy e
a esia was assessed using a semiquan i a i e scale: 0- no mal gonad, 1-
less han a hal o ollicles a e a ec ed, 2- abou hal o ollicles a e
a ec ed, 3- mo e han hal o ollicles a e a ec ed and 4- all ollicles a e
a ec ed [86]. In ensi y was calcula ed as Sp/NH, whe e Sp is he sco e
co esponding o he in ensi y o a esia and NH is he numbe o
specimens wi h a esia [83].
2.10. Whole o ganism esponses
Condi ion index was assessed acco ding o Na a o e al. [88]. So
issues o 7 animals pe ank we e excised om he shells, d ied a 80 ◦C
o 24 h and weigh ed. A e wa ds, mussel shell leng hs we e eco ded
wi h a Ve nie calipe and condi ion index was calcula ed as issue d y
weigh (g) / [shell leng h (cm)]
3
.
2.11. Da a analysis
S a is ical analyses we e ca ied ou wi h he aid o he s a is ical
package SPSS 24 (IBM Analy ics, A monk, NY), and he signi icance
le el was 0.05. All da a se s we e es ed o no mali y and homogenei y
o a iance using Kolmogo o -Smi no ´s and Le ene’s es s, espec-
i ely. No mally dis ibu ed da a, which me he assump ions o ho-
mogenei y o a iances, we e assessed ia one-way ANOVA. When
signi ican di e ences we e ound, he Tukey’s pos -hoc es was used o
Fig. 3. A: Neu al ed up ake (gi en as abso bance/10
6
cells); B: ca alase ac i i y (gi en as mM H
2
O
2
/min/mg p o ) in hemocy es o con ol mussels and in mussels
exposed o 7 days o GO, GO+BaP and BaP. Box-plo s show median alue (ho izon al line), 25%−75% qua iles (box) and s anda d de ia ion (whiske s). Do s
deno e ou lie s. Le e s indica e signi ican di e ences among ea men s (one-way ANOVA wi h Tukey’s pos -hoc, p <0.05); C-J: Ligh mic og aphs o mussel
hemocy es du ing he mic onucleus assay showing C) no mal hemocy e o a con ol mussel; D) hemocy e o a mussel exposed o GO showing a mic onucleus (a ow);
E) hemocy e o a mussel exposed o GO showing a nuclea bud; F) binuclea ed cell wi h nucleoplasmic b idge o a mussel exposed o GO; G) binuclea hemocy e o a
mussel exposed o BaP; H) hemocy e o a mussel exposed o GO+BaP showing a mic onucleus (a ow); I) hemocy e o a mussel exposed o GO+BaP showing a
nuclea bud; J) binuclea ed cell wi h nucleoplasmic b idge o a mussel exposed o GO+BaP. Scale ba : 10 µm.
Table 2
F equency (‰) o mic onuclei, binuclea ed cells, binuclea ed cells wi h nucle-
oplasmic b idges and nuclea buds in con ol mussels (N =8) and mussels
exposed o 7 days o GO (N =7), GO+BaP (N =6) and BaP (N =8). Le e s
deno e s a is ical di e ences among g oups (p <0.05 a e K uskal-Wallis es
ollowed by Dunn’s pos -hoc).
Mic onuclei Binuclea ed
cells
Binuclea ed cells wi h
nucleoplasmic b idges
Nuclea
buds
Con ol 0
A
0.125 ±0.35 2.38 ±2.67 2.63
±2.13
A
GO 1.86
±1.35
B
0.14 ±0.38 5.43 ±2.51 8.71
±2.93
B
GO+BaP 3.67
±3.08
B
0.50 ±0.55 6.33 ±2.58 7.17
±1.72
AB
BaP 0.88
±0.83
AB
0.63 ±0.52 3.88 ±2.23 5.88
±1.73
AB
N. Gonz´
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7
iden i y signi ican di e en ea men s. Da a which did no me he
abo e assump ions we e analyzed by he one-way K uskal-Wallis es ,
ollowed by he Dunn’s pos -hoc es when signi ican di e ences we e
ound. Fo his opa hological da a exp essed as pe cen ages, he X
2
es
was used [89]. When no di e ences we e ound be ween he wo
eplica e anks, da a se s we e mixed and displayed as: Con ol, GO,
GO+BaP, BaP. Howe e , when di e ences we e ound be ween epli-
ca es, da a se s we e main ained sepa a e and displayed as: Con ol R1,
Con ol R2, GO R1, GO R2, GO+BaP R1, GO+BaP R2, BaP R1, BaP R2.
3. Resul s
3.1. Bioaccumula ion o BaP
BaP concen a ion in con ol mussels and in mussels exposed o GO
was below he de ec ion limi (Table 1). BaP was bioaccumula ed in
mussels exposed o GO+BaP (23–28 ng/g d y weigh ), bu especially in
mussels exposed o BaP (18,400–18,700 ng/g d y weigh ) (Table 1). In
wa e , BaP was de ec ed only in he anks exposed o BaP alone.
3.2. De e mina ion o g aphene oxide in mussel issues and eces
GO was de ec ed only in he lumen o he diges i e ac o mussels
exposed o bo h GO and GO+BaP (Fig. 2), indica ing ha e en i GO was
in e nalized, i was no accumula ed in he es o mussel issues a e 7
days o exposu e. Acco dingly, GO was de ec ed in eces o mussels
exposed o GO and GO+BaP om day one o exposu e (Fig. S2). Spec a
o mic oalgae Isoch ysis galbana, BaP s ock solu ion and DMSO we e no
obse ed in mussel issues o eces.
3.3. Cellula bioma ke s in hemocy es
Viabili y o hemocy es dec eased in mussels exposed o GO+BaP and
BaP in compa ison o con ols (Fig. 3A). No di e ences we e obse ed
among g oups in he ac i i y o ca alase (Ca ) (Fig. 3B).
Rega ding geno oxici y, highe mic onuclei equency was obse ed
in hemocy es o mussels exposed o GO and GO+BaP in compa ison o
con ols (Table 2, Fig. 3D, G). In addi ion, highe equency o nuclea
buds was obse ed in hemocy es o mussels exposed o GO han in
con ol mussels (Table 2, Fig. 3E, H). No di e ences we e obse ed
among g oups in binuclea ed cells (Table 2, Fig. 3J) and binuclea ed
cells wi h a nucleoplasmic b idge (Table 2, Fig. 3F, I).
3.4. Enzyme ac i i ies in mussel issues
The e we e s a is ically signi ican di e ences in he ac i i y o
ace ylcholines e ase (AChE) be ween eplica es and hus, da a we e
ea ed sepa a ely (Fig. 4A). Ac i i y o AChE was lowe in one o he
eplica es exposed o GO+BaP (R1) and in bo h eplica es exposed o
BaP in compa ison o one o he con ol eplica es (R1) (Fig. 4A). Inhi-
bi ion o isoci a e dehyd ogenase (IDH) was obse ed in mussels
exposed o BaP in compa ison o mussels exposed o GO and GO+BaP
(Fig. 4B). Simila ly, inhibi ion o Glu a hione-S-T ans e ase (GST) was
obse ed in he diges i e gland o mussels exposed o GO+BaP and BaP
in compa ison o con ol mussels (Fig. 4C), while no esponse was
obse ed in gills (Fig. S3A).
(cap ion on nex column)
Fig. 4. Ace ylcholines e ase (AChE) ac i i y in he aduc o muscle (gi en as
nmol/min/mg p o ); B) Isoci a e dehyd ogenase (IDH) ac i i y in he diges i e
gland (gi en as nmol/min/mg p o ); C) Glu a hione-S-T ams e ase (GST) ac-
i i y in he diges i e gland (gi en as nmol/min/mg p o ) con ol mussels and
in mussels exposed o 7 days o GO, GO+BaP and BaP. Box-plo s show median
alue (ho izon al line), 25%−75% qua iles (box) and s anda d de ia ion
(whiske s). Do s deno e ou lie s. Le e s deno e s a is ical di e ences among
means (K uskal-Wallis es ollowed by Dunn’s pos -hoc in AChE and GST and
one-way ANOVA ollowed by Tukey’s pos -hoc in IDH, p <0.05).
N. Gonz´
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Jou nal o Haza dous Ma e ials 452 (2023) 131280
8
Rega ding an ioxidan enzymes, induc ion o Ca ac i i y was
obse ed in he diges i e gland o mussels exposed o GO+BaP in
compa ison o con ols (Fig. 5A), bu no esponse was obse ed in gills
(Fig. S3B). No di e ences we e ound in he ac i i y o glu a hione
pe oxidase (GPx) in he diges i e gland (Fig. S3C), bu inhibi ion was
obse ed in gills o mussels exposed o GO in compa ison o mussels
exposed o BaP (Fig. 5B).
Supe oxide dismu ase (SOD) was inhibi ed in he diges i e gland o
mussels exposed o BaP in compa ison o con ol mussels and mussels
exposed o GO+BaP, while induc ion o SOD was obse ed in mussels
exposed o GO+BaP in compa ison o mussels exposed o GO and BaP
(Fig. 5C). Howe e , no clea end was obse ed in gills (Fig. S3C).
3.5. His opa hology o diges i e gland
S a is ical di e ences we e ound be ween eplica es o some his-
opa hological al e a ions. In hose cases da a se s o he wo eplica es
we e ea ed sepa a ely (Table 3). Fib osis (Fig. 6A) was widesp ead in
all mussels including con ols. All g oups showed high p e alences,
signi ican ly highe in BaP R1 han in GO R1 (Table 3). P e alence o
hemocy ic in il a ion (Fig. 6B) was simila in con ol R2 and all expo-
su e g oups bu i was signi ican ly highe in he GO R2 g oup han in
con ol R1 (Table 3). O e all, agg ega ion o b own cells (Fig. 6C, D)
we e mo e equen in exposed g oups han in con ols, a leas in one o
he eplica es (Table 3). Agg ega ion o b own cells in he connec i e
issue was signi ican ly highe in mussels exposed o GO, GO+BaP and
BaP han in con ols (Table 3) while in he diges i e ac epi helium, i
was signi ican ly highe in GO R2 and GO+BaP R1 han in con ol R1
(Table 3). In addi ion, agg ega ion o b own cells in he diges i e ac
epi helium was signi ican ly highe in GO+BaP R1 han in GO R1
(Table 3).
A eas o nec osis o diges i e ubule epi helium we e ound in bo h
con ol and exposed mussels. P e alence o nec osis was signi ican ly
highe in GO+BaP R2 and BaP R2 compa ed o con ol R2 (Table 3).
Nec osis o diges i e ubule epi helium appea ed o be associa ed o he
occu ence o an in acellula cilia ed p o ozoan. I was ound in he
diges i e epi helium o mussels o all expe imen al g oups, wi h i s
highes p e alence in con ols, especially in compa ison o mussels
exposed o BaP (Table 3). In addi ion, he p o ozoan Nema opsis sp. was
he mos common pa asi e in he diges i e gland. I appea ed in he
connec i e issue o almos all mussels, wi h high p e alences in all he
eplica es (Table 3). O he pa asi es such as My ilicola in es inalis we e
also obse ed in he diges i e gland o some mussels, bu wi h low
p e alences (Table 3).
3.6. Game e de elopmen , gonad index and his opa hology o mussel
gonad
Mussels om di e en g oups we e in a simila game e de elopmen
s age. Spawning was he p edominan s age (Fig. S4) as expec ed o he
expe imen al pe iod. The e we e no di e ences in he sex a io and
gonad index among g oups (Fig. S4).
Fib osis (Fig. 6F), hemocy ic in il a ion (Fig. 6G) and agg ega ion o
b own cells (Fig. 6H) we e widely obse ed in he gonad o bo h con ol
and exposed mussels. P e alence o ib osis was signi ican ly highe in
BaP R2 mussels han in Con ol R2 mussels (Table 4). Hemocy ic
(cap ion on nex column)
Fig. 5. Ca alase (Ca ) ac i i y in he diges i e gland (gi en as mM H
2
O
2
/min/
mg p o ); B: Glu a hione Pe oxidase (GPx) ac i i y in gills (gi en as nmol/min/
mg p o ); C: Supe oxide dismu ase (SOD) ac i i y in he diges i e gland (U/mg
p o ) o con ol mussels and in mussels exposed o 7 days o GO, GO+BaP and
BaP. Box-plo s show median alue (ho izon al line), 25%−75% qua iles (box)
and s anda d de ia ion (whiske s). Do s deno e ou lie s. Le e s deno e s a is-
ical di e ences among means (one-way ANOVA ollowed by Tukey’s pos -hoc
in Ca and SOD, K uskal-Wallis es ollowed by Dunn’s pos -hoc in
GPx, p <0.05).
N. Gonz´
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Jou nal o Haza dous Ma e ials 452 (2023) 131280
9
in il a ion occu ed bo h in he connec i e issue and wi hin he gonad
ollicles and p e alences we e highe in GO+BaP R1 and BaP R2 han in
bo h con ol eplica es and in GO R1 (Table 4). Agg ega ion o b own
cells, bo h in he connec i e issues and gonad ollicles, was highe in
mussels exposed o GO and o BaP han in con ols (Table 4). Howe e ,
he main his opa hological al e a ion obse ed was oocy e a esia
(Fig. 6F) wi h p e alences o e 75% in all con ol and exposed g oups
(Table 4). Con ol mussels showed low in ensi y o a esia which can be
linked o he de elopmen al s age o he gonad (Table 4, Fig. S4). On he
o he hand, s ages 2 and 3 o oocy e a esia we e only eco ded in
exposed mussels and he highes in ensi y was ound in mussels exposed
o GO+BaP (Table 4). Nema opsis sp. p o ozoan was ound in he con-
nec i e issue o he gonad o almos all mussels (Table 4).
3.7. Whole o ganism esponses
No di e ences we e eco ded among g oups in mussel condi ion
index (Fig. S5).
4. Discussion
In his wo k, he oxic e ec s o sho - e m (7 days) exposu e o GO
alone (500 µg/L) and o GO wi h so bed BaP (500 µg/L GO incuba ed
wi h 100 µg/L BaP) has been assessed using a wide selec ion o biolog-
ical esponses, om molecula o o ganism le els. Fo compa ison, he
e ec s o exposu e o BaP alone a a concen a ion o 96.7 µg/L,
equi alen o ha so bed in GO+BaP p epa a ions [63], we e also
assessed. In pa allel, he a e o GO nanopla ele s in mussels was s udied
by Raman spec oscopy, as well as BaP bioaccumula ion.
A e 20 h o exposu e, BaP o GO could no be de ec ed in he wa e
o anks exposed o GO+BaP, sugges ing ha mussels had up aken he
GO nanopla ele s wi h so bed BaP. Li le is known abou he deso p ion
dynamics o PAHs om GO and o he GFNs in he ma ine en i onmen .
In aqueous solu ions, adso p ion o phenan h ene in o magne ic g a-
phene is mos ly i e e sible [90]. The s abili y o GO wi h so bed BaP
could explain ha BaP was unde ec able in wa e in GO+BaP anks as
mussels would in e nalize BaP adso bed in o GO nanopla ele s. How-
e e , he concen a ion o BaP bioaccumula ed in mussels was much
lowe in mussels exposed o GO+BaP compa ed o hose exposed o
dissol ed BaP. Sanchis e al. [41] epo ed ha he T ojan ho se e ec is
mo e likely o occu wi h o ganic pollu an s ha p esen an in e media e
a ini y o ca bon-based NMs, han wi h non pola compounds showing
a oma ic ings such as BaP, as he mos non pola compounds would be
i e e sibly bound o he ca bon-based NMs dec easing hei bioa ail-
abili y. Al e na i ely, i could be ha BaP bioaccumula ion was low in
mussels exposed o GO+BaP because o he exc e ion o GO nano-
pla ele s wi h so bed BaP. This idea is suppo ed by he de ec ion o GO
in eces o mussels exposed o GO and o GO+BaP.
A s udying he s abili y o GO in anks wi h and wi hou oys e s,
Khan e al. [34,91] concluded ha he animals we e ac i ely emo ing
he GO om he wa e , in line wi h ou esul s. Fil a ion is he common
pa hway o he in e naliza ion o di e en NMs du ing he eeding
p ocess o il e - eeding o ganisms such as mussels. A e a i s con ac
wi h he gills, NM agg ega es a e inges ed and, depending on hei size,
hey can be accumula ed in he diges i e gland and/o ansloca ed o
hemolymph and hen dis ibu ed o o he o gans [92]. In he p esen
wo k GO was no de ec ed in gills, p obably due o he sho con ac
be ween GO and gills. This could explain he lack o e ec s on enzyme
ac i i ies s udied in gills o mussels exposed o GO and GO+BaP in
compa ison o con ols. P esence o GO was con i med h ough Raman
spec oscopy in he lumen o he diges i e ac and eces o mussels
exposed o bo h GO and GO+BaP, sugges ing ha GO ends o accu-
mula e in o gans ela ed wi h ood in ake, as p e iously epo ed by
Josende and colleagues [93]. The p esence o GO in he diges i e ac
has been p e iously epo ed in exposu es o o he aqua ic in-
e eb a es, such as nema odes [36] and c us aceans [93–98], bu no in
bi al es. To he bes o ou knowledge his is he i s ime ha he
p esence o GO in he diges i e ac o bi al es was con i med h ough
Raman spec oscopy. As he diges i e gland is one o he mos impo an
o gans in mussels, esponsible o in acellula diges ion and also o
an ioxidan de ense and pollu an seques a ion and de oxi ica ion,
signi ican damages in his o gan could lead o impac a he o ganism
le el [92,99,100]. GO could hen be ansloca ed om he diges i e
sys em o he es o o gans [101,102]. Howe e , Bo olozzo e al. [36]
obse ed ha ca boxyl g oups in GO edges can a oid pe meabili y o GO
nanopla ele s h ough he in es ine in nema odes. This could explain he
p esence o GO in eces a e each diges i e cycle and he absence o GO
ou o he diges i e ac in his wo k, al hough he e is ye no conclusi e
da a on he dis ibu ion and exc e ion s a egies o GFNs inside animal
bodies [5].
E en hough GO was no de ec ed in hemocy es by Raman spec-
oscopy, cy o oxici y occu ed in hemocy es o mussels exposed o
GO+BaP and BaP wi h espec o con ols. Fu he , geno oxic e ec s
we e obse ed in hemocy es o mussels exposed o GO and GO+BaP in
compa ison o con ols. DNA damage has been p e iously epo ed in
o he o ganisms exposed o GO [103–106], bu he e is a single wo k
epo ing geno oxici y o GFNs o mussels [107]. Geno oxici y may be
p o oked by he physical damage caused by nanopla ele s and/o
h ough oxida i e s ess [93,101]. In mos publica ions he come assay
has been used o measu e geno oxici y a e exposu e o GFNs, bu DNA
s and b eaks de e mined by he come assay can be e e sible [108].
Flasz e al. [105] epo ed ha DNA damage measu ed in c icke s Ache a
domes icus by he come assay a e 5 and 25 days o GO exposu e was
comple ely e e sed a e 10 days o depu a ion. On he o he hand,
Table 3
P e alence o his opa hological al e a ions in he diges i e gland o bo h eplica es (R1 and R2) o con ol mussels and mussels exposed o GO, GO+BaP and BaP o 7
days. Da a a e shown in pe cen ages o 10 mussels pe expe imen al eplica e ank. Le e s deno e s a is ical di e ences o each al e a ion (X2 es , p <0,05).
In lamma o y esponses Pa asi es
Fib osis Hemocy ic
in il a ion
B own cells in he
connec i e issue
B own cells in he
diges i e ac
epi helium
Nec osis o diges i e
ubule epi helium
In acellula cilia ed
p o ozoan
Nema opsis
sp.
My ilicola
in es inalis
Con ol
R1
90
AB
10
A
0
A
10
A
50
AB
80
A
80 10
Con ol
R2
90
AB
40
AB
30
A
30
ABC
80
A
70
A
100 20
GO R1 80
A
50
AB
60
B
20
AB
50
AB
60
AB
90 10
GO R2 60
AB
60
B
60
B
60
CB
60
AB
30
AB
100 0
GO+BaP
R1
90
AB
40
AB
60
B
70
C
60
AB
60
AB
80 20
GO+BaP
R2
90
AB
50
AB
40
B
20
AB
30
B
40
AB
80 10
BaP R1 100
B
40
AB
70
B
10
A
80
A
40
B
90 10
BaP R2 90 A
B
50
AB
60
B
30
ABC
20
B
40
B
70 20
N. Gonz´
alez-So o e al.
Jou nal o Haza dous Ma e ials 452 (2023) 131280
16
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