Ci a ion: Ma ínez-Ál a ez, I.; Le
Menach, K.; De ie , M.-H.;
Caja a ille, M.P.; Budzinski, H.;
O bea, A. Sc eening o he Toxici y o
Polys y ene Nano- and Mic oplas ics
Alone and in Combina ion wi h
Benzo(a)py ene in B ine Sh imp
La ae and Zeb a ish Emb yos.
Nanoma e ials 2022,12, 941. h ps://
doi.o g/10.3390/nano12060941
Academic Edi o s: Lau a Canesi and
Ila ia Co si
Recei ed: 31 Janua y 2022
Accep ed: 8 Ma ch 2022
Published: 12 Ma ch 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.
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nanoma e ials
A icle
Sc eening o he Toxici y o Polys y ene Nano- and
Mic oplas ics Alone and in Combina ion wi h Benzo(a)py ene
in B ine Sh imp La ae and Zeb a ish Emb yos
Ignacio Ma ínez-Ál a ez 1,2, Ka yn Le Menach 2, Ma ie-Hélène De ie 2, Mi en P. Caja a ille 1,
Hélène Budzinski 2and Amaia O bea 1,*
1CBET Resea ch G oup, Depa men o Zoology and Animal Cell Biology, Resea ch Cen e o Expe imen al
Ma ine Biology and Bio echnology PiE and Science and Technology Facul y, Uni e si y o he Basque
Coun y (UPV/EHU), E-48940 Leioa, Spain; [email p o ec ed] (I.M.-Á.);
[email p o ec ed] (M.P.C.)
2Uni e si y o Bo deaux, UMR 5805 CNRS, EPOC, Labo a o y o Physico- and Toxico-Chemis y o he
En i onmen , CEDEX, F-33405 Talence, F ance; [email p o ec ed] (K.L.M.);
[email p o ec ed] (M.-H.D.); helene.budzinski@u-bo deaux. (H.B.)
*Co espondence: [email p o ec ed]; Tel.: +34-946-012-735
Abs ac :
The occu ence o nanoplas ics (NPs) and mic oplas ics (MPs) in aqua ic ecosys ems and
hei capaci y o so b hyd ophobic pollu an s is nowadays an issue o g ea conce n. This s udy aimed
o assess he po en ial bioa ailabili y and acu e oxici y o polys y ene (PS) NPs (50 and 500 nm) and
o MPs (4.5
µ
m), alone and wi h so bed benzo(a)py ene (B(a)P), in he emb yo/la al s ages o b ine
sh imps and zeb a ish. Exposu e o p is ine plas ics up o 50.1 mg PS/L did no cause signi ican
impac on b ine sh imp su i al, while some ea men s o plas ics-B(a)P and all concen a ions o
B(a)P (0.1–10 mg/L) esul ed acu ely oxic. In zeb a ish, only he highes concen a ions o MPs-B(a)P
and B(a)P caused a signi ican inc ease o mal o ma ion p e alence. Inges ion o NPs was obse ed
by 24–48 h o exposu e in he wo o ganisms ( om 0.069 o 6.87 mg PS/L). In b ine sh imps, NPs we e
obse ed o e he body su ace and wi hin he diges i e ac , associa ed wi h eces. In zeb a ish, NPs
we e localized in he eyes, yolk sac, and ail a 72 h, showing hei capaci y o ansloca e and sp ead
in o he emb yo. MP inges ion was only demons a ed o b ine sh imps. In zeb a ish emb yos
exposed o plas ics-B(a)P, B(a)P appea ed in he yolk sac o he emb yos. The p esence o B(a)P was
also no iceable in b ine sh imps exposed o 500 nm NPs-B(a)P. In conclusion, NPs en e ed and sp ead
in o he zeb a ish emb yo and PS NPs, and MPs we e success ul ec o s o B(a)P o b ine sh imp and
zeb a ish emb yos. Pa icle size played a signi ican ole in explaining he oxici y o plas ics–B(a)P.
Ou s udy p o ides suppo o he idea ha plas ics may pose a isk o aqua ic o ganisms when
combined wi h pe sis en o ganic pollu an s such as B(a)P.
Keywo ds:
polys y ene; nanoplas ics; mic oplas ics; benzo(a)py ene; zeb a ish emb yos; b ine
sh imp la ae; acu e oxici y; bioa ailabili y
1. In oduc ion
Due o hei du abili y and widesp ead use in popula ed a eas, he p esence o nano-
(NPs) and mic oplas ics (MPs) in aqua ic ecosys ems is epo ed wo ldwide [
1
]. The
sou ces o NPs and MPs a e nume ous [
2
], wi h ligh e NPs and MPs being mo e p one
o a i e o aqua ic ecosys ems. The polyme ypes mos ly p esen in aqua ic ecosys ems
include polye hylene (PE), polyp opylene (PP), polys y ene (PS), poly inyl chlo ide (PVC),
polyu e hane (PUR), and polye hylene e eph hala e (PET). PS has an in e media e densi y
(1.05 g/cm
3
) among he p e iously men ioned polyme s and is close o wa e densi y
(1–1.03 g/cm
3
depending on salini y). This makes PS MPs beha e di e en ly in wa e s
o di e en salini y and become bioa ailable o aqua ic o ganisms, om su ace wa e o
bo om wa e s o sedimen s [3].
Nanoma e ials 2022,12, 941. h ps://doi.o g/10.3390/nano12060941 h ps://www.mdpi.com/jou nal/nanoma e ials
Nanoma e ials 2022,12, 941 2 o 23
Se e al s udies ha e al eady epo ed ha m ul e ec s o NPs and MPs on aqua ic
bio a [
4
]. As e iewed o 28 axonomic o de s o zooplank on, MPs p o oke nega i e
e ec s on he eeding beha io , g ow h, de elopmen , and li e span o some a h opods
and ish la ae, wi h MP size and densi y being he main cha ac e is ics con olling hei
bioa ailabili y [
5
]. Non-selec i e il e - eede s, such as b ine sh imp, ha e been p oposed
as a sui able biological model in nanoeco oxicology, due o hei cos -e ec i eness [
6
], and
s anda d guidelines, such as ISO/TS 20787 [
7
], ha e been de eloped o assess he oxici y
o nanoma e ials. B ine sh imps showed inges ion and elimina ion o NPs and MPs [
8
–
11
],
which impai ed ood up ake. Howe e , acu e e ec s o PS MPs we e no epo ed in b ine
sh imps. No signi ican mo ali y was obse ed in o ganisms exposed o up o 100 mg/L
o 0.05, 0.1 o 10
µ
m- PS MPs o 24 o 48 h [
8
,
9
,
12
,
13
]. In con as , exposu e o PP MPs (size
ange 11.86–44.62
µ
m) caused oxida i e s ess and mo ali y in b ine sh imp nauplii (LC50
40.947 µg/mL) [11].
O ganisms such as ish ha occupy a highe posi ion in he ophic chain also inges
and accumula e MPs [
14
,
15
], he ea ly li e s ages being especially sensi i e o NP and MP
impac [
16
]. Zeb a ish emb yos ha e been widely used o es he oxici y o dissol ed
chemicals and nanoma e ials [
17
,
18
]. Mo e ecen ly, zeb a ish emb yos ha e also been
u ilized o es he oxicological e ec s o NPs and MPs [
19
–
23
]. Sub-le hal e ec s, such
as changes in locomo o ac i i y, hea bea a e, oxida i e s ess, and al e a ions in he
ne ous sys em, ha e been obse ed in zeb a ish emb yos 120 h pos e iliza ion (hp ),
a e exposu e o 0.1–10 mg/L o 0.025, 0.05, 0.1, o 45
µ
m PS MPs/NPs [
10
,
22
–
24
], and
mo ili y educ ion has been eco ded a e exposu e o 0.01–1 mg/L o PS MPs o 1
µ
m [
19
].
In addi ion o he impac o NPs and MPs, ano he conce n has been aised ega ding hei
abili y o so b o he pollu an s [
25
,
26
], especially pe sis en o ganic pollu an s (POPs), ea-
u ing a high hyd ophobici y and esis ance o deg ada ion (pe sis ence) in he ecosys ems.
Mos POPs a e well-known oxic compounds o aqua ic o ganisms, and many o hem
display ca cinogenic p ope ies [
27
]. This is he case o some o he ubiqui ous polycyclic
a oma ic hyd oca bons (PAHs) [
28
]. Apa om polyme ype, pa icle size is a key ac o
d i ing he so p ion capaci y o plas ics; o e all, he smalle he size o he plas ics, he
highe he so p ion capaci y [
29
]. Pollu an s so bed o NPs and MPs can be eleased inside
o ganisms a e assimila ion [30].
Few s udies ha e add essed he impac s p oduced by PS NPs/MPs combined wi h
POPs in non-selec i e il e - eede s [
29
,
31
]. The po en ial ans e o C
14
phenan h ene
(0.05–1.2 mg/L) in co-exposu e wi h wo di e en sized PS pa icles (50 nm NPs and 10
µ
m
MPs; 2.5–14.5 mg/L and 2.5–50 mg/L, espec i ely) was e alua ed in Daphnia magna. The
esul s showed ha co-exposu e o 2, 7, and 14 days wi h NPs p o oked highe C14
phenan h ene bioaccumula ion han co-exposu e wi h MPs, due o he highe adso p ion
capaci y o NPs o phenan h ene [
29
]. D. magna was also co-exposed o 100 nm PS NPs
(0.01–75 mg/L) and polychlo ina ed biphenyls (PCBs, 0.640 mg/L). The inc ease in he PS
NP concen a ion, combined wi h a ixed concen a ion o PCBs, inc eased he mo ali y o
D. magna, indica ing ha NPs enhanced he PCB oxici y [31].
Fish la ae, including zeb a ish emb yos, ha e also been s udied o e alua e he
po en ial e ec o PS NPs/MPs combined wi h POPs. Panne ie e al. [
32
] assessed he
po en ial anspo o B(a)P in o medaka emb yos, using MPs collec ed om beaches. A
mix u e wi h 1% o <600
µ
m MPs (PP, PE, and PS) coa ed wi h B(a)P (250
µ
g o B(a)P/g o
MPs) caused high mo ali y (81%) o medaka emb yos a e 48 h o exposu e, as well as
an inc ease o 7-e hoxy eso u in-O-dee hylase (EROD) ac i i y. Smalle size MPs (200–250
µ
m, 400 mg/L) we e also analyzed o hei capaci y o anspo 17
α
-e hynyles adiol
(EE2, 0.001–1
µ
g/L) and phenan h ene (0.1–0.5 mg/L) in o zeb a ish emb yos [
33
]. A
dec ease o EE2 and phenan h ene bioa ailabili y, indica ed by gene exp ession analysis
in zeb a ish emb yos, was due o he sedimen a ion o MPs. When MPs and NPs we e
compa ed o po en ial ans e o compounds o zeb a ish emb yos, a highe EE2 bioaccu-
mula ion was obse ed in zeb a ish co-exposed o 50 nm NPs (1 mg/L) and EE2 (2 and
20
µ
g/L) han in hose co-exposed o 45
µ
m PS MPs (1 mg/L), which esul ed in la al
Nanoma e ials 2022,12, 941 3 o 23
hypoac i i y. This change in la al ac i i y was no obse ed in emb yos exposed o EE2
alone, indica ing he capaci y o NPs o enhance oxici y when combined wi h EE2 [
20
].
Highe bioaccumula ion o phenan h ene was also obse ed in emb yos co-exposed wi h
small NPs (20 nm) han in emb yos co-exposed wi h la ge NPs (500 nm) o exposed o
phenan h ene alone [
34
].While, 44 nm PS NPs (10 mg/L) alone and combined wi h PAHs
om a sedimen ex ac p o oked a dys unc ion o he mi ochond ial unc ion in zeb a ish
emb yos. The epo ed mi ochond ial dys unc ion was no longe obse ed in he emb yos
exposed o he co esponding PAH mix u e alone (5073 ng/mL), while ca dio oxici y and
impai ed b ain ascula i y we e obse ed [35].
The aim o he p esen s udy was o es he po en ial bioa ailabili y and acu e oxici y
o PS NPs and o MPs alone and wi h so bed B(a)P, as a model py oly ic PAH, in he em-
b yo/la al s ages o wo model aqua ic o ganisms, b ine sh imps and zeb a ish. The e o e,
he speci ic objec i es we e (1) o assess he de elopmen al oxici y o ‘p is ine’ PS plas ic
pa icles o di e en sizes (50 nm, 500 nm, and 4.5
µ
m) and o dissol ed B(a)P on b ine
sh imp la ae and zeb a ish emb yos; (2) o assess he oxici y o NPs (500 nm) and MPs
(4.5
µ
m) wi h so bed B(a)P; and (3) o e alua e he a ailabili y o NPs and o MPs wi h
so bed B(a)P o b ine sh imp la ae and zeb a ish emb yos. As he aim o he s udy was o
es he accumula ion and e ec s o B(a)P so bed o he plas ics, a oiding co-exposu e o
dissol ed B(a)P and plas ics, pa icles we e il e ed a e incuba ion wi h B(a)P. Owing o
his, 50 nm NPs we e no combined wi h B(a)P, due o he echnical di icul y o il e ing
and e icien ly eco e ing 50 nm NPs a e incuba ion, as was done o he o he pa icle
sizes.
2. Ma e ials and Me hods
2.1. Chemicals and Plas ic Pa icles
Benzo(a)py ene (B(a)P, pu i y
≥
96%), benzo(a)py ene d
12
(B(a)P d
12
,
≥
98%), and
dime hylsul oxide (DMSO, pu i y
≥
96%) we e pu chased om Sigma-Ald ich (S . Louis,
MO, USA). Fluo escen Fluo esb i e
®
ca boxyla e 50 nm PS NPs (exci a ion/emission
wa eleng hs o 360/407 nm), non- luo escen PS NPs (50 and 500 nm), and MPs (4.5
µ
m) in
aqueous suspensions we e pu chased om Polysciences Inc. (Wa ing on, PA, USA). The
concen a ion o he comme cial s ocks was 2.5% (3.64
×
10
14
pa icles/mL o 50 nm NPs,
3.64 ×1011 pa icles/mL o 500 nm NPs and 4.99 ×108pa icles/mL o 4.5 µm MPs).
2.2. P epa a ion and Analysis o Exposu e Media
B(a)P concen a ions used o oxici y assays we e 0.1, 0.5, and 1 mg/L B(a)P in 0.01%
DMSO and 5 and 10 mg/L B(a)P in 0.1% DMSO. An ini ial s ock solu ion o 10 g/L (50 mg o
B(a)P in 5 mL pu e DMSO) was p epa ed and used o p epa e he o he wo s ock solu ions
o 5 and 1 g/L o B(a)P in 100% DMSO. These s ock and in e media e solu ions we e s o ed
a
−
20
◦
C in closed glass ials. All he s ock solu ions, in e media e solu ions, and inal
dilu ions we e dispe sed o 10 min in an ul asounds ba h (VWR, Radno , PA, USA) be o e
being used. Each s ock solu ion was dilu ed 1:1000 in he exposu e medium equi ed o
each expe imen (MilliQ wa e o chemical analysis o B(a)P concen a ion, emb yo wa e
o zeb a ish emb yo oxici y es o sal wa e o b ine sh imp immobiliza ion es ), in
o de o ob ain he h ee highes B(a)P exposu e concen a ions (10, 5, and 1 mg/L B(a)P)
in 0.1% ( / ) DMSO). Then, 0.5 and 0.1 mg/L B(a)P we e p epa ed in 0.01% DMSO by
dilu ing he p e ious ones 1:10 in exposu e media.
Immedia ely a e being p epa ed, an aliquo om each concen a ion was collec ed
o chemical analysis and dilu ed wi h MilliQ wa e up o 1
µ
g/L in 10 mL glass ials.
Pe deu e a ed B(a)P was added as an in e nal s anda d p io he analysis, in o de o pe -
o m quan i ica ion by iso opic dilu ion. Analysis was pe o med by gas ch oma og aphy
and mass spec ome y (GC/MS) a e solid phase mic oex ac ion (SPME), using selec ed
ion moni o ing mode (elec onic impac ioniza ion). SPME consis ed in a hea ing p ocess
a 40
◦
C wi h 35 min s i ing pe iod a 250 pm o he polydime hylsiloxane (PDMS) ibe
(Supelco, Sigma-Ald ich, Sou h A ica). The ibe (100
µ
m) was he mally deso bed in o
Nanoma e ials 2022,12, 941 4 o 23
he GC/MS sys em (Agilen GC 7890A/Agilen MSD 5975C, Agilen Technologies, San a
Cla a, CA, USA) o 10 min a 280
◦
C. The GC/MS sys em was ope a ed wi h an ene gy o
ioniza ion o 70 eV in ioniza ion mode o elec onic impac equipped wi h an HP5MS-UI
column (5% phenyl me hylpolysiloxane; 30 m
×
0.25 mm i.d.; 0.25
µ
m so ben , Agilen
Technologies). Injec ion in he GC/MS sys em was done wi h he inle empe a u e a
280
◦
C, in he pulsed spli less mode; a pulse p essu e o 25 psi was main ained o 1.5 min,
he pu ge low o spli en was 60 mL/min a e 1.5 min. The injec ion olume was 1
µ
L,
wi h a helium (pu i y 6.0) cons an low a e o 1.3 mL/min. The column empe a u e was
ini ially held a 50
◦
C o 2 min, and was hen inc eased o 250
◦
C a 10
◦
C/min o 1 min,
o 280
◦
C o 2 min and o 310
◦
C a 10
◦
C/min, whe e i was held o 3 min. Compound
de e mina ion was ope a ed in he selec ed ion moni o ing (SIM). B(a)P was quan i ied by
iso ope dilu ion (B(a)P d12).
In o de o assess whe he he e was any B(a)P loss du ing expe imen al exposu es,
2 mL o each concen a ion we e placed in duplica e in a 24-well mic opla e. The mic opla e
was main ained unde a pho ope iod simula ing he es condi ions (12 h ligh /12 h da k,
20
◦
C). Aliquo s we e aken a 24, 48, and 120 h, dilu ed up o 1
µ
g/L, and p ocessed as
desc ibed be o e.
Exposu e concen a ions o PS NPs and MPs o he h ee sizes, in e ms o numbe
o pa icles and in e ms o PS mass used in he oxici y assays, a e shown in Table 1. To
p epa e 500 nm PS NPs and 0.45
µ
m PS MPs wi h so bed B(a)P, 50 mg PS/L was incuba ed
in glass bo les wi h 100
µ
g/L B(a)P (in 0.01% DMSO) p epa ed in MilliQ wa e . The
incuba ion olume used o MPs con amina ion wi h B(a)P depended on he amoun o
MPs equi ed o he assays, always keeping he PS concen a ion used in p e ious so p ion
expe imen s (50 mg/L). PS suspensions we e w apped wi h aluminum and shaken a
300 pm o 24 h a 20
◦
C. Then, samples we e il e ed h ough a polye he sul one il e
(0.45-
µ
m il e po e, Sa s ed AG & Co., Nümb ech , Ge many) and washed wo imes
wi h 10 mL o MilliQ wa e . PS pa icles we e eco e ed om he il e s using sal wa e o
emb yo wa e o b ine sh imps o zeb a ish, espec i ely. These NPs-B(a)P and MPs-B(a)P
suspensions we e dilu ed wi h he co esponding medium o p epa e he i e exposu e
concen a ions (Table 1). The eco e y o 4.5
µ
m MPs a e il a ion was measu ed using a
Coul e Coun e (Z-coun e , Beckman Coul e , B ea, CA, USA) (Figu e S1).
Table 1.
Rela ionship be ween he plas ic pa icle concen a ions, in e ms o mass and in e ms o
numbe o pa icles o he h ee sizes used.
50 nm NPs 500 nm NPs 4.5 µm MPs
Pa icles/mL mg/L Pa icles/mL mg/L Pa icles/mL mg/L
1060.000069
5×1030.00034
1070.00069 0.00069
5×1020.025
1090.069 1060.069 1030.069
1010 0.687 1070.687 1070.687
1011 6.87 1086.87 1086.87
10650.1
2.3. Acu e Toxici y Assays
2.3.1. B ine Sh imp Cul u es and Immobiliza ion Tes
One g am o b ine sh imp cys s (A emia Ko al GmbH, Nü nbe g, Ge many) was incu-
ba ed wi h igo ous ae a ion in a empe a u e-con olled oom a 26
◦
C unde con inuous
illumina ion in 30
‰
sal wa e p epa ed om comme cial sal (Se a, Heinsbe g, Ge many).
Ha ched la ae we e ha es ed wi h a glass pipe e, a oiding collec ing unha ched cys s,
and ans e ed o a eshly p epa ed sal medium, whe e la ae we e main ained o 24 o
48 h, be o e s a ing he oxici y es s. A 24 o 48 h pos ha ch (hph), cul u es we e mo ed
o some hou s o a empe a u e-con olled oom a 20
◦
C o acclima e indi iduals o he
Nanoma e ials 2022,12, 941 5 o 23
oxici y assays, which we e pe o med ollowing he p ocedu e p e iously desc ibed in
Laca e e al. [
36
], using immobiliza ion as a c i e ion o acu e oxici y. The exposu es o
non- luo escen PS NPs and MPs we e un in co e ed 24-well mic opla es a 20
±
1
◦
C,
wi h a pho ope iod o 12 h ligh /12 h da k. Fi e b ine sh imps we e placed in each well
wi h 2 mL o exposu e medium, and 6 wells we e used pe concen a ion (30 indi iduals
pe expe imen al g oup and 30 indi iduals as con ols). The selec ion o indi iduals was
made unde a s e eoscopic mic oscope (Nikon smz800, Kanagawa, Japan) paying a en ion
o he mo phology co esponding o he 24 hph and 48 hph s ages. A e 24 and 48 h o
exposu e, he amoun o immobilized la ae was eco ded. A la a was conside ed as
immobile when i was no able o mo e a e shaking he pla e. The oxici y o 0.01, 0.05,
and 0.1% DMSO was es ed in pa allel.
2.3.2. Zeb a ish Main enance, Egg P oduc ion, and Emb yo Toxici y Tes
The zeb a ish (wild ype AB Tübingen) s ock was main ained in a empe a u e-
con olled oom a 28
◦
C wi h a 14 h ligh /10 h da k cycle in 100 L anks p o ided wi h
mechanical and biological il e s, ollowing s anda d p o ocols o zeb a ish cul u e. Condi-
ioned wa e (600
µ
S/cm and 7–7.5 pH) was p epa ed om deionized wa e and comme cial
sal . Fish we e ed wice pe day wi h Vipag an baby (Se a, Heinsbe g, Ge many) and
b ine sh imp nauplii o 24 hph, cul u ed as desc ibed abo e. B eeding emale ish we e
selec ed and main ained sepa a ely in ish b eeding ne s inside he same anks, in o de o
a oid con inuous spawning. The day be o e he assay, one emale and wo male zeb a ish
we e placed sepa a ely in a b eeding ap, p e iously loca ed in a 2-L ank con aining
condi ioned wa e . Fish we e le o e nigh and, jus be o e he ligh was swi ched on, he
sepa a ion was emo ed. The e ilized eggs we e collec ed in a Pe i dish wi h he help o
a Pas eu pipe e. The eggs selec ed as iable unde a Nikon smz800 (Nikon, Kanagawa,
Japan) s e eoscopic mic oscope we e ans e ed o he exposu e mic opla es.
The oxici y es s we e ca ied ou in co e ed 24-well mic opla es, placing one emb yo
pe well in 2 mL o es solu ion made in emb yo wa e (600–800
µ
S/cm, 6.5–6.8 pH) [
37
]
ollowing he OECD guideline TG236 [
38
]. In each mic opla e, wo di e en concen a ions
we e es ed (10 emb yos pe concen a ion). Fou con ol emb yos we e placed in emb yo
wa e in he emaining wells. Fo each exposu e condi ion, h ee mic opla es we e p epa ed,
esul ing in 30 emb yos exposed o each concen a ion and 36 con ol emb yos. Newly
e ilized emb yos (<2 hp ) we e exposed o he non- luo escen PS NPs and MPs up o
120 hp . The es was only conside ed alid when he su i al o he con ol g oup o each
eplica e was
≥
90%. The oxici y o 0.01, 0.05, and 0.1% DMSO was es ed in pa allel. Daily
and up o he end o he es , emb yos we e examined o de e mine su i al a e (as he pe -
cen age o ali e emb yos a 120 hp ), ha ching ime (as he ime ha emb yos need o ha ch),
and mal o ma ion p e alence (as he pe cen age o mal o med emb yos o e su i ing
emb yos a 120 h). No mal emb yo mo phology and mal o ma ions we e based on Fako
and Fu geson [
39
]. De elopmen al abno mali ies sco ed as mal o ma ions we e spinal co d
lexu e, caudal in al e a ion, ail mal o ma ion, pe ica dial edema, yolk sac edema, eye
abno mali y, and s un ed body. Mal o ma ions we e eco ded and pho og aphed unde a
Nikon AZ100 s e eoscopic mic oscope.
2.4. Analysis o Bioa ailabili y o Plas ic Pa icles and B(a)P
Dis ibu ion o luo escen 50 nm PS NPs alone, and o non- luo escen 500 nm PS NPs
and 4.5
µ
m MPs wi h so bed B(a)P, wi hin b ine sh imp la ae and de eloping zeb a ish
emb yos was examined unde a con ocal mic oscope (Olympus Fluo iew FV500, Tokyo,
Japan). All he indi iduals used o hese analyses we e exposed a he same ime as
hose used o oxici y assays and a he same exposu e concen a ions. T ansmi ed
and luo escence con ocal images o Z-s acks se ies we e acqui ed using a 10x UPLAPO
NA0.45 lens. Fluo escence images we e ob ained by exci a ion a 405 nm and emission
a
430–460 nm
. Fluo escence emi ed by B(a)P accumula ed in b ine sh imp la ae and
zeb a ish emb yos exposed o B(a)P alone was analyzed a he end o he oxici y assays
Nanoma e ials 2022,12, 941 6 o 23
using a Cy a ion 5 mic opla e eade (Bio ek Ins umen s Inc., Winooski, VT, USA) p o ided
wi h a blue il e (360/460 nm exci a ion/emission wa eleng hs). Fo imaging, b ine sh imp
la ae and zeb a ish emb yos we e anes he ized wi h 2% ( / ) chlo o o m in sal wa e
and wi h 200 mg/L benzocaine p epa ed in emb yo wa e , espec i ely.
2.5. S a is ical Analyses
Da a on su i al o b ine sh imp la ae and o zeb a ish emb yos and p e alence o
mal o ma ions in zeb a ish emb yos we e analyzed by binomial logis ic eg ession [
36
,
40
].
Odd a ios we e calcula ed, in o de o es ima e and compa e he isk associa ed wi h he
exposu es. EC
50
and LC
50
alues wi h con idence in e als (CI) o 5 o 95% we e calcula ed
using a P obi model using he R package (R Founda ion o S a is ical Compu ing, Vienna,
Aus ia). Ha ching ime o zeb a ish emb yos was es ed o no mali y (Kolmogo o -
Smi no es ) and homogenei y o a iances (Ba le es ). Then, da a we e analyzed by
one-way ANOVA, ollowed by he Tukey pos hoc es (p< 0.05), using he G aphPad P ism
e sion 5.00 o Windows (G aphPad So wa e, La Jolla, CA, USA).
3. Resul s
3.1. Analysis o he B(a)P Exposu e Media
Measu ed B(a)P concen a ions o e ime o he i e exposu e concen a ions a e
shown in Table 2. B(a)P concen a ion measu ed a 0 h was always lowe (29–63%) han he
nominal concen a ion. B(a)P concen a ion was cons an du ing he i s 24 h, wi h he
excep ion o 5 mg/L o B(a)P, whose concen a ion d opped om 1.496
±
0.707 a 0 h o
1.173
±
0.505 mg/L a 24 h. A 48 h, he B(a)P concen a ions d opped in all cases, excep
o 10 mg/L B(a)P, which emained qui e s able un il 120 h. A his ime, he lowes B(a)P
concen a ion (0.1 mg/L) p esen ed he highes ela i e loss (up o 48.3% o he ini ially
measu ed concen a ion). In e media e concen a ions showed simila o e en highe
alues han hose measu ed a 48 h.
Table 2.
B(a)P concen a ion (mg/L) o e ime o he i e exposu e concen a ions. Pe cen age o
eco e ed B(a)P compa ed o ha measu ed a ime 0 is indica ed in b acke s. Values a e gi en as
mean ±S.D. (n= 2).
Nominal Concen a ion Measu ed Concen a ion
0 h 24 h 48 h 120 h
0.1 0.029 ±0.005 0.029 ±0.006 (100%) 0.022 ±0.002 (75.9%) 0.014 ±0.001 (48.3%)
0.5 0.315 ±0.142 0.432 ±0.084 (137.1%) 0.2578 ±0.011 (81.8%) 0.282 ±0.012 (89.5%)
1 0.496 ±0.111 0.503 ±0.006 (101.4%) 0.301 ±0.001 (60.7%) 0.265 ±0.025 (53.4%)
5 1.496 ±0.707 1.173 ±0.505 (78.4%) 0.738 ±0.299 (49.3%) 1.118 ±0.053 (74.7%)
10 4.922 ±1.995 5.023 ±0.397 (102.1%) 4.927 ±0.283 (100.1%) 5.269 ±1.095 (107%)
3.2. Toxici y Assays in B ine Sh imp La ae
E ec on su i al o he exposu e o NPs and o MPs alone and wi h so bed B(a)P o
24 h and 48 h o 24 hph and 48 hph b ine sh imp la ae is shown in Table 3. La ae exposed
o p is ine NPs and MPs did no show any signi ican di e ence on su i al compa ed o
con ols a any exposu e concen a ion o ime. Su i al a e o exposed la ae anged
om 75% in he exposu e o 48 hph la ae o 48 h, o 50.1 mg/L o 500 nm NPs o 100%.
Exposu e o 24 hph la ae o 0.00034 mg/L, 0.00069 mg/L, and 6.87 mg/L o 500 nm
NPs-B(a)P o 48 h caused a signi ican dec ease in su i al a e compa ed o con ol la ae.
The 48 hph la ae exposed o 0.00034 mg/L o 500 nm NPs-B(a)P o 48 h also showed a
signi ican educ ion in su i al compa ed o con ol la ae. B(a)P alone esul ed he mos
oxic ea men , excep o 24 hph b ine sh imp la ae exposed o 24 h, which did no show
any de imen al e ec . Howe e , exposu e o 48 h o all B(a)P concen a ions p o oked a
signi ican educ ion in su i al compa ed o con ol la ae, declining up o 59.4% in la ae
exposed o he highes concen a ion. In he case o 48 hph la ae, exposu e o any B(a)P
concen a ion o 24 o 48 h p o oked 100% mo ali y. Exposu e o b ine sh imp la ae o
Nanoma e ials 2022,12, 941 7 o 23
24 hph o 48 hph up o 0.1% DMSO o 24 h o 48 h did no p o oke any acu e oxic e ec
(Table S1).
Table 3.
E ec s on su i al (pe cen age o e he whole sample, n= 30 indi iduals) o he 24 h and
48 h exposu es o NPs and o MPs alone and wi h so bed B(a)P o 24 hph and 48 hph b ine sh imp
la ae.
Concen a ion
(mg/L)
24 hph La ae 48 hph La ae
24 h 48 h 24 h 48 h
50 nm NPs
0 100.0 96.8 93.8 100.0
0.000069 93.3 90.0 100.0 100.0
0.00069 97.0 97.0 96.8 93.3
0.069 96.9 96.9 93.3 100.0
0.687 96.7 90.0 100.0 100.0
6.87 93.1 89.7 100.0 93.3
500 nm NPs
0 96.8 93.5 100.0 100.0
0.00034 93.5 96.8 100.0 96.8
0.00069 93.3 93.3 100.0 100.0
0.069 96.8 87.1 100.0 91.2
0.687 90.9 87.9 100.0 97.0
6.87 97.1 94.3 100.0 96.9
4.5 µm MPs
0 100.0 96.8 100.0 93.3
0.0251 100.0 96.8 100.0 96.8
0.0501 96.8 93.5 100.0 80.0
0.501 100.0 93.3 96.9 93.8
5.01 96.7 93.3 96.7 87.1
50.1 100.0 96.7 100.0 75.0
500 nm
NPs-B(a)P
0 96.7 86.7 100.0 80.0
0.00034 93.3 80.0 * 93.3 53.3 *$
0.00069 100.0 60.0 *$ 100.0 60.0
0.069 96.7 83.3 100.0 56.7
0.687 100.0 73.3 100.0 56.7 $#
6.87 93.3 36.7 *$# 100.0 60.0 $#
4.5 µm
MPs-B(a)P
0 96.7 76.7 93.3 80.0
0.0251 90.0 60.0 $ 96.7 90.0
0.0501 100.0 83.3 100.0 86.7
0.501 86.7 56.7 $ 100.0 90.0
5.01 90.0 70.0 100.0 70.0
50.1 100.0 63.3 $ 93.3 90.0
B(a)P
0 97.2 91.7 93.3 80.0
0.1 100.0 65.6 * 0.0 0.0
0.5 94.3 51.4 * 0.0 0.0
1 97.0 57.6 * 0.0 0.0
5 87.9 45.5 * 0.0 0.0
10 90.6 59.4 * 0.0 0.0
As e isks (*) indica e s a is ically signi ican di e ences (p< 0.05) compa ed o he con ol g oup acco ding o
he binomial logis ic eg ession. Dolla s ($) indica e s a is ically signi ican di e ences (p< 0.05) compa ed o
he exposu e o NPs and MPs o he same size wi hou B(a)P a he same concen a ion. Hashes (#) indica e
s a is ically signi ican di e ences (p< 0.05) be ween 500 nm NPs-B(a)P and 4.5
µ
m MPs-B(a)P a he same
exposu e concen a ion. The co esponding odd a ios and con idence in e als a e gi en in Table S2.
In he case o b ine sh imp la ae o 24 hph exposed o 48 h o 500 nm NPs-B(a)P,
es ima ed LC
50
alue was 4.75
±
1.03 mg/L (95% CI: 2.7–6.81). Fo he o he ea men s
wi h plas ic pa icles, LC
50
alues we e always highe han he highes es ed concen a ion.
Fo B(a)P alone, calcula ed LC
50
alues o b ine sh imp la ae o 24 hph we e also highe
han he highes es ed concen a ion (nominal concen a ion 10 mg B(a)P/L, measu ed
ini ial concen a ion 4.92 mg/L), while o b ine sh imp la ae o 48 hph LC
50
alues o
0.004
±
0.197 mg B(a)P/L (95% CI:
−
0.390–0.397) and 0.002
±
0.119 mg B(a)P/L (95% CI:
Nanoma e ials 2022,12, 941 8 o 23
−
0.236–0.240) o 24 h and 48 h o exposu e, espec i ely, we e calcula ed based on he
measu ed concen a ions.
Compa ison be ween exposu e ea men s e ealed some signi ican e ec s o he
so bed B(a)P and o he plas ic pa icle size in he su i al a e o b ine sh imp la ae.
Rega ding he e ec o he so bed B(a)P, exposu e o 24 hph la ae o 0.00069 mg/L and
6.87 mg/L o 500 nm NPs-B(a)P o 48 h caused a signi ican dec ease in su i al compa ed
o he exposu e o 500 nm NPs alone (Table 3, Table S2). Simila ly, exposu e o 48 hph
la ae o 0.00034 mg/L, 0.687 and 6.87 mg/L o 500 nm NPs-B(a)P o 48 h caused a
signi ican dec ease in su i al, compa ed o he exposu e o 500 nm NPs alone. Signi ican
di e ences we e obse ed in su i al a e o 24 hph la ae exposed o 48 h o 0.025 mg/L,
0.501 mg/L and 50.1 mg/L o 4.5 µm MPs-B(a)P, compa ed o la ae exposed o he same
concen a ion o 4.5
µ
m MPs alone. Rega ding he e ec o he pa icle size, in he case o
plas ic pa icles wi h so bed B(a)P, 500 nm NPs had a s onge e ec han 4.5
µ
m MPs, he
di e ence being s a is ically signi ican in he case o 24 hph la ae exposed o 6.87 mg/L
o 500 nm NPs-B(a)P, and o 48 hph b ine sh imp la ae exposed o 6.87 and 0.687 mg/L
o 500 nm NPs-B(a)P o 48 h.
3.3. Toxici y Assays in Zeb a ish Emb yos
The esul s o he de elopmen al pa ame e s o zeb a ish emb yos a e p esen ed
in Table 4and Table S3. Exposu e o PS NPs and o MPs alone o in combina ion wi h
B(a)P did no p o oke any signi ican e ec on zeb a ish emb yo su i al a 120 hp o in
ha ching ime. Only he exposu e o emb yos o 50.1 mg/L o 4.5
µ
m MPs-B(a)P caused a
signi ican inc ease (up o 56.7%) o mal o med emb yos a 120 hp . A simila p e alence o
mal o med emb yos was eco ded a e exposu e o 5 mg/L (50%) and 10 mg/L (58.6%) o
B(a)P alone. Exposu e o B(a)P caused a concen a ion-dependen inc ease o mal o ma ion
p e alence. P e ious oxici y assays wi h di e en DMSO concen a ions showed ha
he DMSO concen a ion p esen in he B(a)P exposu e was no dele e ious o zeb a ish
emb yos (Table S4). The EC
50
alues es ima ed o mal o ma ion p e alence a 120 hp
o emb yos exposed o NPs and o MPs alone and o 500 nm NPs-B(a)P we e always
abo e he highes exposu e concen a ion. EC
50
alues calcula ed o emb yos exposed o
4.5
µ
m MPs-B(a)P and o B(a)P alone we e 45.57
±
9.12 mg/L (95% CI: 27.34–63.81) and
3.55 ±0.68 mg/L (95% CI: 2.183–4.915), espec i ely.
Table 4. Resul s o he de elopmen al pa ame e s o zeb a ish emb yos exposed o NPs and o MPs
alone and in combina ion wi h B(a)P. Da a on su i al and mal o ma ion p e alence a 120 hp a e
gi en as pe cen ages o he whole sample (n= 30–36 indi iduals). Da a on ha ching ime a e shown
as means ±s anda d de ia ions.
Exposu e Conc.
(mg/L) Su . (%) Ha ching
Time (h)
Mal o m.
(%)
Type o Mal o m. (%)
SC PE YE EA
50 nm NPs
0 97.2 72 11.4 11.4 0 0 0
0.000069 96.7 72 20.7 20.7 0 0 0
0.00069 100 72 6.7 6.7 0 0 0
0.069 93.3 72 7.1 3.6 3.6 0 0
0.687 96.7 72 10.3 10.3 0 0 0
6.87 90 72 3.7 3.7 0 0 0
500 nm
NPs
0 94.4 72 8.8 8.8 0 0 0
0.00034 93.3 72 10.7 10.7 0 0 0
0.00069 90 71.1 ±4.6 11.1 11.1 0 0 0
0.069 96.7 72 17.2 17.2 0 0 0
0.687 96.7 72 20.7 20.7 3.5 3.5 3.5
6.87 93.3 72 21.4 14.3 10.7 0 0
Nanoma e ials 2022,12, 941 9 o 23
Table 4. Con .
Exposu e Conc.
(mg/L) Su . (%) Ha ching
Time (h)
Mal o m.
(%)
Type o Mal o m. (%)
SC PE YE EA
4.5
µ
m MPs
0 94.4 71.3 ±4.1 2.9 2.9 0 0 0
0.0251 96.7 72 10.3 6.9 3.4 0 0
0.0501 100 72 16.7 10 6.7 0 0
0.501 93.3 70.3 ±6.3 7.1 7.1 0 0 0
5.01 100 72 16.7 13.3 3.3 0 0
50.1 100 70.4 ±6.1 20 16.6 6.7 0 0
500 nm
NPs-B(a)P
0 94.4 58.6 ±12.1 16.7 13.9 2.8 0 0
0.00034 93.3 55.7 ±11.4 16.7 10 6.7 0 0
0.00069 90 54.2 ±10.7 26.7 26.7 6.6 3.3 0
0.069 96.7 57.1 ±11.9 16.7 13.3 3.3 0 0
0.687 96.7 55.4 ±11.3 16.7 10 6.7 0 0
6.87 93.3 56.6 ±12.0 10 6.7 6.7 0 0
4.5 µm
MPs-B(a)P
0 100 71.3 ±4 11.1 11.1 0 0 0
0.0251 100 72 16.7 16.7 0 0 0
0.0501 100 72.0 16.7 16.7 3.3 0 0
0.501 100 70.4 ±6.1 13.3 10 3.3 0 0
5.01 100 72 26.7 20 10 3.3 0
50.1 100 70.4 ±6.1 56.7 *$ 36.7 30 6.7 0
B(a)P
0 97.2 67.2 ±9.7 11.6 8.6 2.9 0 0
0.1 96.7 69.5 ±8.3 13.8 13.8 0 0 0
0.5 86.7 69.6 ±7.8 24.1 23.1 7.7 0 0
1 86.7 69.2 ±7.8 26.9 23.1 7.7 3.8 0
5 96.7 70.3 ±6.2 50 * 27.5 20.6 3.4 0
10 96.7 70.3 ±6.2 58.6 * 44.8 24.1 0 0
Conc. = concen a ion; EA: eye abno mali y; Mal o m. = mal o ma ion; PE = pe ica dial edema; SC = spinalco d
lexu e; Su . = su i al; YE = yolk sac edema. As e isks (*) indica e s a is ically signi ican di e ences (p< 0.05)
compa ed o he con ol g oup acco ding o he binomial logis ic eg ession. Dolla s ($) indica e s a is ically
signi ican di e ences (p< 0.05) compa ed o he exposu e o NPs and MPs o he same size wi hou B(a)P a he
same exposu e concen a ion. The alues o he co esponding odd a ios and con idence in e als a e gi en in
Table S4.
The p e alence o indi idual mal o ma ions is also shown in Table 4, and Figu e 1
illus a es some o he mal o ma ions ound. Spinal co d lexu e was he mos p e alen
mal o ma ion ound in emb yos exposed o all he ea men s (Figu e 1B–D). Those exposed
o 4.5
µ
m MPs-B(a)P (Figu e 1D) and o B(a)P p esen ed he highes p e alence, wi h alues
o 36.7% and 44.8%, espec i ely. High p e alence o pe ica dial edema (24.1% and 30%,
espec i ely) was obse ed in emb yos exposed o he highes concen a ions o B(a)P alone
(Figu e 1E) o 4.5
µ
m MPs-B(a)P (Figu e 1D). Yolk sac edema (Figu e 1C) was only obse ed
in one o wo indi iduals pe concen a ion and ea men , such as in emb yos exposed o
5.1 and 50.1 mg/L o 4.5
µ
m MPs-B(a)P (3.3% and 6.7%, espec i ely), and o 1 and 5 mg/L
o B(a)P (3.8% and 3.4%, espec i ely) (Figu e 1D,F). Only one case o eye abno mali y was
obse ed in an emb yo exposed o 0.687 mg/L o 500 nm NPs.
Nanoma e ials 2022,12, 941 16 o 23
yolk sac a exposu e concen a ions abo e 0.501 mg/L. A simila signal in he yolk sac was
obse ed in 96 hp zeb a ish emb yos exposed o 5.01 and 50.1 mg/L o 4.5
µ
m MPs-B(a)P
(Figu e 6G,H). The 120 hp emb yos p esen ed luo escence a exposu e concen a ions
highe han 0.501 mg/L o 4.5
µ
m MPs-B(a)P (Figu e 6I) and up o 5.01 mg/L (Figu e 6J).
As i can be seen, he luo escence in ensi y was highe o zeb a ish emb yos exposed o
500 nm NPs-B(a)P (Figu e 5) han o 4.5
µ
m MPs-B(a)P (Figu e 6). The 120 hp zeb a ish em-
b yos exposed o di e en B(a)P concen a ions (0.1 and 0.5 mg/L, Figu e 6K,L) p esen ed
luo escence wi h a simila in ensi y o emb yos exposed o MPs con amina ed wi h B(a)P.
4. Discussion
In he p esen s udy, we aimed o assess he po en ial acu e oxici y and bioa ailabili y
o PS NPs and MPs in b ine sh imp la ae and zeb a ish emb yo de elopmen and whe he
hese plas ic pa icles can ac as ca ie s o PAHs. Th ee plas ic sizes we e used, in o de o
in es iga e he e ec o his pa ame e on hei oxici y and bioa ailabili y. Comme cial PS
NPs and MPs we e chosen o his s udy, due o hei a ailabili y in di e en and homoge-
neous sizes, which makes hem e y sui able o s udying size-dependen e ec s, and due
o hei widesp ead use in labo a o y expe imen s [
41
], and in spi e o hei lowe en i on-
men al ele ance compa ed o o he polyme s such as PP o PVC [
42
,
43
] o en i onmen al
NPs and MPs [
44
]. Especially in he case o Fluo esb i e
®
50 nm NPs, which, acco ding
o he manu ac u e ’s in o ma ion, a e designed as s anda ds o ins umen calib a ion
and p obes o cellula s udies, luo escen labelling g ea ly acili a es hei localiza ion
and dis ibu ion wi hin he o ganisms by non-in asi e mic oscopical echniques, wi hou
in e e ing in he indi iduals’ de elopmen .
P e ious s udies ha es ed he oxici y o PS NPs and MPs in di e en species o b ine
sh imps epo ed no signi ican e ec s on su i al [
8
,
9
,
12
,
45
,
46
]. In A emia pa henogene ica,
acu e e ec s we e no obse ed a e 14 days o exposu e o low concen a ions o 10
µ
m
PS MPs (1–1000 pa icles/mL o 0.55–550
µ
g/L). Howe e , al e a ions a cell le el, such as
abno mal ul as uc u e o in es inal epi helial cells and appea ance o au ophagosomes,
which could a ec he ene ge ic sys em, we e desc ibed [
9
]. Simila esul s we e ob ained
in A emia anciscana. Exposu e o 100 nm PS NPs did no cause acu e oxici y wi h a
LC
50
alue highe han he highes es ed concen a ion (100 mg/L) [
12
]. Fo smalle
plas ics, such as unc ionalized PS NPs (40 nm PS-COOH and PS-NH2), oxici y es s we e
pe o med in A. anciscana la ae, and no e ec was eco ded on mo ali y, e en o he
highes exposu e concen a ion (100 mg/L) [
8
]. Ne e heless, ecen s udies indica ed
ha o he polyme s, such as PP can exe mo e oxic e ec s on b ine sh imps. Jeya ani
and cowo ke s epo ed an inc eased oxida i e bu s and mo ali y (LC
50
40.95 mg/L) in
nauplii exposed o 11.86–44.62
µ
m PP MPs [
11
]. In he p esen s udy, LC
50
alues highe
han he highes es ed concen a ions, 50.1 mg/L o 4.5
µ
m PS MPs and 6.9 mg/L o
50 nm and 500 nm PS NPs, we e es ima ed, in ag eemen wi h he p e iously men ioned
esul s epo ed in he li e a u e.
Su i al a e, ha ching ime, and mal o ma ion p e alence in zeb a ish emb yos ex-
posed o he di e en sized MPs and o NPs did no show any signi ican change compa ed
o he con ol alues. Acu e e ec s in zeb a ish emb yos ha e no been epo ed in he li e -
a u e, and suble hal e ec s ha e no always been epo ed [
19
,
21
,
22
,
47
,
48
]. B un e al. [
22
]
in es iga ed he po en ial oxici y o PS NPs (25 nm) on he immune sys em o he skin and
in es ine o zeb a ish emb yos a e injec ion o 1 nL o 1 mg NPs/L a 30 hp . NP injec ion
did no p o oke signi ican changes in he su i al o ha ching a e o 54 hp zeb a ish
emb yos, bu up egula ion o immune sys em- ela ed genes (in e leukin1
β
and chemokine
(C-C mo i ) ligand 20a) was obse ed. Ka ami e al. [
21
] exposed zeb a ish emb yos o a
mix u e o low-densi y PE agmen s (5–500
µ
g/L) o di e en sizes (<17.6
µ
m). No e ec
was obse ed a e 10 days o exposu e o he assessed genes (casp8, casp3a, sod1, gs p1, and
ca ), while a signi ican down egula ion o ca ,casp3a, and casp9 a e 20 days o exposu e,
which esul ed in minimal impac on he o ganisms, was epo ed. Ne e heless, e ec s
on swimming mobili y o zeb a ish emb yos a e exposu e o 1 mg/L o 1
µ
m PS MPs
Nanoma e ials 2022,12, 941 17 o 23
we e obse ed, wi hou signi ican changes in ha ching a e, compa ed o con ols [
19
]. In
addi ion, up egula ion o in lamma ion- (in e leukin1
β
) and oxida i e s ess- ela ed genes
(ca alase) in emb yos exposed o 1 mg/L o PS MPs was obse ed. Thus, o e all, NPs and
MPs a e epo ed as non-acu ely oxic ma e ials.
NPs and non-con amina ed MPs did no cause acu e oxic e ec s, bu he exposu e o
ea ly li e s ages o b ine sh imps o NPs and MPs wi h so bed B(a)P p o oked signi ican
e ec s on su i al. The 500 nm NPs-B(a)P we e oxic o 24 hph b ine sh imp la ae a e
48 h o exposu e wi h a LC
50
alue o 4.75 mg/L. To he bes o ou knowledge, hese
e ec s o NPs associa ed wi h POPs on b ine sh imp la ae ha e no been p e iously
epo ed. In D. magna, ano he aqua ic b anchiopod commonly used o aqua ic oxici y
assessmen , highe le hali y was eco ded in indi iduals co-exposed o 48 h o PS NPs (100
nm) and PCBs (0.64 mg/L) han in hose exposed o PCBs alone. The combined oxici y
o D. magna depended on he ela i e concen a ion o NPs and PCBs, wi h PCBs being
less oxic when combined wi h low concen a ions o NPs (<1 mg/L), while highe NP
concen a ions enhanced he le hali y [
31
]. Wa e bo ne exposu e o D. magna o la ge PS
NPs o up o 1
µ
m (3
×
10
5
pa icles/mL o 0.038 mg/L), in combina ion wi h p e iously
known oxic concen a ions o he insec icides dime hoa e and del ame h in, did no lead
o any e ec on he s udied endpoin s, such as su i al and mobili y [
49
]. A e being
in e nalized, B(a)P so bed o he plas ic pa icles could be eleased o he b ine sh imp
diges i e sys em, inc easing i s oxici y [
50
]. In he p esen s udy, signi ican di e ences
we e ound be ween he exposu e o b ine sh imp la ae o a simila concen a ion o
4.5
µ
m MPs-B(a)P (5.01 mg/L) and o 500 nm NPs-B(a)P (6.87 mg/L), showing ha he isk
o dea h inc eased wi h he dec ease o con amina ed plas ic pa icle size. Thus, esul s
om he li e a u e and om he p esen s udy sugges ha in he case o con amina ed
NPs and MPs, size plays a c i ical ole in oxici y, wi h smalle plas ic pa icles being mo e
oxic o aqua ic o ganisms han la ge ones, when combined wi h o he oxic pollu an s.
This seems o be due o he highe su ace/ olume a io o smalle pa icles, which con e s
hem a highe capaci y o ca y B(a)P [29].
P e ious s udies ha e add essed he po en ial oxici y o NPs and MPs combined
wi h o ganic compounds on zeb a ish de elopmen [
20
,
33
,
51
]. In zeb a ish emb yos o
3 hp , co-exposu e o 48 h and 72 h o 45
µ
m PS MPs o 50 nm PS NPs wi h EE2 (2
µ
g/L
and 20
µ
g/L) p oduced an inc ease o oxici y a he highes EE2 concen a ion, showing
an inc ease in ca alase (CAT) ac i i y and glu a hione (GSH) con en compa ed o con ol
emb yos. A high concen a ion o EE2 combined wi h MPs was, in e ms o oxida i e
s ess, mo e oxic han a high concen a ion o EE2 combined wi h NPs, showing ha
he bioa ailabili y o EE2 was highe in he p esence o MPs han o NPs [
20
]. On he
con a y, co-exposu e o 200–250
µ
m PVC MPs and EE2 o phenan h ene educed he
bioa ailabili y o he o ganic compounds o zeb a ish emb yos, esul ing in no e ec s o
he es ed ea men s [
33
]. The in e media e MP size (11–13
µ
m PE, 100 mg/L) spiked
wi h B(a)P (16.87
µ
g/g) p o oked an inc ease o bio ans o ma ion me abolism (EROD
ac i i y and cy och ome P450 1A (cyp1a) ansc ip ion le els), indica ing he success ul
ans e o B(a)P ia MPs o he emb yos, bu no acu e emb yo oxici y [
51
]. In he p esen
s udy, only exposu e o 4.5
µ
m MPs wi h so bed B(a)P caused oxici y o 120 hp emb yos
(EC
50
= 45.57
±
9.12 mg/L o 9.1
×
10
6
pa icles/mL), while no signi ican e ec s we e
eco ded, e en a he highes es ed concen a ion o 500 nm NPs-B(a)P (EC
50
> 6.87 mg/L
o 10
8
pa icles/mL). The ype o mal o ma ions obse ed in 120 hp zeb a ish emb yos
exposed o 4.5
µ
m MPs-B(a)P we e simila o hose p o oked by B(a)P exposu e. Wi h 30%
o he emb yos exposed o 52.5 mg/L o 4.5
µ
m MPs-B(a)P p esen ing pe ica dial edema.
As p e iously men ioned, ca dio oxici y is a common e ec o PAHs om c ude oil in
zeb a ish emb yos [52].
B(a)P is a well-known oxic compound o aqua ic o ganisms [
53
–
55
]. In his s udy,
es ima ed LC
50
alues o 48 hph b ine sh imp la ae exposed o 24 and 48 h we e
0.004
±
0.197 mg/L and 0.002
±
0.119 mg/L, espec i ely, whe eas he EC
50
alue, based
on mal o ma ion appea ance, o zeb a ish emb yo exposed o 120 h was 3.55
±
0.68 mg/L.
Nanoma e ials 2022,12, 941 18 o 23
B ine sh imps p esen ed an inc eased sensi i i y h oughou he de elopmen al s ages
(24 hph o 96 hph) when exposed o B(a)P. This inc eased sensi i i y could be due o he
p og essi e loss o ene gy ese oi s p esen in he i s de elopmen al s ages and he
lack o eeding du ing he assay. The LC
50
alues o 48 hph b ine sh imp la ae exposed
o B(a)P we e simila o hose epo ed o D. magna. Exposu e o D. magna o 48 h o
1–32
µ
g/L B(a)P wi hou eeding esul ed in a LC
50
alue o 4.7
µ
g/L [
55
]. PAHs, oge he
wi h o he o ganic compounds, such as PCBs, a e amongs he mos oxic compounds o
plank onic c us aceans (b anchiopod, copepod, and os acod), p ima ily due o he highe
oxici y and g ea e pe sis ence o hei me aboli es, han o he pa en compounds [
54
]. The
obse ed mo ali y in ea ly s ages o b ine sh imps a low B(a)P exposu e concen a ions
shows he sensi i i y o his o ganism o PAH exposu e; being a sui able model o oxici y
e alua ion o hese compounds.
Zeb a ish emb yos we e less sensi i e o B(a)P exposu e han b ine sh imp la ae.
Ne e heless, B(a)P has also been p o en o be a oxic PAH o zeb a ish emb yo de el-
opmen [
56
–
59
]. Knech e al. [
58
] obse ed a non-signi ican inc ease o mo ali y a e
and mal o ma ion p e alence in 120 hp zeb a ish emb yos exposed o 0.1 and 1 mg/L o
B(a)P (1% DMSO), bu he al e a ion o he swimming beha io esul ed in hype ac i i y
o emb yos exposed o B(a)P. Fu he mo e, inc easing B(a)P concen a ion up o 2.5 mg/L
caused mo ali y in 72 hp zeb a ish emb yos, wi h a LC
50
alue o 1.285 mg/L and a EC
50
o mal o ma ions o 0.131 mg/L [
56
]. A highe B(a)P concen a ion (500
µ
M = 12.6 mg/L)
p o oked 40–50% o mal o med emb yos (72 hp ) wi hou u he changes in hei hea
a e, bu oxida i e damage o DNA was obse ed in 72 hp zeb a ish emb yos exposed
o a lowe B(a)P concen a ion (1
µ
M = 0.25 mg/L) [
57
]. Exposu e o 1
µ
M B(a)P also
caused geno oxici y in 96 hp emb yos [
59
]. The highe sensi i i y obse ed o b ine
sh imps exposed o B(a)P han o zeb a ish emb yos was possibly due o he e iciency o
aqua ic e eb a es, e en a he ini ial s ages o de elopmen , o de oxi ica ion o o ganic
compounds compa ed o in e eb a es [60].
P e ious wo ks epo ed he use o B(a)P concen a ions anging om 0.2 o 25.2 mg/L
o de elopmen al and suble hal oxici y s udies in zeb a ish emb yos [
56
,
58
]. The low
solubili y o B(a)P in wa e (1.64
µ
g/L) [
61
] makes i di icul o each acu ely oxic concen-
a ions o o ganisms [
62
,
63
], which a e usually abo e solubili y poin , e en using DMSO
as ehicle. Due o his low solubili y, B(a)P in wa e ends o agg ega e, leading o analy ical
de ia ions. The analy ical esul s epo ed by Cos a e al. [
62
] and Zhao e al. [
63
] showed
ha none o he B(a)P solu ions eached he expec ed nominal concen a ion. Mo eo e ,
B(a)P is an hyd ophobic compound ha ends o be so bed on he plas ic ma e ial used
o biological es s, especially on uncoa ed PS mic opla es [
64
,
65
]. Acco ding o Fische
e al. [
65
], exposu e o PAHs wi h pa i ion coe icien s (log K
ow
) highe han 6, such as
B(a)P, should be ca e ully moni o ed, especially o low concen a ions, due o he p edic ed
losses highe han 80% om ini ial concen a ion, as a esul o adso p ion o PS mic opla es.
Chlebowski e al. [
64
] also epo ed a high so p ion o di e en PAHs ( luo an hene, py ene,
ch ysene, and B(a)P) a he highes exposu e concen a ion used (0.32
µ
M o each PAH
s udied). In he case o B(a)P, 48% o he o al (80.64
µ
g/L = 0.32
µ
M) emained in he wa e ,
39% was so bed on o he walls, and 13% was assimila ed by he zeb a ish emb yos. In
ag eemen , ou esul s showed ha he B(a)P concen a ion dec eased when he es media
we e placed in o he PS mic opla e wells, likely due o bo h, deg ada ion, and so p ion o
he mic opla e walls, wi h he loss pe cen age being highe o he lowe concen a ions.
Con ocal mic og aphs showed he abundan p esence o 50 nm luo escen NPs wi hin
he b ine sh imp diges i e ac . Non- luo escen ly labeled 4.5
µ
m MPs we e also easily
obse ed in b ine sh imp diges i e ac , whe eas 500 nm NPs we e no dis inguishable
unde he ligh mic oscope. Ne e heless, aking in o accoun ha 50 nm NPs and 4.5
µ
m
MPs we e inges ed by b ine sh imp, assimila ion o 500 nm NPs was also expec ed o
occu . Localiza ion o plas ic pa icles in he diges i e ac o b ine sh imp has been
epo ed o NPs and MPs o di e en sizes [
8
,
13
]. Fo smalle plas ics, such as 40 nm
anionic ca boxyla ed (PS-COOH) and 50 nm ca ionic amino (PS-NH2) luo escen PS NPs,
Nanoma e ials 2022,12, 941 19 o 23
inges ion by A. anciscana was obse ed o concen a ions anging om 5 o 100 mg/L,
al hough luo escence was only de ec ed on he su ace o he eces o b ine sh imp [
8
].
Inges ion was obse ed o a concen a ion anging om 1 o 1000 pa icles/mL o 10
µ
m
PS MPs by A. pa henogene ica (<24 h old) o e 24 h [
13
]. As p e iously highligh ed, i is
e iden ha MPs and NPs a e inges ed by b ine sh imps, bu signs o in e naliza ion in o
issues ha e no ye been epo ed. The inges ion o NPs and MPs by zooplank onic species,
such as b ine sh imp, which a e consumed by nume ous o ganisms a highe le els o he
ophic chain, is a po en ial isk o plas ic ans e and accumula ion in he ood web, e en
be ween di e en zooplank onic o ganisms [45,66] in aqua ic ecosys ems.
In he case o zeb a ish, NPs sp ead h ough he body and a e accumula ed in speci ic
o gans (eye, yolk sac, and ail), e en in ea ly de elopmen s ages, mainly due o he la ge
po e size o he emb yo cho ion (600–700 nm) [
67
]. Using con ocal mic oscopy, luo escence
was de ec ed in se e al op ical sec ions, indica ing in e naliza ion o NPs in he eye, as
also epo ed by an Pome en e al. [
68
] o 25 and 50 nm PS NPs, bu no o la ge NPs
(200 nm). O he nanoma e ials, such as 27 nm SiO
2
luo escen nanopa icles ha e also
been localized in o zeb a ish emb yo eyes a 120 hp [
69
]. The p esence o nanopa icles
in he yolk sac is usually epo ed o zeb a ish emb yos [
23
,
41
,
69
]. Zeb a ish emb yos
exposed o luo escen PS NPs p esen ed a highe in e naliza ion o NPs wi h he inc ease
o la al age [
23
,
70
], wi h luo escence being loca ed on he su ace o he cho ion, in he
yolk sac (24 hp ), and in he head [
23
]. A di e en o gan accumula ion o NPs, in he
panc eas and li e , was obse ed in 120 hp zeb a ish emb yos [
23
]. In his s udy, a no able
inc ease o luo escence was obse ed nea he ail and he yolk sac, in he o m o i egula
deposi s. As seen by E ensen e al. [
71
], an injec ion o luo escen PS NPs (100 nm) in he
ca dinal ein p oduced an accumula ion o NPs on mac ophages, wi h simila s uc u e
and dis ibu ion o hose ound in ou s udy. A simila dis ibu ion o luo escen PS NPs
was obse ed a neu omas le el, localized inside mac ophages [
22
]. NPs can be a po en ial
sou ce o damage o ne ous and diges i e sys ems, due o hei epo ed accessibili y o
di e en o gans. The p esence o NPs in he eyes and b ain leads o, o example, locomo o
al e a ions [
20
,
72
]. Thus, u he s udies on he long- e m e ec s in ju eniles and adul s
a e an acu e exposu e e en a emb yo s age would be o g ea in e es .
Inges ion o in e naliza ion o MPs o con amina ed MPs was no obse ed in zeb a ish
emb yos in he p esen s udy, bu B(a)P luo escence was obse ed all o e he zeb a ish
emb yos exposed o con amina ed MPs, om e y ea ly s ages. This luo escence could be
due o he up ake o B(a)P deso bed om he MPs o he medium o by di ec con ac o
MP-B(a)P wi h he emb yo’s su ace.
Resul s o b ine sh imp and zeb a ish exposu es showed a success ul anspo o
B(a)P om MPs o he o ganisms, in a wide ange o concen a ions (0.00069 o 6.9 mg/L
o 500 nm NPs and 0.501 o 50.1 mg/L o 4.5
µ
m MPs). In b ine sh imps, he esul s o
he exposu es o 500 nm NPs and o 4.5
µ
m MPs-B(a)P di e ed. In he case o 500 nm
NPs-B(a)P, luo escence was only de ec ed in he diges i e ac and in he eces, while o
4.5
µ
m MPs-B(a)P, luo escence was only obse ed all o e he body and in he diges i e
ac . As shown by Ba el e al. [
73
], 1–5
µ
m and 10–20
µ
m PE MPs-B(a)P eleased he B(a)P
all o e he body o b ine sh imps and hen B(a)P was ans e ed om b ine sh imps o
adul zeb a ish ia hei die . Due o he capaci y o b ine sh imps o il e la ge amoun s o
wa e , exposu e o MPs wi h so bed B(a)P can lead o inc eased exposu e o B(a)P compa ed
o o he o ganisms ha a e mo e selec i e in e ms o eeding, ep esen ing a isk o hese
o ganisms. In zeb a ish emb yos, B(a)P luo escence was obse ed on he su ace o he
cho ion and in he yolk sac. The success ul anspo o B(a)P in o emb yos by MPs o bo h
sizes was obse ed, e en h ough he cho ion wall, p o ing he isk o MPs as ec o s o
o he compounds. Howe e , i was no possible o deciphe whe he he anspo o B(a)P
ook place by deso p ion o B(a)P om MPs in he exposu e media o by B(a)P deso bed
a e inges ion.
Nanoma e ials 2022,12, 941 20 o 23
5. Conclusions
In summa y, NPs and MPs alone did no cause acu e e ec s in zeb a ish emb yos and
b ine sh imp la ae, e en i hey we e inges ed by bo h o ganisms. Toxic e ec s occu ed
when NPs (500 nm) and MPs (4.5
µ
m) we e con amina ed wi h B(a)P, and exposu e o
NPs and MPs wi h so bed B(a)P esul ed in o B(a)P accumula ion in b ine sh imp la ae
and zeb a ish emb yos. MP size played a signi ican ole in explaining he oxici y o MPs
wi h so bed B(a)P, implying a po en ial isk o b ine sh imps when MP size dec eased,
due o he inc ease o su ace o olume a io, which esul s in a highe quan i y o B(a)P
so bed o po en ial ans e . Thus, polys y ene NPs and MPs o di e en size a e likely o
ac as ec o s o PAHs in he aqua ic en i onmen , modula ing hei bioa ailabili y and
p o oking oxic e ec s in o ganisms ha play impo an oles in ecosys ems. Howe e , a
lo o wo k emains o be done, especially o s anda diza ion o MP expe imen s, as well as
o he use o en i onmen ally ele an MPs a ealis ic concen a ions, in o de o e alua e
he h ea posed by NP and MP con amina ion. S udies o he long- e m consequences a
physiological and beha io al le els a e equi ed.
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a : h ps:
//www.mdpi.com/a icle/10.3390/nano12060941/s1, Figu e S1: Measu ed concen a ions om a
nominal concen a ion o 2.51 mg/L o 5
×
10
4
pa icles/mL o 4.5
µ
m MPs using a cell coun e
be o e and a e il a ion using a polye he sul one il e (0.45
µ
m il e po e). Ba s ep esen he mean
o h ee ins umen al eplica es wi h hei co esponding s anda d de ia ions. The pe cen age o
MPs measu ed om he nominal concen a ion is gi en abo e each ba . Figu e S2. Mic og aphs o
zeb a ish emb yos exposed o simila masses o plas ics o di e en sizes. (A) 48 hp emb yo exposed
o 6.87 mg/L o 50 nm NPs; (B) 48 hp emb yo exposed o 6.87 mg/L o 500 nm NPs; (C) 48 hp
emb yo exposed o 5.01 mg/L o 4.5
µ
m MPs; (D) non-ha ched 120 hp ali e emb yo exposed o
5.01 mg/L o 4.5 µm MPs-B(a)P. Scale ba s: 100 µm. Table S1: E ec on su i al (%) o he exposu e
o 24 hph and 48 hph b ine sh imp la ae o DMSO o 24 h and 48 h. Table S2: Odd a io alues
indica ing he isk o dea h (immobiliza ion) o b ine sh imp la ae exposed o NPs and MPs alone
o in combina ion wi h B(a)P. Table S3: Odd a io alues indica ing he isk o mal o ma ion in 120 hp
zeb a ish emb yos exposed o 4.5
µ
m MPs alone o in combina ion wi h B(a)P o o B(a)P alone.
Table S4: E ec s o 120 h DMSO exposu e on de elopmen al pa ame e s o zeb a ish emb yos.
Au ho Con ibu ions:
Concep ualiza ion, M.P.C., H.B. and A.O.; Me hodology, I.M.-Á. and K.L.M.;
Valida ion, K.L.M., H.B. and A.O.; Fo mal Analysis, I.M.-Á. and K.L.M.; In es iga ion, I.M.-Á.;
Resou ces, K.L.M., M.-H.D. and A.O.; W i ing—O iginal D a P epa a ion, I.M.-Á.; W i ing—Re iew
& Edi ing, A.O., H.B., M.-H.D. and M.P.C.; Visualiza ion, I.M.-Á. and A.O.; Supe ision, H.B. and
A.O.; P ojec adminis a ion, M.P.C., M.-H.D., H.B. and A.O.; Funding acquisi ion, M.P.C., H.B. and
A.O. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This wo k was unded by UPV/EHU (p edoc o al g an o IMA), Basque Go e nmen
(consolida ed esea ch g oup IT810-13 and IT1302-19), Spanish MINECO p ojec NACE (CTM2016-
81130-R), F ench ANR (No.–10–IDEX-03-02 and Clus e o Excellence COTE (ANR-10-LABX 45). This
wo k was pe o med wi hin he amewo k o he Cen e o Ad anced S udies (CAS) p ojec “H2020
CAS6 Nanoplas ics” unded by he Eu opean Commission- Join Resea ch Cen e (JRC/A/05).
Ins i u ional Re iew Boa d S a emen :
No applicable. The s udy was conduc ed wi h species and
li e s ages no conside ed wi hin he cu en Eu opean legisla ion on he p o ec ion o animals used
o expe imen a ion (Di ec i e 2010/63/UE).
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : Da a a e con ained wi hin he a icle.
Acknowledgmen s:
The au ho s gi e hanks o he echnical and human suppo p o ided by
SGIke (UPV/EHU/ ERDF, EU).
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Nanoma e ials 2022,12, 941 21 o 23
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