Chemosphe e 311 (2023) 136935
A ailable online 26 Oc obe 2022
0045-6535/© 2022 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
Inno a i e in i o and in i o bioassays o he es ablishmen o oxici y
h esholds o pollu an s in sedimen quali y assessmen using polychae es
and hei immune cells
N. Ga cia-Velasco
a
,
b
,
*
, J.A. Ca e o
c
, E. U ionaba ene xea
a
,
b
, L. Doni
b
, B. Zaldiba
a
,
b
,
U. Izagi e
a
,
b
, M. So o
a
,
b
a
Cell Biology in En i onmen al Toxicology (CBET) Resea ch G oup, Dep . Zoology and Animal Cell Biology, Facul y o Science and Technology, Uni e si y o he Basque
Coun y (UPV/EHU), Basque Coun y, Sa iena Auzoa Z/g, 48940, Leioa, Basque Coun y, Spain
b
Resea ch Cen e o Expe imen al Ma ine Biology and Bio echnology PIE-UPV/EHU, Uni e si y o he Basque Coun y (UPV/EHU), A ea za Pasealekua, 48620,
Plen zia, Basque Coun y, Spain
c
Dep . o Applied Chemis y, Facul y o Chemis y, Uni e si y o he Basque Coun y (UPV/EHU), Manuel de La dizabal Pasealekua 3, 20018 Donos ia, Basque
Coun y, Spain
HIGHLIGHTS GRAPHICAL ABSTRACT
•The bioassays wi h H. di e sicolo accu-
a ely assess sedimen -bounded pollu -
an s e ec s.
•The oxici y o Cu and AgNPs we e
en isaged a e y sho -exposu e imes
wi h coelomocy es.
•In i o app oaches wi h coelomocy es
showed g ea po en ial o sedimen
eco oxicology.
•The oxic esponses o H. di e sicolo
we e simila o he h esholds ob ained
wi h SET.
ARTICLE INFO
Keywo ds:
Hedis e di e sicolo
Sedimen oxici y es
Bioma ke s
In i o
Coelomocy es
ABSTRACT
Sedimen oxici y es ing has become a c ucial componen o assessing he isks posed by con amina ed sedi-
men s and o he de elopmen o sedimen quali y assessmen s a egies. Commonly used o ganisms o bio-
assays wi h es ua ine sedimen s include amphipods, A enicola ma ina polychae es and echinoids. Among he
la e , he Sea U chin Emb yo es (SET) is he mos widely used. Howe e , one ele an limi a ion o his
bioassay is he una ailabili y o game es all yea - ound, pa icula ly ou side he na u al spawning seasons.
Consequen ly, he es ablishmen o an app op ia e and complemen a y model o ganism o a con inuous
assessmen o sedimen quali y is ecommended. A eliable assessmen o he haza ds esul ing om pollu an s in
sedimen s o po e wa e , can be achie ed wi h ecologically ele an species o sedimen such as he polychae e
Hedis e di e sicolo , which is widesp ead in es ua ies and has he capaci y o accumula e pollu an s. The aim o
his wo k was o de elop eliable in i o and in i o bioassays wi h H. di e sicolo and i s coelomocy es (immune
cells) o de e mine he oxici y h esholds o di e en con aminan s bounded o sedimen s o esuspended in o
wa e . Polychae es we e exposed o suble hal concen a ions o CuCl
2
(in i o) and a non-in asi e me hod o
* Co esponding au ho . Cell Biology in En i onmen al Toxicology (CBET) Resea ch G oup, Dep . Zoology and Animal Cell Biology, Facul y o Science and
Technology, Uni e si y o he Basque Coun y (UPV/EHU), Basque Coun y, Sa iena Auzoa z/g, 48940, Leioa, Basque Coun y, Spain.
E-mail add ess: [email p o ec ed] (N. Ga cia-Velasco).
Con en s lis s a ailable a ScienceDi ec
Chemosphe e
jou nal homepage: www.else ie .com/loca e/chemosphe e
h ps://doi.o g/10.1016/j.chemosphe e.2022.136935
Recei ed 30 Ap il 2022; Recei ed in e ised o m 7 Oc obe 2022; Accep ed 16 Oc obe 2022
Chemosphe e 311 (2023) 136935
2
collec ion o polychae es coelomocy es was applied o he in i o bioassay, exposing cells o a se ies o CuCl
2
and AgNPs concen a ions. Same e e ence oxican s we e used o expose Pa acen o us li idus ollowing he SET
(ICES Nº 51; Bei as e al., 2012) and ob ained oxici y h esholds we e compa ed be ween he wo species. In i o
exposu e o polychae es o high concen a ions o Cu p oduced weigh loss and his opa hological al e a ions.
A e in i o app oaches, a signi ican dec ease in coelomocy es iabili y was eco ded o bo h oxican s, in a
mono onic dose- esponse cu e, a e y sho -exposu e imes (2 h). The oxici y h esholds ob ained wi h
polychae es we e in line wi h he ones ob ained wi h he SET, concluding ha hei sensi i i y is simila . In
conclusion, in i o and in i o bioassays de eloped wi h H. di e sicolo a e accu a e oxici y sc eenings o pol-
lu an s ha could be bounded o sedimen s o dissol ed in he po e wa e , and may complemen he SET ou side
he spawning pe iod o he echinode ms. The bioassays he ein de eloped could be applied no only o es ablish
he oxici y h esholds o indi idual compounds o mix u es, bu also o assess he oxici y o ield collec ed
sedimen s.
1. In oduc ion
Es ua ies ha e unde gone impo an inpu s o di e en chemicals
o e yea s due o indus ial exploi a ion and u ban was es elease
(B and e al., 2018; Ruiz e al., 2020). As a esul , many es ua ine sed-
imen s can be conside ed as ese oi s o pe sis en oxic subs ances
in oduced in coas al en i onmen s by human ac i i ies, ac ing as a sink
and sou ce o con aminan s ha can pose a isk o he en i onmen
(I abien e al., 2018). A eliable assessmen o he haza ds esul ing om
pollu an s in sedimen s can be achie ed a e in eg a ing chemical
analysis and eco oxicological s udies (Hansen e al., 2007; Chapman
e al., 2013; Boehle e al., 2017). Sedimen oxici y es s expose o -
ganisms o sedimen s unde con olled condi ions in o de o gi e an
es ima ion o he le el o oxici y o con amina ed sedimen s in he ield
(Simpson e al., 2017) and ha e he ad an age o e lec he bioa ailable
ac ion o con aminan s, which can be e y di e en om he o al
amoun de e mined by chemical analysis (Ba , 2005). Despi e signi ican
ad ances ha e been made in he use o biological e ec s based oxici y
indices in isk assessmen (Al enbu ge e al., 2019; De Baa e al., 2019),
he conside a ion o he isk o ben hic bio a is s ill poo ly ep esen ed
in sedimen quali y assessmen s a egies and imp o emen s could be
implemen ed. Sedimen oxici y es ing wi h ecologically ele an spe-
cies should become a c ucial componen o assessing he isks posed by
con amina ed sedimen s and o he de elopmen o sedimen quali y
assessmen s a egies (OECD, 2007; Sc imshaw e al., 2007; De Baa
e al., 2019) and d edged ma e ial egula ions (Hansen e al., 2007;
CIEM, 2015).
The mos commonly used ben hic o ganisms o bioassays o assess
he oxici y o ma ine and es ua ine sedimen s include amphipods (EPA,
1994; Casado-Ma inez e al., 2006), A enicola ma ina polychae es (Ba ,
1998; Thain and Bi ield, 2001) and echinoids (Hansen e al., 2007),
being each model ep esen a i e o di e en o ms o exposu e (e.g.
solid o liquid phase). Wi h echinoids, he sea u chin emb yo es (SET)
has been wo ldwide used as a apid, sensi i e, and cos -e ec i e bio-
logical ool o he e alua ion o seawa e quali y (Bei as e al., 2001;
Losso e al., 2007; Saco-´
Al a ez e al., 2010). The SET has been also
desc ibed o sedimen oxici y sc eenings by exposing emb yos o elu-
ia es coming om he sedimen and using size inc ease as a quan i-
a i e endpoin (Bei as e al., 2012). In addi ion, de elopmen al
abno mali ies can be measu ed in la ae a e exposu e o sedimen
elu ia es, since a high sensi i i y o skele ogenesis o speci ic con am-
inan s has been demons a ed (Ca ballei a e al., 2012). Some ele an
limi a ions o his bioassay a e he una ailabili y o sea u chins game es
all yea - ound, pa icula ly ou side he na u al spawning seasons, a
leas in empe a e and/o cold en i onmen s, and he need o 48 h o
pe o m he es . Consequen ly, he es ablishmen o an app op ia e and
complemen a y model o ganism o a con inuous assessmen o sedi-
men quali y is ecommended. Mos o he endoben hic o ganisms and
polychae e wo ms in pa icula , a e in close con ac wi h sedimen s and
inges subsu ace sedimen s ha could ha e po en ial oxican s boun-
ded. Among hem, he common agwo m Hedis e di e sicolo , a
deposi - eede ound in bo h sandy and muddy sedimen s o he
Eu opean and he No h Ame ican coas o he A lan ic Ocean (Smi h
e al., 1996), ole a es a b oad ange o salini ies and empe a u es in
es ua ies (Scaps, 2002; Ashley and Budd, 2016). They a e usually
a ailable h oughou he yea and play impo an oles in he ood chain
and consequen ly, in he sedimen communi y o ganisa ion (Ba , 2005).
The bio u ba ion due o hei ac i i y is an impo an de e minan o
sedimen biogeochemis y and elemen cycling, and also o oxygen
a ailabili y in bu ow en i onmen s ha may a ec g ow h and popu-
la ion sizes o he associa ed meioo ganisms (Palomo and I iba ne,
2000; Scaps, 2002). The easy sampling and main enance unde labo a-
o y condi ions o agwo ms ( ole ance o empe a u e, salini y, and
oxygen le els), and hei high deg ee o esponsi eness (quick esponse
o en i onmen al s esso s), make H. di e sicolo pa icula ly sui able o
eco oxicological s udies, likely o up ake me als h ough me al-bound
pa icles and om po e wa e (Gh ibi e al., 2019; Sil a e al., 2020).
Hence, se e al wo ks demons a ed H. di e sicolo o be sensi i e o
common con aminan s (Mo ei a e al., 2006; Bou aoui e al., 2016), bu
also o chemicals o eme ging conce n in he sedimen o in he wa e
column (Cong e al., 2014; Fonseca e al., 2017; Sil a e al., 2020;
Abouda e al., 2022). These cha ac e is ics make his a sui able species
o biomoni o ing campaigns (Du ou e al., 2007), o acu e and me al
accumula ion pa e n bioassays (Bu linson and Law ence, 2007; Cong
e al., 2014) and o he applica ion o mul i-bioma ke app oaches
(Bu e e al., 2013; Ca alano e al., 2012; De Ma chi e al., 2017; Fon-
seca e al., 2017; F ei as e al., 2017; Gh ibi e al., 2019). The bioma ke s
we e mainly associa ed wi h cy o oxici y, oxida i e s ess, changes in
me abolism, lipid pe oxida ion and neu o and geno oxici y; all in ol ed
in he oxic esponse o common an h opogenic chemicals. Simila ly,
obse a ion o his ological al e a ions and/o damage o soma ic issues
such as he diges i e epi helia ( issue-le el bioma ke s) could be ex-
pec ed in H. di e sicolo unde pollu ion condi ions. De ailed his ological
s udies abou he mic oana omy o he diges i e epi helia in polychae es
a e sca ce (Rod igo e al., 2015) and, mo eo e , his opa hological al-
e a ions ha e no been widely used as issue-le el bioma ke s o assess
biological e ec s o pollu ion like in o he ma ine species (i.e. mussels;
Ga mendia e al., 2010).
A a ie y o s anda dized immunoassays ha e been de eloped wi h
soil ea hwo ms in o de o assess he immuno oxic po en ial o oxi-
can s based on cell-media ed and humo al esponses (Hayashi and
Engelmann, 2013; I iza e al., 2014, 2015; Engelmann e al., 2016;
Ga cia-Velasco e al., 2019). Ma ine polychae es possess simila immune
mechanisms and cells (coelomocy es) o hose o ea hwo ms which
could se e o he de elopmen o adequa e and sensi i e bioassays o
oxici y sc eening o sedimen s (Cu illie -Ho e al., 2014; A edondo
e al., 2020). In ac , in p e ious wo ks wi h H. di e sicolo cellula
endpoin s ha e been quan i ied in coelomocy es a e in i o exposu e,
wi h good esul s o en i onmen al heal h assessmen (Ca alano e al.,
2012; Cong e al., 2014; Fonseca e al., 2017, 2019). Mo eo e , he use
o coelomocy e p ima y cul u es would be a p omising ool o de e mine
sedimen pollu ion e ec s in a apid, simple and non-in asi e o m,
since hese cells, unlike sea u chin emb yos, a e a ailable all along he
yea . The aim o his wo k was o de elop quick, eliable and inno a i e
N. Ga cia-Velasco e al.
Chemosphe e 311 (2023) 136935
3
in i o and in i o sedimen oxici y es s wi h ecologically ele an
species such as H. di e sicolo and i s coelomocy es. Two model oxican s
CuCl
2
and sil e nanopa icles (AgNPs) we e used as es subs ances, and
he esul s o he bioassays we e compa ed wi h he oxici y h esholds
ob ained by he s anda dized bioassay SET (Bei as e al., 2012) pe -
o med wi h species ecommended in egula o y guidelines, he sea
u chin Pa ace o us li idus. The imp o emen o ecologically ele an
bioassays could con ibu e o ende mo e obus sedimen oxici y
es s/p o ocols: combining mul iple species o be de eloped all yea
ound, a ying exposu e pa hways and including ou comes (his opa-
hology and cy o oxici y) complemen a y o acu e endpoin s (mo ali y,
g ow h, beha iou ).
2. Ma e ials and me hods
2.1. Tes o ganisms
H. di e sicolo polychae es, pu chased om he comme cial deale
Cebocuc® (Galicia, Spain) we e ecei ed in he labo a o y one day a e
manual collec ions in he Galician coas s and eadily placed in he
acclima ion anks (30 L). The la e we e illed wi h a laye o clean
sedimen (6 cm) collec ed in Plen zia (43◦40′6.746′′ N, 2◦95′1.060′′ W;
Bu oe es ua y) and seawa e di ec ly aken om he Plen zia Bay,
na u ally il e ed by sand in he up ake wells aided wi h a pump ha
sen he wa e o he ma ine s a ion. The anks had a cons an seawa e
low wi h a salini y ange o 32–33‰ and cons an ae a ion. The accli-
ma ion labo a o y had a con olled empe a u e o 18 ◦C, wi h a
pho ope iodic sys em o 12/12 h o a i icial ligh /da kness. Ragwo ms
we e kep in he acclima ion anks o 7 days p io o he bioassay, being
ed wi h comme cial ish ood (Vipag am g anula , 41.9% p o ein and
8.7% a ) wice a week.
Adul P. li idus we e collec ed in a ocky sho e in A min za
(43◦43′3.625′′ N, 2◦89′9.262′′ W; Bay o Biscay), in Ap il 2019. The
o ganisms we e anspo ed ali e o he labo a o y and we e placed in
aqua ia wi h seawa e a con olled empe a u e (16 ±1 ◦C) o 2–3
weeks p io expe imen a ion. Mac oalgae we e gi en weekly as ood
supply un il spawning induc ion.
2.2. In i o expe imen s wi h H. di e sicolo
2.2.1. Sedimen spiking p ocedu e and cha ac e iza ion
Tes sedimen s we e collec ed in he same sampling poin as hose
used o he acclima ion anks o polychae es (Plen zia, Bu oe es ua y).
Acco ding o p e ious me al cha ac e iza ion o sedimen s om Plen zia
collec ed in Sep embe 2018 (Supplemen a y Ma e ial-Table S1) and in
2019 (Table 1), and o Blanco-Ray´
on e al. (2019), his si e showed no
signi ican me allic con amina ion and e en showed educed alues o
all he me al analysed be ween bo h yea s. A physico-chemical cha ac-
e iza ion, including oxidable o ganic ma e (OM) con en , ex u e (%
o sand, sil and clay) and wa e holding capaci y (WHC) was pe o med
in CSR labo a o ies (Ja´
en, Spain). In addi ion, pH, elec ical conduc-
i i y (EC) and salini y we e also de e mined (Table 2).
Sedimen s we e sampled and s o ed acco ding o Thain and Bi ield
(2001) and King e al. (2004) wi h some modi ica ions (sie ed a 4 mm
a e ai d ying). Pa o he d ied sedimen s we e a i icially con ami-
na ed wi h coppe (Cu) by spiking hem wi h il e ed seawa e solu ions
con aining Cu sal s (CuCl
2
⋅2H
2
O, Sigma Ald ich, p epa ed in olume ic
lasks, 1 M HCl-washed glasswa e) in di e en quan i ies o ob ain he
desi ed concen a ions (0, 50, 150 and 300 mg Cu/kg). Coppe was
selec ed as model con aminan and because is he e e ence oxican in
sea u chin emb yo es . Coppe concen a ions we e selec ed on he
basis ha 50 mg Cu/kg ep esen low pollu ion le els (Menchaca e al.,
2012), and 150 mg Cu/Kg and 300 mg Cu/kg p oduced ch onic and
acu e oxic e ec s, espec i ely (Blaise and F´
e a d, 2013; Wa son e al.,
2018). Spiked sedimen s we e ho oughly homogenized wi h a clean
polye hylene spa ula and we e le o s abilize o 14 days, by mixing
hem e e y 2 days (King e al., 2004). Coppe concen a ions we e
measu ed in spiked sedimen s (58, 134, 235 mg/kg) and we e simila o
he nominal ones (50, 150, 300 mg/kg) (Table 1), indica ing a p ope
spiking p ocedu e and s abiliza ion pe iod.
2.2.2. Expe imen al se up and pe o mance
The expe imen al p ocedu e was designed ollowing he guidelines
ou lined by Thain and Bi ield (2001), wi h small modi ica ions. Tes
anks (5 L, polye hylene) we e illed wi h 1 Kg o s abilized sedimen s
( esul ing in 3 cm laye ) and 3.75 L o seawa e (0.2
μ
m- il e ed, 30
‰-3.5 L seawa e +250 mL dis illed wa e - o ob ain a inal salini y o
32–33‰ a e con ac wi h sedimen ), eaching 12 cm in heigh and
ob aining a sedimen -o e lying wa e dep h a io o 1:4. All es anks
we e allowed o se le o e a pe iod o 24 h.
H. di e sicolo polychae es o simila weigh (3.0–3.3 g) we e
weigh ed in pools o i e indi iduals, and main ained in anks wi h
con ol (CT) and a i icially pollu ed sedimen s wi h Cu, o 7 days.
Th ee eplica es we e p epa ed o each ea men . Con inuous ai low,
18 ◦C and pho ope iod (12/12 h ligh : da k) condi ions we e kep du ing
he expe imen , being he physico-chemical pa ame e s o he seawa e
(pH, empe a u e, salini y and dissol ed oxygen-DO-) moni o ed wi h a
mul ipa ame ic p obe (YSI P o essional Plus). Ragwo ms we e no ed
du ing he 7 days o exposu e.
A he end o he expe imen , sedimen samples we e collec ed o
de e mine he concen a ion o 20 me als (Li, Mo, Ag, Sn, Sb, Ba, W, Hg,
Table 1
Me al cha ac e iza ion o sedimen s collec ed in Plen zia, showing me al con-
cen a ions o 20 elemen s in con ol and a i icially pollu ed sedimen s wi h
h ee CuCl
2
concen a ions (50, 150 and 300 mg Cu/Kg).
Me al cha ac e iza ion
(mg/kg)
Con ol
(Plen zia)
50 mg Cu/
kg
150 mg
Cu/kg
300 mg
Cu/kg
Cu 25.87 ±3.07 57.77 ±
3.33
134.25 ±
5.88
235.93 ±
16.76
Li 16.00 ±0.12 15.76 ±
0.72
16.87 ±
1.57
15.51 ±
2.7
Mo 1.28 ±0.37 0.85 ±
0.04
1.60 ±
0.86
0.85 ±
0.22
Ag 0.18 ±0.03 0.16 ±
0.01
0.18 ±
0.01
0.18 ±
0.02
Sn 4.28 ±0.94 4.46 ±
0.95
4.06 ±
0.13
4.33 ±
3.53
Sb 0.36 ±0.04 0.20 ±
0.12
0.40 ±
0.09
0.41 ±
0.04
Ba 83.2 ±3.87 89.15 ±
8.41
94.24 ±
8.51
80.88 ±
6.57
W 0.25 ±0.00 0.20 ±
0.02
0.35 ±
0.15
0.25 ±
0.18
Hg 0.46 ±0.15 0.38 ±
0.04
0.41 ±
0.06
0.42 ±
0.05
Tl 0.17 ±0.01 0.18 ±
0.01
0.17 ±
0.02
0.17 ±
0.02
Pb 29.90 ±1.95 28.68 ±
0.77
30.01 ±
4.94
29.93 ±
0.80
Ti 104.12 ±
1.74
109.54 ±
8.20
116.20 ±
16.03
119.75 ±
7.69
Co 5.55 ±1.74 6.37 ±
1.64
5.83 ±
0.33
5.74 ±
0.34
Zn 138.33 ±
9.52
134.77 ±
6.32
146.35 ±
6.2
142.77 ±
2.21
As 13.83 ±0.44 16.45 ±
2.76
17.36 ±
0.07
14.35 ±
0.88
Cd 0.32 ±0.10 0.41 ±
0.19
0.32 ±
0.09
0.35 ±
0.07
V 20.61 ±0.28 20.72 ±
0.71
22.33 ±
0.90
20.23 ±
2.12
C 12.7 ±0.21 12.89 ±
0.47
13.91 ±
0.65
12.78 ±
1.11
Ni 9.29 ±0.11 10.43 ±
2.00
9.76 ±
0.47
9.54 ±
0.31
Se 0.62 ±0.09 0.88 ±
0.10
0.89 ±
0.11
0.76 ±
0.08
N. Ga cia-Velasco e al.
Chemosphe e 311 (2023) 136935
4
Tl, Pb, Ti, Co, Zn, As, Cd, V, C ; Mn, Ni, Se) by ICP-MS (NexION 300,
Pe kinElme ). B ie ly, abou 0.5 g o each d ied and sie ed sample was
subjec ed o an ex ac ion p ocedu e wi h 3.9 mL o ni ic acid (69%,
T acepu , Me ck), 6.2 mL o hyd ochlo ic acid (37%, T acepu , Me ck)
and 9.8 mL Milli-Q quali y wa e (Millipo e, Bille ica, MA, USA). Ul-
asound ene gy was applied o 6 min by means o a HD 2070 Sonopuls
Ul asonic Homogenize (Bandelin, Ge many) equipped wi h a 6 mm
glass ip. The acid ex ac was il e ed using a 0.45
μ
m PVDF il e and
dilu ed in Milli-Q wa e . Di e en dilu ions we e p epa ed in o de o
ma ch he elemen concen a ions o he dynamic ange o he ins u-
men . In all he cases he concen a ion o HNO
3
in he dilu ions was
adjus ed o 1%.
2.2.3. O ganisms and issue le el endpoin s
A he end o he expe imen , polychae es we e emo ed om he
anks, mo ali y checked and pools o o ganisms we e again weigh ed o
de e mine weigh loss. A midgu body po ion (segmen s 20–25) o he
polychae es (n =5) was dissec ed ou , ixed in o malin (10% com-
me cial Fo maldehyde in Phospha e Bu e Saline-PBS-wi h 0.17 M
NaCl, s o ed a 4 ◦C) and ou inely p ocessed o his ology (Ma oja and
Ma oja-Pie son, 1970). His opa hological al e a ions we e add essed in
ans e sal pa a in sec ions s ained wi h Haema oxylin-Eosin, analysed
unde a ligh mic oscope (Nikon ECLIPSE Ni). Polychae es collec ed
om he acclima ion ank a he beginning o he expe imen ( e e ed
as CT0) and a e 7 d in non-spiked sedimen s (CT7) we e used as
e e ence o he ana omical and physiological s a es o he pu chased
animals. In addi ion, me al accumula ion in issues was measu ed in 5
polychae es o all ea men s.
2.3. In i o app oaches wi h H. di e sicolo coelomocy es
2.3.1. Coelomocy e ex usion and seeding in mic opla e
Coelomocy es we e ex uded oge he wi h coelomic luid ollowing
a non-in asi e p ocedu e based on a p e iously de eloped me hod o
ea hwo ms (I iza e al., 2014). Polychae es we e kep oiding hei gu
con en s in cons an seawa e low du ing 24 h and we e ho oughly
cleaned o emo e any sedimen pa icle a ached o he body. O gan-
isms we e indi idually placed in glass pe i dishes and coelomocy es
we e ex uded by elec ic s imula ion (9 V) using 0.35 M NaCl PBS, wi h
0.02% EDTA and 1% an ibio ics (An ibio ic an imyco ic solu ion
(×100): 10,000 uni s penicillin, 10 mg s ep omycin and 25
μ
g
ampho e icin B pe mL). Ex usion solu ion was adjus ed o he salini y
o polychae es coelomic luid (0.17 M NaCl) aking in o accoun he
dilu ion ac o a e exposu e in he mic opla e wells. The p esence o
game es in he solu ion was checked unde ligh mic oscope and only
he cell solu ions ex uded om emales (con aining oocy es) we e
collec ed. In o de o a oid he oocy es, he coelomocy e solu ion was
il e ed h ough a 40
μ
m s aine in a cen i uga ion s ep (500 pm, 2
min). Cell solu ions ex uded om 8 o 10 emales we e pooled oge he ,
coelomocy es we e coun ed wi h he aid o a Neubaue chambe and he
cellula concen a ion adjus ed o 5 ×10
5
cells/mL. Coelomocy es in
coelomic luid based medium we e hen seeded in mic opla es (100
μ
L,
5 ×10
4
cells/well) and we e le s abilizing o 1 h a 18 ◦C be o e
exposu e. Fo each pollu an (Cu, AgNPs) and exposu e ime (2 and 24
h), h ee mic opla es we e p epa ed ( illed wi h cell solu ions om
di e en pools, Supplemen a y Ma e ial-Fig. S1).
2.3.2. Coelomocy e exposu e and iabili y assays
H. di e sicolo coelomocy e esponses in i o we e ob ained by
exposing cells o a ange o concen a ions (0, 0.05, 1, 5, 10, 50 mg/L) o
CuCl
2
and o AgNPs. AgNPs we e PVP-PEI coa ed wi h a size o 5.08 ±
2.03 nm, and Z-po en ial o 18.6 mV. Exposu es we e pe o med by
addi ion o 100
μ
L o exposu e solu ion, illing eigh wells pe concen-
a ion. As con ol, 0.17 M NaCl PBS was used. A e 2 and 24 h o
exposu e, coelomocy e iabili y (Calcein-AM) was assessed h ough a
mic opla e assay. The schema ic diag am o he expe imen al design
ollowed o he in i o exposu e o H. di e sicolo coelomocy es o Cu
and AgNPs is illus a ed in SM-Fig. S1.
The Calcein-AM Viabili y assay p o ides a simple, apid, and accu-
a e me hod o measu e cell iabili y and cy o oxici y. The calcein
ace oxyme hyl es e (Calcein AM) pe mea es li e cells and is hyd olyzed
by in acellula es e ase o calcein, a hyd ophilic and s ongly luo es-
cen compound ha is well e ained in he cell cy oplasm (B a osin e al.,
2005). The Calcein-AM iabili y es was pe o med acco ding o Ga -
cia-Velasco e al. (2019), wi h sligh modi ica ions ( emo al o washing
s eps). In hal o he pla es wells, 2
μ
l o Calcein-AM Wo king solu ion
(2.5
μ
M Calcein/well) we e added. In he emaining wells, 2
μ
l o PBS
we e added in o de o o e come backg ound noise (SM-Fig. S1). A e
40 min o incuba ion (18 ◦C, da kness), luo escence was ead a 490/20
nm exci a ion, and 520/20 nm emission in Cy a ion 3 Cell Imaging
mul i-mode eade (BioTek Inc.). Supposing ha he con ol g oup has
he maximum calcein e en ion capaci y (100%), he ela i e alues
we e calcula ed. In addi ion, he median le hal concen a ion (LC
50
) was
calcula ed.
2.4. Sea u chin emb yo es (SET): e e ence bioassay in sedimen
oxicology
In pa allel, he SET (Bei as e al., 2012) was pe o med, exposing
e ilized eggs o P. li idus, o he same e e ence oxican s a a ange o
exposu e concen a ions (CuCl
2
: 0, 0.005, 0.05, 0.5, 5 mg Cu/L; AgNPs:
0, 0.005, 0.05, 0.5, 5, 50 mg/L). A e 48 h, emb yo size inc ease and
de elopmen al abno mali ies we e measu ed. Size inc ease consis ed on
he measu emen s o he maximum dimension o 50 emb yos pe ial,
while mal o ma ions we e eco ded on 100 emb yos/ ial ollowing
Ca ballei a e al. (2012). A gene al index o oxici y (IT), ha weigh he
deg ee o de elopmen al abno mali ies by he equency, was hen
calcula ed o each ea men . Wi h size inc ease esul s, he median
e ec concen a ion (EC
50
) was calcula ed o each oxican .
2.5. S a is ical analysis
The s a is ical analysis o he da a was ca ied ou wi h he aid o he
SPSS s a is ical package (IMB SPSS S a is ics 23). Shapi o-Wilk (n <30)
and Le ene’s es s we e pe o med o s udy no mali y and equali y o
a iances o he da ase s, espec i ely. Da ase s we e analysed wi h
K uskal-Wallis ollowed by Dunn’s pos -hoc es and signi ican di e -
ences we e es ablished a p <0.05. In o de o es ima e he median le-
hal and e ec concen a ion (LC
50
, EC
50
) he P obi model was used.
Table 2
Physico-chemical cha ac e iza ion (oxidizable o ganic ma e in %p/p, ex u e as % o sand, sil and clay, Wa e Holding Capaci y-WHC in %p/p, pH, elec ical
conduc i i y in mS/cm and salini y in g NaCl/L) o he sedimen used o expe imen a ion.
SEDIMENT
SITE
SAMPLING
DATA
OXIDIZABLE
O.M (%p/p)
TEXTURE WHC (%p/p) pH EC (mS/cm) SALINITY (g/L)
Sand Sil Clay USDA Soil Class.
Plen zia Sep 2018 3.07 53 35 12 Loamy-Sand 22.3 7.59 15.79 8.83
N. Ga cia-Velasco e al.
Chemosphe e 311 (2023) 136935
5
3. Resul s
3.1. In i o expe imen s wi h H. di e sicolo
3.1.1. Sedimen cha ac e iza ion
The sedimen s collec ed in Plen zia and used o he in i o exposu e
o polychae es we e classi ied as a loamy o sand ma ix (USDA, 1999),
p esen ing 3.07% o ganic ma e and a wa e holding capaci y o 22.3%
(Table 2). The concen a ions o me als measu ed in hese sedimen s
(Table 1) we e low and simila o hose measu ed p e iously in sedi-
men s collec ed in Sep embe 2018 (SM-Table S1), ein o cing his si e
as a clean si e o me allic con amina ion (Lego bu u e al., 2013;
Blanco-Ray´
on e al., 2019). Conce ning he sedimen s om Plen zia
spiked wi h Cu, he eal exposu e concen a ions we e linea ly co e-
la ed wi h he nominal ones. Hence, a he end o he exposu e a nominal
concen a ion o 50 mg Cu/kg esul ed in 58 mg/kg, 150 mg Cu/kg in
134 mg/kg and 300 mg Cu/kg esul ed in 236 mg/kg (Table 1).
3.1.2. O ganism le el e ec s
In i o exposu e o H. di e sicolo o Cu caused inc eased mo ali y
(a ound 30%) a e exposu e o 150 mg Cu/Kg, and a signi ican dose-
esponse weigh loss compa ed wi h he con ol g oup, wi h a
maximum (>30%) o he highes dose (300 mg Cu/Kg, Fig. 1).
Ne me al accumula ion did no occu in con ol polychae es (CT7)
main ained in sedimen s o Plen zia compa ed wi h CT0. In spiked
sedimen s, H. di e sicolo accumula ed mainly Cu, Cd and Ag in issues
(Fig. 2). The concen a ion o Cu measu ed in he o ganisms main ained
in 50 mg Cu/Kg ea men we e simila (p >0.05) o CT0 alues (14.94
±3.90
μ
g/g and 8.25 ±1.42
μ
g/g, espec i ely). A e exposu e o 150
and 300 mg Cu/kg, highe Cu concen a ions we e measu ed, 48.65
μ
g/
g and 51 ±8
μ
g/g, espec i ely (Fig. 2 A). En ichmen s o Cd and Sn
occu ed in agwo ms main ained in he highes concen a ion o Cu
(Fig. 2 B and D), while highes Ag accumula ions we e eco ded in 50
and 300 mg Cu/Kg ea men s (Fig. 2 C).
3.1.3. E ec s a issue-le el
Con ol polychae es (CT7) exhibi ed a no mal body wall s uc u e
( egumen , epide mis) and diges i e epi helium in eg i y a e 7 days o
expe imen (Fig. 3A). Exposu e o he highes Cu concen a ions (150
and 300 mg Cu/Kg) showed clea blood accumula ion in he in es inal
a eas when compa ed o CT0 and con ol (CT7) polychae es (Fig. 3). In
he case o polychae es exposed o 300 mg Cu/Kg, conspicuous b own
deposi s we e clea ly obse ed in diges i e epi helia (Fig. 3D). Pa asi es
(spo es o a Mesomyce ozoea pa asi e, unicellula amoeba-like p o is s;
Fig. 3D) we e obse ed in he diges i e epi helium o agwo ms i e-
spec i e o ea men s. The in ec ion wi h pa asi es does no seem o be
se ious o he animals since no in lamma ion signs we e eco ded.
3.2. In i o app oaches wi h H. di e sicolo coelomocy es
The Calcein-AM iabili y assay pe o med in coelomocy es ex uded
om H. di e sicolo and exposed o CuCl
2
and AgNPs showed a signi i-
can dose- esponse dec ease in calcein e en ion (Fig. 4). Cells exposed
o longe ime (24 h) showed less e en ion capaci y han a e 2 h o
exposu e o Cu and AgNPs (Fig. 4). Coelomocy es iabili y signi ican ly
dec eased om 5 o 0.5 mg/L onwa ds o Cu (Fig. 4A) and AgNPs
(Fig. 4B), espec i ely. The calcula ed LC
50
alues a e Cu exposu e
we e 13.4 (2 h) and 4.2 mg/L (24 h), and o AgNPs 2.2 (2 h) and 0.43
mg/L (24 h).
3.3. Sea u chin emb yo es (SET)
The SET showed ha he exposu e o CuCl
2
caused dec ease in em-
b yo size, being signi ican a e exposu e o 5 mg Cu/L (Fig. 5A). A e
exposu e o AgNPs, emb yos showed mo e ma ked e ec s a lowe
concen a ions, wi h a signi ican dec ease in size s a ing a 0.05 mg/L
(Fig. 5B). The EC
50
alues o size impai men s we e es ablished a 0.38
mg/L and 0.086 mg/L o Cu and AgNPs, espec i ely. The alues o he
Index o oxici y (IT), we e highe a e exposu e o he highes con-
cen a ions o Cu (5 mg Cu/Kg) and om 0.5 onwa ds in he case o
exposu e o AgNPs (Fig. 5C and D).
4. Discussion
In he las 15 yea s an exponen ial g ow h in he unde s anding o
con aminan s in es ua ine sedimen s has occu ed. Acco dingly, sedi-
men quali y guidelines (SQG) ha e ecen ly been in oduced in
di e en coun ies and egions al hough he applica ion o hese
guidelines is no based on a scien i ic consensus. SQG can be unde s ood
as igge alues o h eshold concen a ions (Chapman and Ande son,
2005). Below his h eshold he possibili y o p oduce dele e ious bio-
logical e ec s is e y low. Mo eo e , Chapman and Ande son (2005)
Fig. 1. Mo ali y and weigh loss (in %) o H. di e sicolo a e in i o exposu e o con ol and Cu spiked (50, 150 and 300 mg/kg) sedimen s. Values a e ep esen ed
as a e age ±s anda d de ia ions and signi ican di e ences (p <0.05 wi h K uskal-Wallis) be ween ea men s a e ep esen ed by as e isk.
N. Ga cia-Velasco e al.

Chemosphe e 311 (2023) 136935
6
Fig. 2. Me al accumula ion (Cu, Cd, Ag, Sn) in issues o H. di e sicolo a e 7 d in i o exposu e o CuCl
2
(50, 150 and 300 mg Cu/Kg) spiked in he sedimen s.
Values a e ep esen ed as a e age ±s anda d de ia ions.
Fig. 3. Diges i e ac in ans e sal sec ions o H. di e scolo a he beginning o he expe imen ( 0) (A), a e 7 days in con ol sedimen (B), and a e 7 d exposu e
o 150 mg Cu/Kg (C) and 300 mg Cu/Kg (D). Blood i iga ion is labelled wi h an as e isk (*) and b own deposi s wi h an a ow. Epi helium (E), Oocy es (Oo),
Pa asi es (P). Scale ba : 100
μ
m. (Fo in e p e a ion o he e e ences o colo in his igu e legend, he eade is e e ed o he Web e sion o his a icle.)
N. Ga cia-Velasco e al.
Chemosphe e 311 (2023) 136935
7
sugges ed ha in es iga ions should combine sedimen chemis y
(including con aminan bioa ailabili y and bioaccumula ion), ben hic
ecology and oxici y es ing. All his in o ma ion will o e a apid and
accu a e ool in o de o pe o m a p ope managemen o pollu ed
sedimen s.
The wo ld-wide used SET es p o ides a gene al iew o he oxici y
exe ed by chemicals bioa ailable in seawa e by quan i ying (in e ms
o plu eus la ae size) he deg ee o de elopmen al comple ion achie ed
by he emb yos (Bei as e al., 2012). This bioassay endpoin is a quan-
i a i e, obse e -independen , au oma ically eadable esponse, and
ha e been s anda dized and amended unde se e al in e na ional
guidelines and di ec i es (ASTM, 1990; USEPA, 1995; En i onmen
Canada, 1997; CETESB, 1999; ASTM, 2002). P esen ly, SET was highly
sensi i e o de e mine he oxici y h esholds o CuCl
2
and AgNPs a e
48 h o exposu e. In he case o Cu exposu e, he EC
50
( o he size in-
c ease endpoin ) was es ima ed in 0.38 mg/L. This alue is e y simila
o he no malized SQGs calcula ed o he Basque coas using h ee
oxici y es s including SET, AMBI and Mic o ox ha was 0.55 mg Cu/L
(Menchaca e al., 2012). In he same line, Chapman e al. (1999) and
Choue i e al. (2009) p oposed ha SQG should be used in he egion
whe e hey we e de eloped o be e p edic he oxici y o con ami-
nan s o each speci ic coas al en i onmen . The coincidence o his
calcula ion sugges ha his alue is he e e ence h eshold o Cu in he
Basque Coas . The case o AgNPs is no so clea due o he lack o wo ks
ega ding oxici y h esholds calcula ed on he basis o oxici y es s.
P esen ly, he EC
50
o AgNPs was es ima ed in 0.086 mg/L, much lowe
ha o CuCl
2
, sugges ing ha he SQGs should be mo e es ic i e o
his con aminan . In conclusion, he sensi i i y o ea ly de elopmen al
Fig. 4. Cell iabili y (Calcein AM iabili y assay, ep esen ed by calcein e en ion in % ela i e o he con ol) in p ima y cul u es o H. di e sicolo coelomocy es
exposed o CuCl
2
(A) and AgNPs (B) (n =3). Values a e ep esen ed as means ±s anda d de ia ions and he signi ican di e ences (p ≤0.05 wi h K uskal-Wallis) a e
ep esen ed by le e s (a,b,c o 2 h and A,B,C o 24 h).
Fig. 5. Size inc ease (% o con ol; A, B) and he index o oxici y (IT; C, D) calcula ed om he equency o mal o ma ions de ec ed in emb yos exposed o Cu (A, C)
and AgNPs (B, D). Signi ican di e ences (p ≤0.05 wi h K uskal-Wallis) a e ep esen ed by le e s (a,b,c).
N. Ga cia-Velasco e al.
Chemosphe e 311 (2023) 136935
8
s ages o sea u chin allows o e alua e he po en ial e ec s o Cu and
AgNPs ha may po en ially become a ailable o he wa e column (o
elease o he wa e column due o sedimen esuspension) and o
es ablish oxici y h esholds o bo h pollu an s.
One o weaknesses, when planning oxici y es ing wi h sea u chins
in he amewo k o biomoni o ing p og ams, is he lack o con inuous
a ailabili y o sui able biological ma e ial (game es) ou side he na u al
spawning season o he species. The e o e, he de elopmen o bioassays
wi h species ha could complemen he SET in seasons ou side he
spawning pe iod would be e y welcome. One ecommenda ion is he
use o epi-/ben hic es species (ECHA, 2014), which a e in di ec con-
ac wi h he sedimen and po e wa e , wi h a b oad geog aphical dis-
ibu ion, compa ible wi h selec ed exposu e me hods and endpoin s
and ha e a oxicological da abase demons a ing sensi i i y o a ange o
con aminan s (Simpson e al., 2005). Endoben hic aqua ic polychae es
such as H. di e sicolo ul il abo e men ioned equi emen s. Hence, in
i o and in i o assays ha e been de eloped wi h polychae es exposed o
e e ence oxican s (CuCl
2
, AgNPs), being he sui abili y o he assays
alida ed h ough compa ison wi h he SET e e ence bioassay (Bei as
e al., 2012).
A e he in i o bioassay, in which H. di e sicolo we e exposed
du ing 7 d o loamy sedimen s spiked wi h di e en Cu concen a ions,
hey accumula ed Cu in issues ollowing a dose-dependen pa e n. The
massi e accumula ion was ela ed wi h mo ali y a es in 150 mg/kg
exposu e and weigh losses eco ded o doses anging 150–300 mg Cu/
Kg, sugges ing he onse o acu e oxici y esponses. Weigh losses ha e
been p e iously ela ed wi h induced dehyd a ion o specimens as a
esul o enhanced mucus gene a ion and exc e ion (exocy osis) om he
egumen o o m a p o ec i e laye o e i s su ace unde s ess con-
di ions (Be he e al., 2003; Mu ay e al., 2012). Con e sely, lowe Cu
concen a ions (50 mg Cu/Kg) did no p oduce any e ec (mo ali y o
weigh loss). In con as , exposu e o o he polychae e species such as
A. ma ina o doses sligh ly highe han 14 mg Cu/Kg induced le hali y
(LC
50
=20 mg/kg; Ba and Ra aelli, 1998). Mo eo e , Cu spiked in he
wa e column induced mo ali y in Ne eis a concen a ions highe han
2.3 mg Cu/L (B yan, 1976). This migh sugges di e en sensi i i y o
polychae es o pollu an insul depending on hei eeding habi s
(mac ophagic eede s-A enicola- s. p eda o s –Ne eis-), o he phase in
which he pollu an is spiked (sedimen s. wa e column).
Unde some speci ic condi ions compe i i e displacemen eac ions
occu ed a e he addi ion o me als, such as Cu and Cd in es ua ine
sedimen s (Simpson e al., 2000). In e es ingly, Cu exposu e (300 mg
Cu/kg) p oduced a concomi an inc ease in Cd and Sn le els al hough
he le els eached we e e y low. Simpson e al. (2000) sugges ed ha in
he Cu-spiked sedimen s, Cd (p esen in he o iginal sedimen ) was
displaced in o he po e wa e s becoming mo e a ailable o polychae es.
Howe e , i canno be disca ded ha he bu owing o bio u ba ion
ac i i y o polychae es can inc ease he elease o con aminan s in sed-
imen s o he wa e column (Scaps, 2002; Ma inez-Ga cia e al., 2015).
The ex en o which me al addi ions a ec sedimen pH and edox
condi ions, and dis up he equilib ium pa i ioning o sedimen con-
s i uen s, is poo ly unde s ood and dese es u he in es iga ion
(Luoma and B yan, 1982; Simpson e al., 2000).
Assessmen o he his opa hological al e a ions p oduced in a ge
o gans o polychae es a e exposu e o pollu an s has been p e iously
epo ed al hough he amoun o esea ch in his ield is s ill e y low
(Ge aci ano e al., 2004; Mu ay e al., 2012). Ana omically he poly-
chae e possess a la ge cen al diges i e epi helium su ounded by he
coelom ca i y illed wi h game es and coelomocy es, he ne e co d, he
do sal and en al essels (Rod igo e al., 2015). A e exposu e o Cu
(high dose), an inc eased blood i iga ion close o he diges i e ac and
massi e accumula ion o b own deposi s (p obably lipo uscin g anules)
we e obse ed in diges i e epi helia. These deposi s a e
memb ane-bound s uc u es ha can con ain by-p oduc s o he diges-
i e p ocess ha a e eleased o he lumen o diges i e ac a mo e
ad anced s ages o diges ion (Cos a e al., 2014). His ochemical
examina ions o H. di e sicolo a e exposu e o ace ylsalicylic acid ha e
shown impo an inc ease o mucous cells in he egumen (Gomes e al.,
2019), bu up o now, hese kind o his opa hological al e a ions ha e
no been eco ded in he diges i e sys em o polychae es. These al e -
a ions may indica e ha changes a issue le el migh occu unde
en i onmen al s ess si ua ions and dese es mo e a en ion o u u e
sedimen oxicological wo ks.
The p esence o pa asi es is indica i e o a dep essed immunological
sys em due o a a ie y o en i onmen al ac o s including pollu an s in
aqua ic animals (Beni o e al., 2019). Pa asi es we e obse ed, mainly in
he diges i e epi helium o he gu , al hough hey we e p esen in he
majo i y o indi iduals ega dless he ea men . The e is a lack o
li e a u e on polychae es pa asi es, howe e , acco ding o hei size,
gene al aspec (unicellula amoeba-like p o is s) and smoo h capsule,
he pa asi es ound in polychae es seem like he ac i e spo es o a
Mesomyce ozoea, acco ding o D . Ped o Cos a (No a Uni . Lisbon,
pe s. comm).
The use o p ima y cul u es o immune cells o in e eb a es o
in es iga e he impac o pollu an s in he en i onmen ha e been
ecen ly ou lined wi h p omising esul s. In ac , annelids (oligo and
polychae es) ha e de eloped cellula immuni y (phagocy osis, encap-
sula ion agains pa asi es) and possess humo al componen s o immu-
ni y which a e essen ially ep esen ed by an ibac e ial p o eins
(Dhainau and Scaps, 2001; Ca alano e al., 2012; Hayashi and Engel-
mann, 2013; Cong e al., 2014; Cu illie -Ho e al., 2014; I iza e al.,
2015; Ga cia-Velasco e al., 2019). P esen ly, in i o app oaches we e
op imized o ma ine wo ms (polychae es H. di e sicolo ) based on a
p e iously de eloped me hod o ea hwo ms (I iza e al., 2014). The
modi ica ions in he p o ocol included he use o coelomic based me-
dium (wi h PBS adjus ed o polychae es coelom osmola i y) o main ain
he cells in cul u e, he sho ening o he exposu e o 2 and 24 h
(a oiding medium eplacemen s) and he emo al o washing s eps
when pe o ming he Calcein-AM iabili y es . In i o exposu e o
coelomocy es o a se ies o Cu concen a ions p oduced a signi ican
dose- esponse dec ease in he iabili y o cells. Likely, ch onic coppe
exposu e impai ed he immunological esponse o he polychae e Eu -
y hoe complana a wi h changes in coelomocy es iabili y, numbe and
phagocy osis capaci y (Nuse i e al., 1998). Exposu e o AgNPs exe ed
mo e oxic e ec s han Cu acco ding o LC
50
alues a e 2 and 24 h.
Simila ly, exposu e o AgNPs caused cy o oxici y in coelomocy es o
Eisenia e ida being EC
50
6 mg AgNP/L (Ga cia-Velasco e al., 2019).
Addi ionally, hese au ho s ound a selec i e oxici y in amoebocy es
mo e han in eleocy es (subpopula ions o coelomocy es), ha was he
opposi e o non-pa icula ed con aminan s (ionic Cu) in which he
a ge we e he eleocy es (I iza e al., 2015). Mo e wo k is needed o
assess he di e en ial ole in me al handling ega ding he wo sub-
popula ion o coelomocy es in polychae es, al hough i can be concluded
he capaci y o he es o p edic impai men s caused by pollu an s
( oxici y h esholds) and p o ide apid and aluable in o ma ion o
(cy o) oxicology.
The sensi i i y o his inno a i e in i o bioassay based in coelo-
mocy e iabili y e lec ed he same esponse pa e n ob ained using he
SET wi h simila sensi i i y. Tha is, Cu exposu e exe ed a signi ican
oxici y e ec on cell iabili y al eady a e exposu e o 10 mg Cu/L,
while SET showed oxici y be ween 5 and 10 mg Cu/L. The esponse o
coelomocy es could be en isaged a e 2 h (wi h simila esul s a e 24
h) while SET was showing oxici y a e he 48 h o du a ion o he es .
The esul s ob ained a e exposu e o AgNPs we e simila bu wi h mo e
powe o disc imina ion o SET s. coelomocy e cul u ing: 0.5 mg
AgNP/L s. 1–5 mg AgNP/L, espec i ely. Maybe, dissimila dissolu ion
a es o AgNPs in seawa e media and coelomic luid based cul u e
media (10‰ salini y) could be esponsible o his sligh disc epancy
be ween me hods. Ne e heless, he alues o he IT, calcula ed om he
equency o mal o ma ions de ec ed in exposed sea u chin emb yos,
we e in line wi h he esponses ob ained o coelomocy es. Hence, i can
be concluded ha he sensi i i y o he size endpoin in SET is sligh ly
N. Ga cia-Velasco e al.
Chemosphe e 311 (2023) 136935
9
highe al hough coelomocy e bioassay showed as e esponses ha can
be used o a quick sc eening o oxici y. Fu he mo e, p ima y cul u es
o coelomocy es may ende high- h oughpu amoun o da a in a sho
ime when cells a e seeded and exposed in wells and ead in a mic o-
pla e. Finally, i is sugges ed ha his oxici y sc eening could be applied
no only o es ablish he oxici y h esholds o indi idual compounds o
mix u es, bu also o assess he oxici y o elu ia es coming om ield
collec ed sedimen s.
4. Conclusions
The bioassays de eloped wi h Hedis e di e sicolo (in i o) and hei
coelomocy es (in i o) a e use ul ools o assess he e ec s o pollu an s
ha can be bounded o sedimen s o esuspended in o wa e . The
oxici y h esholds ob ained wi h bo h assays we e in line wi h he ones
ob ained wi h he sea u chin emb yo es (SET), concluding ha hei
sensi i i y is simila . The op imiza ion o he me hodology o p ima y
cul u es o H. di e sicolo coelomocy es (non-in asi e ex usion me hod,
use o coelomic luid based medium, iabili y assay wi h emo al o
washing s eps) can be o g ea impo ance o sedimen oxici y assess-
men since i allows pe o ming apid and simul aneous sc eening o
oxican s (including indi idual compounds o mix u es and elu ia es o
eal sedimen s). In i o and in i o bioassays wi h polychae es and hei
coelomocy es may be cos -e ec i e ools o oxici y sc eening and may
complemen he SET ou side he spawning pe iod o he echinode ms
when game es a e no a ailable o pe o m he es .
Au ho con ibu ions s a emen
Ga cia-Velasco, Ne ea: In es iga ion (samplings, in i o and in i o
expe imen design and de elopmen ), Me hodology (op imiza ion o in
i o me hod wi h coelomocy es), Da a acquisi ion and cu a ion (g aphs,
ables and s a is ics), W i ing (o iginal d a , e iews and edi ing).
Ca e o, Jose An onio: In es iga ion (chemical sample p ocessing and
analysis), Me hodology, Da a cu a ion ( ables), W i ing ( e iew and
edi ing). U ionaba ene xea, E ik: In es iga ion (samplings, suppo
o in i o expe imen ), Me hodology (suppo in he op imiza ion o in
i o me hod wi h coelomocy es), W i ing ( e iew). Doni, Lucia:
In es iga ion (samplings, suppo o in i o expe imen ), W i ing
(suppo o he o iginal d a ). Zaldiba , Be˜
na : Da a cu a ion, Su-
pe ision, W i ing ( e iew and edi ing), Izagi e, U zi: In es iga ion
(b eeding o labo a o y animals, expe imen al design in aqua ia), Da a
cu a ion, Supe ision, W i ing ( e iew and edi ing). So o, Manu:
Funding acquisi ion, P ojec adminis a ion, Resou ces, Supe ision,
Da a cu a ion, W i ing (o iginal d a , e iews and edi ing).
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
No da a was used o he esea ch desc ibed in he a icle.
Acknowledgemen s
The au ho s hank Basque Go e nmen (G an o Consolida ed
Resea ch G oups; IT1302-19, IT1213-19) and Spanish Minis y o
Economy and Compe i i eness (SEAdimenTOX p ojec , CTM 2017-
87766-R).
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.o g/10.1016/j.chemosphe e.2022.136935.
Table S1
Ini ial me al cha ac e iza ion o he sedimen s collec ed in Plen zia in Sep embe 2018.
SEDIMENT METAL
CHARACTERIZATION (mg/kg)
Cu Li Mo Ag Sn Sb Ba W Hg Tl
32.34 ±
3.92
21.07 ±
0.12
1.52 ±
0.18
0.17 ±0.01 5.35 ±
0.44
0.82 ±
0.06
111.62 ±
15.15
0.19 ±
0.03
0.53 ±
0.04
0.27 ±
0.02
Pb Ti Co Zn As Cd V C Ni Se
42.59 ±
5.24
345.59 ±
25.84
16.31 ±
0.86
243.45 ±
32.32
19.32 ±
3.00
0.36 ±
0.00
57.02 ±
1.99
38.29 ±
3.64
24.83 ±
2.38
3.67 ±
0.23
Re e ences
Abouda, S., Missawi, O., Cappello, T., Bougha as, I., De Ma co, G., Maisano, M.,
Banni, M., 2022. Toxicological impac o en i onmen al mic oplas ics and benzo[a]
py ene in he seawo m Hedis e di e sicolo unde en i onmen ally ele an exposu e
condi ions. En i on. Pollu . 310, 119856.
Al enbu ge , R., B ack, W., Bu gess, R.M., Busch, W., Esche , B.I., Focks, A., Ma k
Hewi , L., Jacobsen, B.N., de Alda, M.L., Ai - Aissa, S., Backhaus, T., Gineb eda, A.,
Hilsche o ´
a, K., Hollende , J., Holle , H., Neale, P.A., Schulze, T., Schymanski, E.L.,
Teodo o ic, I., Tindall, A.J., de A ag˜
a o Umbuzei o, G., V ana, B., Zonja, B.,
K auss, M., 2019. Fu u e wa e quali y moni o ing: imp o ing he balance be ween
exposu e and oxici y assessmen s o eal-wo ld pollu an mix u es. En i on. Sci.
Eu . 31, 12.
A edondo, L., Nuse i, O., Lemus, M., 2020. Me hod o collec ing coelomocy es om
Eu y hoe complana a (Annelida: amphinoidae) wi h po en ial applica ion in
oxicology. Biol. 12 (1).
Ashley, M., Budd, G.C., 2016. Hedis e di e sicolo in li o al mud. In: Tyle -Wal e s, H.,
Hiscock, K. (Eds.), Ma ine Li e In o ma ion Ne wo k: Biology and Sensi i i y Key
In o ma ion Re iews. Ma ine Biological Associa ion o he Uni ed Kingdom,
Plymou h [on-line].
ASTM, 1990. P oposed S anda d Guide o Conduc ing Toxici y Tes s wi h Spe m and
Eggs o Sea U chins and O he Echinoids. ASTM Subcommi ee E-47.01 on Aqua ic
Toxicology (Chai man G.A. Chapman), ol. 4. Ame . Soc. Tes ing and Ma e ials,
Philadelphia, PA, D a No, p. 61.
ASTM, 2002. S anda d Guide o Conduc ing S a ic Acu e Toxici y Tes s wi h Echinoid
Emb yos. In: Annual Book o ASTM S anda ds, ol. 11. Ame ican Socie y o Tes ing
and Ma e ials, Wes Conshohocken, PA, pp. E1563–E1598, 936-E1598.
Ba , L., 1998. In luence o sedimen on hea y me al up ake by he polychae e A enicola
ma ina. T . J. Zoology 22 (4), 341–350.
Ba , L., Ra aelli, D., 1998. Sedimen oxici y es ing: a bioassay app oach using he
amphipod Co ophium olu a o and he polychae e A enicola ma ina. J. Exp. Ma .
Biol. Ecol. 226, 217–239.
Ba , L., 2005. A e iew o sedimen oxici y bioassays using he amphipods and
polychae es. Tu k. J. Fish. Aqua . Sci. 5, 119–139.
Bei as, E., V´
azquez, J., Bellas, J., Lo enzo, N., Fe n´
andez, G., Macho, J., Ma i˜
no, C.,
Casas, L., 2001. Sea-u chin emb yo bioassay o in si u e alua ion o he Biological
Quali y o coas al seawa e . Es ua . Coas Shel Sci. 52, 29–32.
Bei as, R., Du ´
an, I., Bellas, J., S´
anchez-Ma ín, P., 2012. Biological e ec s o
con aminan s: Pa acen o us li idus sea u chin emb yo es wi h ma ine sedimen
elu ia es. ICES Tech. Ma . En i on. Sci. 51, 13.
Beni o, D., Ah o, A., Nuu inen, J., Bilbao, D., Saenz, J., E xeba ia, N., Lekube, X.,
Izagi e, U., Leh onen, K.K., Ma ig´
omez, I., Zaldiba , B., So o, M., 2019. In luence o
season-depending ecological a iables on bioma ke baseline le els in mussels
(My ilus ossulus) om wo Bal ic Sea sub egions. Sci. To al En i on. 689,
1087–1103.
N. Ga cia-Velasco e al.