Innovative in vivo and in vitro bioassays for the establishment of toxicity thresholds of pollutants in sediment quality assessment using polychaetes and their immune cells.

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.