Assessment of oxidative stress biomarkers in Palaemon varians exposed to deep eutectic systems

Vol.:(0123456789) En i onmen al Science and Pollu ion Resea ch (2024) 31:57959–57972 h ps://doi.o g/10.1007/s11356-024-34983-3 RESEARCH ARTICLE Assessmen o oxida i e s ess bioma ke s inPalaemon a ians exposed odeep eu ec ic sys ems MªPila Ga alaga1· InesFe ei a2,5· Lau aLomba1· Elisabe Pi es3,4· Sa aG acia‑Ba be án3,4· AnaRi aC.Dua e2· Má ioDiniz5,6 Recei ed: 30 May 2024 / Accep ed: 10 Sep embe 2024 / Published online: 21 Sep embe 2024 © The Au ho (s) 2024 Abs ac In ecen yea s, he e has been ex ensi e esea ch wi hin he scien i ic communi y on deep eu ec ic sys ems due o hei ema kable e sa ili y in solubilizing di e se subs ances and se ing as e ec i e sol en s in ca aly ic p ocesses. While ini ially ega ded as non- oxic, a comp ehensi e oxicological assessmen is essen ial o comp ehend hei beha io wi hin o ganisms. In his s udy, se en dis inc sys ems, composed o N,N,N- ie hyl-N-(2,3-dihyd oxyp opyl)ammonium chlo ide (N00Cl) and glyce ol-de i ed e he s wi h alkyl chains o a ying leng hs (100, 200, 3F00, 300, 3i00, and 400), in a 1:2 mola a io we e in es iga ed o hei aqua ic oxici y in sh imp (Palaemon a ians). The assessmen in ol ed analyzing oxida i e s ess bioma ke s such as glu a hione S- ans e ase, glu a hione pe oxidase, ca alase, supe oxide dismu ase, o al an ioxidan capaci y (TAC), and lipope oxida ion (MDA con en ). Resul s show an odd–e en e ec o LC50 alues being N00Cl-300, he sys em showing highe alues. Rega ding oxida i e s ess, an imbalance be ween eac i e oxygen species (ROS) and an ioxidan capaci y in he o ganisms has been obse ed, sugges ing signi ican oxici y o sh imps due o he changes in oxida i e s ess bioma ke s a high concen a ions. Howe e , a 100mg/l all sys ems can be conside ed en i onmen ally sa e, and no nega i e impac s a e expec ed on aqua ic ecosys ems. Keywo ds Glyce ol-de i ed e he s· Aqua ic oxicology· Sh imp· Reac i e oxygen species· En i onmen ally sa e In oduc ion Eu ec ic sys ems a e o med by combining subs ances ha in e ac a he molecula le el o lowe he mel ing poin (Chak abo y e al. 2021). In o he wo ds, hey a e combi- na ions o wo o mo e subs ances, which oge he ha e a lowe mel ing poin han he indi idual componen s (Abbo e al. 2003). Due o hei unique physicochemical p ope ies, hese mix u es ha e a ac ed inc easing in e es in a ious indus ial and echnological a eas. The applica ions o hese deep eu ec ic sys ems (DES) a e di e se and widely di e si- ied: one o he mos common uses is in he pha maceu i- cal indus y, whe e hey a e used as sol en s and ehicles o d ug deli e y (Chak abo y e al. 2021). They can also be ound in he ood indus y (Mišan e al. 2020) as addi- i es and ex u e imp o e s (Hayyan e al. 2012), as well as being used in o he biological and chemical p ocesses such as c yop ese a ion (Ho nbe ge e al. 2021), bioca aly- sis (Xu e al. 2017), syn hesis (Zhang e al. 2012; Yu e al. 2022), ex ac ion (Duan e al. 2016), gas ch oma og aphy (Momo ko e al. 2022). Responsible Edi o : B uno Nunes * Ana Ri a C. Dua e adua [email p o ec ed] Má io Diniz [email p o ec ed] 1 Uni e sidad San Jo ge. Campus Uni e si a io, Au o A23 Km 299, 50830Za agoza, Villanue adeGállego, Spain 2 LAQV-REQUIMTE, Depa men o Chemis y, School o Science andTechnology, NOVA Uni e si y Lisbon, 2829-516Capa ica, Po ugal 3 Ins i u o de Sín esis Química y Ca álisis Homogénea (ISQCH), Facul ad de Ciencias, CSIC-Uni e sidad de Za agoza, C/ Ped o Ce buna, 12, 50009Za agoza, Spain 4 Dep o. Química O gánica, Facul ad de Ciencias, Uni e sidad de Za agoza, C/Ped o Ce buna, 12, 50009Za agoza, Spain 5 Depa men o Chemis y, UCIBIO, NOVA School o Science andTechnology, Uni e sidade NOVA de Lisboa, Quin a da To e, 2829-516Capa ica, Po ugal 6 Associa e Labo a o y i4HB, Ins i u e o Heal h andBioeconomy, NOVA School o Science andTechnology, Uni e sidade NOVA de Lisboa, 2819-516Capa ica, Po ugal 57960 En i onmen al Science and Pollu ion Resea ch (2024) 31:57959–57972 Addi ionally, he e has been g owing in e es in he an i- mic obial and cy o oxic p ope ies o DESs (Ma chel e al. 2022). Such esea ch highligh s he po en ial o DESs in applica ions such as an imic obial agen s, which opens new a enues o hei use bu also aises conce ns ega ding hei sa e y and en i onmen al impac . As he indus ial and echnological in e es in hese eu ec ic sys ems inc eases, i becomes c ucial o unde s and and assess he po en ial isks o human and en i onmen- al heal h. Recen s udies ha e ques ioned whe he deep eu ec ic sol en s migh con ibu e o en i onmen al ha m, conside ing hei ola ili y and po en ial o elease ha m- ul subs ances unde ce ain condi ions (Janjhi e al. 2023). Simila ly, he possibili y o c oss-con amina ion o wa e bodies by DESs is ano he signi ican en i onmen al issue. Hyd ophobic deep eu ec ic sol en s (HDESs), which a e inc easingly used as ex ac an s o emo ing pollu an s om wa e and was ewa e , could pose a isk o in oducing hese sol en s in o aqua ic sys ems, leading o con amina ion (Ma chel e al. 2023). These conce ns unde sco e he need o comp ehensi e en i onmen al assessmen s o DESs, pa - icula ly as hei indus ial applica ions expand. Eco oxicological and oxicological es ing a e essen ial wi hin he REACH amewo k (Ga alaga e al. 2022b), In he Eu opean con ex , companies a e equi ed o pe - o m (eco) oxicological es ing, depending on he le el o p oduc ion, o ensu e adequa e isk managemen and o allow he placing o new chemicals subs ances on he ma ke . These es s may a y depending on he quan i y o he subs ance, i s in ended use, and he le el o conce n associa ed wi h i s p ope ies (acu e oxici y, ch onic oxic- i y, geno oxici y, ca cinogenici y, e c.). Gene ally, conduc - ing mul iple aqua ic assays, bo h in c us aceans and algae, ish, and aqua ic plan s, is a common p ac ice o e alua e he en i onmen al impac o he compounds unde s udy. The aqua ic oxici y o some o hese mix u es has al eady been s udied in ma ine biomodels such as Daphnia magna (Pe ales e al. 2017; E azquin e al. 2021), Alii ib io is- che i (Ven u a e al. 2014; Ga cía e al. 2015; Ga alaga e al. 2022a), o algae (Lapeña e al. 2021; Ga alaga e al. 2022a). Thei oxicological po en ial has also been in es- iga ed in ish (Juneidi e al. 2016; Lapeña e al. 2021) and o he aqua ic o ganisms such as sh imps (Hayyan e al. 2013b, a) and ma ine plan s (Wen e al. 2015). Palaemon a ians is an in e eb a e aqua ic o ganism used as a bio- model in aqua ic oxici y s udies (Jeliński e al. 2019). I is a species o sh imp ha is pa icula ly sensi i e o chemical pollu an s in wa e . The use o his biomodel has nume ous ad an ages: bo h collec ion and main enance a e ela i ely simple. I is a species on which physiological esponses such as he concen a ion o an ioxidan enzymes can be measu ed. Finally, i plays a c ucial ole in aqua ic ecosys ems by ul illing indispensable unc ions wi hin he ood chain, such as he decomposi ion o o ganic ma e ial o he con- ol o o he ma ine popula ions (Sabo owski e al. 2022). Oxida i e s ess esul s om an imbalance be ween he p oduc ion o eac i e oxygen species (ROS) and he an ioxidan capaci y o he o ganism. Inc eased ROS p o- duc ion in a species is indica i e o cell damage and is he e o e a quan i iable pa ame e in (eco) oxici y s udies. Some o he mos used an ioxidan enzymes o quan i y he an ioxidan capaci y o a subs ance a e supe oxide dis- mu ase (SOD), ca alase (CAT), and pe oxidase. On he o he hand, i is also common o ind assays in which glu- a hione (in acellula an ioxidan ) le els a e measu ed. An ioxidan enzyme s udies p o ide a apid and easily quan i iable me hod o assessing an ioxida i e capaci y h ough he emo al o p oduc s p oduced by oxida i e s ess. The in o ma ion ob ained om hese s udies makes i possible o s udy a a molecula and cellula le el how a subs ance can a ec a pa icula o ganism. In o de o unde s and he e ec s o eu ec ic mix u es on aqua ic o ganisms and ecosys ems, he eco oxici y o se en DES consis ing o N,N,N- ie hyl-N-(2,3-dihyd oxyp opyl) ammonium chlo ide (N00Cl) as HBA and glyce ol-de i ed e he s as (HBA) a di e en concen a ions will be analyzed in his ial. In addi ion, bioma ke s o oxida i e s ess (ca - alase, glu a hione-S- ans e ase, glu a hione pe oxidase, supe oxide dismu ase, lipid pe oxida ion, and o al an ioxi- dan capaci y) we e measu ed o de e mine he biochemical impac o hese subs ances on he P. a ians biomodel. These sys ems p esen in e es ing physicochemical p op- e ies and e y low eco- oxici y (Pe ales e al. 2017); hese glyce ol-de i ed sol en s a e being used in eplacing glyc- e ol as he HBD componen in eu ec ic sol en s (Leal-Duaso e al. 2017b). These DES ha e success ully been applied o he design o eco e able homogeneous ca aly ic sys ems (Leal-Duaso e al. 2020, 2021) and a e cu en ly unde s udy as solubiliza ion media o bioac i e compounds. The e o e, i is c ucial o conduc eco oxicological s udies o hese sys- ems o assess po en ial esidues ha could be gene a ed a an indus ial le el in he e en o a spill in o a i e o e lu- en . The concen a ions es ed a e high o unde s and he consequences o ele a ed le els in hese sys ems. Ma e ials andme hods P epa a ion o DES Sigma-Ald ich supplied glycidol, 3-chlo op opane-1,2-diol, and ie hylamine. Scha lab p o ided MeOH, E OH, i P OH, and po assium o sodium hyd oxide. Al a Aesa was he sou ce o BuOH, 2,2,2- i luo oe hanol, phenol, 2-me hoxy- phenol, and choline chlo ide. The p ocedu e o p epa ing 57961En i onmen al Science and Pollu ion Resea ch (2024) 31:57959–57972 deep eu ec ic sys ems (DES) has been de ailed in p io desc ip ions (Leal-Duaso e al. 2017a, b). B ie ly, DES we e ob ained by mixing N,N,N- ie hyl-N-(2,3-dihyd oxyp opyl) ammonium chlo ide (N00Cl) as HBA and 100, 200, 3F00, 300, 3i00, and 400 glyce ol e he s as HBD in a 1:2 mola a io. The mix u es we e s i ed in a glass ial a 70°C un il a anspa en mix u e was o med, a e which hey we e cooled down o oom empe a u e. Be o e each essay, all he sys ems we e d ied unde acuum o 24h, due o he high hyg oscopici y, be o e use. Exposu e ials Sh imps (P. a ians), kindly p o ided by a local aquacul u e (Ribei a das Enguias), se ed as he oxicological biomodel o es ing deep eu ec ic sys ems (DES). Following a 24-h acclima iza ion pe iod in labo a o y condi ions, he sh imps we e alloca ed o 5-l aqua iums equipped wi h a il e ed seawa e sys em. The exposu e es s we e pe o med wi h il e ed seawa e (Vib an Sea, Seachem, USA) and salin- i y, 33g/l. Th oughou he expe imen , pH (8 ± 0.1) and empe a u e (23.0 ± 0.1°C) we e ca e ully con olled. The sh imps expe ienced a 12-h ligh and 12-h da k pho ope iod, along wi h con inuous oxygena ion (> 6mg/l dissol ed oxy- gen). Fo each expe imen , a andom selec ion o 5 sh imps (n = 30; 231.3 ± 121mg) was exposed o i e di e en con- cen a ions (5000mg/l, 2500mg/l, 1000mg/l, 500mg/l, and 100mg/l) o he chosen DES, dis ibu ed by 2-l polys y ene aqua iums. The sh imps ecei ed daily eedings o comme - cial d y ood (Se a b and), and labo a o y pa ame e s such as pH and empe a u e we e moni o ed daily. Each expe imen was conduc ed in duplica e o e 7days, wi h sh imp mo al- i y eco ded h oughou he p ocess. Upon comple ing he expe imen al ial, he sh imps we e placed in Eppendo ubes (1.5ml), weighed, and subsequen ly ozen a − 80°C o p ese a ion un il u he analysis o oxida i e s ess bioma ke s. Biochemical essays Sample ea men The ozen sh imps we e homogenized in 2ml o PBS, and a e g inding wi h he aid o a issue homogenize (Tissue Mas e 125, Omni, Kennesaw, GA, USA), he homogena e was cen i uged a 15,000g o 10min a 4°C (VWR, CT 15RE, Hi achi Koki, Tokyo, Japan). The esul ing supe - na an was collec ed and ozen a − 80°C o subsequen sample e alua ions. To acili a e da a analysis, samples we e dilu ed (1/10 dilu ion) in phospha e saline bu e (PBS). P io o conduc ing any enzyma ic assays, p o ein quan i ica- ion was pe o med using he B ad o d me hod o no malize he biochemical esul s (B ad o d 1976). Glu a hione S‑ ans e ase (GST) The me hodology used o assessing glu a hione S- ans- e ase (GST) ac i i y (EC 2.5.1.18) was based on he p o- cedu e ini ially desc ibed by Habig e al. (Ka o and Nai o 1974). I in ol es measu ing he inc ease in abso bance a 340nm, a ibu ed o he o ma ion o he conjuga e be ween educed glu a hione (GSH) and 1-chlo o-2,4-dini obenzene (cDNB). The me hod was adap ed o a 96-well pla e, whe e 20µl o he cen i uged supe na an and 180µl o he sub- s a e solu ion (phospha e bu e saline wi h 100mM cDNB and 200mM GSH om Sigma-Ald ich) we e added o each well. Enzyma ic ac i i y was moni o ed e e y minu e o 6min a 340nm using a mic opla e eade (Syne gy HTX, BioTek, Winooski, VT, USA). Resul s a e exp essed in nmol.min−1·mg−1 o al cy osolic p o ein o he sample. GST ac i i y = (Δ A340 sample ∕ min −Δ A340 blank ∕ min )× V o al ( ml )× dilu ion 0.0053 ×Vsample (ml) Equa ion1 shows he de e mina ion o glu a hione-S- ans e ase ac i i y. Glu a hione pe oxidase (GPx) The glu a hione pe oxidase (GPx) ac i i y (EC 1.11.1.9) is based on he me hodology employed by Law ence and Bu k (1976). In b ie , 20µl o each p e iously desc ibed sample ( e e o he “Sample ea men ” sec ion) was combined wi h a 96-well pla e con aining 120µl o a bu e solu ion (com- p ising 5mM EDTA and 50mM phospha e bu e ) and 50µl o a co-subs a e mix u e, consis ing o 1mM nico inamide adenine dinucleo ide phospha e (NADPH, Sigma-Ald ich, Ge many), 4mM educed glu a hione (GSH, Sigma-Ald ich, Ge many), 4 U/ml glu a hione educ ase (GSSG- educ ase, Sigma, Ge many), 1mM nico inamide adenine dinucleo ide 57962 En i onmen al Science and Pollu ion Resea ch (2024) 31:57959–57972 phospha e (NADPH, Sigma-Ald ich, Ge many), and 4mM sodium azide (Sigma-Ald ich, Ge many). The ini ia ion o he enzyma ic eac ion in ol ed adding 20µl o 15mM hyd ope oxide cumene (Sigma-Ald ich, Ge many). Enzy- ma ic ac i i y, exp essed as nmol.min−1·mg−1 o al p o ein, was de e mined by measu ing he dec ease in abso p ion pe minu e a 340nm using a mic opla e eade (Syne gy HTX, BioTek, Winooski, VT, USA), which is p opo ional o he educ ion o β-NADPH. Equa ion2 shows he glu a hione pe oxidase ac i i y de e mina ion. Ca alase (CAT) The me hod in ol es assessing ca alase ac i i y (EC 1.11.1.6) by he colo ime ic measu emen o he o malde- hyde p oduced by he ca alase eac ion. The me hodology was adap ed o a 96-well pla e om Johansson and Håkan Bo g (1988). To assess ca alase ac i i y in each sample, a calib a ion cu e anging om 0 o 75µM o maldehyde (Sigma-Ald ich, Ge many) was gene a ed. In a nu shell, 70µl o sh imp homogena e supe na an was combined wi h 50µl o a bu e solu ion (100mM po assium phos- pha e, pH 7.0) and 30µl o me hanol (Scha lau, Spain) in each well o a 96-well pla e. Subsequen ly, 20µl o 0.035M hyd ogen pe oxide (Sigma-Ald ich, Ge many) was added o ini ia e he enzyma ic eac ion. Following 20min o pla e incuba ion unde agi a ion (Fishe b and mic opla e shak- ing), 30µl o 10M po assium hyd oxide (Chem-Lab, Bel- gium) and 30µl o Pu pald solu ion (34.2M in 0.5M HCl, Sigma-Ald ich, Ge many) we e added. The pla e unde wen a 10-min incuba ion wi h a ligh co e and cons an shaking. Finally, 10µl o po assium pe ioda e (65.2mM, Chem-Lab, Belgium) was added o hal he enzyma ic eac ion. A e a 5-min incuba ion, he pla e was spec opho ome ically ead a 540nm using a pla e eade (Syne gy HTX, BioTek, Winooski, VT, USA). Ca alase ac i i y is de e mined by he o maldehyde p oduced in each sample, and he esul s a e exp essed by no malizing o he o al p o ein mass (nmol. min−1·mg−1 o al p o ein). Supe oxide dismu ase (SOD) The assay o his enzyme (SOD, EC 1.15.1.1) in ol es he inhibi ion o ni oblue e azolium educ ion (NBT) and was pe o med acco ding o Sun e al. (1988) a e being adap ed o a 96-well pla e. SOD acili a es he dismu a- ion o adicals (O2−) in compe i ion wi h NBT. In sum- ma y, each well o a 96-well pla e ecei ed 10µl o 3mM GPx ac i i y = Abs340 / 𝑚𝑖𝑛×V(ml)×dilu ion 0.00373 ×Vsample(ml) xan hine (Sigma-Ald ich, Ge many), 200µl o phospha e bu e (50mM; pH 8.0), 10µl o 0.075mM NBT (Sigma- Ald ich, Ge many), 10µl o 3mM EDTA, and 10µl o he sample. Subsequen ly, 10µl o 10 U/ml xan hine oxi- dase (Sigma-Ald ich, Ge many) was added o each well. The abso bance was measu ed a 535nm e e y 2min o a o al du a ion o 10min using a pla e eade (Syne gy HTX, BioTek, Winooski, VT, USA). Nega i e con ols (dis illed wa e and PBS) we e included in he expe imen o ep esen he maximum inc ease in abso bance. The pe cen age inhibi- ion pe minu e e lec s SOD ac i i y and is exp essed as % inhibi ion pe o al p o ein concen a ion. Equa ion3 shows he supe oxide dismu ase inhibi ion (%). To al an ioxidan capaci y (TAC) This spec opho ome ic me hod elies on he educ ion o he 2,2′-azino-bis-3 e hylbenzo hiazoline-6-sul onic acid (ABTS) ca ion adical, ollowing he me hod ou lined by Kambayashi e al. (2009). T olox se ed as he s anda d an ioxidan ( anging om 0 o 0.33mM) o build a calib a- ion cu e. In summa y, each well o a 96-well mic opla e ecei ed 10µl o 90µM myoglobin (Sigma, Ge many), 150µl o 600µM ABTS (Al a Aesa , Ge many), and 10µl o he sample. The eac ions we e ini ia ed by adding 40µl o hyd ogen pe oxide (500µM, Sigma-Ald ich, Ge many). A e a 10-min incuba ion pe iod, abso bance was meas- u ed a 415nm using a mic opla e eade (Syne gy HTX, BioTek, Winooski, VT, USA). The ob ained esul s a e exp essed no malized o he p o ein mass o each sample (nmol·mg−1 o al p o ein). Lipid pe oxida ion The hioba bi u ic acid (TBA) me hod ollowed he p o- ocol es ablished by Uchiyama and Miha a (1978). The assay mix u e, p epa ed in 2-ml mic o ubes, in ol ed com- bining 5µl o each sample (supe na an a e cen i uga- ion), 93.5µl o ichlo oace ic acid (20%, Pan eac, Spain), 93.5µl o hioba bi u ic acid (Sigma-Ald ich, Ge many), 12.5µl o SDS (8.1%, Sigma-Ald ich, Ge many), 45µl o a phospha e bu e (pH 7–7.4), and 50.5µl o MQ-g ade ul apu e wa e . A e igo ous agi a ion, he mic o ubes we e subjec ed o boiling wa e (100°C o 5min), a e punc u ing caps, o ini ia e he eac ion and p omp ly cooled on ice. Subsequen ly, 62.5µl o MQ-g ade ul apu e wa e was added. Then, he mic o ubes we e ho oughly shaken, % SOD inhibi ion = (Δ A560 nega i e con ol∕min −ΔA560 sample∕min) ΔA 560 nega i e con ol∕min 57963En i onmen al Science and Pollu ion Resea ch (2024) 31:57959–57972 and 150µl o each mic o ube’s con en was ans e ed o each well in a 96-well mic opla e. The MDA con en o he samples was de e mined by measu ing abso bance a 530nm using a mic opla e eade (Syne gy HTX, BioTek, Win- ooski, VT, USA). A calib a ion cu e wi h malondialdehyde bis(dime hylace al) (MDA, Me ck) s anda ds acili a ed he quan i ica ion o lipid pe oxida ion (0–0.1µM). The esul s we e exp essed as nmol·mg−1 o al p o ein. S a is ical analysis S a is ical analysis was pe o med using P ism 9.0 (G aph- Pad So wa e). All esul s a e exp essed as mean ± s and- a d de ia ion (SD). Compa isons we e ca ied ou h ough he K uskal–Wallis es o one-way ANOVA. Addi ionally, Dunne ’s mul iple compa isons es was used o analyze each expe imen . Resul s anddiscussion Mo ali y a e LC50 alues, along wi h hei co esponding s anda d de ia- ions, a e compiled in Table1. Mo eo e , EC50 alues o al e na i e aqua ic biomodels (A. ische i and R. subcapi- a a) a e also p o ided, enabling a compa ison o he beha - io ac oss h ee di e en species used as biological models in oxici y s udies. Addi ionally, he mo ali y a e o sh imps exposed o he s udied DES was e alua ed. Figu eS1 shows he no malized mo ali y a e o he s udied DES a a i- ous concen a ions. The e is a posi i e co ela ion be ween sh imp mo ali y and exposu e concen a ion. In all com- pounds, he e is an inc ease in he numbe o dea hs wi h inc easing concen a ion. I is impo an o no e ha in some concen a ions es ed no sh imp su i ed. Fo ins ance, N00Cl-000, N00Cl-100, N00Cl-200, and N00Cl-300 sys ems showed su i ing sh imp we e obse ed a all concen a ions es ed; howe e , in he case o N00Cl-400, N00Cl-3F00, and N00Cl-3i00, no su i ing animals we e obse ed a 2500 and 5000mg/l. When he EC50 da a ob ained a e analyzed, i is obse ed ha in he sys ems wi h e en chains, he EC50 alues dec ease as he chain leng h inc eases being he less oxic N00Cl-000 ollowed by N00Cl-200 and N00Cl-400. How- e e , his end is no obse ed in he case o he odd chain, whe e he oxici y is highe o N00Cl-100 han N00Cl-300. Addi ionally, he oxici y o he N00Cl-300 sys em is sligh ly highe han o he N00Cl-3i00, showing ha when ami ica ions a e included in he HBD alkyl chain, an inc ease o he EC50 alue has been obse ed and he e o e a dec ease in DES oxici y. Fu he mo e, he in luence o he p esence o luo ine a oms can be obse ed when compa ing N00Cl-200 and N00Cl-3F00 mix u es. When luo ine a oms a e inco po- a ed in o he s uc u e o he HBD componen , an inc ease in oxici y is obse ed; his end is only obse ed o he P. a ians biomodel. Fo he o he wo aqua ic biomodels p esen ed in Table1, i can be obse ed ha he EC50 o compound N00Cl-3F00 is highe , in bo h cases, han he compound N00Cl-200, indica ing lowe oxici y. The impac o luo ide on algae and bac e ia can a y, ei he inhibi - ing o p omo ing hei g ow h depending on ac o s such as luo ide concen a ion, du a ion o exposu e, and he spe- ci ic species in ol ed. Aqua ic plan s ha e shown po en ial in e ec i ely emo ing luo ide om pollu ed wa e in bo h con olled labo a o y se ings and na u al en i onmen s; howe e , in aqua ic animals, luo ide ends o accumula e in he exoskele ons o in e eb a es and in he bone issues o ish (Cama go 2003). Fo all he s udied sys ems, a concen a ion-dependen oxici y has been ob ained. This ela ionship has been s ud- ied in he ield o oxicology and o o he DES (Zwa e al. 1990; Inaya e al. 2022). Rega ding LC50 alues, he e is a clea end be ween oxici y and inc eased alkyl chain in HBD o e en chain DES. This end has been obse ed in he aqua ic bioin- dica o s, R. subcapi a a, bu no o A. ische i. Ne e he- less, his end is no obse ed o he case o he odd ones, whe e he oxici y dec eases as he alkyl chain inc eases. The “e en–odd e ec ” in he oxici y o alkyl chains has been al eady epo ed and e e s o he obse a ion ha o ganic compounds wi h an e en numbe o ca bon a oms end o exhibi di e en oxic p ope ies compa ed o hose wi h an odd numbe (Adachi e al. 1995). A p oposed explana ion o his e ec is based on he physicochemical p ope ies and molecula s uc u e o he alkyl chains. I is sugges ed ha alkyl chains wi h an e en leng h may exhibi g ea e sym- me y and mo e e icien packing in cell memb anes o a biological binding si es compa ed o odd-leng h chains. This inc eased symme y can a ec anspo p ope ies ac oss Table 1 Values o P. a ians, LC50 (mg/l) and alues o EC50 (mg/l) o s udies wi h A. ische i and R. subcapi a a exposed o DES DES LD50 (mg/l) EC50 (mg/l) P. a ians A. ische i [16] R. subcapi a a [16] N00Cl-000 3835 ± 994 83277 ± 4282 7015 ± 170 N00Cl-100 4434 ± 1716 93192 ± 4487 11,423 ± 924 N00Cl-200 2924 ± 510 8089 ± 128 7343 ± 567 N00Cl-300 5002 ± 315 16976 ± 2766 8087 ± 523 N00Cl-400 1052 ± 248 3446 ± 1132 5828 ± 666 N00Cl-3F00 1157 ± 284 34957 ± 4525 12,560 ± 196 N00Cl-3i00 2015 ± 1026 24754 ± 1205 9597 ± 1205 57964 En i onmen al Science and Pollu ion Resea ch (2024) 31:57959–57972 biological memb anes and a ini y o biological ecep o s (Zein and Win e 2000; De Meye e al. 2008). In addi ion o hese compa isons, i is also necessa y o analyze how mo ali y changed when compa ing he N00Cl-200 and N00Cl-3F00 sys ems. The bioindica o s A. ische i and R. subcapi a a show ha oxici y dec eases by including ca bon a oms in he chemical s uc u e; how- e e , his end is con a y o ha obse ed in sh imp. When compa ing N00Cl-300 and N00Cl-3i00 sys ems, he same end obse ed in o he bioindica o s is no ed: he p esence o adicals dec eases oxici y in di e en biomodels. This may be ela ed, as men ioned in o he a icles (Ga cía e al. 2015), o he inc eased di icul y in c ossing biological ba - ie s due o b anching and molecula weigh inc ease o he DES. Howe e , his e ec is less p onounced in he case o he P. a ians biomodel compa ed o he bac e ial model and he algae. F om he h ee aqua ic biomodels es ed, i can be obse ed ha , al hough oxici y ini ia es a e y high con- cen a ions and acco ding o he Passino and Smi h Clas- si ica ion (PSC) (Passino and Smi h 1987), hese sys ems could be ca ego ized as non-haza dous o aqua ic medium; howe e , P. a ians exhibi s a highe sensi i i y o hese sys ems. This is pa icula ly highligh ed by he in oduc ion o luo ides wi hin he eu ec ic sys em, whe e he di e ence in EC50/LD50 is up o 30 imes o A. ische i and 10 imes o R. subcapi a a. This could be due o hese biomodels ep- esen ing di e en ophic le els, wi h sh imps being a much mo e complex biomodel han bac e ia and algae. Al hough eco oxicological da a o hese compounds in P. a ians a e no a ailable, Pe ales e al. de e mined he eco oxici y o he HBD compounds (glyce ol e he s) in he c us acean Daph- nia magna (Pe ales e al. 2017). The componen s o ming he HBD appea o indi idually p esen lowe oxici y han he eu ec ic mix u e. Howe e , we should no o e look he di e ence in biomodels and he ac ha he e is no oxici y da a o he HBA (N00Cl), making i impossible o de e - mine i syne gies in oxici y exis . In gene al, we obse e ha o all biomodels, al hough he sensi i i y o sys ems may no be he same, an e en–odd e ec in oxici y is e iden . Addi ionally, as seen in mos oxicological s udies conduc ed on eu ec ic mix u es, modi- ica ion o he HBD con ibu es o changes no only in he physicochemical p ope ies o he mix u e bu also in i s eco- oxicological p ope ies. These compounds ha e only been p e iously s udied in wo o he aqua ic biomodels, making i s ill challenging o es ablish a s uc u e– oxici y ela ionship (Ga alaga e al. 2022a). Howe e , i is c ucial o no e he dis inc i eness be ween his species, comp ising bac e ia and mic oalgae, and sh imp, as hey may exhibi a ying esponses o DES expo- su e. While algae and bac e ia ha e been u ilized in assess- ing DES oxici y, hey se e as unicellula models, whe eas sh imp ep esen a mo e biologically complex o ganism (B own e al. 2020; Sabo owski e al. 2022). Mo eo e , he pa ame e s such as pH, empe a u e, and cul u e media di e be ween he A. ische i and R. subcapi a a es s and sh imp es s, po en ially in luencing DES in e ac ions wi h he medium and o ganisms, hus yielding di e en e ec s on he assayed models (El Achka e al. 2019). Nume ous s udies ha e in es iga ed he impac o acid- i y/alkalini y and wa e con en on DES physicochemical p ope ies and beha io . Fo ins ance, Jançiko á e al. no ed a linea dec ease in DES pH wi h ising empe a u e. The ype o hyd ogen bond dono s signi ican ly a ec s DES acid- i y, while wa e con en in luences p ope ies like pola i y and solubiliza ion capaci y, as co obo a ed by Skulco a e al. (Skulco a e al. 2019). Rega ding sys em bioa ailabil- i y, besides pH, conside a ions such as chemical s uc u e, ionic s eng h, and co-sol en p esence play c ucial oles, as highligh ed by Smulek e al. in hei bac e ial s udies, wi h po en ial ex apola ion o o he o ganisms (Smułek and Kaczo ek 2022). In he case o sh imp, i is no ewo hy ha his biomodel eadily adap s o en i onmen al changes, pa icula ly in sal concen a ions. A s udy by Missiona io e al. demons a ed hei adep ness in hype - and hypo-osmo egula ion, which is pi o al o main aining cellula unc ion ac oss a ying salini ies (Missioná io e al. 2023). Glu a hione S‑ ans e ase (GST) In Fig.1, glu a hione S- ans e ase ac i i y, a di e en DES concen a ions, is depic ed. In gene al, in all cases, i is obse ed ha he ac i i y dec eases as he concen a ion o DES used inc eases. I is obse ed ha o N00Cl-000, he e a e no sig- ni ican di e ences wi h he con ol up o concen a ions o 2500mg/l, bu in he case o N00Cl-100, his end is obse ed up o concen a ions o 1000mg/l. The N00Cl-300 sys em is he one ha shows no di e ences wi h espec o he con ol a any concen a ion excep 2500mg/l. Finally, in he N00Cl-200, N00Cl-400, N00Cl-3F00, and N00Cl-3i00 sys ems, i is obse ed ha he only concen a ion ha does no show di e ences wi h espec o he con ol is 100mg/l, indica ing ha hese sys ems a e p obably no as sa e as hey gene a e modi ica ions in he enzyme. The Spea man’s co ela ion coe icien was de e mined, e ealing a signi ican nega i e co ela ion ( < 0; p < 0.05) o N00Cl-000, N00Cl-100, and N00Cl-200 sys ems ac oss all o he enzyma ic assays conduc ed (GPx, CAT, TAC, LPO, and SOD). In he case o N00Cl-300, his nega i e co ela ion became signi ican o TAC assays (p = 0.04; = − 0.46), LPO assays (p = 0.004; = − 0.61), and SOD assays (p = 0.03; = − 0.63). A simila end was obse ed o N00Cl-400, bu wi h GPx (p = 0.005; = − 0.71), CAT 57965En i onmen al Science and Pollu ion Resea ch (2024) 31:57959–57972 Fig. 1 Glu a hione-S- ans e ase (GST) ac i i y (mean ± SD) in P. a ians. The concen a ions es ed we e 0mg/l (black ba ), 100mg/l (whi e ba ), 500mg/l ( ose ba ), 1000mg/l ( ed ba ), 2500mg/l (da k ed ba ), and 5000mg/l (g ey ba ) Fig. 2 Glu a hione pe oxidase (GPx) ac i i y (mean ± SD) in P. a ians. The concen a ions es ed we e 0mg/l (black ba ), 100mg/l (whi e ba ), 500mg/l ( ose ba ), 1000mg/l ( ed ba ), 2500mg/l (da k ed ba ), and 5000mg/l (g ey ba ) 57966 En i onmen al Science and Pollu ion Resea ch (2024) 31:57959–57972 (p = 0.009; = − 0.68), and LPO (p = 0.015; = − 0.64) assays. Finally, o N00Cl-3F00 and N00Cl-3i00 sys ems, his co ela ion occu ed wi h GPx (p = 0.004; = − 0.67 and p = 0.01; = − 0.61, espec i ely) and LPO (p = 0.02; = − 0.62 and p = 0.03; = − 0.58, espec i ely). These esul s a e shown in Figu eS2 o he Supplemen a y ma e ial. Glu a hione pe oxidase (GPx) The a e age concen a ions o GPx ac i i y in sh imp a e shown in Fig.2. The esul s indica e ha o N00Cl-100 and N00Cl-300 sys ems, no signi ican di e ences we e obse ed a any concen a ion, so he maximum exposu e concen a ion was 5000mg/l. Howe e , o N00Cl-200, he concen a ion inc eases a 1000mg/l. Fo he N00Cl-400 and N00Cl-3F00 sys ems, no signi ican di e ences wi h espec o he con ol we e obse ed in he case o 100mg/l, and inally, o N00Cl-3i00, no signi ican di e ences wi h espec o he con ol we e obse ed in any case, so he enzy- ma ic ac i i y was no al e ed wi h espec o he con ol, no e en a he highes concen a ion a which he sh imps su i ed (1000mg/l) o his DES. Sys ems N00Cl -200, N00Cl-400, and N00Cl-3F00 should be used in mode a ion, as i was obse ed ha he e we e signi ican di e ences wi h he con ol, hus modi ying he enzyma ic ac i i y o GPx. Fo he N00Cl-100 and N00Cl-300 sys ems, he highes concen a ion o GST ac i i y has been ound a 5000mg/l (7.48 nmol/min/mg o al p o ein o N00Cl-100 and 6.58nmol/min/mg o al p o ein o N00Cl-300, espec- i ely), whe eas he lowes concen a ion was measu ed a 100mg/l (5.12nmol/min/mg o al p o ein and 5.64nmol/ min/mg o al p o ein, espec i ely). Fo GPx, as p e iously men ioned, a signi ican nega i e co ela ion is obse ed o all sys ems excep o N00Cl- 300 in GST. On he o he hand, bo h N00Cl-000 and N00Cl-400 show a signi ican inc easing end, such ha an inc ease in GST ac i i y leads o a signi ican inc ease in CAT ac i i y (p = 0.01; = 0.49 and p = 0.02; = 0.63, espec i ely). We only obse e a signi ican posi i e co - ela ion o he lipope oxida i e ac i i y (LPO) in N00Cl- 3i00 (p = 0.001; = 0.802). N00Cl-100 and N00Cl-200 sys ems sha e he same posi i e co ela ion wi h CAT (p = 8.42e − 005; = 0.68 and p = 0.001; = 0.59, espec- i ely) and TAC (p = 0.002; = 0.58 and p = 2.89e − 004; = 0.64, espec i ely). Ca alase (CAT) In Fig.3, he concen a ion o ca alase ac i i ies in he cy osol is p esen ed. When analyzing he igu es, he da a ob ained is supe io o he con ol, bu when analyzing he sys ems sepa a ely, i can be seen ha in he case o he N00Cl-000, N00Cl-100, and N00Cl-300, only he concen- a ion o 5000mg/l causes signi ican changes in enzy- ma ic ac i i y. Fo N00Cl-200, in addi ion o he 5000mg/l concen a ion, he 2500mg/l concen a ion also causes Fig. 3 Ca alase ac i i y (mean ± SD) in P. a ians. The concen a ions es ed we e 0mg/l (black ba ), 100mg/l (whi e ba ), 500mg/l ( ose ba ), 1000mg/l ( ed ba ), 2500mg/l (da k ed ba ), and 5000mg/l (g ey ba ) 57967En i onmen al Science and Pollu ion Resea ch (2024) 31:57959–57972 signi ican modi ica ions. No e ha in he case o N00Cl- 400, all concen a ions es ed show signi ican di e ences, indica ing ha his sys em should be used wi h cau ion. Finally, N00Cl-3F00 shows signi ican di e ences a con- cen a ions o 1000mg/l, and N00Cl-3i00 shows no di e - ences and e y low enzyma ic ac i i y. In his case, a signi ican posi i e co ela ion is obse ed be ween ca alase ac i i y and GPx, TAC, LPO, and SOD o N00Cl-000, N00Cl-100, and N00Cl-200. N00Cl-300 exhibi s he same posi i e co ela ion, bu only o TAC (p = 0.015; = 0.57) and SOD (p = 2.2e − 005; = 0.814). On he o he hand, no co ela ion is obse ed wi h o he enzyma ic assays o N00Cl-3i00, while bo h N00Cl-400 and N00Cl-3F00 posi i ely co ela e wi h lipid pe oxida- ion, such ha an inc ease in ca alase ac i i y co esponds o an inc ease in lipid pe oxida ion (p = 0.02; = 0.518 and p = 0.01; = 0.654, espec i ely). Supe oxide dismu ase (SOD) The supe oxide dismu ase ac i i y (% o inhibi ion) is shown in Fig.4. In his case, he SOD inc eases acco ding o DES concen a ions. Highe alues a e ound o N00Cl-000 (48.2%), N00Cl-100 (59.6%), and N00Cl-300 (73.4%) a 5000mg/l. The highes obse ed SOD o N00Cl-200 occu s a 2500mg/l (46.9%) while o N00Cl-400 and N00Cl- 3i00, he highes le el is exp essed a 500mg/l (44.1%) and 1000mg/l (35.5%), espec i ely. When luo ine a oms a e in oduced in he HBD, he inhibi ion o he enzyme dec eases. Lowe alues o N00Cl-3i00 we e ound when i was compa ed o N00Cl-300. When de e mining he Spea man co ela ion coe i- cien (Supplemen a y Ma e ial 1), i was obse ed ha he inc ease in SOD co esponded also o an inc ease in GPx, CAT, TAC, and LPO ac i i y o N00Cl-100 and N00Cl- 200 sys ems and N00Cl-300, indica ing a signi ican posi- i e co ela ion (p < 0.05; > 0). Fo N00Cl-000 and N00Cl- 400, his posi i e co ela ion occu s be ween SOD and TAC and LPO ac i i y (p = 4.98e − 007; = 0.79 and p = 0.001; = 0.56 o N00Cl-000 and p = 0.02; = 0.53 and p = 0.013; = 0.57, espec i ely), while in he case o N00Cl-3i00, no signi ican co ela ion is obse ed. To al an ioxidan capaci y (TAC) The a e age concen a ion o TAC esul s in sh imps is p esen ed in Fig.5. Fo N00Cl-000, N00Cl-100, N00Cl- 200, and N00Cl-300, i is obse ed ha he TAC inc eases as he DES concen a ion inc eases, ob aining alues highe han he con ol. On he con a y, in N00Cl-400, N00Cl-3F00, and N00Cl-3i00, some o he TAC alues ob ained a e lowe han hose p esen ed by he con ol. The highes alue o TAC is ob ained o N00Cl-200 a 5000mg/l (75.13nmol/mg o o al p o ein). When compa ing he N00Cl-200 and N00Cl-3F00 sys ems, i is obse ed ha somewha highe alues a e Fig. 4 Supe oxide dismu ase ac i i y as a pe cen age o inhibi ion (mean ± SD) in P. a ians. The concen a ions es ed we e 0mg/l (black ba ), 100mg/l (whi e ba ), 500mg/l ( ose ba ), 1000mg/l ( ed ba ), 2500mg/l (da k ed ba ), and 5000mg/l (g ey ba )