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Eco oxicological s udy o Glucose:Choline chlo ide and So bi ol:Choline chlo ide a di e en
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con en o wa e
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Au ho s:
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Lau a Lomba.
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Facul ad de Ciencias de la Salud, Uni e sidad San Jo ge. Campus Uni e si a io, Au o A23 km 299,
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50830. Villanue a de Gállego Za agoza. Spain
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Diego E azquin.
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Facul ad de Ciencias de la Salud, Uni e sidad San Jo ge. Campus Uni e si a io, Au o A23 km 299,
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50830. Villanue a de Gállego Za agoza. Spain
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Pila Ga alaga.
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Facul ad de Ciencias de la Salud, Uni e sidad San Jo ge. Campus Uni e si a io, Au o A23 km 299,
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50830. Villanue a de Gállego Za agoza. Spain
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Noelia López.
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Facul ad de Ciencias de la Salud, Uni e sidad San Jo ge. Campus Uni e si a io, Au o A23 km 299,
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50830. Villanue a de Gállego Za agoza. Spain
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Co esponding au o :
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Bea iz Gine .
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Facul ad de Ciencias de la Salud, Uni e sidad San Jo ge. Campus Uni e si a io, Au o A23 km 299,
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50830. Villanue a de Gállego Za agoza. Spain
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e-mail: [email p o ec ed], phone: 0034 976 060 100, ax 0034 976 077 584
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Abs ac : The sea ch o new sol en s is cu en ly ocused on Deep Eu ec ic Sol en s (DES). Howe e ,
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he e a e no many eco oxicological s udies in di e en biomodels o DES ha allow knowing how hese
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chemicals a ec o he en i onmen along he ophic chain. In his manusc ip , wo DES a di e en
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p opo ion o wa e ha e been p epa ed and cha ac e ized om he eco oxicological poin o iew. These
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sol en s a e: glucose:chlo ide choline (2:5) and so bi ol:chlo ide choline (3:2) a di e en con en o wa e .
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To ca y ou he eco oxicological s udy, h ee biomodels ha e been used: bac e ia Alli ib io ishe i (A.
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ishe i), c us acean Daphnia magna (D. magna) and algae Raphidocelis subcapi a a (R. subcapi a a). The
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ob ained esul s show ha he eco oxici y o hese chemicals depends on he biomodel used and he amoun
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o wa e , being oxici y alues lowe o chemicals wi h highe wa e con en . Howe e , i is impo an o
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highligh ha he eco oxici y o all chemicals is qui e low wi h e ec i e concen a ions, EC50 alues abo e
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1000 mg/L in all he s udied cases.
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Keywo ds: eco oxici y, DES, glucose so bi ol, Alii ib io ische i, Daphnia magna, Raphidocelis
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subcapi a a.
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3
1. In oduc ion
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Deep Eu ec ic Sol en s (DES) and Na u al Deep Eu ec ic Sol en s (NADES) a e mix u es o med
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by wo o mo e chemicals a solid s a e a oom empe a u e bu , when combined in a speci ic mola a io,
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o m a liquid solu ion. This is mainly due he s ong and complex ne wo k o in e molecula o ces
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s ablished ha cause a sha p diminu ion o he mel ing poin (Lau a Lomba 2021a).
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DES can be conside ed as “design sol en s” since he possibili y o combina ions o di e en
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componen s and composi ions is almos unlimi ed. The goal is se ing he igh mix u e o ob aining he
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physicochemical p ope ies equi ed o a speci ic chemical p ocesses(Mi a e al. 2019).
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DES a e o med by a hyd ogen bond accep o (HBA) and a hyd ogen bond dono (HBD) and he
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ypical componen s a e na u al chemicals such as: o ganic acids, alcohols, suga s, amino acids, u ea, small
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hyd ophilic molecules o na u al me aboli es among o he s(Nys ed e al. 2021).
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DES a e usually low cos , a e also easy o p epa e (Ben enu i e al. 2020, Ben enu i e al. 2019),
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p esen low ola ili y, a e biodeg adable (Wils e al. 2021) and no mally non- eac i e wi h wa e . The label
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o non- oxic mix u es has o be e iewed and la ely he e is some con o e sy in he scien i ic communi y
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in his ega d; he e a e an impo an numbe o s udies ha show low oxici y o eu ec ic mix u es o he
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en i onmen o human bo h in i o and in i o s udies (Lau a Lomba 2021b), while some o he s s udies
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demons a e he oxici y o DES abo e a ce ain limi o concen a ion (To eg osa-C espo e al. 2020).
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Ne e heless, he oxici y o DES depends on se e al ac o s ha include no only he na u e o he
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componen s o he mix u e bu also he o ganisms on which oxici y is s udied, hei p e-adap a ion pe iods
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o e en he s e iliza ion p ocesses ca ied ou du ing he measu emen o oxici y. This may be caused by
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he acidi ica ion o he medium caused by he DES hyd olysis (To eg osa-C espo e al. 2020). Thus, i is
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equi ed mo e speci ic s udies abou he e ec o DES on di e en endpoin s, co e ing se e al ophic
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le els, o ganisms, cells o issues and he use o mo e app op ia e oxicological echniques (Ma chel e al.
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2022b, Ruesgas-Ramon e al. 2017).
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I is also said ha p esen good physicochemical p ope ies o se e al chemical p ocesses hanks
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o hei wide pola i y ange and low apou p essu e o chemical s abili y, among o he s(Ben enu i e al.
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2020, Lau a Lomba 2021a, Lau a Lomba 2021b, Sil a e al. 2019). Fo hese easons DES can be used as
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s a iona y phases in ch oma og aphy (Momo ko e al. 2021, 2022) and memb anes (Cas o-Munoz e al.
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2022, Khaja ian e al. 2022) and addi ionally, hey a e a ac i e o se e al indus ial p ocesses such as
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bioca alysis (Zhou e al. 2021), c yop ese a ion(C a ei o e al. 2021), biochemical applica ions(Sil a e
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al. 2019, Yang 2019), ex ac ion medium(Ruesgas-Ramon e al. 2017, Ruka ina e al. 2021), biological
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assays (Liu e al. 2018), pha maceu ical indus y(Liu e al. 2021, Mo ison e al. 2009) o cosme ic
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indus y(Benoi e al. 2021).
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Howe e , he main ba ie o he widesp ead use o DES is he lack o in o ma ion ega ding
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a ious physiochemical and oxic p ope ies. This limi a ion a ises om he e y na u e o he DES; he
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mul iple combina ions be ween componen s and composi ions (AlOma e al. 2016, Lapena e al. 2020,
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Lapena e al. 2019b, a) makes di icul o ha e a comple e se o p ope ies ha allow knowing he speci ic
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cha ac e is ics o a mix u e (componen s and concen a ion) (Li e al. 2022) o e en o p edic he beha iou .
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Howe e , i is impo an no ice ha he he mal s abili y o DES can be diminished a high
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empe a u e. Mos o he s udies abou his issue analyse he he mal s abili y o DES using he onse
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4
decomposi ion empe a u es (Tonse ). These empe a u es, ha we e no mally ob ained om dynamic
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he mog a ime y (TGA) unde di e en expe imen al condi ions ha , in gene al, lead o he
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o e es ima ion o he onse decomposi ion empe a u e. I is impo an o bea in mind ha he e a e some
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scien i ic s udies ela ed o he he mal s abili y o DES ha ha e ound, in some cases, ha DES can
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decompose in o HBA and HBD due o he weaking o he hyd ogen bonds. This p ocess can occu in wo
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s eps: he i s one, he DES componen wi h a lowe boiling poin o li le s abili y unde goes ola iliza ion
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o decomposi ion (no mally HBD) and he second one, he decomposi ion o he o he DES a a highe
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empe a u e (no mally he HBA). Thus, he ole o he hyd ogen bonds in he he mal s abili y seems o be
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de e minan . (Gu ie ez e al. 2010, Ma chel e al. 2022a). Addi ionally, i is impo an o no e ha he
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oxici y o DES may a y when he hyd ogen bond ne is modi ied; new sup amolecula s uc u es can be
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c ea ed, and hus, he oxicological ac i i y may be also di e en .(Gu ie ez e al. 2010, Ma chel e al.
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2022b, a)
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Wi h he aim o inc easing he in o ma ion a ailable on hese subs ances, we ha e p e iously
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explo ed he eco oxici y o some o hem: eline, e haline and glyceline owa ds se e al aqua ic biomodels
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(algae, c us aceans and bac e ia)(Lapena e al. 2021). We ounded ha he eco oxic e ec clea ly depended
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on he s udied biomodel and he ype o es used as a ool o moni o oxici y. Fu he mo e, he wa e
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con en did no ollow he expec ed end and s udied DES beha ed e a ically wi h ega ds o he wa e
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composi ion(Lapena e al. 2021).
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Thus, wi h he objec i e o deepening he knowledge o he eco oxic beha iou o DES, in his
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wo k we ha e s udied he eco oxici y o se e al eu ec ic mix u es con aining suga s and chlo ide choline
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a di e en concen a ions o wa e : glucose:chlo ide choline (2:5) and so bi ol:chlo ide choline (3:2) wi h
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di e en wa e con en . Conc e ely, we ha e ob ained he oxic e ec ha hese mix u es p oduce in a ious
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aqua ic o ganisms, co e ing he ophic chain: bac e ia Alli ib io ishe i (A. ishe i), c us acean Daphnia
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magna (D. magna) and algae Raphidocelis subcapi a a (R. subcapi a a).
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Besides, i has been p e iously epo ed ha iscosi y is an impo an p ope ies key o explain
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oxici y in some cells. An inc ease o oxici y is no mally associa ed wi h highe cell le hali y a es(Hayyan
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e al. 2016). Fo ha eason, we ha e also measu ed he kinema ic iscosi y o he s udied DES. Toxici y
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esul s ha e i s been analysed in an independen way, conside ing bo h he DES and he biomodel. They
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we e hen conside ed oge he and he inal conclusions abou he eco oxic beha io ha e been eached.
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2. Ma e ial and me hods
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2.1 Chemicals and p epa a ion o Deep Eu ec ic Sol en s
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In Table 1, he used chemicals o he DES p epa a ion a e ga he ed. Choline chlo ide has been
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d ied unde acuum o 24 h p io o use. Mix u es wi h wa e ha e been p epa ed conside ing he p e ious
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amoun o wa e o each o he componen s o he mix u es wi h Milli-Q wa e ( esis i i y less han 18.2
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M·cm) using a Sa o ius En is 5201-1S balance (unce ain y ±10-1 g). Once each subs ance has been
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weighed, has been ans e ed o a closed lask (250 ml) wi h a magne ic nucleus inside. The lask has been
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placed on a hea ing pla e and hea ed o a empe a u e o app oxima ely 80ºC wi h con inuous s i ing. The
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p ocess has been comple ed wi hin one hou and a homogeneous liquid has been obse ed. In Table 2,
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s udies DES, composi ion, and abb e ia ion a e shown.
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Table 1. Cha ac e is ics o he used chemicals
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Name
CAS
Supplie
Mass ac ion pu i y
Fo mula
Mola mass
(g/mol)
Glucose anhyd ous
50-99-77
Pan eac
0.975
C6H12O6
180.16
So bi ol
50-70-4
Sigma-Ad ich
0.980
C6H14O6
182.17
Choline Chlo ide
67-48-1
Sigma-Ald ich
0.993
C5H14ClNO
139.63
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Table 2. S udied DES: composi ion and abb e ia ion
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HBD
HBA
Add-on
Mola a io
Abb e ia ion
Glucose
Chloline chlo ide
Wa e
5:2:5
Glu5
5:2:7.5
Glu7.5
5:2:10
Glu10
So bi ol
Chloline chlo ide
wa e
2:3:5
So 5
2:3:7.5
So 7.5
2:3:10
So 10
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2.2 Viscosi y measu emen s
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Kinema ic iscosi ies, ν, ha e been ob ained using a Schoo -Ge ä eAVS-440 au oma ic measu ing
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uni along wi h se e al Ubbelohde capilla y iscosime e s. The unce ain y o he ime low measu emen s
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is 0.01 s, and kine ic ene gy co ec ions ha e been applied o he expe imen al da a. The empe a u e has
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been con olled a 25ºC by means o a Schoo -Ge ä e CT 1150/2 he mos a , wi h a empe a u e unce ain y
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o 0.01K.
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2.3 A. ische i eco oxici y es
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The lyophilized A. ische i (s ain NRRL-B-11177 and e e ence 945006) ha e been supplied by
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Macha ey-Nagel. This es has been based on UNE-11348-3(UNE-11348-3 2009). All solu ions ha e been
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p epa ed wi h 2% o NaCl and he pH has been adjus ed using 0.1 M HCl o 0.1 M NaOH in 2 % NaCl.
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Addi ionally, posi i e con ols such as phenol and zinc sul a e ha e been used(Jennings e al. 2001) and
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nega i e con ol has been cul u ing medium (Bio ix Lumi medium o eeze-d ied luminous bac e ia by
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DIN EN USO 11348–3, Mache ey-Nagel, Du en, Ge many).
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Luminescence has been measu ed in acu e mode (Bio ox B) using a Bio ix Lumi-10 luminome e
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(Mache eyNagel) a e 30 minu es o exposu e and a 15ºC. Th ee eplica es o each con ol ha e been
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es ed. Mo e de ails o his es can be ound in ou p e ious wo ks(Ga cia e al. 2015, Lomba e al. 2014).
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2.4 D. magna eco oxici y es
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D. magna eco oxici y es ha e been based on he guidelines OECD 202(202 2004). The D. magna
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ephippia (Toxki , Daph oki F Magna, e . DM090812) ha e been s o ed a 4ºC and pu chased om
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Vid a oc. The eggs ha e been incuba ed wi h cul u ing medium conside ing he speci ica ions o he
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supplie , du ing 72 h a 22ºC wi h 6000 lux in a Toxki CH-0120D-AC/DC incuba o (supplied by Eco es )
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and ed wi h Spi ulina algae 2 h be o e s a ing he expe imen . The pH has been adjus ed 7-7.5 be o e
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exposu e. Fu he mo e, posi i e con ol (sodium dich oma e) and nega i e con ols ha e been es ed.
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A o al o 20 newbo n daphnids (aged less han 24 h) ha e been exposed o he di e en dilu ions
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o he DES in da k o 24 h a 20ºC pe compound and concen a ion. The c us aceans ha e been sepa a ed
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in o ou g oups o i e o ganisms, ou eplica es pe concen a ion exposu e. The es has been epea ed
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in iplica e. Daphnids ha e been conside ed immobilized i hey ha e been no able o swim o 15 s a e
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agi a ion. Mo e de ails o his p o ocol can be ound in bibliog aphy(Pe ales e al. 2017).
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2.5 R. subcapi a a eco oxici y es
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R. subcapi a a es ha e been ca ied ou acco ding o OECD 201(OECD 1984). Algae ha e been
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pu chased om ECOTEST (SC2B1214). All he expe imen s ha e been epea ed in iplica e. Mo eo e ,
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cul u ing medium and de-mobiliza ion o algae ha e been p epa ed using he supplie speci ica ions.
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The algae cells ha e been incuba ed a 23ºC in 100 mL beake con aining 50 mL o cul u ing
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medium wi h 10000 lux o illumina ion. Cells densi ies o ha e been co ela ed wi h op ical densi y (OD)
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a 670 nm basing on he supplie speci ica ions. The algae ini ial concen a ion has been 3·105 cells/ml.
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The pH o he solu ions has been adjus ed in he ange o 7.9 and 8.2 using 0.1 M NaOH o 0.1 M
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HCl solu ions. The assay has been ca ied ou in a 96 well pla e. To minimize e apo a ion, he ou e wells
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o he pla e ha e been illed wi h 0.3 mL o dis illed wa e and only he inne wells ha e been illed wi h
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con ols and es solu ions. The ini ial OD has been measu ed a 670 nm. The well pla e has been incuba ed
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a 23ºC o 72 hou s. Nex , all pla es ha e been e-suspended o a oid he se ling o algae and he inal OD
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has been measu ed.
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2.6 S a is ics ea men
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Da a ha e been analysed using a non-linea eg ession- ep esen ing log (inhibi o ) e sus
166
inhibi ion.
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To ob ain he EC50 alues o each compound, he expe imen al esul s ha e been i ed o he
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ollowing unc ion:
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( )
( )
bCa log
101
100
I% −
+
=
(4)
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whe e %I deno es % inhibi ion o luminescence o A. ische i, % o immobiliza ion o D. magna and %
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o inhibi ion o g ow h o R. subcapi a a; C is he concen a ion in mg/L and Log EC50 and a a e he
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adjus able pa ame e s.
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3. Resul s and discussion
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In his sec ion, he ob ained esul s o he s udies compounds a e shown. Rega ding o he
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p epa ed DES, i is impo an o no e ha all DES o med by so bi ol (So 5, So 7.5 and So 10) and glucose
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(Glu10) ha e been ob ained in a unique a clea phase a oom empe a u e (25ºC), howe e , in he case o
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glucose DES (Glu5, Glu7) i has been necessa y o sligh ly hea (30ºC) o ob ain ha liquid phase be o e
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he es s.
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In his sec ion, esul s ha e been analyzed om he pe spec i e o he DES (o DES + wa e
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mix u es) i sel , as a whole. Howe e , i is impo an o e ise he na u e o he componen s o he eu ec ic
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mix u es om he oxicological poin o iew. Fo ins ance, choline chlo ide is a known componen o
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Vi amin B, and plays impo an me abolic unc ions; choline is usually he chosen cellula aw ma e ial o
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he syn hesis o cellula phospholipidic memb anes (phospha idylcholine and sphingomyelin) (Hayyan e
185
al. 2016). On he o he hand, om a cellula pe spec i e glucose and so bi ol a e essen ial ca bohyd a es;
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hei me abolism p o ides he equi ed ene gy o di e en cellula unc ions. A e adso p ion, glucose
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unde go glycolysis when ene gy is equi ed in he cell, o he wise is s o ed as glycogen. The so bi ol
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me abolism is an accesso y pa hway in he glucose ou e. Th ough a chain o eac ions, he so bi ol is an
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in e media e ha con e s glucose in o uc ose. The glycoly ic pa hway o glucose and uc ose leads o
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syn hesis o nucleic acids h ough he pen ose phospha e pa hway, he ene gy p oduc ion in he
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mi ochond ia h ough ica boxylic acid pa hway and he a y acid syn hesis h ough he lipogenesis
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p ocess. Fo hese easons, i is expec ed high cellula ole ance o he s udied DES o med by choline and
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hese suga s. Howe e , du ing he eu ec ic mix u e o ma ion p ocess and he es ablishmen o he hyd ogen
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bond ne wo k, an impo an modi ica ion o he chemical and physical p ope ies is made, and his includes
195
he oxicological beha io o he eu ec ic mix u es.
196
Re u ning o he analysis o he esul s ob ained by he DES s udied in he biomodels, alues o
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EC50 owa ds A. ische i, D. magna and R. subcapi a a a e shown in Table 3. Fo all he s udied chemicals,
198
he e is a dependence ela ionship be ween he oxici y and he concen a ion; i.e, he oxic e ec inc eases
199
as he concen a ion does. The gene al oxici y end obse ed o all biomodels is he ollowing: Glu 5 >
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Glu 7.5 > Glu10 > So 5 > So 7.5 > So 10. In Figu e 1, he oxici y o ob ained alues o all he s udied
201
biomodels a e p esen ed. The esul s indica e ha he mos sensi i e species was D. magna ollowed by R.
202
subcapi a a and A. ische i.
203
I is impo an o highligh ha none o he s udies mix u es can be conside ed oxic o he
204
en i onmen ( alues o EC50 do no exceed he gene al limi o 1000 mg/L). The Globally Ha monized
205
Sys em o classi ica ion And labelling o Chemicals (GHS) (Na ions 2021) has been used o ca ego ize he
206
oxici y o subs ances haza dous o he aqua ic en i onmen , Sho - e m (acu e) as ollow: Acu e 1 (EC50
207
≤ 0.1 mg/L), acu e 2 ( EC50 > 1 bu ≤ 10 mg/L), acu e 3 ( EC50 > 10 bu ≤ 100 mg/L). Fo his GHS
208
classi ica ion, algae, c us acea and ish, a e conside ed su oga e species. Howe e , da a on o he o ganisms
209
may also be conside ed and he classi ica ion can be applied oo. The eco oxici y esul s ob ained in his
210
wo k indica es ha o all s udied mix u es he e is no need o classi y o sho - e m (acu e) haza d. I is
211
wo h men ioning he case o A. ische i whe e some mix u es exhibi ed alues o EC50 highe han 100000
212
mg/L indica ing e y low oxici y o his biomodel (Figu e 1).
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8
214
Figu e 1. Compa ison o he ob ained esul s o each biomodel. The solid line a Log EC50 = 3
215
mg/L co espond o he gene al oxici y limi 1000 mg/L.
216
217
The lipophilia is one o he key p ope ies adi ionally used o explain he oxic beha io : he
218
highe lipophilic cha ac e , he highe abili y o he molecules o c oss h ough he biological memb anes
219
and p oduce a wha e e e ec on he ac i i y. In his case, ocusing o he suga s o ming pa o he DES,
220
glucose and so bi ol, ou oxici y esul s a e in close ag eemen wi h lipophilic end desc ibed by hei
221
co esponding oc anol-wa e pa i ion coe icien s, log Kow = -3,17 and -4.67 espec i ely (Chemspide ,
222
accessed a Ap il 13 d 2022).
223
In Table 3, alues o kinema ic iscosi y a e also ga he ed. Fo hese mix u es, he iscosi y has
224
been highe han he iscosi y o some ypical molecula liquids such as wa e o some o he low molecula
225
weigh o ganic sol en s. In he case o glucose mix u es, he highe iscosi y has been ob ained o Glu5
226
ollowed by Glu7 and Glu10. In he case o DES con aining so bi ol, he end is he same as o glucose
227
mix u es, being he mos iscous So 5 ollowed by So 7.5 and So 10. This p ope y is clea ly a ec ed by
228
he na u e o i s componen s, empe a u e, o mola a io. The con en o wa e is impo an oo, no only in
229
physicochemical p ope ies bu also in he in eg i y o DES changing hei sup amolecula in e ac ions (El
230
Achka e al. 2021). As men ioned be o e, iscosi y and con en o wa e may explain he ob ained esul s;
231
i has been obse ed ha high iscosi y a e associa ed wi h inc eased le hali y(Hayyan e al. 2016).
232
233
Table 3. Values o EC50 (mg/L) o A. ische i, D. magna and R. subcapi a a and alues o kinema ic
234
iscosi y a 25ºC.
235
DES
A. ische i
D. magna
R. subcapi a a
Kinema ic
Viscosi ies
30 min
24 h
72 h
ν, (mm/s)
Glu5
34196 ± 3965
2433 ± 31
1021 ± 122
787.69
Glu7.5
39955 ± 2455
2527 ± 22
1305 ±114
375.22
Glu10
69591 ± 3073
3048 ± 24
7544 ± 475
185.02
So 5
149748 ± 1279
5153 ± 85
12496 ± 663
3333.9
So 7.5
161857 ± 812
5942 ± 283
17545 ± 295
613.01
So 10
171209 ± 789
6557 ± 242
31325 ± 131
503.51
236
Glu5 Glu7.5 Glu10 So 5 So 7.5 So 10
0
1
2
3
4
5
6
Log EC50 (mg/L)
A. ische i
D. magna
R. subcapi a a
9
A. ische i bac e ia is G am-nega i e, lagella ed, non-pa hogenic and od-shaped bac e ium,
237
which is dis ibu ed in ma ine en i onmen s(Abbas e al. 2018). The bioluminescence mechanism is based
238
in wo subs a es: luci e in and a long chain a y aldehyde. Exogenous agen s ( educed) p oduce he
239
educ ion o FMN ( la in mononucleo ide oxida ed) o FMNH2 ( la in mononucleo ide educed) h ough
240
luci e ase ( la in mono-oxygenase oxido educ ase) ac ion which eac s wi h oxygen o ming an
241
in e media e chemical called 4a-pe oxy- la in. This compound oxidizes he a y aldehyde o ming i s
242
co esponding acid and a luci e ase–hyd oxy la in complex. This in e media e is decomposed slowly and
243
emi ing a blue-g een ligh wi h i s highes in ensi y a 490 nm(Meighen 1991). This p ocess is comple ely
244
linked o espi a ion, h ough he elec on anspo chain, and gi es an idea abou he me abolic s a us as a
245
chemical oxici y. The p esence o oxic subs ances diminishes he esul an luminescence. The inhibi ion
246
o bac e ial me abolism is mani es ed by a enua ion o ligh emi ance which co esponds o he oxici y
247
le el o he es ed subs ance (Abbas e al. 2018, Bulich 1982).
248
Fo he s udied mix u es, he cu e dose- esponse o A. ische i a 30 min a e shown in Figu e 2.
249
Fo his biomodel, he alues o EC50 a e e y high a ying be ween 30000-172000 mg/L, indica ing a low
250
oxici y o he s udied mix u es owa ds his biomodel; howe e , DES con aining glucose a e mo e oxic
251
han DES con aining so bi ol, conc e ely be ween 4.1 and 2.5 imes mo e oxic, depending on he DES. In
252
all cases, oxici y dec eases wi h wa e con en . In he case o DES wi h so bi ol, his dec ease o oxici y
253
is almos linea while o glucose DES, he he dec ease is mo e p onounced i he wa e con en inc eases.
254
255
256
Figu e 2. Cu e dose- esponse in A. ische i o Glu and So a 30 min. Con en o : 5 o wa e ( ), 7.5 o
257
wa e ( ) and 10 o wa e ( ).
258
259
D. magna is one o he oldes o ganisms used in biological esea ch (Tkaczyk e al. 2021). They
260
can il e bac e ia, algae, p o ozoans o o he small aqueous pa icles. Daphnia a e qui e impo an o he
261
eshwa e aqua ic ood chain, as a p ima y consume o p eda o y in e eb a es and ish. Addi ionally,
262
daphnids consume algae and hus, imp o e wa e quali y (Die ich e al. 2010, Tkaczyk e al. 2021). The
263
Daphnia mo ili y is easy o obse e and he e o e, his ea u e is commonly used in immobiliza ion,
264
le hali y and ep oduc ion es s(Bownik 2020). In addi ion, swimming beha io pa ame e s (swimming
265
ac i i y, swimming ime, swimming speed, beha io al s eng h and hopping equency) can be used as
266
sensi i e endpoin s (Bownik 2017). Howe e , i is impo an o con ol he assay condi ions because
267
2 3 4 5 6 7
0
25
50
75
100
Log C (mg/L)
% Inhibi ion o luminescence
Glucose:choline chlo ide:wa e
4.0 4.5 5.0 5.5 6.0
0
25
50
75
100
Log C (mg/L)
% Inhibi ion o luminescence
So bi ol:choline chlo ide: wa e
