Antagonistic Toxic Effects of Surfactants Mixtures to Bacteria Pseudomonas putida and Marine Microalgae Phaeodactylum tricornutum

Ci a ion: Ríos, F.; Lechuga, M.; Loba o-Gua nido, I.; Fe nández-Se ano, M. An agonis ic Toxic E ec s o Su ac an s Mix u es o Bac e ia Pseudomonas pu ida and Ma ine Mic oalgae Phaeodac ylum ico nu um.Toxics 2023,11, 344. h ps://doi.o g/10.3390/ oxics11040344 Academic Edi o : Łukasz Ch zanowski Recei ed: 4 Ma ch 2023 Re ised: 31 Ma ch 2023 Accep ed: 3 Ap il 2023 Published: 5 Ap il 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). oxics A icle An agonis ic Toxic E ec s o Su ac an s Mix u es o Bac e ia Pseudomonas pu ida and Ma ine Mic oalgae Phaeodac ylum ico nu um F ancisco Ríos * , Manuela Lechuga, Ismael Loba o-Gua nido and Me cedes Fe nández-Se ano Depa men o Chemical Enginee ing, Facul y o Sciences, Uni e si y o G anada, Campus Fuen e Nue a s/n, 18071 G anada, Spain *Co espondence: ios@ug .es; Tel.: +34-958-244-075 Abs ac : Su ac an s can be ound in an e e -widening a ie y o p oduc s and applica ions, in which he combina ion o se e al ypes o su ac an s is used o ein o ce hei p ope ies, looking o syne gis ic e ec s be ween hem. A e use, hey end o be disca ded in o was ewa e , ending up in aqua ic bodies wi h conce ning ha m ul and oxic e ec s. The aim o his s udy is he oxicological assessmen o h ee anionic su ac an s (e he ca boxylic de i a i e, EC) and h ee ampho e ic su ac an s (amine-oxide-based, AO), indi idually and in bina y mix u es o hem (1:1 w/w), o bac e ia Pseudomonas pu ida and ma ine mic oalgae Phaeodac ylum ico nu um. C i ical Micelle Concen a ion (CMC) was de e mined o demons a e he capaci y o educe su ace ension and he oxici y o he su ac an s and mix u es. Ze a po en ial ( ζ -po en ial) and micelle diame e (MD) we e also de e mined o con i m he o ma ion o mixed su ac an micelles. The Model o Toxic Uni s (MTUs) was used o quan i y he in e ac ions o su ac an s in bina y mix u es and o p edic i he concen a ion addi ion o esponse addi ion p inciple can be assumed o each mix u e. The esul s showed a highe sensi i i y o mic oalgae P. ico nu um o he su ac an s es ed and hei mix u es han bac e ia P. pu ida. An agonism oxic e ec s ha e been de ec ed in he mix u e o EC + AO and in one bina y mix u e o di e en AOs; his is o say, he mix u es showed lowe oxici y han expec ed. Keywo ds: oxici y; amine-oxide-based su ac an s; e he ca boxylic de i a i e su ac an s; mix u es; Pseudomonas pu ida;Phaeodac ylum ico nu um; an agonism; model o oxic uni s 1. In oduc ion Su ac an s a e commonly used in a wide ange o p oduc s, including soaps, de e - gen s, shampoos, and cleaning agen s, o in ag icul u al, indus ial, o o he comme cial applica ions. The global demand o su ac an s has inc eased by a ound 300%, exceeding he cu en wo ld p oduc ion o 3 million ons. pe yea [ 1 ]. A e use, mainly in aqueous solu ions, hey a e discha ged in o wa e s eams, eaching was ewa e ea men plan s and aqua ic bodies [ 2 ]. Due o hei su ace-ac i e p ope ies, and depending on hei concen a ion, hey a e oxic o aqua ic li ing o ganisms causing memb ane dis up ion, depola iza ion o cellula memb anes o in e ac ion wi h i al enzyma ic p o eins [ 3 – 6 ]. This makes he e alua ion o he oxic e ec s o su ac an s manda o y o ensu e ha hey a e sa ely and esponsibly used. Eco oxici y s udies a e necessa y o unde s and he isks associa ed wi h he use o hese compounds and o de elop sa e and mo e sus ainable al e na i es o adi ional su ac an s. Mix u es o di e en su ac an s (anionic, non-ionic and ampho e ic su ac an s) a e commonly used in o mula ions because o hei be e pe o mance compa ed wi h indi- idual su ac an s. Sui able mix u es o su ac an s a e mos ly used o inc ease e iciency and s abili y o o educe cos s and he po en ial o he p oduc s o cause skin and eye i i a ion [ 7 , 8 ]. Consequen ly, a e hei use, mix u es o su ac an s o a di e en na u e a e discha ged oge he in wa e s eams. Toxics 2023,11, 344. h ps://doi.o g/10.3390/ oxics11040344 h ps://www.mdpi.com/jou nal/ oxics Toxics 2023,11, 344 2 o 15 In ecen yea s, se e al oxicological s udies conce ning pollu an s ha e ocused on he join oxicological assessmen o co-pollu an s, e ealing ha mul iple in e ac ions be ween oxics can appea and need o be desc ibed [ 9 – 13 ]. In he case o su ac an s, while indi idual s udies o hem can p o ide aluable in o ma ion, hey may no e lec eal-wo ld exposu e o mul iple su ac an s a once. As happens wi h hei physicochemi- cal p ope ies, syne gis ic o an agonis ic e ec s can appea on he combined oxici y o su ac an s, meaning ha he combined e ec s a e g ea e o lowe han he sum o he in- di idual e ec s [ 14 – 17 ]. The oxici y o indi idual su ac an s has been ex ensi ely s udied on se e al aqua ic o ganisms, such as bac e ia, Vib io ishe i, mic oalgae, Pseudoki chne iella subcapi a a, o mic oc us aceans, Daphnia magna, especially o he adi ional and mo e common su ac an s [ 2 , 18 , 19 ]. Ne e heless, he e is a lack o esea ch ha s udies he combined e ec s o mix u es o su ac an s and e alua es he possibili y o syne gis ic, addi i e, o an agonis ic e ec s. In he case o mix u es ha exhibi syne gis ic e ec s, su ac an s may in e ac wi h each o he , and he combined oxici y o he su ac an s is g ea e han he sum o hei indi idual oxici ies. I he e is an addi i e e ec , i means ha he combined oxici y is equal o he sum o hei indi idual oxici ies. On he o he hand, in case o an an agonis ic e ec , su ac an s may in e ac being he mix u e less oxic han he sum o hei indi idual oxici ies, which is e y ele an om an eco- iendly poin o iew [20]. To add ess his gap in knowledge, i is impo an o conduc esea ch ha examines he e ec s o mul iple su ac an s and ocuses on he elucida ion o he ype o ac ion o hese mix u es on aqua ic o ganisms. The model o oxic uni s (MTU) [ 20 – 22 ] is used o quan i y he in e ac ions o oxican s in bina y mix u es o chemicals and o p edic i he concen a ion/dose addi ion p inciples can be assumed, meaning ha bo h su ac an s ac by he equal mode o ac ion (MoA). Equally, i allows us o p edic i he esponse addi ion p inciple can be expec ed; his is, su ac an s ac independen ly om each o he , usually by di e en MoAs ha do no in luence each o he [15]. Among comme cial su ac an s, e he ca boxylic de i a i e su ac an s a e equen ly used in cleaning, pe sonal ca e, and cosme ic o mula ions, p o iding an excellen oaming capaci y and low eye and skin i i a ion po en ial [ 23 ]. Amine-oxide-based su ac an s a e excellen oaming s abilize s in mix u es o anionic and ampho e ic su ac an s. I s addi- ion, hey inc ease he de e gency le el o he mix u e, being commonly used in cosme ic o mula ions, washing up liquids, and cleane s in gene al. The indi idual oxici y o bo h amilies o su ac an s o bac e ia V. ische i, eshwa e mic oalgae, and mic oc us aceans ha e been p e iously epo ed [ 16 , 18 , 23 ]. Ne e heless, oxici y o o he ele an and ep esen a i e species, such as Pseudomonas o ma ine mic oalgae, has no been s udied, and mo e impo an ly, he mode o ac ion and possible addi i e, syne gis ic o an agonis ic e ec s o hese su ac an mix u es has no e e been add essed. In his s udy, we e alua ed he oxici y o h ee anionic su ac an s (e he ca boxylic de i a i e, EC) and h ee ampho e ic su ac an s (amine-oxide-based, AO), indi idually and in bina y mix u es (1:1 w/w), o bac e ia Pseudomonas pu ida and ma ine mic oalgae Phaeodac ylum ico nu um.P. pu ida, which a e G am-nega i e bac e ia ha can be ound in su ace wa e s and a e commonly used in en i onmen al s udies because o hei capaci y o deg ade a wide ange o o ganic compounds and hei esis ance o en i onmen al s esso s [ 24 , 25 ]. They a e equen ly used o oxici y assessmen s, and a s anda d oxici y p ocedu e has been p oposed o he e alua ion o he g ow h inhibi ion o hese bac e ia caused by oxican s [ 26 , 27 ]. They can also be conside ed o be ep esen a i e he e o ophic mic oo ganisms in eshwa e and as a simpli ied model o an ac i a ed sludge bio ea men . In addi ion, he use o P. pu ida is p e e able o some o he Pseudomonas species as i is a sa e species o bac e ia, unlike P. ae uginosa, o example, which is an oppo unis ic human pa hogen. Ma ine mic oalgae Phaeodac ylum ico nu um a e impo an p ima y p oduce s in ma ine ecosys ems and a e sensi i e indica o s o en i onmen al s esso s, including oxic subs ances. Toxici y es ing wi h ma ine mic oalgae P. ico nu um is a common Toxics 2023,11, 344 3 o 15 me hod used o assess he po en ial impac o subs ances on ma ine aqua ic en i onmen s in a apid, sensi i e, and cos -e ec i e way [28–32]. This s udy ex ends he knowledge conce ning he oxici y o indi idual anionic su ac- an s (EC) and ampho e ic su ac an s (AO) and bina y mix u es o hem (anionic/anionic, anionic/ampho e ic and ampho e ic/ampho e ic) wi h wo ep esen a i e o ganisms om esh and ma ine wa e . Addi ionally, especially, i con ibu es o inc easing he knowledge conce ning he ype o ac ion o he su ac an s and iden i ies po en ial in e ac ions be ween su ac an s, p o iding mo e accu a e in o ma ion conce ning he isks associa ed wi h ex- posu e o aqua ic o ganisms o mix u es o su ac an s. This b ings aluable in o ma ion o he selec ion o su ac an s in he o mula ion o mo e eco- iendly p oduc s. 2. Ma e ials and Me hods 2.1. Su ac an s The oxici y o h ee anionic su ac an s (e he ca boxylic de i a i e su ac an s) and h ee ampho e ic su ac an s (amine-oxide-based su ac an s) supplied by Kao Chemicals Eu ope (Ba celona, Spain) was es ed. Amine-oxide-based su ac an s a e a special class o su ac an s ha ha e ca ionic beha io unde acid condi ions and a non-ionic cha ac e in neu al and basic mediums (pH ange o he oxici y es s la e desc ibed). Figu e 1shows hei molecula s uc u e, and Table 1shows hei abb e ia ion name, INCI name, CAS egis y numbe , leng h alkyl chain (R), e hoxyla ion deg ee (E), and % ac i e ma e . Toxics 2023, 11, x FOR PEER REVIEW 3 o 15 po an p ima y p oduce s in ma ine ecosys ems and a e sensi i e indica o s o en i on- men al s esso s, including oxic subs ances. Toxici y es ing wi h ma ine mic oalgae P. ico nu um is a common me hod used o assess he po en ial impac o subs ances on ma- ine aqua ic en i onmen s in a apid, sensi i e, and cos -e ec i e way [28–32]. This s udy ex ends he knowledge conce ning he oxici y o indi idual anionic su - ac an s (EC) and ampho e ic su ac an s (AO) and bina y mix u es o hem (anionic/ani- onic, anionic/ampho e ic and ampho e ic/ampho e ic) wi h wo ep esen a i e o ganisms om esh and ma ine wa e . Addi ionally, especially, i con ibu es o inc easing he knowledge conce ning he ype o ac ion o he su ac an s and iden i ies po en ial in e - ac ions be ween su ac an s, p o iding mo e accu a e in o ma ion conce ning he isks associa ed wi h exposu e o aqua ic o ganisms o mix u es o su ac an s. This b ings al- uable in o ma ion o he selec ion o su ac an s in he o mula ion o mo e eco- iendly p oduc s. 2. Ma e ials and Me hods 2.1. Su ac an s The oxici y o h ee anionic su ac an s (e he ca boxylic de i a i e su ac an s) and h ee ampho e ic su ac an s (amine-oxide-based su ac an s) supplied by Kao Chemicals Eu ope (Ba celona, Spain) was es ed. Amine-oxide-based su ac an s a e a special class o su ac an s ha ha e ca ionic beha io unde acid condi ions and a non-ionic cha ac e in neu al and basic mediums (pH ange o he oxici y es s la e desc ibed). Figu e 1 shows hei molecula s uc u e, and Table 1 shows hei abb e ia ion name, INCI name, CAS egis y numbe , leng h alkyl chain (R), e hoxyla ion deg ee (E), and % ac i e ma e . Figu e 1. Molecula s uc u e (a) e he ca boxylic de i a i e su ac an s, EC-R8E8, EC-R12–14E3, EC- R12–14E10, (b) amine-oxide-based su ac an s, AO-R14 and AO-R12, (c) amine-oxide-based su ac an , AO-Cocoamido. R: leng h alkyl chain; E: e hoxyla ion deg ee, R’:R12. Table 1. Chemical iden i ica ion and echnical speci ica ions o he su ac an s es ed. Abb e ia ion INCI Name 1 CAS 2 R 3 E 4 % Ac i e Ma e EC-R8E8 Cap yle h-9 ca boxylic acid 53563-70-5 8 8 92.8 EC-R12–14E3 Lau e h-4 ca boxylic acid 27306-90-7 12–14 3 92.5 EC-R12–14E10 Lau e h-11 ca boxylic acid 27306-90-7 12–14 10 89.1 AO-R12 Lau yl dime hyl amine oxide 1643-20-5 12 - 29.4 AO-R14 My is yl dime hyl amine oxide 3332-27-2 14 - 30.8 AOP-Cocoamido Cocoamidop opyl dime hyl amine oxide 68155-09-9 R’ = 12 - 34.3 1 INCI: in e na ional nomencla u e o cosme ics ing edien s. 2 CAS: Chemical Abs ac s Se ice eg- is y numbe . 3 R: alkyl chain leng h, n-CiH2i + 1-. 4 E: deg ee o e hoxyla ion–(OCH2CH2)nO. 2.2. Reagen s The eagen s used in he oxici y es s we e o analy ical g ade, supplied by Me cK KGaA (Da ms ad , Ge many). The solu ions we e p epa ed in ul apu e MilliQ® wa e ( esis i i y 18.2 MΩ·cm a 25 °C). The glasswa e in con ac wi h su ac an s was ca e ully cleaned wi h a dissolu ion o 16 gL−1 o ammonium pe oxydisul a e in sul u ic acid (98%). Figu e 1. Molecula s uc u e ( a ) e he ca boxylic de i a i e su ac an s, EC-R 8 E 8 , EC-R 12–14 E 3 , EC- R 12–14 E 10 , ( b ) amine-oxide-based su ac an s, AO-R 14 and AO-R 12 , ( c ) amine-oxide-based su ac an , AO-Cocoamido. R: leng h alkyl chain; E: e hoxyla ion deg ee, R’:R12. Table 1. Chemical iden i ica ion and echnical speci ica ions o he su ac an s es ed. Abb e ia ion INCI Name 1CAS 2R3E4% Ac i e Ma e EC-R8E8Cap yle h-9 ca boxylic acid 53563-70-5 8 8 92.8 EC-R12–14E3Lau e h-4 ca boxylic acid 27306-90-7 12–14 3 92.5 EC-R12–14E10 Lau e h-11 ca boxylic acid 27306-90-7 12–14 10 89.1 AO-R12 Lau yl dime hyl amine oxide 1643-20-5 12 - 29.4 AO-R14 My is yl dime hyl amine oxide 3332-27-2 14 - 30.8 AOP-Cocoamido Cocoamidop opyl dime hyl amine oxide 68155-09-9 R’ = 12 - 34.3 1 INCI: in e na ional nomencla u e o cosme ics ing edien s. 2 CAS: Chemical Abs ac s Se ice egis y numbe . 3R: alkyl chain leng h, n-CiH2i + 1-.4E: deg ee o e hoxyla ion–(OCH2CH2)nO. 2.2. Reagen s The eagen s used in he oxici y es s we e o analy ical g ade, supplied by Me cK KGaA (Da ms ad , Ge many). The solu ions we e p epa ed in ul apu e MilliQ ® wa e ( esis i i y 18.2 M Ω· cm a 25 ◦ C). The glasswa e in con ac wi h su ac an s was ca e ully cleaned wi h a dissolu ion o 16 gL −1 o ammonium pe oxydisul a e in sul u ic acid (98%). 2.3. Ac i e Ma e Analysis The ac i e ma e o he su ac an s es ed was measu ed by in a ed adia ion (model AD-4714A, A&D, Tokyo, Japan). The empe a u e o d ying was se a 105 ◦ C. The wa e Toxics 2023,11, 344 4 o 15 con en in he sample was egis e ed e e y 30 s o 90 min as he di e ence om he ini ial weigh . Ac i e ma e is calcula ed as 100% minus he wa e con en de e mined (Table 1). 2.4. C i ical Micelle Concen a ion The su ac an concen a ion a which su ac an molecules can agg ega e and o m micelles is known as he c i ical micelle concen a ion (CMC). The su ace ension o a concen a ion se ies ( anging om 0.1 o 1 × 10 4 mgL −1 ) was measu ed in o de o calcula e he CMC. Su ace ension plo s, as a unc ion o su ac an concen a ion in a semi-log plo , esul in a quick linea decline in su ace ension, ollowed by a g adual decline. The p oduc ion o micelles (CMC) is indica ed by he b eakpoin . The Wilhelmy Pla e Me hod was used o measu e su ace ension using a ensiome e model K11 (K üss GmbH, Hambu g, Ge many) i ed wi h a 2 cm pla inum pla e, in acco dance wi h he BS EN 14370:2004 guidelines [ 33 ]. Be o e each measu emen , he pla e was ca e ully cleaned and lame-d ied wi h a Bunsen bu ne . By sinking a e ical pla e in o he solu ion and hen pulling i ou again, he su ace ension was measu ed. The s anda d de ia ion did no su pass 0.1 mNm −1 du ing he six subsequen obse a ions. The jacke ed cell was illed wi h he mos a ed wa e , which main ained he empe a u e a 25 ± 0.5 ◦ C. CMC de e mina ions we e pe o med in iplica e o ob ain a mean CMC and i s con idence in e al (95%). 2.5. Ze a Po en ial (ζ-Po en ial) and Micelle Diame e (MD) The Ze a po en ial (a measu e o he magni ude o he elec os a ic o cha ge epul- sion/a ac ion be ween micelles) o he indi idual and mix u es o su ac an s (1:1 w/w) unde he condi ions o he oxici y es o P. pu ida ( eshwa e ) and P. ico nu um (ma ine medium) was analyzed using a Ze asize Ul a (Mal e n Panaly ical L d., Mal e n, UK) by he echnique o Elec opho e ic Ligh Sca e ing (ELS). The Ze asize sys em was also used o measu e he a e age micella hyd odynamic diame e (MD) o he su ac an s es ed abo e he CMC in he wo-cul u e media by dynamic ligh sca e ing (DLS). The de ec ion limi o he equipmen is 0.3 nm o 10 mm. The ins umen se ings we e op imized au o- ma ically by means o he ZS XPLORER so wa e (Mal e n Panaly ical L d., Mal e n, UK). All o he expe imen s we e pe o med a a con olled empe a u e (25 ± 1 ◦ C) in iplica e. 2.6. Pseudomonas Pu ida Toxici y Tes The P. pu ida oxici y es was ca ied ou ollowing he guideline ISO 10712:1995 (Wa e quali y—Pseudomonas pu ida g ow h inhibi ion es [ 26 ]. A monocul u e s ain Pseudomonas pu ida CECT 324, p o ided by he Spanish Type Cul u e Collec ion (València, Spain), was used in he oxici y es s. Bac e ia we e supplied in a lyophilized o m. These cul u es we e e i ed on a solid aga Pe i dish a pH 7.2, 1 gL −1 bee ex ac , 2 gL −1 yeas ex ac , 5 gL −1 pep one, 5 gL−1NaCl, and 15 gL−1aga powde . P. pu ida was incuba ed on he su ace o he solid s ock cul u e (1 gL −1 NaNO 3 , 0.24 gL −1 K 2 HPO 4 , 0.12 gL −1 KH 2 PO 4 , 0.1 gL −1 yeas ex ac , 10 gL −1 glucose, 0.4 gL −1 MgS0 4· 7H 2 O, 0.001 gL −1 e ic ci a e and 18 gL −1 aga powde ) in slan ubes o 24 h. A e he incuba ion pe iod, he cells we e insed wi h a p ecul u e medium (0.5 gL −1 NaNO 3 , 0.12 gL −1 K 2 HPO 4 , 0.06 gL −1 KH 2 PO 4 , 0.05 gL −1 yeas ex ac , 2 gL −1 glucose, 0.2 gL −1 MgS04 · 7H 2 O and 0.0005 gL −1 e ic ci a e) and ans e ed in o a lask wi h a u bidi y o 10 FNU (A610 nm, 0.02) using p ecul u e medium. This inoculum was incuba ed o 7 days a 25 ± 1 ◦ C wi h cons an agi a ion. The inoculum was added o he p ecul u e medium and incuba ed o 5 ± 0.5 h a 23 ± 1 ◦ C in a shake . When he incuba ion ended, he bac e ial suspension was dilu ed o 50 FNU (A610 nm, 0.1). Fu he mo e, 10 mL o his p ecul u e was used o inocula e he lasks wi h di e en su ac an concen a ions. The pH o he solu ions was adjus ed o 7.4 ± 0.3 wi h ei he 1 N HCl o 1 M NaOH be o e he assay was ini ia ed. Toxics 2023,11, 344 5 o 15 The lasks we e incuba ed in he da k o 16 h a 23 ± 1 ◦ C in a shake . A e he incuba ion, he u bidi y in he es lask was measu ed using a spec opho ome e a 610 nm. The g ow h inhibi ion was e alua ed as he change in he abso bance o he bac e ial cul u e be o e and a e exposu e. The pe cen age o he inhibi ion o cell mul iplica ion (I) o each concen a ion o he es ed su ac an was calcula ed using he ollowing equa ion: I(%)=BC−Bn BC−B0 ·100 (1) whe e Iis he cell mul iplica ion inhibi ion [%], B n is he u bidi y o he biomass measu ed a e he es ime o he ump een h concen a ion in he es lask, B c is he u bidi y o he biomass measu ed a e he es ime in he con ol lask, and B 0 is he alue o he u bidi y o he biomass measu ed a ime 0 in he con ol lask. The alues o Iwe e plo ed agains he su ac an concen a ions. The oxici y alues we e measu ed as EC 50 , which is he su ac an concen a ion ha inhibi s 50% a e 16 h o exposu e. 2.7. Ma ine Algae Toxici y Tes The 72 h algal g ow h-inhibi ion es wi h he ma ine algal Phaeodac ylum ico nu um was adminis e ed acco ding o he Guideline ISO 10253:2006 (Wa e quali y—Ma ine algal g ow h inhibi ion es wi h Skele onema cos a um and Phaeodac ylum ico nu um) [ 34 ]. The mic oalgae inoculum, supplied by Mic obio es Inc. (Gen , Belgium), was incuba ed o 3 days a 20 ± 2 ◦ C, wi h cons an uni o m illumina ion (10,000 lux) in a g ow h medium p epa ed wi h he addi ion o 1 L: 22 g NaCl, 9.7 g MgCl 2· 6H 2 O, 3.7 g Na 2 SO 4 , 1.0 g CaCl 2 , 0.65 g KCl, 0.2 g NaHCO 3 and 40.1 mg H 3 BO 3 , 0.72 mg FeCI 3· 6H 2 O, 2.16 mg MnCI 2· 4H 2 O, 0.68 mg ZnSO 4· 7H 2 O, 2.4 µ g CuSO 4· 5H 2 O, 6.1 µ g CoCI 2· 6H 2 O, 15 mg Na 2 EDTA, 25 µ g Thiamin hyd ochlo ide, 0.05 µ g bio in, 0.05 µ g i amin B 12 , 3 µ g K 3 PO 4 , 40 µ g NaNO 3 , and 14.9 µ g Na 2SiO 3· 5H 2 O. A e 3 days o incuba ion, he inoculum o he algae was added o he ials wi h he subs ance o be es ed and o he con ol ials o ob ain an algae concen a ion o 1 · 10 4 algae cells mL −1 . Fi e concen a ions o he a ge subs ance and con ol we e pe o med in iplica e in 10 cm pa h leng h ials. The ials we e kep a 20 ±2◦C unde cons an uni o m illumina ion (10,000 lux) o 72 h, and he pH was se o 8.0 ±0.2 wi h ei he 1 N HCl o 1 M NaOH. A e 24, 48, and 72 h, he op ical densi y o he cul u e a 670 nm was de e mined o es ablish whe he g ow h had been inhibi ed o s imula ed wi h espec o he con ol. Speci ic g ow h a es (µ) we e calcula ed using Equa ion (2). µih−1=lnNL−lnN0 L− 0 (2) whe e 0 is he ime o he es s a , L is he ime o he es de e mina ion (24, 48, and 72 h ), N0is he nominal ini ial cell densi y, and NLis he measu ed cell densi y a ime L. The pe cen age inhibi ion o each es concen a ion (I µi ) was calcula ed om Equa ion (3): Iµi(%)=µc−µi µc ·100 (3) whe e µcis he mean g ow h a e o he con ol and µi he g ow h a e o es ial i. A linea ela ionship can be deduced be ween he inhibi ion and he concen a ion o he su ac an used in he ollowing o m: log(i)=A·I+B(4) EC 50 ep esen s he concen a ion o su ac an ha caused a 50% g ow h inhibi ion a e 72 h o exposu e. Th ee eplica e es s we e pe o med o ob ain a mean EC 50 and i s con idence in e al (95%). Toxics 2023,11, 344 6 o 15 2.8. Model o Toxic Uni s The model o oxic uni s (MTU) [ 20 – 22 ] was used o quan i y he in e ac ions o he oxican s and o p edic i he concen a ion/dose o esponse addi ion p inciples can be assumed. TU i is he ela ion o he concen a ion o he oxican in a mix u e [i] and i s hal maximal e ec i e concen a ion (EC 50i ), (Equa ion (5)) being he oxic uni o he mix u e (TUmix), he sum o TUio he single componen s (Equa ion (6)). TUi=[i] EC50i (5) TUmix =TUA+TUB=[A] EC50A +[B] EC50B (6) Acco ding o he bibliog aphy and p e ious s udies, simple addi i i y (concen a ion addi ion) is cha ac e ized by 0.8 < TU mix < 1.2, while TU mix ≤ 0.8 indica es syne gism (mo e han addi i e) and TUmix ≥1.2 ep esen s an agonism (less han addi i e) [35–37]. I oxican s wo k independen ly om each o he ( esponse addi ion), he p edic ed oxic uni (TU ) needs o be calcula ed by Equa ions (7) and (8). When TU ≈ 1, he esponse addi ion can be expec ed [33]. I TUA>TUBTU =1+TUB TUA (7) I <TUBTU =1+TUA TUB (8) 2.9. S a is ical Analysis S a is ical analysis was pe o med using he s a is ical p og am S a g aphics 5.1. The da a we e subjec ed o analysis o a iance (ANOVA). Tukey’s mul iple ange es [ 38 ] was used o de e mine i signi ican di e ences exis ed be ween he g oups o oxici y da a o bac e ia P. pu ida and mic oalgae P. ico nu um. The di e ences be ween he mean alues we e conside ed signi ican a a le el o con idence o 95%. 3. Resul s 3.1. C i ical Micelle Concen a ion (CMC) CMC is a e y ele an pa ame e in he case o oxici y s udies o su ac an s, as i indi- ca es i he su ace ension in he solu ion has been educed o he minimum, concen a ion abo e (CMC), o su ac an monome s a e only si ua ed in he ai –wa e in e ace (below CMC), which can a ec he a ailabili y o su ac an molecules o pa i ion in o cellula memb anes and cause oxici y. Highe oxici y is co ela ed wi h highe hyd ophobici y [ 39 ] bu is also dependen on CMC since oxic e ec s, speci ically o non-ionic su ac an s, ake place a su ac an concen a ions nea o abo e he CMC [ 40 , 41 ]. The CMC alues o he su ac an s s udied ha e been epo ed in p e ious s udies [ 16 , 23 ]; ne e heless, CMCs ha e been again de e mined wi h he ba ch o su ac an s es ed in his s udy. CMCs o he mix u es (1:1 w/w) ha e also been de e mined, and he pa ame e o he CMCU (C i ical Micelle Concen a ion Uni ) has been calcula ed as he ela ionship be ween he CMC ob ained o he mix u e and he a e age CMC alues o he indi idual su ac an s. Table 2shows he CMC and CMCU alues de e mined o he indi idual su ac an s and he mix u es es ed. Toxics 2023,11, 344 7 o 15 Table 2. CMC (95% CI) and CMCU o su ac an s and su ac an s mix u es (1:1 w/w). Su ac an CMC (mgL−1) CMCU EC-R8E8214.2 ±5.2 - EC-R12–14E328.6 ±4.8 - EC-R12–14E10 66.9 ±7.2 - AO-R12 156.0 ±10.5 - AO-R14 102.4 ±3.2 - AOP-Cocoamido 301.3 ±5.1 - EC-R8E8+ EC-R12–14E10 100.2 ±1.57 0.71 EC-R8E8+ AO-Cocoamido 442.7 ±7.6 1.71 AO-R12 + AO-Cocoamido 403.8 ±12.4 1.76 AO-R14 + AO-Cocoamido 157.9 ±6.1 0.78 3.2. Ze a Po en ial and Micelle Diame e The ζ -po en ial and a e age MD o he indi idual and su ac an mix u es (1:1 w/w) in he g ow h medium o P. pu ida and P. ico nu um ha e been measu ed o con i m he o ma ion o mixed micelles and o unde s and he e ec o he g ow h medium on he s abili y and size o he micelle o med. The esul s a e shown in Table 3, including he s anda d de ia ion (SD) o h ee eplica es. Table 3. ζ -po en ial and MD o su ac an s and su ac an s mix u es (1:1 w/w) in cul u e media o P. pu ida and P. ico nu um. Su ac an ζ-Po en ial P. pu ida Medium, (±SD) MD P. pu ida Medium, (nm ±SD) ζ-Po en ial P. ico nu um Medium, (±SD) MD P. ico nu um Medium, (nm ±SD) EC-R8E8−13.6 ±0.5 123.3 ±19.2 −3.5 ±1.3 162.6 ±22.0 EC-R12–14E3−51.9 ±2.2 19.9 ±2.3 −28.7 ±1.1 18.1 ±1.3 EC-R12–14E10 −9.0 ±1.1 5.6 ±0.4 −2.4 ±1.3 6.7 ±0.1 AO-R12 −27.8 ±1.5 52.4 ±0.8 −11.0 ±0.6 195.0 ±4.5 AO-R14 −14.7 ±1.3 8.8 ±0.1 −6.9 ±0.7 7.1 ±1.1 AOP-Cocoamido −12.7 ±0.2 54.1 ±8.7 −9.1 ±0.4 326.9 ±19.8 EC-R8E8+ EC-R12–14E10 −11.3 ±1.1 7.0 ±0.5 −5.9 ±0.6 6.4 ±0.5 EC-R8E8+ AO-Cocoamido −12.8 ±1.1 123.4 ±12.0 −7.1 ±0.2 36.0 ±1.1 AO-R12 + AO-Cocoamido −23.3 ±3.4 48.2 ±8.2 −10.4 ±2.1 68.6 ±2.7 AO-R14 + AO-Cocoamido −12.0 ±1.3 10.8 ±2.6 −10.0 ±1.5 8.9 ±1.6 3.3. Toxici y o Indi idual Su ac an s The oxici y o he indi idual anionic and he ampho e ic su ac an s o bac e ia P. pu ida (exposu e ime: 16 h) and ma ine mic oalgae P. ico nu um (exposu e ime: 72 h ) was de e mined. The esul s a e shown in Table 4. The oxici y alues a e indica ed as EC 50 (concen a ion o he subs ance es ed in wa e causing a 50% o g ow h inhibi ion). The alues anged om 28.8 mgL −1 o 443.95 mgL −1 o P. pu ida and om 4.27 mgL −1 o 93.05 mgL−1 in he case o P. ico nu um. The esul s e eal a highe sensi i i y o he ma ine mic oalgae P. ico nu um han bac e ia P. pu ida o he su ac an s es ed. Toxics 2023,11, 344 8 o 15 Table 4. Toxici y alues (95% CI) o indi idual su ac an s o bac e ia P. pu ida (exposu e ime: 16 h and mic oalgae P. ico nu um (exposu e ime: 72 h). Su ac an EC50,P. pu ida mgL−1EC50,P. ico nu um mgL−1 EC-R8E8443.95 ±10.24 93.05 ±8.21 EC-R12–14E3331.54 ±15.77 13.14 ±2.25 EC-R12–14E10 370.37 ±15.02 53.14 ±1.22 AO-R12 93.48 ±4.76 29.72 ±2.91 AO-R14 28.8 ±2.48 4.27 ±1.03 AOP-Cocoamido 311.48 ±9.54 57.66 ±5.48 3.4. Toxici y o Su ac an s Mix u es Tables 5and 6show he oxici y alues o he mix u es (1:1 w/w) o he su ac an s es ed o bac e ia P. pu ida (exposu e ime: 16 h) and ma ine mic oalgae P. ico nu um (exposu e ime: 72 h), espec i ely. They include a mix u e o wo anionic su ac an s (EC- R 8 E 8 + EC-R 12–14 E 10 ), a mix u e o an e he ca boxylic de i a i e su ac an and he less oxic amine-oxide-based su ac an s (EC-R 8 E 8 + AO- Cocoamido ), and wo bina y mix u es o he ampho e ic su ac an s (AO-R 12 + AO- Cocoamido and AO-R 14 + AO- Cocoamido ). The oxici y o mix u es using he anionic su ac an EC-R 12–14 E 3 is no included since he solu ions o his su ac an wi h he o he e he ca boxylic de i a i e su ac an s and amine-oxide-based su ac an s showed u bidi y in he concen a ion ange o he es s, making i impossible o de e mine he g ow h inhibi ion o bac e ia o mic oalgae by spec opho ome ic measu emen s. In he same way, i was no possible o de e mine he oxici y o he mix u e o AO-R12 and AO-R14. Using he model o Toxics Uni s p e iously desc ibed [ 20 – 22 ], he oxic uni s o he single componen s (TU A , TU B ), oxic uni s o he mix u es (TU mix ), and he p edic ed oxic uni s (TU ) we e calcula ed. The alues, oge he wi h he conclusions on he ype o ac ion o he mix u es es ed o bac e ia P. pu ida and mic oalgae P. ico nu um, a e included in Tables 5and 6, espec i ely. Table 5. Toxici y alues and MTU pa ame e s o su ac an s mix u es (1:1 w/w) o bac e ia P. Pu ida (exposu e ime 16 h). Su ac an A Su ac an B EC50,P. pu ida mgL−1(95% CI) TUATUBTUmix TU Type o Ac ion EC-R8E8EC-R12–14E10 414.71 ±18.33 0.47 0.56 1.03 1.83 Concen a ion addi ion EC-R8E8AO-Cocoamido 461.74 ±22.15 0.52 0.74 1.26 1.70 Less han addi i e (an agonism) AO-R12 AO-Cocoamido 210.98 ±11.49 1.13 0.34 1.47 1.30 Less han addi i e (an agonism) AO-R14 AO-Cocoamido 41.89 ±3.87 0.73 0.07 0.79 1.09 Response addi ion Table 6. Toxici y alues and MTU pa ame e s o su ac an s mix u es (1:1 w/w) o ma ine mic oalgae P. ico nu um (exposu e ime: 72 h). Su ac an A Su ac an B EC50,P. ico nu um mgL−1(95% CI) TUATUBTUmix TU Type o Ac ion EC-R8E8EC-R12–14E10 55.3 ±10.10 0.30 0.52 0.82 1.57 Concen a ion addi ion EC-R8E8AO-Cocoamido 69.91 ±4.45 0.38 0.61 0.98 1.62 Concen a ion addi ion AO-R12 AO-Cocoamido 65.24 ±14.70 1.10 0.57 1.66 1.52 Less han addi i e (an agonism) AO-R14 AO-Cocoamido 12.58 ±4.18 1.47 0.11 1.58 1.07 Response addi ion Toxics 2023,11, 344 9 o 15 4. Discussion 4.1. Ze a Po en ial and Micelle Diame e o Indi idual and Mix u es o he Su ac an Solu ions All o he su ac an solu ions es ed showed a nega i e ζ -po en ial anging om − 51.9 o − 2.4, showing he lowes alue o he indi idual anionic su ac an EC-R 12–14 E 3 in he P. pu ida medium (Table 3). As expec ed, he ζ -po en ial is highe o su ac an s in he ma ine mic oalgae medium, indica ing he lowe s abili y o he su ac an micelles caused by he shielding e ec [ 42 , 43 ]. Compa ing he ζ -po en ial o he su ac an mix u es wi h he ζ -po en ial o he indi idual su ac an s, i can be obse ed ha app oxima ely in e media e alues we e ob ained o bo h cul u e media (P. pu ida and P. ico nu um), and his indica es he o ma ion o mixed micelles in he mix u e solu ions. I can only be app ecia ed ha o he mix u e o AO-R 14 and AO- Cocoamido , he ζ -po en ial in he P. pu ida medium is sligh ly abo e he ζ -po en ial o he indi idual su ac an s, and he ζ -po en ial o he mix u e EC-R 8 E 8 and EC-R 12–14 E 10 in he P. ico nu um medium is sligh ly below he ζ -po en ial o he indi idual su ac an s. Ne e heless, conside ing he s anda d de ia ion and expe imen al e o s, hese di e ences can be conside ed o be negligible. In he case o he MD o he indi idual su ac an s (Table 3), he esul s show a highe MD o he e he ca boxylic de i a i e su ac an wi h he sho e alkyl chain and lowe hyd ophobici y (EC-R 8 E 8 ), whe eas he MD o he e he ca boxylic de i a i e wi h he longes alkyl chain (EC-R 12–14 E 10 ) shows he lowes alue in bo h cul u e media. The concen a ion o sal s in he medium had a di ec e ec on he a e age MD o mos o he su ac an s; his is, he MD o he indi idual su ac an s is highe in he P. ico nu um medium han in he P. pu ida medium. This could be ela ed o he highe s abili y o he micelles (lowe ζ -po en ial) in he P. pu ida medium; consequen ly, he possibili ies o in e ac ions be ween micelles o o m bigge micelles a e p e en ed. On he o he hand, i is especially ema kable ha he g ea MD o he amine-oxide-based su ac an AO- Cocoamido in he P. ico nu um medium, which is six imes highe han i s MD in he P. pu ida medium, which could be in luenced by he in e ac ion o sal s wi h he amide g oup exis ing in he a y alkyl chain. Rega ding he a e age MD o he mix u es o he su ac an s, o mos o hem, he MD is lowe han he MD o he indi idual su ac an s o close o he lowes alue o he indi idual su ac an s, e ealing he p e e ence o he su ac an s monome s o o m smalle micelles i hey a e included in mixed micelles. I is ema kable ha in he mix u e EC-R 8 E 8 + AO- Cocoamido , he MD in a ma ine medium is much smalle han he MD o he indi idual su ac an s. I is wo h no ing ha , in he case o mix u es, he MD is he a e age o mul iple possible combina ions o mixed micelles con aining monome s om su ac an s A and B. 4.2. Toxici y o Indi idual Su ac an s O e all, he su ac an s es ed a e mo e oxic o ma ine mic oalgae P. ico nu um han o bac e ia P. pu ida (Table 4). The alues o EC 50 o P. pu ida we e be ween 3 and 40 imes highe han EC 50 o P. ico nu um. Especially ele an is he di e ence o he anionic su ac an EC-R 12–14 E 3 . The esul s con as wi h some esul s ound in simila s udies wi h o he species o he same ophic le els. In p e ious s udies [ 16 , 35 , 44 ], we ound highe ox- ici y o polyoxye hylene glyce ol es e s, e he ca boxylic de i a i es, alkylpolyglucosides and amine-oxide-based su ac an s o ma ine bac e ia V. ische i compa ed wi h eshwa e mic oalgae P. subcapi a a. The e a e no published oxici y da a a ailable ega ding he su ac an s and species es ed in his s udy o compa ison. Howe e , he di e ences could be ela ed o he di e en beha io o he su ac an s on saline and eshwa e mic oalgae mediums, co obo a ed by measu emen s o he CMC, ζ -po en ial and MD in he di e en media (Tables 2and 3). In a medium wi h a highe concen a ion o sal s, su ac an s can easily dec ease he su ace ension a a lowe su ac an concen a ion [ 45 ], which is di ec ly ela ed o he oxici y e ec on mic oo ganisms. In he case o mic oalgae, su ac an s can induce mo phological changes in he cells a ec ing he s uc u e and in eg i y o lipid mem- b anes, dena u ing he memb ane p o eins, and consequen ly, inc easing he pe meabili y