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