Chemosphe e 312 (2023) 137028
A ailable online 30 Oc obe 2022
0045-6535/© 2022 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
Eco oxicological assessmen o he e ec s o luoxe ine on Daphnia magna
based on acu e oxici y, mul igene a ional ep oduc ion e ec s, and
a ac ion- epellence esponses
Helmu S emmel
a
,
b
,
*
, Linda Weiss
b
, Gema Pa a
c
, Eloísa Ramos-Rod íguez
d
,
C is iano V.M. A aújo
a
,
**
a
Depa men o Ecology and Coas al Managemen , Ins i u e o Ma ine Sciences o Andalusia (ICMAN – CSIC), 11510, Pue o Real, Cadiz, Spain
b
Depa men o Animal Ecology, E olu ion and Biodi e si y, Ruh Uni e si ¨
a Bochum, 44801 Bochum, Ge many
c
Animal Biology, Plan Biology and Ecology Depa men , Uni e si y o Ja´
en, 23071 Ja´
en, Spain
d
Ins i u e o Wa e Resea ch, Uni e si y o G anada, 18071 G anada, Spain
HIGHLIGHTS GRAPHICAL ABSTRACT
•E ec s o luoxe ine on D. magna we e
s udied: su i al, beha iou , and
ep oduc ion.
•Fluoxe ine had no clea impac on
ep oduc ion in mul igene a ional
expe imen s.
•P e ious exposu e o luoxe ine did no
al e he colonisa ion esponse.
•Fluoxe ine igge ed ea ly p oduc ion o
neona es in he 21-day ch onic es .
•An a ac ion a highe concen a ions
was obse ed in he a oidance
expe imen s.
ARTICLE INFO
Keywo ds:
A oidance
Colonisa ion
Mul igene a ional
Rep oduc ion
SSRI
ABSTRACT
Fluoxe ine, a common pha maceu ical used as an an idep essan , is al eady conside ed po en ially haza dous o
bio a due o i s inc easing use and de ec ion in Eu opean, No h Ame ican, and Asian i e s. We s udied he
e ec s o luoxe ine on Daphnia magna, as we hypo hesized ha luoxe ine migh ha e ha m ul e ec s, sho and
long- e m, a di e en le els: su i al, beha iou , and ep oduc ion (o sp ing p oduc ion). We applied wo
di e en app oaches: (i) a scena io a en i onmen ally ele an concen a ions (0.1–1.0
μ
g/L) and (ii) a scena io
simula ing a u u e wo sening o con amina ion (1–800
μ
g/L) un il he each o le hal concen a ions. In he
o me , we examined whe he he e a e mul igene a ional e ec s on ep oduc ion and on he a oidance/colo-
nisa ion beha iou in p e iously exposed popula ions. In he la e , h ee esponses we e assessed: su i al,
a oidance beha iou and ep oduc ion. We did no de ec di e ences in he ep oduc ion ou pu o D. magna
among he ea men s o e he h ee gene a ions examined. I espec i e o he mul igene a ional ea men , D.
magna colonised he en i onmen s wi h luoxe ine in a simila way. In he second scena io, we de e mined he
le hal concen a ion o 50% o he popula ion (96 h-LC
50
=365
μ
g/L), which, in spi e o he oxic e ec , was
* Co esponding au ho . Depa men o Ecology and Coas al Managemen , Ins i u e o Ma ine Sciences o Andalusia (ICMAN – CSIC), 11510, Pue o Real, Cadiz,
Spain.
** Co esponding au ho .
E-mail add esses: [email p o ec ed] (H. S emmel), [email p o ec ed] (C.V.M. A aújo).
Con en s lis s a ailable a ScienceDi ec
Chemosphe e
jou nal homepage: www.else ie .com/loca e/chemosphe e
h ps://doi.o g/10.1016/j.chemosphe e.2022.137028
Recei ed 30 Augus 2022; Recei ed in e ised o m 19 Oc obe 2022; Accep ed 24 Oc obe 2022
Chemosphe e 312 (2023) 137028
2
a ac i e o o ganisms du ing he a oidance es s (24 h); in ac , D. magna we e a ac ed (no epellence) e en o
he highes concen a ions o luoxe ine es ed (800
μ
g/L). Las ly, in a 21-day ch onic oxici y es he ep o-
duc ion ou pu o D. magna inc eased wi h highe concen a ions o luoxe ine. This e ec migh be ela ed o he
ac ha he o ganisms in he con amina ed ea men began hei i s ep oduc ion ea lie , when compa ed o
ha in he con ol ea men s. In conclusion, his s udy discusses an iden i ied haza d o aqua ic bio a due o he
luoxe ine a ac ion e ec and a p edic i e assessmen o he consequences expec ed i i s indisc imina e use
inc eases.
1. In oduc ion
Fluoxe ine ( adema k P ozac®) is a pha maceu ical compound,
adminis e ed as a acemic mix u e, which belongs o he g oup o
Selec i e-Se o onin-Reup ake Inhibi o s (SSRIs) and is widely p e-
sc ibed in i s wa e soluble chemical o m as a hyd ochlo ide sal
( luoxe ine hyd ochlo ide) (Wen hu e al., 2013; Risley and Bopp,
1990). Fluoxe ine is a neu oac i e subs ance ha inhibi s he eup ake
o se o onin, which leads o an inc easing ex acellula se o onin le el
(Bla di e al., 2002; Bymas e e al., 2002); he e o e, i is widely used as
a ea men o dep essions, ea ing-diso de s, compulsi e beha iou and
panic diso de among o he s (U.S. FDA, 2021). In he yea 2018,
luoxe ine anked 23 in he USA on he lis o he mos p esc ibed d ugs
(Kane, 2021). I s use has inc eased o e he yea s, as documen ed by he
U.S. Depa men o Heal h and Human Se ices (2021), and he e is a
g owing conce n ega ding he p esence o luoxe ine in some aqua ic
ecosys ems. In ac , luoxe ine has been de ec ed in un ea ed was e-
wa e s and e luen s o was ewa e ea men plan s (WWTP) a con-
cen a ions ha a y be ween 0.012 and 3.5
μ
g/L (B ooks e al., 2003;
Schul z e al., 2010; W i e e al., 2013; Ribei o e al., 2014). Fluoxe ine
canno be elimina ed comple ely by WWTPs as shown by Yuan e al.
(2013) and Cao e al. (2020). In ac , Ma e al. (2018) epo ed ha he
e luen s o WWTPs had an e en highe concen a ion; up o 300%. A
g adien om ea ed was ewa e s, eshwa e and sal wa e sys ems
has shown maximum concen a ions o 2.7
μ
g/L, 0.3
μ
g/L and 0.03
μ
g/L, espec i ely (Mole and B ooks, 2019). To ci e some examples, he
p esence o luoxe ine has been eco ded in some US i e s, s eams and
was ewa e s (E icson e al., 2002; W i e e al., 2013), in Spanish i es
(Fe n´
andez e al., 2010; L´
opez-Rold´
an e al., 2010), in i e s and ap
wa e in Poland (Giebuł owicz e al., 2014), in wells ha a e a wa e
supply o he popula ion in India (Fick e al., 2009) and in sewage and
in ecei ing wa e s in No way (Vasskog e al., 2008) and China (Wu
e al., 2017; Ma e al., 2018). The Global Bu den o Disease S udy has
shown an inc ease o 50% o diagnosed dep essions wo ldwide om
1990 o 2017 (Liu e al., 2020), so i is o be expec ed ha concen a ions
o an idep essan s such as luoxe ine will inc ease u he in aqua ic
sys ems, which equi es mo e s udies ega ding i s en i onmen al e -
ec s. In spi e o luoxe ine being a globally used d ug o almos 50
yea s, he da a a ailable abou he concen a ion o luoxe ine, i s a e in
he en i onmen and he impac i has on he aqua ic ecosys ems a e
a he spa se.
Al hough he e a e ew s udies on he e ec s o luoxe ine on non-
a ge o ganisms, he esul s show haza ds a di e en le els. Fo
ins ance, G zesiuk and Pawelec (2021) ound a delay in he esponse
ime o a possible dange ous si ua ion (e.g. a p eda o ) in Neogobius
lu ia ilis (monkey goby) and Dua e e al. (2020) showed ha luoxe ine
was accumula ed in ish issue (A gy osomus egius; Salmon-bass)
causing DNA damage in li e cells, a ec ing g ow h, and igge ing
an ioxidan de ence esponses. In he clam Tegilla ca g anosa (blood
clam), luoxe ine educes he haemocy e iabili y and imposes physio-
logical s ess, which leads o a supp essed immune esponse (Shi e al.,
2019). The accumula ion o luoxe ine in he b ain issue o he ish
Ca os omus comme sonii (whi e sucke ) was epo ed by Schul z e al.
(2010). Fong (1998) showed an e ec o induced spawning in D eissena
polymo pha (zeb a mussels) by luoxe ine. E ec s s udied on Ce -
iodaphnia dubia ha e shown a educ ion in he numbe o neona es and
b oods p oduced (Hen y e al., 2004). In D. magna, luoxe ine in e e es
wi h sexual ep oduc ion by inc easing he clu ch size, and leading o
smalle -sized o sp ing (Campos e al., 2012, 2016). I was epo ed by
Campos e al. (2012) ha he e ec ha luoxe ine has on D. magna
ep oduc ion is simila o hose p oduced by low ood quali y and
a ailabili y. Aulseb ook e al. (2022) showed ha he e ec s o luoxe-
ine on ecundi y, body size and he in insic g ow h a e o D. magna a e
dependen on geno ypes and ha in combina ion wi h di e en em-
pe a u es he e ec s may di e ma kedly. Fluoxe ine accumula ed in
D. magna wi hin 48 h and inc eased he il a ion a e as epo ed by
Ding e al. (2017).
Conside ing he e idence o he en i onmen al isks o luoxe ine
conce ning non- a ge o ganisms, we hypo hesized ha luoxe ine
migh ha e nega i e e ec s, in he sho and e en long e m, on di e en
li e-his o y ai s (su i al and ep oduc ion) and on di e en beha -
iou al esponses (colonisa ion and a oidance). The e o e, wo scena ios
we e simula ed: (i) a eal scena io a en i onmen ally ele an con-
cen a ions and (ii) a u u e wo se scena io a highe concen a ions
simula ing a se e e con amina ion e en un il he each o le hal con-
cen a ions. The goals in he i s scena io we e o assess how luoxe ine
a ec s D. magna ep oduc ion and colonisa ion beha iou a e mul i-
gene a ional exposu e o low and high le els o luoxe ine. To achie e
his, we analysed he e ec s o luoxe ine on o sp ing p oduc ion,
ephippia p oduc ion ( es ing eggs) and mo ali y a es o e h ee gen-
e a ions (F1, F2 and F3). The beha iou al e ec on he F3 gene a ion
was analysed by assessing he capaci y o o ganisms, p e iously exposed
o luoxe ine, o colonise habi a s con amina ed wi h di e en concen-
a ions o luoxe ine. In he second scena io, h ee app oaches we e
employed: (a) acu e exposu e o ob ain he le hal concen a ion (LC
50
)
o luoxe ine o e a pe iod o 96 h, (b) beha iou al e ec s using
a oidance assays o de e mine any epellen e ec o luoxe ine and (c)
sub-le hal consequences o assess how luoxe ine a ec s he ep oduc-
ion o D. magna using a 21-day ch onic oxici y es (OECD, 2004;
OECD, 2012).
2. Ma e ial and me hods
2.1. Fluoxe ine
Fluoxe ine-Hyd ochlo ide (CAS-No 56296-78-7, LOT-No LRAC3045,
o analy ical s anda d, 100% pu i y pu chased om Sigma-Ald ich,
made in he USA) was employed in he expe imen s. We p epa ed a
100 mL solu ion o 20 mg/L (nominal concen a ion) wi h Milli-Q wa e
and p ese ed i in a glass bo le, a 4 ◦C. Concen a ions o luoxe ine
we e de e mined ollowing he me hods desc ibed by Baena-Nogue as
e al. (2016). The ex ac ion o aqueous samples was ca ied ou by using
on-line ex ac ion (OLE), di ec ly coupled o Ul a Pe o mance Liquid
Ch oma og aphy andem mass spec ome y analysis (UPLC-MS/MS)
using a B uke EVOQ Eli e sys em (B uke ). The samples we e il e ed
using 0.22
μ
m poly e a luo oe hylene (PTFE) il e s (Teknoch oma,
Ba celona, Spain) o p e en he possible en y o pa icles h ough he
LC sys em. The online-SPE p og am consis ed o i e phases including
condi ioning, equilib a ion, loading, washing, and ex ac ing. Wa e and
me hanol we e used as washing sol en s (Scha lab, Ba celona, Spain).
A e washing, he luoxe ine cap u ed on he ca idge (C18 T ap, 30
mm ×2.1 mm i. d., B uke , USA) was elu ed in back lush mode using
H. S emmel e al.
Chemosphe e 312 (2023) 137028
3
he ch oma og aphic g adien (o e all un ime 8 min). A e he
elu ion, he al e was swi ched back o he loading posi ion o he nex
injec ion. The ch oma og aphic sepa a ion was ob ained using an
ACQUITY UPLC BEH C18 Column (2.0
μ
m, 2.1 mm ×100 mm, Wa e s,
Mil o d, MA). The limi o de ec ion was o 0.075
μ
g/L. Fu he in o -
ma ion on he UPLC analysis can be ound in Baena-Nogue as e al.
(2016).
2.2. Tes species and s ock cul u e condi ions
The es o ganism was he commonly used D. magna. We used an uni-
clonal s ock cul u e o D. magna p o ided by he Uni e si y o Ja´
en
(Spain), sampled om Laguna G ande (Ja´
en, Spain). The cul u e was
main ained o six mon hs in ou labo a o y unde a empe a u e o 23
±1 ◦C and a pho ope iod o 12:12 h (ligh :da k). The medium ( enewed
weekly) used o he s ock cul u e o D. magna consis ed o bo led sp ing
wa e (Fon Na u a®, Sie a de Loja, G anada, Spain) en iched wi h
bio in (0,75
μ
g/L; CAS-58-85-5), hiamine (75
μ
g/L; CAS-67-03-8), so-
dium seleni e (2
μ
g/L; CAS-10102-18-8) and i amin B12 (2
μ
g/L; CAS-
68-19-9) as sugges ed by Cas illo Mo ales (2004). The o ganisms we e
ed h ee imes a week wi h he g een unicellula algae Scenedesmus sp.
(concen a ion o 10
6
cells/o ganism), main ained in a non-ma ine
op imal Haema occus medium (F´
ab egas e al., 2000).
2.3. Expe imen al design
The expe imen al app oach was di ided in wo g oups: (i) en i on-
men ally ele an concen a ions simula ing a eal scena io and (ii) a
u u e wo s scena io o se e e con amina ion (see scheme in Fig. 1). In
he ealis ic scena io, he concen a ions o luoxe ine a ied om 0.1 o
1
μ
g/L and wo endpoin s we e measu ed: he mul igene a ional
ep oduc ion esponse and colonisa ion beha iou . In he wo s sce-
na io, he concen a ions o luoxe ine we e in he ange o 1–800
μ
g/L
and he endpoin s s udied we e: acu e mo ali y, a oidance beha iou
and ch onic oxici y ep oduc ion. Fo he expe imen s in which wo
ea men s o luoxe ine we e used, he le els o luoxe ine a e named as
FLX
L
and FLX
H
o low and high le els, espec i ely. As epo ed con-
cen a ions a y be ween 0.012 and 3.5
μ
g/L (B ooks e al., 2003;
Schul z e al., 2010; W i e e al., 2013; Ribei o e al., 2014) we decided
o pe o m he a oidance es s wi h concen a ions (0, 1, 5, 10, 50 and
100
μ
g/L) and o use highe concen a ions (800
μ
g/L) o ind he
h eshold o mo ali y and hen o calcula e he LC50 alue.
2.3.1. Mul igene a ional ep oduc ion assessmen
Fo he mul igene a ional expe imen s, h ee ea men s o luoxe-
ine [con ol, 0.1 (FLX
L
) and 1
μ
g/L (FLX
H
)] we e es ed (see scheme in
Fig. S1). Fo each ea men , 25 indi iduals (no olde han 48 h) om
he s ock cul u e we e in oduced in o a 1 L glass con aine . Each
ea men was es ed wi h i e eplica es. The pa en al gene a ion (P0)
was exposed o 14 days, and he o sp ing we e coun ed a e 9 and 14
days. The solu ions o luoxe ine and i amins o he cul u e medium
we e comple ely enewed e e y week. A e wo weeks, ano he 25 in-
di iduals (<48 h) o each eplica e we e again ans e ed o a new 1 L
glass con aine o s a he nex gene a ion. To make su e ha neona es
selec ed we e <48 h, we did coun and emo ed any neona es p esen on
day 12. This p ocedu e (coun ing o o sp ing and he enewal o he
medium) was simila ly epea ed o he neona es p oduced in F1 and F2.
Du ing he expe imen wi h P0, he o ganisms we e ed Scenedesmus
(~6 ×10
4
cells/ml) h ee imes a week (Monday, Wednesday, and
F iday); howe e , due o a delay in ep oduc ion, indi iduals om F1
and F2 we e ed wi h ~5 ×10 cells/mL h ee imes a week and ~3 ×
10
4
cells/mL wice a week (Tuesday and Thu sday). The exposu e
condi ions we e simila o hose desc ibed o he cul u e condi ions and
he eplica es we e andomly dis ibu ed a leas wo imes a week.
2.3.2. Colonisa ion beha iou assay a e mul igene a ional exposu e
O ganisms (10 days old) om he las gene a ion (F3) o e e y
ea men [con ol, 0.1 (FLX
L
) and 1
μ
g/L (FLX
H
)] o he mul igene a-
ional expe imen we e es ed o he colonisa ion esponse using he
HeMHAS e sion 3 (Figs. S2 and S3). HeMHAS is a sys em cha ac e ised
Fig. 1. Schema ic display o he isk assessmen app oaches used in he s udy: (i) en i onmen ally ele an concen a ions and (ii) concen a ions simula ing a wo se
con amina ion scena io. The colou s g een, shades o yellow, and ed in he es ecipien s ep esen di e en and inc easing le els o con amina ion by luoxe ine.
The ba s wi h di e en colou s (▬▬, ▬▬, and ▬) indica e D. magna popula ions p e iously exposed o he con ol, 0.1
μ
g/L and 1
μ
g/L o luoxe ine, espec i ely. (Fo
in e p e a ion o he e e ences o colou in his igu e legend, he eade is e e ed o he Web e sion o his a icle.)
H. S emmel e al.
Chemosphe e 312 (2023) 137028
4
by mul iple, in e connec ed compa men s, ha con ain di e en
con amina ion scena ios. The sys em was cons uc ed using ace al
(polyoxyme hylene), an enginee ing he moplas ic wi h a high densi y
and a low chemical eac i i y, o p e en adso p ion o con aminan s.
The HeMHAS has a dimension o 110 ×74 ×12.5 cm (leng h, wid h,
and heigh ) and is o med by 24 compa men s (4 ×6) wi h 6 cm o
heigh and capaci y o 320 mL, which a e connec ed o he adjacen
compa men s by gy a o y doo s. The opening and closing o he doo s
among he compa men s a e con olled elec onically h ough he
HeMHAS So wa e. Each compa men has a lid o educe e apo a ion.
A g adien wi h ou concen a ions o luoxe ine (0, 0.1, 0.5 and 1.0
μ
g/L) was c ea ed in hese expe imen s, o p o ide a he e ogeneous
en i onmen h ough which he o ganisms could mo e. Be o e placing
he concen a ions in o he compa men s o he HeMHAS, he doo s
connec ing he adjacen compa men s we e closed. Solu ions (320 mL)
o luoxe ine we e pu in o he compa men s o ming a g adien o
con amina ion. A e wa ds, 40 o ganisms we e in oduced in o he
con ol (no con aminan ) compa men and he connec ing doo s we e
opened using he elec onical in e ace; hen, he o ganisms we e able o
mo e eely. The posi ion o he o ganisms was eco ded du ing 24 h;
e en hough we ha e da a h oughou 24 h we conside ed he esul s
a e 24 h he mos impo an as his is he ime equi ed o o ganisms o
colonise all he compa men s in he sys em. The expe imen s we e
pe o med (wi h F3-o ganisms om he p eceding mul igene a ional
expe imen a e i was ended) wice wi h wo eplica es each (n =4 in
o al). The exposu e occu ed in da kness. No ood was p o ided du ing
he expe imen s.
2.3.3. A oidance- epellence esponse assay
Th ee independen a oidance expe imen s (each N =4) we e pe -
o med o assess he po en ial o luoxe ine o igge a oidance in D.
magna. The expe imen s di e ed ega ding he g adien o luoxe ine:
assay #1 (0, 1, 5, 10, 50 and 100
μ
g/L), assay #2 (0, 5, 20, 50, 100 and
200
μ
g/L) and assay #3 (0, 100, 200, 400, 600 and 800
μ
g/L) (Fig. S4).
As in he a oidance assay #1 a displacemen o o ganisms owa ds he
highes concen a ion (100
μ
g/L) was obse ed (see esul s), we decided
o inc ease he concen a ion o 200
μ
g/L and hen o 800
μ
g/L o check
i he a ac ion e ec o luoxe ine would be obse ed e en a le hal
concen a ions. A e placing he concen a ions in o he compa men s
in HeMHAS, indi iduals (10 days old) om a con ol ea men (no
luoxe ine) we e dis ibu ed in e e y compa men o he HeMHAS (n =
10 o ganisms pe concen a ion) while he connec ions we e closed. This
di e s om colonisa ion, in which all he o ganisms a e pu in he i s
uncon amina ed compa men o see i hey a e able o colonise all he
sys em. A e opening he connec ions, he o ganisms we e able o mo e
eely along he g adien . The h ee expe imen s we e pe o med in
quad uplica e. Du ing he 24 h pe iod, he spa ial dis ibu ion o he
o ganisms along he g adien was pe iodically (1, 2, 3, 4 and 24 h)
assessed. The exposu e occu ed in da kness. No ood was p o ided
du ing he expe imen s.
2.3.4. Acu e mo ali y/immobili y es
Le hali y es s we e pe o med ollowing he s anda d p ocedu e
acco ding o OECD 202 guidelines (OECD, 2004), excep ega ding he
age o he indi iduals. Fi e indi iduals (8–10 days old) we e in oduced
in 50 mL con aine s, which con ained a dis inc luoxe ine concen a ion
(0, 100, 200, 400, 600 and 800
μ
g/L). Checks o any dead o ganisms
we e pe o med a each 24 h du ing 96 h. Each ea men was es ed
using i e eplica es. The labo a o y condi ions we e simila o he
cul u e condi ions. No ood was p o ided du ing he expe imen s.
2.3.5. Ch onic oxici y es
The ch onic oxici y es on ep oduc ion was ca ied ou ollowing
he OECD 211 guidelines (OECD, 2012). O ganisms (be ween 6 and <24
h) we e exposed indi idually in 100 mL lasks con aining di e en
luoxe ine ea men s: 0, 10 (FLX
L
) and 100
μ
g/L (FLX
H
). Fi e eplica es
we e pe o med o each ea men . The o ganisms we e ed ~3 ×10
6
cells/D. magna (~3 ×10
4
cells/mL) h ee imes a week (Monday,
Wednesday, and F iday) and ~1.5 ×10
6
cells/D. magna (~1.5 ×10
4
cells/mL) we e p o ided wo imes a week (Tuesday and Thu sday).
Neona es we e coun ed daily. The es las ed 21 days a a empe a u e o
20 ±1 ◦C and a pho ope iod o 12:12 (ligh :da k). The medium was
enewed a e 7 and 14 days. The eplica es we e dis ibu ed andomly
on a daily basis.
2.4. Da a analysis
Fo s a is ical analysis we es ed o no mal dis ibu ion, when ou
da a ollowed a pa ame ic da a se we used a one-way ANOVA, using
RS udio® e sion 1.4.17 wi h R e sion 4.1.0 (R Co e Team, 2021) and
o de e mine LC
50
we did a P obi analysis (Sakuma, 1998) by P iP o-
bi ® So wa e e sion 1.63.
2.4.1. Mul igene a ional ep oduc ion assessmen
We used a One-Way-Analysis o Va iance (ANOVA) o compa e he
means (o he same gene a ion and he same pe iod (9 o 14 days))
among he h ee ea men s. I he ANOVA showed a s a is ically sig-
ni ican di e ence a pos -hoc Tukey es was pe o med. The signi i-
cance le el was se a 5%.
2.4.2. Colonisa ion
The po en ial o he o ganisms om he las gene a ion o he
mul igene a ional expe imen o colonise en i onmen s (% o colo-
nise s: N
C
) wi h luoxe ine was calcula ed ollowing he o mula (Islam
e al., 2019):
Nc= (NO/NE)x 100
whe e he numbe o o ganisms obse ed (N
O
) is he numbe o o gan-
isms in a gi en compa men including he o ganisms obse ed in he
ollowing compa men s wi h highe concen a ion, whe eas N
E
(num-
be o expec ed o ganisms) was calcula ed as ollows: we in oduced 40
o ganisms in he i s compa men (con ol) and we had ou com-
pa men s, we di ided hose o ganisms h ough 4 compa men s, which
esul ed in 10, as a hypo he ical uni o m dis ibu ion. Then, N
E
con-
side s he o ganisms expec ed in a gi en compa men plus he o gan-
isms expec ed in he compa men s wi h highe concen a ions: o he
i s compa men (con ol wi hou luoxe ine) N
E
was 40, and o he
ollowing compa men s i was 30 and 20, and in he las compa men i
was 10 (see Islam e al., 2019).
We calcula ed a one-way ANOVA a e 24 h o each concen a ion
and each popula ion. I he ANOVA showed a s a is ically signi ican
di e ence, a pos -hoc es was pe o med. The signi icance le el was se
a 5%.
2.4.3. A oidance
Fo he a oidance es s, he numbe o a oide s (N
A
) was calcula ed
ollowing he o mula desc ibed by A aújo e al. (2018):
NA=NE– NO
Whe e N
E
(numbe o o ganisms expec ed) is he numbe o o ganisms
eleased in o a gi en compa men plus he o ganisms in oduced in o
he compa men s wi h highe concen a ions, whe eas N
O
( he numbe
o o ganisms obse ed) is he numbe o o ganisms eco ded in a
compa men plus he o ganisms obse ed in he compa men s wi h
highe concen a ions. Fo ins ance, as we in oduced 10 o ganisms in
e e y compa men and we had a g adien wi h six compa men s, N
E
o he i s compa men (con ol wi hou luoxe ine) was 60, and o
he ollowing compa men s i was 50, 40, 30 and 20, and in he las
compa men i was 10. Then, he numbe o a oide s was di ided by he
numbe o o ganisms expec ed, acco ding o he ollowing o mula, o
calcula e he pe cen age o a oidance:
H. S emmel e al.
Chemosphe e 312 (2023) 137028
5
A oidance (%) = (NA/NE)x 100
We calcula ed a one-way ANOVA a e 24 h o each concen a ion
and each popula ion. I he ANOVA showed a s a is ically signi ican
di e ence, a pos -hoc Tukey es was pe o med. The signi icance le el
was se a 5%.
2.4.4. Acu e mo ali y
The pe cen age o dead o ganisms was calcula ed o each concen-
a ion a e 96 h o exposu e and he le hal concen a ion o 50% o he
D. magna popula ion, LC
50,
and he 95% con idence in e als, we e
de e mined using P iP obi ®. We also de e mined he LC
20
as his alue
could be conside ed he h eshold o an ini ial isk o he popula ion
exposed.
2.4.5. Ch onic oxici y es 21 d
The sum o neona es o e e y o ganism accumula ed each day along
he 21 days o exposu e was calcula ed o each o ganism. The ea -
men s we e compa ed using a one-way ANOVA. I he ANOVA showed a
s a is ically signi ican di e ence, a pos -hoc Tukey es was pe o med.
The signi icance le el was se a 5%.
3. Resul s
3.1. Mul igene a ional ep oduc ion assessmen
The e was no mo ali y o ephippia obse ed in any expe imen al
ea men du ing he mul igene a ional es s. The esul s showed ha
he mul igene a ional ep oduc ion was no a ec ed by luoxe ine
(Fig. 2). The compa isons o he neona es p oduced pe D. magna among
he di e en ea men s showed no signi ican di e ences, ega dless o
he days o exposu e (9 o 14) o gene a ion (P0, F1 o F2); excep o he
14-day exposu e o he F1, when a signi ican di e ence be ween he
con ol and he high exposu e ea men was obse ed (F
2,12
=4.3 (F
C i
=3.89) and p <0.05) (Fig. 2).
3.2. Colonisa ion beha iou a e mul igene a ional exposu e
D. magna exposed o low and high luoxe ine le els showed a simila
abili y o colonise he luoxe ine-con amina ed en i onmen s, as
obse ed in he con ol popula ion du ing a 24 h expe imen (Fig. 3). In
gene al, he colonisa ion o he lowes concen a ion (0.1
μ
g/L) was
achie ed by 40% o he o ganisms expec ed o colonise i , while only
a ound 20% o he expec ed popula ions colonised he highes concen-
a ion (1.0
μ
g/L).
3.3. A oidance- epellence esponse
In all h ee a oidance assays, he o ganisms we e shown o be
a ac ed o luoxe ine and, in wo assays (#1 and #3), he o ganisms
we e signi ican ly (p <0.05) a ac ed o he highes concen a ion o
luoxe ine (Fig. 4). Assay #1 shows a ound 15% o he o ganisms in he
con ol compa men , wi h a sligh end o a oiding luoxe ine un il he
concen a ion o 10
μ
g/L. Howe e , ha end changed in o a ac ion o
he highes concen a ions, wi h a ound 50% o he o ganism p e e ing
he las wo compa men s (50 and 100
μ
g/L). In assay #2, he o gan-
isms showed a signi ican (p <0.05) a ac ion o 20
μ
g/L (30% o o -
ganisms), while he highes concen a ions we e no p e e ed (a ound
only 16% o o ganisms mo ed o he compa men wi h he highes
concen a ion, which was almos equal o he con ol compa men ). In
assay #3, o ganisms clea ly showed a signi ican (p <0.05) p e e ence
o he highes concen a ion o luoxe ine; he las wo compa men s
(600 and 800
μ
g/L) a ac ed a ound 22% o o ganisms each, while only
10% o he o ganisms we e ound in he con ol compa men .
3.4. Acu e mo ali y/immobili y es
The mo ali y esponse o D. magna in he 96 h-acu e oxici y es
Fig. 2. Box plo (medium, 1s and 3 d qua iles and maximum and minimal alues) o neona es pe D. magna o e e y ea men and e e y gene a ion a e he 9 h
and 14 h day. S a is ically signi ican di e ences among he luoxe ine ea men s, wi hin each gene a ion a each exposu e pe iod, a e ep esen ed by
di e en le e s.
Fig. 3. The mean (and s anda d de ia ion) o he colonisa ion o he expec ed
popula ion (%) o en i onmen s con amina ed wi h luoxe ine by h ee
di e en popula ions o D. magna, which had been p e iously exposed o
luoxe ine o h ee gene a ions: con ol (no p e ious exposu e o luoxe ine)
and exposu e o luoxe ine: FLX
L
(0.1
μ
g/L) and FLX
H
(1
μ
g/L).
H. S emmel e al.

Chemosphe e 312 (2023) 137028
6
showed a clea and inc easing concen a ion- esponse pa e n (Fig. 5).
The mo ali y a e in he con ol g oup was 8% a e 96 h, while a he
highes concen a ions (600
μ
g/L) he mo ali y a e eached 71%. In
he p e ious exposu e pe iods (24, 48 and 72 h), he mo ali y was al-
ways lowe han 50%. The concen a ion- esponse cu e o he sigmoid
model o a 96 h exposu e pe iod showed a
2
- alue o 0.9948 and he
LC
50
and LC
20
alues (and con idence in e als) we e de e mined as 365
μ
g/L (216 and 570
μ
g/L) and 154 (29–246)
μ
g/L, espec i ely.
3.5. Ch onic oxici y es
The esul s o he numbe o neona es p oduced pe D. magna du ing
he 21-day ep oduc ion es a e shown in Fig. 6. Ou esul s show ha
luoxe ine s imula ed an ea lie p oduc ion o neona es, as he i s
o sp ing occu ed a e 7 days in he o ganisms exposed o luoxe ine,
while he i s neona es we e bo n a e 9 days in he con ol ea men .
In addi ion, he numbe o neona es was highe in he highes concen-
a ion o luoxe ine (100
μ
g/L) and s a is ically di e en om he
con ol in almos all he exposu e pe iod, eaching a mean alue o 58.8
±4.6, while in he con ol and low ea men s he mean neona e p o-
duc ion was 46.4 ±3.4 and 50.6 ±5.9, espec i ely.
4. Discussion
The aim o his s udy was o assess he isks ha he an idep essan luoxe ine migh pose o aqua ic ecosys ems, in pa icula o D. magna.
We ocused on wo di e en app oaches, conside ing (i) en i onmen-
ally ele an concen a ions and (ii) le els o luoxe ine expec ed o
occu i i s use con inues o ise un il le hal le els. A discussion abou he
main indings o he cu en s udy is p esen ed in he ollowing
subsec ions.
4.1. Mul igene a ional esponse
Ou esul s conce ning he mul igene a ional ep oduc ion assess-
men ha e no shown any e ec on he o ganisms ha could be
conside ed o conce n. We did no obse e any e ec on su i al, o
o he signs o s ess such as he p oduc ion o ephippia o a change in
ecundi y du ing he expe imen . O he au ho s ha e shown ha
luoxe ine a highe concen a ions, in luenced he ep oduc ion o
D. magna, simila ly o ood quali y, in a ange o luoxe ine concen a-
ions be ween 10 and 80
μ
g/L (Ba a a e al., 1998; Campos e al., 2012;
Campos e al., 2016). Heyland e al. (2020) showed ha luoxe ine a
0.054 and 0.54
μ
g/L caused signi ican changes such as highe mo al-
i y, dec easing g ow h a e, educ ion in a e age o sp ing size and
highe p oduc ion o ephippia among he gene a ions and ea men s
wi h luoxe ine. Ano he mul igene a ional s udy (Ba bosa e al., 2017)
showed signi ican di e ences be ween he con ol and luoxe ine con-
cen a ions o 0.012 and 0.54
μ
g/L. The ep oduc ion success
Fig. 4. The mean (and s anda d de ia ion) o he pe cen age o o ganisms ound in di e en compa men s wi h a g adien o luoxe ine concen a ions. The o -
ganisms used in he expe imen s had no been p e iously exposed o luoxe ine. S a is ically signi ican di e ences (Tukey es ; p <0.05) o each day a e ep esen ed
by di e en le e s.
Fig. 5. Cu e o concen a ion-mo ali y (and he con idence in e al) in he
acu e es wi h D. magna exposed o luoxe ine du ing 96 h. Values o LC
20
and
LC
50
(le hal concen a ion o 20 and 50% o popula ions, espec i ely) a e
also p esen ed.
Fig. 6. Numbe o neona es pe D. magna accumula ed along a 21-day exposu e
o di e en concen a ions o luoxe ine (con ol, low and high: 0, 10 and 100
μ
g/L). S a is ically signi ican di e ences (Tukey es ; p <0.05) on each day a e
ep esen ed by di e en le e s. No le e was included when he e we e no
s a is ical di e ences.
H. S emmel e al.
Chemosphe e 312 (2023) 137028
7
h oughou h ee gene a ions was s udied and, la e , he ou h gene -
a ion was es ed in ela ion o ep oduc ion combining luoxe ine and
di e en empe a u es. Thei esul s de ec ed a dec ease in neona es in
he ea men s wi h luoxe ine (17% and 29% in he low and high
concen a ions, espec i ely); in addi ion, hose au ho s obse ed ha
luoxe ine in combina ion wi h di e en empe a u es ( ea men s o
20 ◦C o 25 ◦C o a luc ua ion be ween 15 ◦C and 25 ◦C) a ec ed he
ep oduc ion o he ou h gene a ion (Ba bosa e al., 2017). Howe e ,
hey we e using ano he clone lineage (Clone F) and he ood quan i y
pe D. magna was 10 imes lowe compa ed o ou ood condi ions.
Hen y e al. (2022) showed in a 3-yea mul igene a ional s udy wi h he
eshwa e snail Physa acu a ha amoun s as low as 0.03
μ
g/L a ec ed
he egg mass p oduced; luoxe ine exposed g oups had a dec ease in egg
mass, al hough i did no a ec he numbe o emb yos no he a e age
emb yos p oduced pe egg mass.
4.2. Colonisa ion esponse
To ou knowledge, ou s udy is he i s o assess he e ec s o
mul igene a ional exposu e o luoxe ine on he colonisa ion beha iou
o D. magna. We could no ind a s a is ically signi ican di e ence in he
abili y o colonise en i onmen s wi h luoxe ine among any p e iously
luoxe ine-exposed popula ions. Wi hin 24 h he e was only a signi ican
di e ence among all ea men s be ween he con ol compa men and
he compa men s con aining luoxe ine, bu his esul is biased because
he o ganisms we e ini ially in oduced in o he con ol compa men
(see de ails in Fig. 3). O ganisms colonised he comple e sys em wi hin
24 h in he con ol expe imen . The e o e, we can only suspec ha he
exposu e ime in he mul igene a ional expe imen was oo sho o see
any e ec on he colonisa ion esponse ( luoxe ine exposu e o mo e
gene a ions migh be equi ed).
4.3. A oidance- epellence esponse
We expec ed ha D. magna would e en ually a oid luoxe ine, bu
he esul s o he h ee a oidance assays showed ha luoxe ine p o ed
o be a ac i e o o ganisms ins ead. In assays #1 and #3, D. magna was
d awn o he highes concen a ions o 100 and 800
μ
g/L. Some chem-
icals seem o exe an a ac i e e ec on o ganisms despi e hei known
oxic e ec s. Fo ins ance, he a ac ion esponse was obse ed o
O conec es i ilis (c ay ish) exposed o se aline (Woodman e al., 2016
), o Co unix japonica (Japanese quails) exposed o glyphosa e (Ruus-
kanen e al., 2019), he ish Cyp inus ca pio (common ca p) exposed o
diazepam (Jacob e al., 2021). I is known ha some oxic chemicals
migh ha e he abili y o a ac a he han epel, such as e iewed and
discussed by A aújo e al. (2020). Ab eu e al. (2016) showed ha Danio
e io (zeb a ish) we e a ac ed o concen a ions o 25 and 50
μ
g/L o
luoxe ine, bu no a 1
μ
g/L; hey assumed ha he a ac ion was
p obably ia ol ac ion as anosmic ish did no show his beha iou .
Hence, he a ac ion e ec p oduced by po en ially oxic chemicals
could be ela ed o damage o chemo ecep o s (Tie ney e al., 2007;
Che kashin and Blino a, 2011). Ano he possible explana ion migh be
ha he eup ake o se o onin in he ne ous sys em leads o a ‘posi i e’
e ec on he o ganisms’ beha iou , igge ing an ad e se ep oduc ion
esponse (Campos e al., 2016). The e o e, i is impo an o s udy
whe he he a ac ion p oduced by luoxe ine is no ela ed o a alse
bene i , bu ins ead o possible damage caused o he o ganism’s abili y
o ecognise isks chemically; al hough in his la e si ua ion, a andom
dis ibu ion would be mo e expec ed han an a ac ion esponse.
4.4. Le hal e ec
Da a o acu e oxici y o luoxe ine on D. magna showed ha ou
popula ion was highly sensi i e, wi h an LC
50
o 365
μ
g/L a e 96 h.
These da a a e much lowe han hose desc ibed in o he s udies,
al hough he compa ison should be made wi h cau ion due o he
di e ences in he exposu e ime; o ins ance, in a s udy wi h D. magna
(6–24 h neona es) he 48 h-LC
50
was de e mined a 820
μ
g/L by B ooks
e al. (2003), Minguez e al. (2014) epo ed alues o a ound 6 mg/L o
a 48 h-LC
50
and Schlussel e al. (2019) ound a 24 h-LC
50
a a ound 4
mg/L. As in ou s udy, he o ganisms we e six-day-old ju eniles, i is no
easily compa able o he abo e-men ioned s udies. The exposu e ime
was 96 h, because a p e ious exposu e pe iods (24, 48 and 72 h) he
mo ali y obse ed was lowe han 50%. Besides he di e ences in
exposu e ime and age o o ganisms, o he ac o s like he cul u e me-
dium and he clones used migh also a ec he consequences he o -
ganisms expe ience as a esul o chemicals. These esul s e idence ha
he a iabili y o an LC
50
and, he e o e, a low o high isk will depend
no only on he concen a ions es ed, bu also on he di e ences in he
en i onmen al condi ions o he exposu e. In ac , Aulseb ook e al.
(2022) concluded ha no only he geno ype o D. magna bu also he
combina ion wi h en i onmen al empe a u e migh de e mine he e -
ec s o luoxe ine on he li e his o y ai s o D. magna a concen a ions
as low as 0.03, 0.3 and 3
μ
g/L, and which showed a non-mono onic
manne as well.
Fo C. dubia a 48 h-LC
50
o luoxe ine was de e mined a 234
μ
g/L
(B ooks e al., 2003) and 510
μ
g/L (Hen y e al., 2004). The LC
50
alue
epo ed o luoxe ine conce ning Pseudoki chne iella subcapi a a
(mic oalgae) was 24
μ
g/L (B ooks e al., 2003). A ecen s udy by
Rezende e al. (2021) showed LC
50
alues o luoxe ine a e 24, 48, 72
and 96 h o 40, 36, 28 and 26
μ
g/L, espec i ely, o Palaemon pan-
dali o mis (phan om sh imp). This shows ha o he o ganisms a e
clea ly mo e sensi i e o luoxe ine han D. magna. I seems ha he
en i onmen ally ele an concen a ions o luoxe ine as a single sub-
s ance do no imply a uly acu e isk o some aqua ic o ganisms (unde
labo a o y condi ions), ye i is impo an o conside ha he con in-
uous inc ease in he le els o luoxe ine in he en i onmen is indica i e
o an e en highe imminen isk.
4.5. Ch onic oxici y es
Ou da a shows ha ep oduc ion unde a high concen a ion o
luoxe ine (100
μ
g/L) s a ed wo days ea lie and he numbe o neo-
na es in he i s b ood was highe han in he con ol and he FLX
L
(10
μ
g/L). O e he 21 days, i seems ha he e is a highe numbe o
o sp ing in he FLX
H
, bu his migh be a ibu ed o he i s b ood,
which ook place wo days ea lie han in he con ol and FLX
L
. A
ep oduc ion s udy wi h D. magna was also ca ied ou by P´
e y e al.
(2008) wi h concen a ions om 3 o 300
μ
g/L; hey ound a di e ence
in ep oduc ion o 32% and mo ali y o 40% (day 21) a he highes
luoxe ine concen a ion (241
μ
g/L). Mo eo e , in a 21-day es wi h
neona es om he i h b ood, 70% o he neona es exposed o 102
μ
g/L
died and ep oduc ion was educed by 18% a 31
μ
g/L. Such as obse ed
in ou s udy, P´
e y e al. (2008) did no ind di e ences in ep oduc ion a
concen a ions below 241
μ
g/L o he pa en al gene a ion. On he o he
hand, in a 30 day acu e oxici y es , D. magna exposed o 36
μ
g/L o
luoxe ine showed no signi ican di e ence in su i al, g ow h o sex
a io, bu did in he numbe o neona es, wi h a ep oduc ion a e almos
h ee imes highe han in he con ol g oup (Flahe y and Dodson,
2005). These esul s mus also be ea ed wi h cau ion when compa ed
wi h ou s, as he empe a u e hey used was 25 ±1 ◦C, ha migh
signi ican ly a ec he esponse o luoxe ine.
4.6. En i onmen al consequences o con amina ion by luoxe ine
The damage ha luoxe ine can po en ially cause is e y di e se and
complex. Al Shu aiqi e al. (2021) used concen a ions o 0.005–5
μ
g/L
and ound ha he swimming beha iou and eac ion o a conspeci ic
ala m subs ance measu ed a day 7, day 14 and 28 di e s no only wi h
ime bu was also non-mono onic (a non-linea dose- esponse pa e n).
Aulseb ook e al. (2022) obse ed ha he e ec s o luoxe ine on
D. magna migh a y acco ding o geno ypes and he empe a u es o
H. S emmel e al.
Chemosphe e 312 (2023) 137028
8
exposu e.
In ou s udy, e en e y low concen a ions o luoxe ine (20
μ
g/L)
a ac ed he o ganisms, while a e y simila concen a ion (36
μ
g/L)
was shown o induce an o e p oduc ion o o sp ing (Flahe y and
Dodson, 2005). Al hough hese esponses could be conside ed a bene i
o D. magna, an in eg a ed analysis could lead us o unde s and he eal
isk o luoxe ine. As shown by Campos e al. (2016), e en unde con-
di ions o ood dep i a ion, he p esence o luoxe ine s imula es
ep oduc ion, bu in a maladap i e manne (mo e and smalle o sp ing
e en hough his is no necessa y o su i e as i would be unde p e-
da ion p essu e). The e o e, he inc ease in o sp ing and a ac ion o
luoxe ine could lead o se ious consequences o D. magna popula ions.
5. Conclusion
The cu en s udy obse ed ha en i onmen ally ele an concen-
a ions o luoxe ine did no a ec he ep oduc ion ou pu in D. magna
a e exposu e o e h ee gene a ions. I was also obse ed ha in-
di iduals o he las gene a ion did no imp o e hei abili y o colonise
luoxe ine-con amina ed en i onmen s. The e o e, he possibili y o an
adap a ion/acclima ion could no be conside ed. In a second app oach
based on scena ios wi h e y high en i onmen al concen a ions (wo se
scena ios), e ec s we e ound a di e en le els: su i al, a oidance
beha iou and ep oduc ion. Rega ding a oidance beha iou , D. magna
was a ac ed by luoxe ine, e en a concen a ions conside ed
dange ous, much highe han he median le hal concen a ion. Fluoxe-
ine also s imula ed he ep oduc ion o daphnids. Bo h e ec s, he
a ac ion o luoxe ine and he s imula ion o ep oduc ion should be
analysed wi h cau ion, because ins ead o b inging bene i s o o gan-
isms, hey migh indica e a se ious beha iou al, senso ial, and physio-
logical imbalance caused by luoxe ine. Mo e s udies a e equi ed o
unde s and he cellula , biochemical, and gene ic mechanisms in ol ed
in he in e ac ion o luoxe ine wi h aqua ic o ganisms, in line wi h o he
na u al and an h opogenic s esso s, be e .
C edi au ho s a emen
Helmu S emmel: Concep ualiza ion; Me hodology; Fo mal Anal-
ysis; In es iga ion; W i ing – o iginal d a ; Visualiza ion, Linda Weiss:
Resou ces; W i ing – e iew and edi ing, Gema Pa a: Concep ualiza-
ion; Me hodology; W i ing – e iew and edi ing, Eloísa Ramos-
Rod iguez Concep ualiza ion; Me hodology; W i ing – e iew and edi -
ing, C is iano V.M. A aújo: Concep ualiza ion; Me hodology; Fo mal
Analysis; In es iga ion; Resou ces; W i ing – e iew and edi ing; Visu-
aliza ion; Funding acquisi ion.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
Da a will be made a ailable on eques .
Acknowledgemen s
H. S emmel hanks he PROMOS schola ship o he DAAD and he
RUB. C.V.M. A aújo is g a e ul o he g an (Ram´
on y Cajal con ac :
RYC-2017-22324) unded by MCIN/AEI/10.13039/501100011033 and
“ESF In es ing in you u u e”. This esea ch was unded by MCIN/AEI/
10.13039/501100011033 (B ES ess p ojec : PID 2019-105868RA-I00).
The au ho s hank And ea Co de o, Da id Sal a ie a, Gonzalo Ma ínez
o hei labo a o y assis ance and Jon Nesbi o his e ision o he
English ex .
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.chemosphe e.2022.137028.
Re e ences
Ab eu, M.S., Giacomini, A.C.V., Gusso, D., Rosa, J.G., Koakoski, G., Kalichak, F.,
Idalˆ
encio, R., Oli ei a, T.A., Ba cellos, H.H., Bonan, C.D., Ba cellos, L.J., 2016. Acu e
exposu e o wa e bo ne psychoac i e d ugs a ac zeb a ish. Comp. Biochem.
Physiol. C Toxicol. Pha macol. 179, 37–43. h ps://doi.o g/10.1016/j.
cbpc.2015.08.009.
Al Shu aiqi, A., Al-Habsi, A., Ba y, M.J., 2021. Time-, dose- and ansgene a ional
e ec s o luoxe ine on he beha iou al esponses o zeb a ish o a conspeci ic ala m
subs ance. En i on. Pollu . (Ams e dam, Ne h.) 270 (116164), 116164. h ps://doi.
o g/10.1016/j.en pol.2020.116164.
A aújo, C.V.M., Laissaoui, A., Sil a, D.C.V.R., Ramos-Rod íguez, E., Gonz´
alez-
O eg´
on, E., Espíndola, E.L.G., Bald´
o, F., Mena, F., Pa a, G., Blasco, J., L´
opez-
Do al, J., Send a, M., Banni, M., Islam, M.A., Mo eno-Ga ido, I., 2020. No only
oxic bu epellen : wha can o ganisms’ esponses ell us abou con amina ion and
wha a e he ecological consequences when hey lee om an en i onmen ? Toxics 8
(4), 118. h ps://doi.o g/10.3390/ oxics8040118.
A aújo, C.V., Roque, D., Blasco, J., Ribei o, R., Mo ei a-San os, M., To ibio, A.,
Agui e, E., Ba o, S., 2018. S ess-d i en emig a ion in complex ield scena ios o
habi a dis u bance: he he e ogeneous mul i-habi a assay sys em (HeMHAS). Sci.
To al En i on. 644, 31–36. h ps://doi.o g/10.1016/j.sci o en .2018.06.336.
Aulseb ook, L.C., Wong, B.B., Hall, M.D., 2022. Wa me empe a u es limi he e ec s o
an idep essan pollu ion on li e-his o y ai s. P oc. Biol. Sci. 289 h ps://doi.o g/
10.1098/ spb.2021.2701, 1968.
Baena-Nogue as, R.M., Pin ado-He e a, G., M, Gonz´
alez-Mazo, E., La a-Ma ínP, A.,
2016. De e mina ion o pha maceu icals in coas al sys ems using solid phase
ex ac ion (SPE) ollowed by ul a-pe o mance liquid ch oma og aphy – andem
mass spec ome y (UPLC-MS/MS). Cu . Anal. Chem. 12 (3), 183–201. h ps://doi.
o g/10.2174/1573411012666151009193254.
Ba a a, C., Bai d, D.J., 1998. Pheno ypic plas ici y and cons ancy o li e-his o y ai s in
labo a o y clones o Daphnia magna S aus: e ec s o neona al leng h. Func . Ecol. 12
(3), 442–452. h ps://doi.o g/10.1046/j.1365-2435.1998.00201.x.
Ba bosa, M., Inocen es, N., Soa es, A.M., Oli ei a, M., 2017. Syne gy e ec s o luoxe ine
and a iabili y in empe a u e lead o p opo ionally g ea e i ness cos s in Daphnia:
a mul igene a ional es . Aqua . Toxicol. 193, 268–275. h ps://doi.o g/10.1016/j.
aqua ox.2017.10.017.
Bla di, P., de Lalla, A., Leo, A., Au e i, A., Iapichino, S., di Mu o, A., Dell’E ba, A.,
Cas ogio anni, P., 2002. Se o onin and luoxe ine le els in plasma and pla ele s
a e luoxe ine ea men in dep essi e pa ien s. J. Clin. Psychopha macol. 22 (2),
131–136. h ps://doi.o g/10.1097/00004714-200204000-00005.
B ooks, B.W., Fo an, C.M., Richa ds, S.M., Wes on, J., Tu ne , P.K., S anley, J.K.,
Solomon, K.R., Sla e y, M., La Poin , T.W., 2003. Aqua ic eco oxicology o
luoxe ine. Toxicol. Le . 142 (3), 169–183. h ps://doi.o g/10.1016/s0378-4274
(03)00066-3.
Bymas e , F.P., Zhang, W., Ca e , P.A., Shaw, J., Che ne , E., Phebus, L., Wong, D.T.,
Pe y, K.W., 2002. Fluoxe ine, bu no o he selec i e se o onin up ake inhibi o s,
inc eases no epineph ine and dopamine ex acellula le els in p e on al co ex.
Psychopha macology 160 (4), 353–361. h ps://doi.o g/10.1007/s00213-001-0986-
x.
Campos, B., Pi˜
na, B., Fe n´
andez-Sanju´
an, M., Laco e, S., Ba a a, C., 2012. Enhanced
o sp ing p oduc ion in Daphnia magna clones exposed o se o onin eup ake
inhibi o s and 4-nonylphenol. S age- and ood-dependen e ec s. Aqua . Toxicol.
109, 100–110. h ps://doi.o g/10.1016/j.aqua ox.2011.12.003.
Campos, B., Ri e i, C., K ess, T., Ba a a, C., Di cksen, H., 2016. Dep essing
an idep essan : luoxe ine a ec s se o onin neu ons causing ad e se ep oduc i e
esponses in Daphnia magna. En i on. Sci. Technol. 50 (11), 6000–6007. h ps://doi.
o g/10.1021/acs.es .6b00826.
Cao, J., Fu, B., Zhang, T., Wu, Y., Zhou, Z., Zhao, J., Yang, E., Qian, T., Luo, J., 2020. Fa e
o ypical endoc ine ac i e compounds in ull-scale was ewa e ea men plan s:
dis ibu ion, emo al e iciency and po en ial isks. Bio esou . Technol. 310, 123436
h ps://doi.o g/10.1016/j.bio ech.2020.123436.
Che kashin, S.A., Blino a, N.K., 2011. E ec o hea y me als on chemo ecep ion and
beha io o c us aceans (a Re iew). Hyd obiol. J. 47 (4), 83–93. h ps://doi.o g/
10.1615/hyd obj. 47.i4.90.
Ding, J., Zou, H., Liu, Q., Zhang, S., Mami iana Razanaja o o, R., 2017. Bioconcen a ion
o he an idep essan luoxe ine and i s e ec s on he physiological and biochemical
s a us in Daphnia magna. Eco oxicol. En i on. Sa . 142, 102–109. h ps://doi.o g/
10.1016/j.ecoen .2017.03.042.
Dua e, I.A., Reis-San os, P., No ais, S.C., Ra o, L.D., Lemos, M.F., F ei as, A., Pouca, A.S.
V., Ba bosa, J., Cab al, H.N., Fonseca, V.F., 2020. Dep essed, hype ense and so e:
long- e m e ec s o luoxe ine, p op anolol and diclo enac exposu e in a op
p eda o ish. Sci. To al En i on. 712, 136564 h ps://doi.o g/10.1016/j.
sci o en .2020.136564.
E icson, J.F., Laenge, R., Sulli an, D.E., 2002. Commen on “Pha maceu icals, ho mones,
and o he o ganic was ewa e con aminan s in U.S. S eams, 1999−2000: a na ional
econnaissance. En i on. Sci. Technol. 36 (18), 4005–4006. h ps://doi.o g/
10.1021/es0200903.
F´
ab egas, J., Domínguez, A., Reguei o, M., Maseda, A., O e o, A., 2000. Op imiza ion o
cul u e medium o he con inuous cul i a ion o he mic oalga Haema ococcus
H. S emmel e al.
Chemosphe e 312 (2023) 137028
9
plu ialis. Appl. Mic obiol. Bio echnol. 53 (5), 530–535. h ps://doi.o g/10.1007/
s002530051652.
Fe n´
andez, C., Gonz´
alez-Doncel, M., P o, J., Ca bonell, G., Ta azona, J., 2010.
Occu ence o pha maceu ically ac i e compounds in su ace wa e s o he hena es-
ja ama- ajo i e sys em (mad id, Spain) and a po en ial isk cha ac e iza ion. Sci.
To al En i on. 408 (3), 543–551. h ps://doi.o g/10.1016/j.sci o en .2009.10.009.
Fick, J., S¨
ode s ¨
om, H., Lindbe g, R.H., Phan, C., Tysklind, M., La sson, D.J., 2009.
Con amina ion o su ace, g ound, and d inking wa e om pha maceu ical
p duc ion. En i on. Toxicol. Chem. 28 (12), 2522. h ps://doi.o g/10.1897/09-
073.1.
Flahe y, C.M., Dodson, S.I., 2005. E ec s o pha maceu icals on Daphnia su i al,
g ow h, and ep oduc ion. Chemosphe e 61 (2), 200–207. h ps://doi.o g/10.1016/
j.chemosphe e.2005.02.016.
Fong, P.P., 1998. Zeb a mussel spawning is induced in low concen a ions o pu a i e
se o onin eup ake inhibi o s. Biol. Bull. 194 (2), 143–149. h ps://doi.o g/
10.2307/1543044.
Giebuł owicz, J., Nałęcz-Jawecki, G., 2014. Occu ence o an idep essan esidues in he
sewage-impac ed Vis ula and U a a i e s and in ap wa e in Wa saw (Poland).
Eco oxicol. En i on. Sa . 104, 103–109. h ps://doi.o g/10.1016/j.
ecoen .2014.02.020.
G zesiuk, M., Pawelec, A., 2021. Fluoxe ine esul s in misleading conclusions on ish
beha io . Ecol. E ol. 11 (14), 9707–9714. h ps://doi.o g/10.1002/ece3.7797.
Hen y, J., B and, J.A., Bai, Y., Ma in, J.M., Wong, B.B.M., Wlodkowic, D., 2022. Mul i-
gene a ional impac s o exposu e o an idep essan luoxe ine on beha iou ,
ep oduc ion, and mo phology o eshwa e snail Physa acu a. Sci. To al En i on.
814, 152731 h ps://doi.o g/10.1016/j.sci o en .2021.152731.
Hen y, T.B., Kwon, J.W., A mb us , K.L., Black, M.C., 2004. Acu e and ch onic oxici y o
i e selec i e se o onin eup eake inhibi o s in Ce iodaphnia dubia. En i on. Toxicol.
Chem. 23 (9), 2229. h ps://doi.o g/10.1897/03-278.
Heyland, A., Bas ien, T., Halliwushka, K., 2020. T ansgene a ional ep oduc i e e ec s o
wo se o onin eup ake inhibi o s a e acu e exposu e in Daphnia magna emb yos.
Comp. Biochem. Physiol. C Toxicol. Pha macol. 238, 108875 h ps://doi.o g/
10.1016/j.cbpc.2020.108875.
Islam, M.A., Blasco, J., A aújo, C.V.M., 2019. Spa ial a oidance, inhibi ion o
ecoloniza ion and popula ion isola ion in zeb a ish (Danio e io) caused by coppe
exposu e unde a non- o ced app oach. Sci. To al En i on. 653, 504–511.
Jacob, R.S., A aújo, C.V., San os, L.V.D.S., Mo ei a, V.R., Leb on, Y.A.R., Lange, L.C.,
2021. The en i onmen al isks o pha maceu icals beyond adi ional oxic e ec s:
chemical di e ences ha can epel o en ap aqua ic o ganisms. En i on. Pollu .
268, 115902 h ps://doi.o g/10.1016/j.en pol.2020.115902.
Kane, S.P.P., 2021. The op 200 o 2021. Ranking Pha maceu icals US 2018. h ps://c
lincalc.com/D ugS a s/Top200D ugs.aspx.
Liu, Q., He, H., Yang, J., Feng, X., Zhao, F., Lyu, J., 2020. Changes in he global bu den o
dep ession om 1990 o 2017: indings om he global bu den o Disease s udy.
J. Psychia . Res. 126, 134–140. h ps://doi.o g/10.1016/j.jpsychi es.2019.08.002.
L´
opez-Rold´
an, R., de Alda, M.L., G os, M., Pe o ic, M., Ma ín-Alonso, J., Ba cel´
o, D.,
2010. Ad anced moni o ing o pha maceu icals and es ogens in he Llob ega Ri e
basin (Spain) by liquid ch oma og aphy– iple quad upole- andem mass
spec ome y in combina ion wi h ul a-pe o mance liquid ch oma og aphy– ime o
ligh -mass spec ome y. Chemosphe e 80 (11), 1337–1344. h ps://doi.o g/
10.1016/j.chemosphe e.2010.06.042.
Ma, L.D., Li, J., Li, J.J., Liu, M., Yan, D.Z., Shi, W.Y., Xu, G., 2018. Occu ence and sou ce
analysis o selec ed an idep essan s and hei me aboli es in municipal was ewa e
and ecei ing su ace wa e . En i on. Sci. J. In eg . En i on. Res.: P ocess. Impac s
20 (7), 1020–1029. h ps://doi.o g/10.1039/c8em00077h.
Minguez, L., Fa cy, E., Ballandonne, C., Lepailleu , A., Se pen ini, A., Lebel, J.M.,
Bu eau, R., Halm-Lemeille, M.P., 2014. Acu e oxici y o 8 an idep essan s: wha a e
hei modes o ac ion? Chemosphe e 108, 314–319. h ps://doi.o g/10.1016/j.
chemosphe e.2014.01.057.
Mole, R.A., B ooks, B.W., 2019. Global scanning o selec i e se o onin eup ake
inhibi o s: occu ence, was ewa e ea men and haza ds in aqua ic sys ems.
En i on. Pollu . 250, 1019–1031. h ps://doi.o g/10.1016/j.en pol.2019.04.118.
Mo ales, G.C., 2004. Ensayos Toxicol´
ogicos Y M´
e odos de E aluaci´
on de Calidad de
Aguas, i s ed. Cen o In e nacional de In es igaciones pa a el Desa ollo.
OECD, 2004. OECD Guidelines o he Tes ing o Chemicals/Sec ion 2: E ec s on Bio ic
Sys ems Tes No. 202: Daphnia Sp. Acu e Immobilisa ion Tes . Van Ha en
Publishing. h ps://doi.o g/10.1787/9789264069947-en.
OECD, 2012. OECD Guidelines o he Tes ing o Chemicals, Sec ion 2 Tes No. 211:
Daphnia Magna Rep oduc ion Tes . Van Ha en Publishing. h ps://doi.o g/10.1787/
9789264185203-en.
P´
e y, A., Gus , M., Volla , B., Mons, R., Ramil, M., Fink, G., Te nes, T., Ga ic, J., 2008.
Fluoxe ine e ec s assessmen on he li e cycle o aqua ic in e eb a es. Chemosphe e
73 (3), 300–304. h ps://doi.o g/10.1016/j.chemosphe e.2008.06.029.
R Co e Team, 2021. Compu ing. R Founda ion o S a is ical Compu ing. R Co e Team,
Vienna, Aus ia [R: A language and en i onmen o s a is ical. h ps://www.R-p o
jec .o g/.
Rezende, K.F.O., Almeida, G.L.M.D., Hen iques, M.B., Ba bie i, E., 2021. E ec o
luoxe ine hyd ochlo ide on ou ine me abolism o lamba i (Deu e odon iguape,
Eigenmann, 1907) and phan om sh imp (Palaemon pandali o mis, S impson, 1871).
B az. A ch. Biol. Technol. 64 h ps://doi.o g/10.1590/1678-4324-2021200262.
Ribei o, A.R., Maia, A.S., Mo ei a, I.S., A onso, C.M., Cas o, P.M., Ti i an, M.E., 2014.
Enan ioselec i e quan i ica ion o luoxe ine and no luoxe ine by HPLC in
was ewa e e luen s. Chemosphe e 95, 589–596. h ps://doi.o g/10.1016/j.
chemosphe e.2013.09.118.
Risley, D.S., Bopp, R.J., 1990. Fluoxe ine. Anal. P o iles D ug Subs . 193–219. h ps://
doi.o g/10.1016/s0099-5428(08)60368-8.
Ruuskanen, S., Rainio, M.J., Kuosmanen, V., Laihonen, M., Saikkonen, K., Saloniemi, I.,
Helande , M., 2019. Female p e e ence and ad e se de elopmen al e ec s o
glyphosa e-based he bicides on ecologically ele an ai s in Japanese quails.
En i on. Sci. Technol. 54 (2), 1128–1135. h ps://doi.o g/10.1021/acs.es .9b07331.
Sakuma, M., 1998. P obi analysis o p e e ence da a. Appl. En omol. Zool 33 (3),
339–347. h ps://doi.o g/10.1303/aez.33.339.
Schlussel, A., Leininge , E., 2019. Neonico inoid insec icides and selec i e se o onin
eup ake inhibi o s in e ac an agonis ically in Daphnia magna. BIOS 90 (4), 245.
h ps://doi.o g/10.1893/0005-3155-90.4.245.
Schul z, M.M., Fu long, E.T., Kolpin, D.W., We ne , S.L., Schoen uss, H.L., Ba be , L.B.,
Blaze , V.S., No is, D.O., Vajda, A.M., 2010. An idep essan pha maceu icals in wo
U.S. E luen -impac ed s eams: occu ence and a e in wa e and sedimen , and
selec i e up ake in ish neu al issue. En i on. Sci. Technol. 44 (6), 1918–1925.
h ps://doi.o g/10.1021/es9022706.
Shi, W., Han, Y., Guan, X., Rong, J., Su, W., Zha, S., Tang, Y., Du, X., Liu, G., 2019.
Fluoxe ine supp esses he immune esponses o blood clams by educing haemocy e
iabili y, dis u bing signal ansduc ion and imposing physiological s ess. Sci. To al
En i on. 683, 681–689. h ps://doi.o g/10.1016/j.sci o en .2019.05.308.
Tie ney, K.B., Singh, C.R., Ross, P.S., Kennedy, C.J., 2007. Rela ing ol ac o y
neu o oxici y o al e ed ol ac o y-media ed beha io s in ainbow ou exposed o
h ee cu en ly-used pes icides. Aqua . Toxicol. 81 (1), 55–64. h ps://doi.o g/
10.1016/j.aqua ox.2006.11.006.
U.S. Depa men o Heal h and Human Se ices, 2021. Medical Expendi u e Panel
Su ey. Agency o Heal hca e Resea ch and Quali y. h ps://meps.ah q.go /da a
_s a s/download_da a_ iles_ esul s.jsp?cboDa aYea =All&cboDa aTypeY=2%
2CHousehold+E en +File&bu onYea andDa aType=Sea ch&cboPu Numbe =All
&Sea chTi le=P esc ibed+Medicines.
U.S. FDA, 2021. D ugs@FDA: FDA-App o ed D ugs - Fluoxe ine NDA 018936. Fda.Go .
h ps://www.accessda a. da.go /sc ip s/cde /da /index.c m?e en =o e iew.p oc
ess&ApplNo=018936.
Vasskog, T., Ande ssen, T., Pede sen-Bje gaa d, S., Kallenbo n, R., Jensen, E., 2008.
Occu ence o selec i e se o onin eup ake inhibi o s in sewage and ecei ing wa e s
a Spi sbe gen and in No way. J. Ch oma og . A 1185 (2), 194–205. h ps://doi.o g/
10.1016/j.ch oma.2008.01.063.
Wen hu , C.J., Benne , M.R., Lindsley, C.W., 2013. Classics in chemical neu oscience:
luoxe ine (p ozac). ACS Chem. Neu osci. 5 (1), 14–23. h ps://doi.o g/10.1021/
cn400186j.
Woodman, S., S einkey, D., Dew, W., Bu ke , S., B ooks, B., Pyle, G., 2016. E ec s o
se aline on beha io al indices o c ay ish O conec es i ilis. Eco oxicol. En i on. Sa .
134, 31–37. h ps://doi.o g/10.1016/j.ecoen .2016.08.011.
W i e , J.H., Fe e , I., Ba be , L.B., Thu man, E.M., 2013. Widesp ead occu ence o
neu o-ac i e pha maceu icals and me aboli es in 24 Minneso a i e s and
was ewa e s. Sci. To al En i on. 461, 519–527. h ps://doi.o g/10.1016/j.
sci o en .2013.04.099. –462.
Wu, M., Xiang, J., Chen, F., Fu, C., Xu, G., 2017. Occu ence and isk assessmen o
an idep essan s in huangpu i e o shanghai, China. En i on. Sci. Pollu . Con ol
Se . 24 (25), 20291–20299. h ps://doi.o g/10.1007/s11356-017-9293-x.
Yuan, S., Jiang, X., Xia, X., Zhang, H., Zheng, S., 2013. De ec ion, occu ence and a e o
22 psychia ic pha maceu icals in psychia ic hospi al and municipal was ewa e
ea men plan s in Beijing, China. Chemosphe e 90 (10), 2520–2525. h ps://doi.
o g/10.1016/j.chemosphe e.2012.10.089.
H. S emmel e al.