Jou nal o En i onmen al Managemen 297 (2021) 113314
A ailable online 21 July 2021
0301-4797/© 2021 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/).
Moni o ing o pha maceu icals in aqua ic bio a (P ocamba us cla kii) o he
Do˜
nana Na ional Pa k (Spain)
Julia Kazako a, Me cedes Villa -Na a o, Ma ía Ramos-Pay´
an, Noemí A anda-Me ino,
C is ina Rom´
an-Hidalgo, Miguel ´
Angel Bello-L´
opez
*
, Ru Fe n´
andez-To es
**
Depa men o Analy ical Chemis y, Facul y o Chemis y, Uni e sidad de Se illa, c/P o . Ga cía Gonz´
alez, s/n., 41012, Se ille, Spain
ARTICLE INFO
Keywo ds:
Pollu an s o eme ging conce n
Mul i esidue pha maceu icals analysis
En i onmen al p o ec ed a ea
P ocamba us cla kii
En i onmen al wa e
ABSTRACT
In his wo k he p esence o di e en pha maceu icals a Do˜
nana Na ional Pa k (Spain) and hei main en y
sou ces (inpu sou ce o en y poin s) ha e been s a ed o e he 2011–2016 yea s pe iod. Twen y- h ee selec ed
pha maceu icals (co esponding o eigh he apeu ic amilies) we e e alua ed in c ay ish and wa e samples om
Do˜
nana Na ional Pa k (Spain) (six sampling poin s selec ed in o de o co e di e en possible pollu ion sou ces
in o and su ounding he Pa k). The mul i esidue de e mina ion was ca ied ou using enzyma ic-mic owa e
assis ed ex ac ion p io o high pe o mance liquid ch oma og aphy mass spec ome y de ec ion. Sulphona-
mides (sul adiazine, sul ame azine, sul ame hazine, and sul ame hoxazole); ime hop im, an an ibio ic ha is
equen ly co-adminis e ed wi h sul ame hoxazole; amphenicols (chlo amphenicol, lo enicol and hiampheni-
col); luo oquinolones (cip o loxacin, en o loxacin, lumequine, dano loxacin, ga i loxacin, no loxacin, ma -
bo loxacin and g epa loxacin); penicillins (amoxicillin); e acyclines (chlo e acycline and oxy e acycline);
non-s e oidal an i-in lamma o y d ugs (salicylic acid and ibup o en); be a-blocke d ugs (a enolol); and an i-
epilep ics (ca bamazepine) we e analysed. Cip o loxacin, ibup o en, salicylic acid, lumequine, and ca bamaz-
epine we e de ec ed and/o quan i ied a some o he selec ed sampling poin s. A clea eco oxicological isk o
he ecosys em was demons a ed om he occu ence o cip o loxacin in samples ob ained a e he punc ual and
massi e p esence o people inside he Pa k. Fu he mo e, lumequine and ca bamazepine ha e been de ec ed in
P ocamba us cla kii specimens in concen a ions a ound 30 ng g
−1
and 14 ng g
−1
, espec i ely, and hei
occu ence in he specimens could indica e he pe sis ence o he discha ge sou ces. The main sou ce o pha -
maceu icals in o he Pa k migh be he li es ock a ming ac i i ies, and he in luence o u ban was ewa e s om
su ounding illages does no seem o be e y impo an .
1. In oduc ion
Con en ional pollu an s, such as me als o pes icides, ha e o many
yea s been subjec ed o s ic use and discha ge con ols and mos o
hem a e ou inely moni o ed. This ac has usually led o a dec ease on
hei le els in he a eas o in luence o pollu ion sou ces. Howe e ,
pollu an s o eme ging conce n (PECs) ha e no been cu en ly consid-
e ed and consequen ly, hei moni o ing and ad e se e ec s on he
en i onmen , alone o in associa ion wi h con en ional pollu an s, ha e
gone la gely unno iced. Nume ous s udies ha e demons a ed he
pene a ion o hese pollu an s in o appa en ly pollu ion- ee a eas
causing in some cases se e e e ec s including de elopmen o esis an
bac e ial popula ions and exposu e wi h po en ial d ug accumula ion in
aqua ic auna and lo a (Rigos e al., 2004; Cha u edi e al., 2021).
PECs ha e been b oadly de ined as chemical subs ances, o e en
mic oo ganisms, ha adi ionally a e no moni o ed in he en i on-
men in con as o classical pollu an s, ne e heless, i s long- e m
con inuous elease has been widely demons a ed (Molina e al., 2020;
Gonz´
alez-Mi a e al., 2018). Thei pe sis ence in he en i onmen can
esul in bio a and humans exposu e, howe e , many o hese pollu an s
ha e no been de ec ed un il he sensi i i y o analy ical me hods we e
imp o ed. Addi ionally, he use o new chemicals o changes in he use
o exis ing ones mus be conside ed as new sou ces o hei elease in o
he en i onmen .
Pha maceu icals, and hei me aboli es and deg ada ion p oduc s,
a e a well-known g oup o PECs. Thei con inuous discha ge and
* Co esponding au ho .
** Co esponding au ho .
E-mail add esses: [email p o ec ed] (M.´
A. Bello-L´
opez), [email p o ec ed] (R. Fe n´
andez-To es).
Con en s lis s a ailable a ScienceDi ec
Jou nal o En i onmen al Managemen
jou nal homepage: www.else ie .com/loca e/jen man
h ps://doi.o g/10.1016/j.jen man.2021.113314
Recei ed 16 Ma ch 2021; Recei ed in e ised o m 29 June 2021; Accep ed 15 July 2021
Jou nal o En i onmen al Managemen 297 (2021) 113314
2
pseudo-pe sis ence in he en i onmen as well as hei po en ial eco-
oxicological e ec sugges he ecosys em and e luen moni o ing need
(Rodil e al., 2012; Cha u edi e al., 2021). Excep o he in luence o
e luen s om pha maceu ical indus ies in some places, he main
sou ces o hese pollu an s a e u ban was ewa e s discha ges (including
hose unde going ea men ) and e luen s om aquacul u e and li e-
s ock ac i i ies (Vys a na e al., 2012; Bendz e al., 2005; Comeau e al.,
2008). Howe e , he p esence o pha maceu icals, gene ally, is no only
es ic ed o he aqua ic en i onmen close o he abo e-men ioned
sou ces; bu also su ace and g oundwa e s eams can ac as ec o s
o he sp ead o hese pollu an s and hey can be ound a signi ican
dis ances om p ima y discha ges (Fe ando-Climen e al., 2014;
Kandie e al., 2020; Szymczycha e al., 2020; Li e al., 2020).
Se e al s udies ha e been published, so a , dealing wi h he occu -
ence o pha maceu icals in en i onmen al wa e s (Sco e al., 2014;
Rod íguez-Mozaz e al., 2016; Oso io e al., 2016; Rogowska e al., 2020;
Kalka e al., 2020; Majumbe e al., 2019) as well as many ocused on
sedimen s (Vazquez-Roig e al., 2012; Llompa e al., 2019) and soils
(Kodeˇ
so ´
a e al., 2019; Golo ko e al., 2016). Al hough o a lessen
ex en , some s udies ha e a emp ed o assess he isk in he aqua ic
en i onmen by he analysis o pha maceu icals in en i onmen al bio a
(Ace˜
na e al., 2016; Mille e al., 2018, 2019; Hue a e al., 2018;
Al a ez-Mu˜
noz e al., 2015; Mo eno-Gonz´
alez e al., 2016; Boillo e al.,
2015; Dodde e al., 2014; R¨
osch e al., 2016). I is he e o e clea ha
he de e mina ion o pollu an s in a bio a ecosys em p o ides mo e in-
o ma ion and helps o unde s and he cause-e ec ela ionships
ega ding hei en i onmen al impac han he measu emen o disc e e
le els on wa e , soil o ai samples. Fu he mo e, ecological and
biochemical s udies o he exposed bio a allow i s use as an en i on-
men al o ecosys em heal h indica o (bioindica o s). Repo s ocused
on he e alua ion o he en i onmen al pollu ion om di e en sou ces
in he su oundings o he Do˜
nana na ional pa k (DNP) (Ga ido e al.,
2016; Ba ´
on e al., 2014, 2015) ha e been published. The esul s e i-
denced ha bo h, ag icul u al ac i i ies and discha ges om was ewa e
ea men plan s (WWTPs) in highly popula ed ci ies nega i ely a ec
he wildli e sanc ua y. On he o he hand, s udies on ee-li ing Mus
sp e us mice, used as bioindica o s, ha e assessed he pollu ion s a us o
a eas su ounding DNP h ough iTRAQ (isoba ic ags o ela i e and
absolu e quan i ica ion) analysis o hei hepa ic p o eins (Ab il e al.,
2015).
The e a e limi ed s udies in di e se zones o he pa k and su -
ounding a eas, and only some pape s ela ed o measu ing p io i y
pollu an s ha e been published. The e o e, he e a e some da a on le els
o pha maceu icals in su ace wa e s wi hin he pa k. Thus, le els o
some pha maceu icals om en i onmen al wa e s and u ban was e-
wa e ea men plan s e luen s we e epo ed (Camacho-Mu˜
noz e al.,
2010a,b), likewise, some pha maceu ically ac i e compounds we e
measu ed in sedimen s om DNP and su ounding a eas (Camacho--
Mu˜
noz e al., 2013).
In he 70s o he las cen u y, he ame ican ed swamp c ay ish
(P ocamba us cla kii) was in oduced in he su oundings o DNP wi h
he aim o i s exploi a ion as a ishe y esou ce. Acclima iza ion o he
specie was a success and soon s a ed o be exploi ed comme cially. The
economic bene i epo ed and he ease o inding li e c abs on he
ma ke encou aged hei exploi a ion in a eas wi h simila habi a s o
epea he expe ience. Clima ic condi ions on he sou h o he Ibe ian
Peninsula and he absence o na u al p eda o s allow o a wide and as
expansion o his c ay ish specie and, nowadays, P ocambu us cla kii is
sp ead h oughou p ac ically all he Ibe ian Peninsula and se e al zones
o Eu ope. P. cla kii was also a ca ie o a disease caused by he ungus
Aphanomyces as aci, which had a de as a ing e ec on Eu opean c ay ish
(Aus opo amobius pallipes) popula ions, in addi ion o hei nega i e
e ec on he ag icul u al in as uc u es due o digging galle ies in he
a eas i occupies. The na i e c ay ish, al eady ha d hi by he loss o
wa e quali y o he i e s hey inhabi ed, was decima ed by his plague
and oday A. pallipes only su i es in he headwa e s o some clean, cold
i e s inaccessible o P. cla kii. Gi en i s p esence h oughou DNP and
i s physiological ole ance o low le els o dissol ed O
2
(2–14 mg L
−1
),
changes in empe a u e (17-22 ◦C) and high a es o pollu ion, P. cla kii
migh be an app op ia e en i onmen al bioindica o specie. Fu he -
mo e, he p esence o P. cla kii in he pa k makes i possible o sp ead he
con amina ion e ec o o he species.
Some pape s ha e been published using P. cla kii as bioindica o by
co ela ing some biochemical pa ame e s and en i onmen al pollu ion
(Vioque-Fe n´
andez e al., 2007, 2009; Osuna-Jim´
enez e al., 2014;
Fe n´
andez-Cisnal e al., 2014).
In his wo k, specimens o P. cla kii we e sampled wi hin he yea s
2011–2016 om six zones o he DNP which we e selec ed in o de o
co e di e en pollu ion sou ces in o and su ounding he Pa k. Addi-
ionally, wa e samples om he same sampling poin s we e also
collec ed and analysed. C ay ish specimens we e analysed acco ding o a
p e iously desc ibed analy ical p ocedu e (Kazako a e al., 2018) and
23 pha maceu ical ac i e p inciples co esponding o 8 he apeu ic
amilies we e analysed: sulphonamides (sul adiazine, sul ame azine,
sul ame hazine, and sul ame hoxazole); ime hop im, an an ibio ic ha
is equen ly co-adminis e ed wi h sul ame hoxazole; amphenicols
(chlo amphenicol, lo enicol and hiamphenicol); luo oquinolones
(cip o loxacin, en o loxacin, lumequine, dano loxacin, ga i loxacin,
no loxacin, ma bo loxacin and g epa loxacin); penicillins (amoxicillin);
e acyclines (chlo e acycline and oxy e acycline); non-s e oidal
an i-in lamma o y d ugs (salicylic acid and ibup o en); be a-blocke
d ugs (a enolol); and an iepilep ics (ca bamazepine).
The main goal o his wo k is explo ing he use o specimens o
P. cla kii as possible bioindica o s o he en i onmen al p essu e o
pha maceu icals ha he Do˜
nana Na ional Pa k could su e in he
aqua ic media. Addi ionally, he iden i ica ion o possible pollu ion
sou ces will be discussed.
2. Ma e ials and me hods
2.1. Chemicals and s anda ds
Sul adiazine (SDZ), sul ame azine (SMR), sul ame hazine (SMT),
and sul ame hoxazole (SMX), ime hop im (TMP), chlo amphenicol
(CLF), lo enicol (FLF), hiamphenicol(TIF), cip o loxacin (CPR),
en o loxacin (ENR), lumequine (FMQ), dano loxacin (DNF), ga i-
loxacin (GTF), no loxacin (NRF), ma bo loxacin (MRB), g epa loxacin
(GPF), amoxicillin (AMX), chlo e acycline (CLT), oxy e acycline
(OXT), salicylic acid (SAC) ibup o en (IBU), a enolol (ATN) and ca ba-
mazepine (CBZ) (97–99.9% pu i y) we e pu chased om Fluka-Sigma-
Ald ich, S.A. (Mad id, Spain). P o einase-K (20.2 mg mL
−1
) ecombi-
nan PCR g ade was supplied om Roche Diagnosis (Ba celona, Spain).
Ace oni ile and me hanol (LC-MS g ade) and o mic acid (analy ical
g ade) we e pu chased om VWR (Ba celona, Spain). Ul apu e deion-
ized wa e (Milli-Q Plus wa e sys em Millipo e (Bille ica, MA, USA))
was used o all aqueous solu ions and dilu ions.
S ock solu ions we e p epa ed by dissol ing he subs ances in
me hanol, excep CLF, FLF, OXT, CLT and TIF which we e dissol ed in
ul apu e wa e and CPR by a 80:20 / me hanol:wa e mix u e. 400
mg L
−1
s ock solu ions we e p epa ed o all ac i e pha maceu ical
subs ances, excep o CLT, OXT and CPR which 100 mg L
−1
ones we e
p epa ed. All he s ock solu ions we e s able o a leas 2 mon hs a 4 ◦C,
excep CPR, CLT and OXT solu ions ha mus be p epa ed daily and kep
a oom empe a u e due o he low solubili y o hese subs ances.
Wo king solu ions we e daily p epa ed by adequa e dilu ions using ul-
apu e wa e .
2.2. Sampling plan in Do˜
nana Na ional Pa k
DNP is a p o ec ed a ea o e 100.000 ha and i is loca ed in he
Guadalqui i es ua y in Andalusia (Spain). Wi hin his a ea, i is he
Do˜
nana Na u al Rese e wi h 54.252 ha, which is in eg a ed in he
J. Kazako a e al.
Jou nal o En i onmen al Managemen 297 (2021) 113314
3
Ramsa Con en ion o he p o ec ion o we lands and ecognized by
UNESCO, i s as a Biosphe e Rese e and hen as a Wo ld He i age Si e.
DNP has a biodi e si y ha is unique in Eu ope.
A sampling plan ( om 2011 o 2016) o P. cla kii specimens and
su ace wa e samples collec ion was designed in o de o selec he
sampling poin s and he mos app op ia e seasons. Fig. 1 shows he
loca ion o he selec ed sampling poin s, and hei co esponding UTM
coo dina es. B ie ly, he main cha ac e is ics ha de e mined he sam-
pling a eas om an en i onmen al poin o iew a e also desc ibed
below. These sampling poin s we e selec ed in o de o de e mine po-
en ial impac s om eme ging pollu an s and, essen ially, conside ing
he possible en i onmen al p essu e om eme ging pollu an s such as
he selec ed ones. Also, i mus be conside ed ha he wa e s eams ha
c oss he DNP ecei e he e luen s o was ewa e ea men plan s
(WWTPs) o some illages.
Rocina (ROC) co esponds o a sampling poin in he headwa e s o
he s eam “La Rocina”. This si e could be in luenced by li es ock hus-
band y and could be also a ec ed by he in ensi e s awbe ies plan-
a ions in g eenhouses loca ed in he sou oundings. The sampling poin
is close o he illage o “El Rocío”, wi h a ela i ely low s able popu-
la ion which signi ican ly inc eases du ing he weekends, and i should
be no ed ha o one week a yea , i s popula ion g ows d ama ically
exceeding a million inhabi an s due o a popula annual pilg image. The
e ec o he punc ual high popula ion on he le els o eme ging pol-
lu an s has been e i ied acco ding o he esul s ob ained.
Be nab´
e (BER) is also a sampling poin loca ed in he lowe wa e -
shed o he s eam “La Rocina” bu nea o “El Rocío”, and he in luence
o li es ock husband y and ci us c ops mus also be conside ed om an
en i onmen al poin o iew.
Pa ido (PAR) co esponds o a sampling poin in he headwa e s o
he s eam “El Pa ido”. Ci us c ops and he p esence o ho ses mus be
conside ed as wa e pollu ion sou ces.
The sampling poin Ajolí (AJO) is also loca ed in he s eam “El
Pa ido” bu nea o he illage “El Rocío”. On his poin , besides he
in luence o ci us c ops, i is necessa y o conside he p esence o ho se
and ca le a ming ac i i ies.
Ma ochal (MAT) is a sampling poin loca ed nea he Guadiama
i e and he ex e nal wes e n limi s o he Pa k. The p esence o pol-
lu an s in his zone can be condi ioned by he nea by and widesp ead
ice c ops, he Aznalcolla mines in he headwa e s o he Guadiama
i e (in amous o an ecological mining disas e in 1998) and u ban
was ewa e discha ges om he illages: Aznalcaza , Pilas and Isla
Mayo . The in luence o wa e om he Guadalqui i i e mus be also
conside ed.
Lucio del Palacio (LPD) was ini ially conside ed as “clean sampling
poin ” due o i s loca ion in he inne mos pa o he Pa k, and no
in luenced by ex e nal pollu ion sou ces. Howe e , he close p esence o
Do˜
nana’s Singula Scien i ic-Technical In as uc u e (ICTS-RBD) o
DPN pe sonnel and esea che s could condi ion some en i onmen al
p essu e.
The sampling pe iod was also de e mined aking in o accoun he
abundance o he P. cla kii species acco ding o he clima e. Clima o-
logical da a we e ob ained om wo s a ions loca ed in he su oundings
o he DNP: RAIFSE004 “Phy osani a y Wa ning and In o ma ion
Ne wo k (CAPDR)” (Red de Ale a e In o maci´
on Fi osani a ia, Con-
seje ía de Ag icul u a y Pesca, Jun a de Andalucía, Spain) and he s a-
ion RIA2110 “Ag oClima ic In o ma ion Ne wo k (IFAPA)” (Red de
In o maci´
on Ag oClim´
a ica (IFAPA), Ins i u o de In es igaci´
on y Fo -
maci´
on Ag a ia y Pesque a, Jun a de Andalucía, Spain). Table S1 and
Table S2 (supplemen a y elec onic da a) p o ide da a on a e age
eco ded mon hly empe a u e and a e age mon hly ain all o e he
pe iod 2011–2016. DNP has a mild, ypically Medi e anean clima e
wi h A lan ic in luence, ain all ends o be sca ce in summe and ela-
i ely gene ous in win e ; howe e , ain all is qui e a iable. The
a e age ain all (580 mm) is mainly usually dis ibu ed om Sep embe
o Ap il. The mic oscilla ion be ween maximum empe a u es
h oughou he yea is abou 17 ◦C. F om he end o sp ing o he end
summe , he p esence o a sub opical an icyclone loca ed nea Azo es
Islands leads o a d y and qui e ho pe iod. Howe e , ce ain zones o he
Pa k e ain wa e s eams due o ex e nal con ibu ions om i e s and
i ule s. Bo h, empe a u e and ain all a ec he c ay ish abundance
and he dilu ion o concen a ion o possible pollu an s in he wa e
s eams. Fo his eason, sp ing ( om May o June) was he mos
app op ia e season.
2.3. Sample collec ion, p epa a ion and ex ac ion
P. cla kii specimens and su ace wa e samples we e collec ed in he
pe iod om May o June 2011 o 2016 a he six sampling poin s (one
ime/24 h sampling) loca ed in he Na u al Rese e o Do˜
nana desc ibed
in p e ious sec ion and i s su ounding a eas.
The numbe o specimens cap u ed each yea was a iable due o
hyd o-me eo ological condi ions; howe e , an a e age numbe o
app oxima ely 100 specimens pe yea in he se o loca ions we e
collec ed using aps placed in wa e ways and collec ed a e 24 h.
Cap u ed P. cla kii we e shipped unde cool condi ions (<4 ◦C) o ou
labo a o y. Muscle issue wi hou en al ne e co d and he hepa o-
panc eas ( isce a) om each specimen we e ob ained o subsequen
analysis and hey we e s o ed o less han 5 day a −80 ◦C be o e
analysis. F ozen samples we e lyophilized and hen homogenized using
a g inde o ob ain wo se s o sample pools om each sampling poin .
Samples we e kep a −80 ◦C un il hei ex ac ion and analysis.
P. cla kii samples we e ex ac ed acco ding o a p e iously op imized
ex ac ion p ocedu e (Kazako a e al., 2018). Thus, 0.5 g o each sample
pool o lyophilized muscle o isce a samples was placed in o p essu -
ized PTFE mic owa e essels and hen a mix u e con aining 10 mL o
ace oni ile:wa e (1:1, / ), 50
μ
L o comme cial P o einase-K solu ion
and 5
μ
L o concen a ed o mic acid was added. 50 W mic owa e powe
(E hos One, Milles one, So isole (BG), I aly) was applied o 5 min.
Cooled ( oom empe a u e) muscle and isce a samples we e cen i-
uged a 7000 pm o 10 min and 20 min, espec i ely. Fi s , he liquid
ex ac s we e e apo a ed unde a ni ogen s eam, hen dissol ed using
1 mL o 0.1% ( / ) aqueous o mic acid solu ion and il e ed h ough a
0.22
μ
m nylon sy inge il e . Finally, a 10
μ
L aliquo was di ec ly
injec ed in o he ch oma og aphic sys em. Fo isce a samples, an
addi ional cen i uga ion s ep (20 min a 7000 pm) was applied p e-
ious o mic o il a ion.
Addi ionally, wa e samples om each sampling poin we e also
collec ed using p ecleaned polyp opylene (PP) bo les. The collec ed
samples we e shipped a empe a u e <4 ◦C. Wa e samples we e
il e ed h ough a nylon 0.45
μ
m il e p io ex ac ion. Ex ac ion was
ca ied ou , wi h sligh modi ica ions, acco ding o he p oposed p o-
cedu e o G os e al. (2009). Fo SPE ex ac ion, a 12-po Visip ep™
S anda d acuum mani old (Supelco, Sigma-Ald ick, Mad id, Spain) was
used. Oasis HLB ca idges (60 mg) (Wa e s Co po a ion, Mil o d, MA,
USA) we e p e iously condi ioned using 5 mL o me hanol and 5 mL o
analy ical-g ade wa e . 500 mL o il e ed wa e we e passed h ough
he SPE ca idge a a low a e o 4–5 mL min
−1
. The ca idges we e
hen washed wi h 5 mL o wa e and hey we e acuum-d ied o 15 min.
Elu ion was ca ied ou using 2 ×4 mL o me hanol and he ex ac s
we e e apo a ed o d yness unde ni ogen s eam. The esidues we e
econs i u ed in 1 mL o analy ical-g ade wa e and il e ed (0.22
μ
m)
p io injec ion o i s analysis (10
μ
L).
2.4. Ins umen al analysis
Analysis o ex ac ed muscle, isce a and wa e samples we e pe -
o med using a p e ious p ocedu e (Kazako a e al., 2018). B ie ly, an
Agilen 1290 In ini y UPLC sys em was used and he sepa a ion was
achie ed using a e e sed-phase Zo bax Eclipse XDB-C18 analy ical
column (150 mm ×4.6 mm, pa icle size 3
μ
m) p eceded by a
gua d-column Zo bax ODS (4.6 mm ×12.5 mm, pa icle size 5
μ
m)
J. Kazako a e al.
Jou nal o En i onmen al Managemen 297 (2021) 113314
4
Fig. 1. Loca ion o he sampling poin s.
J. Kazako a e al.
Jou nal o En i onmen al Managemen 297 (2021) 113314
5
(Agilen Technologies Spain S.L). Bina y g adien elu ion p og ams a
0.4 mL min
−1
low a e we e pe o med using 0.1% ( / ) o mic acid
aqueous solu ion (A) and ace oni ile (B) as mobile phase. Fo posi i e
ioniza ion mode, he g adien was: (1) 12 min linea elu ion g adien
om 0% o 30% B, (2) 5 min linea g adien o 100% B and (3) 4 min o
isoc a ic s ep. Fo nega i e ioniza ion mode, he used g adien was: (1)
15 min linea elu ion g adien om 10% o 30% B and (2) 6 min linea
g adien o 100% B. In bo h cases, he composi ion was e u ned o
ini ial condi ions (0% B and10% B o posi i e and nega i e ioniza ion
mode, espec i ely) and 5 min we e wai ed be o e he nex injec ion.
De ec ion was pe o med by an Applied Biosys ems 3200 QTRAP LC/
MS-MS ins umen (ABSciex, Fos e Ci y, USA), an hyb id iple quad-
upole linea ion ap (QqQLIT) mass spec ome e . Ioniza ion was
pe o med by an elec osp ay ion sou ce (Tu boSp ay®). Mul iple Re-
ac ion Moni o ing (MRM) mode was used o he ch oma og aphic
de ec ion, selec ing [M+H]
+
and [M-H]
-
as p ecu so ions o posi i e
and nega i e ioniza ion mode, espec i ely. O he mass spec ome e
pa ame e s we e: (1) collision gas (ni ogen) a 4 psi, (2) ion sou ce and
cu ain gases a 30 psi (posi i e ioniza ion) and 20 psi (nega i e ioni-
za ion),(3) elec osp ay (Tu boSp ay®) ol ages: 5.5 kV (posi i e) and
−4.5 kV (nega i e), (4) sou ce empe a u e 500 ◦C o bo h modes. Two
analy ical signal we e used: he mos in ense o quan i ica ion and he
second one o quali a i e con i ma ion.
3. Resul s and discussion
Sec ion 2.2 desc ibes he sampling plan o c ay ish specimens and
wa e samples om DNP and su oundings in o de o ob ain ep e-
sen a i e samples o he di e se en i onmen al si ua ions in he a ea
ela ed o he possible sou ces o pha maceu icals con ibu ions o he
DNP aqua ic media. An excellen epo on he hyd ology o he DNP and
su ounding a eas ha e been published (WWF Wo ld Wildli e Fund
Espa˜
na, 2009).
Fig. 2 shows he hyd ological unc ioning scheme o he s udied a ea
and he loca ions o he u ban was ewa e ea men plan s a ound he
DNP. As can be seen, DNP ecei es su ace wa e s con ibu ions om
Guadalqui i and i e s and some mino wa e s eams. The e o e,
su ace wa e s ha con ain discha ges om u ban WWTPs pene a e o
deep a eas o he Pa k. Addi ionally, despi e he ac ha he Gua-
dalqui i i e ecei es e luen s om a signi ican numbe o WWTPs
h oughou i s en i e ou e, i s low low in o he pa k as well as he in-
luence o idal wa e s means ha i s in luence on he aqua ic media o
he pa k should be conside ed sca ce.
The p esence o pha maceu icals in e luen s om u ban was ewa e
ea men plan s (WWTPs) loca ed in he su oundings o DNP ha e
been p e iously desc ibed (Camacho-Mu˜
noz e al., 2010a; Camacho--
Mu˜
noz e al., 2010b) so a p io i i would be possible o hink ha his
con amina ion could each deep a eas o he pa k.
Acco ding o he analy ical p o ocols desc ibed in he expe imen al
sec ion, Table 1 shows he ob ained esul s o he analysis o wa e and
P. cla kii collec ed samples. Analy ical de ails o he applied me hod
(de ec ion and cuan i a ion limi s, ch oma og ams, …) can be e ised in
ou p e ious published pape (Kazako a e al., 2018). Fi s , i mus be
poin ed ou ha du ing he 2013–2016 pe iod, samples om MAT
sampling poin we e no a ailable because he channel was d y.
Samples om ROC, BER, and LDP showed in gene al he lowe (o
null) alues o mos o he analy es. Howe e , wa e samples om ROC
(in 2014) showed 5.1 ng mL
−1
o CPR and his d ug was also de ec ed in
bo h muscle and isce a P. cla kii samples om his loca ion. Despi e he
high alue de ec ed in he wa e sample, he low le el o CPR in he
c ay ish specimens (<1.0 ng g
−1
) could be indica ing a ecen discha ge.
CPR is also a usual e e ina y d ug hus i s p esence could be jus i ied by
li es ock a ming ac i i ies.
FMQ was ound all yea s, excep ing 2011, in wa e samples and
c ay ish specimens collec ed om AJO sampling poin ; i is no iceable
he high alues ound in he yea s 2014–2016 in he wa e samples wi h
alues o 4200 and 5100 ng mL
−1
, espec i ely. Addi ionally, high le els
o FQM ha e been measu ed o he cap u ed specimens in his loca ion,
which could be indica i e o a con inuous discha ge on sampling da es.
Fo he es o he yea s excep ing 2015, low FMQ wa e le els we e also
accompanied by ela i ely high alues om P. cla kii specimens. In
2015, he low FMQ le el in wa e om AJO (<2 ng mL
-
1) and he
ela i ely high le els o his d ug de ec ed in he muscle and isce a o
cap u ed c ay ishes could be co ela ed o a non- ecen discha ge, being
i s accumula ion in he specimens he con i ma ion o hei selec ion as
bioindica o s. In his loca ion, he impo an equine and bo ine li e-
s ock a ming could jus i y his ac since lumequine is a widely used
e e ina y an ibio ic (Biel-Maeso e al., 2018; Kim e al., 2018; Ma e al.,
2012).
Addi ionally, o he d ugs (IBU and CPR) we e ound a lowe con-
cen a ion le els in wa e samples and/o specimens o e he yea s in
his loca ion showing ha AJO could be conside ed as he loca ion ha
suppo s he g ea e en i onmen al p essu e om pha maceu icals in o
he Do˜
nana Na ional Pa k. The ela i ely high pe sis ence o IBU in he
en i onmen could be indica ing he in luence o he discha ges om
some o he WWTPs ha a e loca ed in he DNP su oundings. I is
no iceable ha his d ug was only de ec ed in his loca ion; i s de ec ion
in wa e samples om LPD could be explained in o he e ms (see below
in he ex ).
In he yea 2014, he sampling was ca ied ou a e he annual
pe eg ina ion o “el Rocio” ha c oss he DNP and ga he s mo e han
one million people in he illage o “El Rocio” o some days. This ac
could explain he de ec ion o impo an le els o cip o loxacin (CPR) in
he wa e s om ROC (close o his illage) due o i is an an ibio ic
widely used o he ea men o se e al in ec ious diseases and impo -
an amoun s o no me abolized d ug is exc e ed h ough he u ine.
The punc ual p esence o ca bamazepine in some sampling poin s
and yea s mus be di ec ly ela ed o he spo adic in luence o e luen s
con aining human exc e ions, p obably om discha ges o he WWTPs
ha a e loca ed a ound he Pa k. Ca bamazepine appea s ecu en ly in
se e al s udies ela ed o he occu ence o PECs in u ban was ewa e s
(Ma ínez-Pie nas e al., 2018; Fekadu e al., 2019). CBZ was de ec ed in
specimens o P. cla kii om MAT (in 2012), BER (in 2015) and PAR (in
2015 and 2016). An impo an le el o CBZ (4.7 ng mL
−1
) was also
measu ed in he wa e sample om BER in 2015 bu his d ug was un-
de ec ed in he c ay ish specimens. I is ema kable ha his d ug was
de ec ed a high le els in e luen s om u ban was ewa e ea men
plan s (WWTPs) loca ed in he su oundings o he DNP (Camacho--
Mu˜
noz e al., 2010a; Camacho-Mu˜
noz e al., 2010b). The low en i on-
men al deg ada ion o CBZ (Bahlmann e al., 2014) could explain i s
p esence in c ay ish specimen in deep a eas o he Do˜
nana Na ional Pa k
om he su ounding owns.
Finally, he de ec ion o salicylic acid and ibup o en in wa e samples
om Lucio de Palacio (yea s 2012, 2015 and 2016) could be ela ed o
spo adic discha ges om DNP scien i ic pe sonnel and some o he pa k
gua ds. Ace ylsalicylic acid and ibup o en a e wo e y common pha -
maceu icals usually de ec ed a high le els in u ban was ewa e s,
including hose loca ed nea DNP (Camacho-Mu˜
noz e al., 2010a;
Camacho-Mu˜
noz e al., 2010b). Bo h d ugs, like o he ac i e pha ma-
ceu ical p oduc s, ha e a medium o high pe sis ence in he en i on-
men , as a ma e o ac ad anced oxida ion p ocesses ha e a isen as
al e na i es o hei deg ada ion in was ewa e s (A hu e al., 2018;
Jallouli e al., 2018; Ellepola e al., 2020).
4. Conclusions
A s udy on he p esence and occu ence o 23 selec ed pha maceu-
icals in c ay ish and wa e samples om Do˜
nana Na ional Pa k (Spain)
was ca ied ou om 2011 o 2016 pe iod. This s udy demons a ed ha
some pha maceu icals we e de ec ed e en a ela i ely high le els. I is
ema kable ha hei occu ence in specimens o P. cla kii could indica e
he pe sis ence o he discha ge sou ces.
J. Kazako a e al.

Jou nal o En i onmen al Managemen 297 (2021) 113314
6
Fig. 2. Hyd ological unc ioning diag am o he Do˜
nana Na ional Pa k and i s su oundings and loca ions o u ban was ewa e ea men plan s (WWTPs).
J. Kazako a e al.
Jou nal o En i onmen al Managemen 297 (2021) 113314
7
Table 1
Le els ound (wa e (ng⋅mL
−1
)/muscle (ng⋅g
−1
)/ isce a (ng⋅g
−1
)) o he selec ed analy es om he di e en sampling poin s.
Sampling poin
Analy e Yea 2011 Yea 2012
ROC BER PAR AJO MAT LDP ROC BER PAR AJO MAT LDP
IBU <3/0/0
CBZ 0/<0.9/<0.9
SMX 0/<0.3/<0.3
FMQ <2/<1.1/<1.1 <2/<1.1/<1.1
SAC <0.8/0/0 <0.8/0/0 <0.8/0/0
CPR <2/0/0 0/<1.0/<1.0
Yea 2013 Yea 2014
ROC BER PAR AJO MAT LDP ROC BER PAR AJO MAT LDP
IBU <3/0/0 ** <3/<1.5/0 **
CBZ ** **
SMX ** **
FMQ 0/<1.1/<1.1 ** 4.2/21.2/1.1 **
SAC ** **
CPR ** 5.1/<1.0/<1.0 **
Yea 2015 Yea 2016
ROC BER PAR AJO MAT LDP ROC BER PAR AJO MAT LDP
IBU ** ** <3/0/0
CBZ 0/10.8/0 4.7/0/0 0/0/14.6 ** 0/9.6/20.8 **
SMX ** **
FMQ <2/30.6/27.6 ** 5.1/26.2/34.2 **
SAC ** <0.8/0/0 **
CPR ** **
0: Unde ec ed in he co esponding sample.
** Wa e samples and P.cla kii specimens no a ailable.
Blank: unde ec ed in any sample.
J. Kazako a e al.
Jou nal o En i onmen al Managemen 297 (2021) 113314
8
The da a analysis co ela ing he pha maceu ical ound and he
en i onmen al issues o he co esponding sampling poin s e eals ha
he main sou ce o pha maceu icals in o DNP migh be he li es ock
a ming ac i i ies, mainly equine and bo ine. Addi ionally, esul s e-
eals ha some sampling poin s migh also be submi ed o he in luence
o u ban a eas since he p esence o some d ugs commonly p esc ibed
o human use ha e been also e i ied. Howe e , despi e he high le els
ound in WWTPs e luen s by some au ho s (Camacho-Mu˜
noz e al.,
2010a; Camacho-Mu˜
noz e al., 2010b) and ha su ace wa e s ha
con ain discha ges om u ban WWTPs pene a e o deep a eas o he
Pa k, he in luence o u ban was ewa e s om su ounding illages does
no seem o be e y impo an acco ding o he da a ob ained in his
s udy.
Finally, a di ec an h opogenic in luence was also con i med by he
da a ob ained du ing he annual pilg image o “el Rocio” in 2014 which
passes h ough DNP and by he da a om LDP sampling poin whe e he
close p esence o DNP s a could exe some en i onmen al p essu e.
The da a om cap u ed P. cla kii specimens e eals ha his o gan-
ism could be a good bioindica o because al hough in some sampling
poin s wa e samples analysis does no e eals he p esence o any o he
selec ed pha maceu icals, c ay ish specimens accumula ed some o hem
in o hei muscle and/o isce a.
C edi au ho s a emen
Julia Kazako a: Valida ion, Fo mal analysis, In es iga ion, Re-
sou ces, W i ing - O iginal D a Me cedes Villa -Na a o: In es iga-
ion, Resou ces, W i ing - O iginal D a Ma ía Ramos-Pay´
an:
In es iga ion, W i ing - O iginal D a . Noemí A anda-Me ino: Vali-
da ion, Fo mal analysis, C is ina Rom´
an-Hidalgo: Valida ion, Fo mal
analysis, Miguel ´
Angel Bello-L´
opez: Concep ualiza ion, W i ing -
O iginal D a , W i ing - Re iew & Edi ing, Visualiza ion, Supe ision,
P ojec adminis a ion, Funding acquisi ion Ru Fe n´
andez-To es*:
Concep ualiza ion, Resou ces, W i ing - O iginal D a , W i ing - Re iew
& Edi ing, Visualiza ion, Supe ision, P ojec adminis a 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 .
Acknowledgemen s
Au ho s a e g a e ul o Se icio Gene al de In es igaci´
on de Mic o-
an´
alisis, CENTRO DE INVESTIGACI´
ON TECNOL´
OGICA E INNOVACI´
ON
(CITIUS). Uni e sidad de Se illa, o he use o he ch oma og aphic
equipmen . This wo k was suppo ed by he P ojec s CTM2012-38720-
C03-01 om he “Di ecci´
on Gene al de In es igaci´
on y Ges i´
on del Plan
Nacional de I+D+i (Minis e io de Economía y Compe i i idad, Spain)”,
CTM2015-67902-C02-01-P om he “Di ecci´
on Gene al de In es-
igaci´
on y Ges i´
on del Plan Nacional de I+D+i (Minis e io de Economía
y Compe i i idad, Spain)” and PGC2018-096608-B-C22 om FEDER/
Minis e io de Ciencia e Inno aci´
on – Agencia Es a al de In es igaci´
on.
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.jen man.2021.113314.
Re e ences
Ab il, N., Chicano-G´
al ez, E., Mich´
an, C., Pueyo, C., L´
opez-Ba ea, J., 2015. iTRAQ
analysis o hepa ic p o eins in ee-li ing Mus sp e us mice o assess he
con amina ion s a us o a eas su ounding Do˜
nana Na ional Pa k (SW Spain). Sci.
To al En i on. 5231, 16–27.
Ace˜
na, J., Heue , N., Ga dinali, P., P´
e ez, S., 2016. Suspec sc eening o pha maceu icals
and ela ed bioac i e compounds, hei me aboli es and hei ans o ma ion
p oduc s in he aqua ic en i onmen , bio a and human using LC-HR-MS Techniques.
Comp . Anal. Chem. 71, 357–378.
Al a ez-Mu˜
noz, D., Rod iguez-Mozaz, S., Maul aul , A.L., Tediosi, A., Fe nandez-
Tejedo , M., Van den Heu el, F., Ko e man, M., Ma ques, A., Ba cel´
o, D., 2015.
Occu ence o pha maceu icals and endoc ine dis up ing compounds in
mac oalgaes, bi al es, and ish om coas al a eas in Eu ope. En i on. Res. 143,
56–64.
A hu , R.B., Bonin, J.L., A dill, L.P., Rou k, E.J., Pa e son, H.H., S emmle , E.A., 2018.
Pho oca aly ic deg ada ion o ibup o en o e BiOCl nanoshee s wi h iden i ica ion o
in e media es. J. Haza d Ma e . 358, 1–9.
Bahlmann, A., B ack, W., Schneide , R.J., K auss, M., 2014. Ca bamazepine and i s
me aboli es in was ewa e : analy ical pi alls and occu ence in Ge many and
Po ugal. Wa e Res. 57, 104–114.
Ba ´
on, E., M´
a˜
nez, M., And eu, A.C., Se gio, F., Hi aldo, F., Elja a , E., Ba cel´
o, D., 2014.
Bioaccumula ion and biomagni ica ion o eme ging and classical lame e a dan s in
bi d eggs o 14 species om Do˜
nana Na u al Space and su ounding a eas (Sou h-
wes e n Spain). En i on. In . 68, 118–126.
Ba ´
on, E., Bosch, C., M´
a˜
nez, M., And eu, A.C., Se gio, F., Hi aldo, F., Elja a , E.,
Ba cel´
o, D., 2015. Tempo al ends in classical and al e na i e lame e a dan s in
bi d eggs om Do˜
nana Na u al Space and su ounding a eas (sou h-wes e n Spain)
be ween 1999 and 2013. Chemosphe e 138, 316–323.
Bendz, D., Paxeus, N.A., Ginn, T.R., Loge, F.J., 2005. Occu ence and a e o
pha maceu ically ac i e compounds in he en i onmen , a case s udy: hoje Ri e in
Sweden. J. Haza d Ma e . 122 (3), 195–204.
Biel-Maeso, M., Baena-Nogue as, R.M., Co ada-Fe n´
andez, C., La a-Ma in, P.A., 2018.
Occu ence, dis ibu ion and en i onmen al isk o pha maceu ically ac i e
compounds (PhACs) in coas al and ocean wa e s om he Gul o Cadiz (SW Spain).
Sci. To al En i on. 612, 649–659.
Boillo , C., Ma inez Bueno, M.J., Muna on, D., D eau, M.L., Ma hieu, O., Da id, A.,
Fene , H., Casellas, C., Gomez, E., 2015. In i o exposu e o ma ine mussels o
ca bamazepine and 10-hyd oxy-10,11-dihyd o-ca bamazepine: bioconcen a ion
and me aboliza ion. Sci. To al En i on. 532, 564–570.
Camacho-Mu˜
noz, D., Ma ín, J., San os, J.L., Apa icio, I., Alonso, E., 2010b. Occu ence,
empo al e olu ion and isk assessmen o pha maceu ically ac i e compounds in
Do˜
nana Pa k (Spain). J. Haza d Ma e . 183 (1–3), 602–608.
Camacho-Mu˜
noz, D., Ma ín, J., San os, J.L., Apa icio, I., Alonso, E., 2013. Dis ibu ion
and isk assessmen o pha maceu ical compounds in i e sedimen s om Do˜
nana
Pa k (Spain). Wa e Ai Soil Pollu . 224, 1665–1680.
Camacho-Mu˜
noz, M.D., San os, J.L., Apa icio, I., Alonso, E., 2010a. P esence o
pha maceu ically ac i e compounds in Do˜
nana Pa k (Spain) main wa e sheds.
J. Haza d Ma e . 177 (1–3), 1159–1162.
Cha u edi, P., Shukla, P., Gi i, B.S., Chowdha y, P., Chand a, R., Gup a, P., Pandey, A.,
2021. P e alence and haza dous impac o pha maceu ical and pe sonal ca e
p oduc s and an ibio ics in en i onmen : a e iew on eme ging pollu an s. En i on.
Res. 194, 110664.
Comeau, F., Su e e, C., B un, G.L., Losie , R., 2008. The occu ence o acidic d ugs and
ca eine in sewage e luen s and ecei ing wa e s om h ee coas al wa e sheds in
A lan ic Canada. Sci. To al En i on. 396, 132–146.
Dodde , N.G., Ma uya, K.A., Fe guson, P.L., G ace, R., Klos e haus, S., La Gua dia, M.J.,
Lauens ein, G.G., Rami ez, J., 2014. Occu ence o pollu an s o eme ging conce n in
mussels (My ilus spp.) along he Cali o nia coas and he in luence o land use, s o m
wa e discha ge, and ea ed was ewa e e luen . Ma . Pollu . Bull. 81 (2), 340–346.
Ellepola, N., Ogas, T., Tu ne , D., Gu ung, R., Maldonado-To es, S., Tello-Abu o, R.,
Pa ida , P., Rogelj, S., Piyasena, M., Rubasinghege, G., 2020. A oxicological s udy
on pho o-deg ada ion p oduc s o en i onmen al ibup o en: ecological and human
heal h implica ions. Eco oxicol. En i on. Sa . 188, 109892.
Fekadu, S., Alemayehu, E., Dewil, R., Van de B uggen, B., 2019. Pha maceu icals in
eshwa e aqua ic en i onmen s: a compa ison o he A ican and Eu opean
challenge. Sci. To al En i on. 654, 324–337.
Fe n´
andez-Cisnal, R., Alhama, J., Ab il, N., Pueyo, C., L´
opez-Ba ea, J., 2014. Redox
p o eomics as bioma ke o assessing he biological e ec s o pollu an s in c ay ish
om Do˜
nana Na ional Pa k. Sci. To al En i on. 490, 121–133.
Fe ando-Climen , L., Rod iguez-Mozaz, S., Ba cel´
o, D., 2014. Incidence o an icance
d ugs in an aqua ic u ban sys em: om hospi al e luen s h ough u ban was ewa e
o na u al en i onmen . En i on. Pollu . 193, 216–223.
Ga ido, E., Camacho-Mu˜
noz, D., Ma ín, J., San os, A., San os, J.L., Apa icio, E.,
Alonso, e., 2016. Moni o in o eme ging pollu an s in Guadiama Ri e basin (Sou h
o Spain): analy ical me hod, spa ial dis ibu ion and en i onmen al isk assessmen .
En i on. Sci. Pollu . Res. 23, 25127–25144.
Golo ko, O., Koba, O., Kodeso a, R., Fedo o a, G., Kuma , V., G abic, R., 2016.
De elopmen o as and obus mul i esidual LC-MS/MS me hod o de e mina ion
o pha maceu icals in soils. En i on. Sci. Pollu . Res. 23, 14068–14077.
Gonz´
alez-Mi a, A., To eblanca, A., Hon o ia, F., Na a o, J.C., Ma˜
nan´
os, E., Va ´
o, I.,
2018. E ec s o ibup o en and ca bamazepine on he ion anspo sys em and a y
acid me abolism o empe a u e condi ioned ju eniles o Solea senegalensis.
Eco oxicol. En i on. Sa . 148, 693–701.
G os, M., Pe o ic, M., Ba cel´
o, D., 2009. T acing pha maceu ical esidues o di e en
he apeu ic classes in en i onmen al wa e s by using liquid ch oma og aphy/
Quad upole-Linea ion ap mass spec ome y and au oma ed lib a y sea ching.
Anal. Chem. 81, 898–912.
Hue a, B., Rod iguez-Mozaz, S., Lazo chak, J., Ba cel´
o, D., Ba , A., Wa hen, J., S ahl, L.,
2018. P esence o pha maceu icals in ish collec ed om u ban i e s in he U.S. EPA
2008-2009 Na ional Ri e s and S eams Assessmen . Sci. To al En i on. 634,
542–549.
J. Kazako a e al.
Jou nal o En i onmen al Managemen 297 (2021) 113314
9
Jallouli, N., Pas ana-Ma ínez, L.M., Ribei o, A.R., Mo ei a, N., Fa ia, J.L., Hen a i, O.,
Sil a, A., Ksibi, M., 2018. He e ogeneous pho oca aly ic deg ada ion o ibup o en in
ul- apu ewa e , municipal and pha maceu ical indus y was ewa e s using a TiO
2
/
UV-led sys em. Chem. Eng. J. 334, 976–984.
Kalka, J., Sochacki, A., Kowalska, K., Bajkacz, S., Ha nisz, M., Ko zeniewska, E., 2020.
An imic obial pha maceu icals in he aqua ic en i onmen -occu ence and
en i onmne al implica ions. Eu . J. Pha macol. 866, 172813.
Kandie, F.J., K auss, M., Becke s, L.M., Massei, R., Fillinge , U., Becke , J., Liess, M.,
To o, B., B acka, W., 2020. Occu ence and isk assessmen o o ganic
mic opollu an s in eshwa e sys ems wi hin he Lake Vic o ia Sou h Basin, Kenya.
Sci. To al En i on. 714, 13674.
Kazako a, J., Fe n´
andez-To es, R., Ramos-Pay´
an, M., Bello-L´
opez, M.A., 2018.
Mul i esidue de e mina ion o 21 pha maceu icals in c ay ish (P ocamba us cla kii)
using enzyma ic mic owa e-assis ed liquid ex ac ion and high-pe o mance liquid
ch oma og aphy- iple quad upole mass spec ome y analysis. J. Pha maceu .
Biomed. Anal. 160, 144–151.
Kim, C., Ryu, H.D., Chung, E.G., Kim, Y., 2018. De e mina ion o 18 e e ina y
an ibio ics in en i onmen al wa e using high-pe o mance liquid ch oma og aphy-
q-o bi ap combined wi h on-line solid-phase ex ac ion. J. Ch oma og. B Analy ical
Technologies in he Biomedical and Li e Sciences 1084, 158–165.
Kodeˇ
so ´
a, R., Klemen , A., Golo ko, O., F´
e , M., Nikodem, A., Koˇ
c´
a ek, M., G abic, R.,
2019. Roo up ake o a enolol, sul ame hoxazole and ca bamazepine, and hei
ans o ma ion in h ee soils and ou plan s. En i on. Sci. Pollu . Res. 26,
9876–9891.
Li, P., Zou, X., Wang, X., Su, M., Chen, C., Sun, X., Zhang, H., 2020. A p elimina y s udy
o he in e ac ions be ween mic oplas ics and ci a e-coa ed sil e nanopa icles in
aqua ic en i onmen s. J. Haza d Ma e . 385, 121601.
Llompa , M., Celei o, M., Dagnac, T., 2019. Mic owa e-assis ed ex ac ion o
pha maceu icals, pe sonal ca e p oduc s and indus ial pollu an s in he
en i onmen . T ends Anal. Chem. 116, 136–150.
Ma, F., Yuan, G., Meng, L., Oda, Y., Hu, J., 2012. Con ibu ions o lumequine and
ni oa enes o he geno oxici y o i e and g ound wa e s. Chemosphe e 88 (4),
476–483.
Majumbe , A., Gup a, B., Gup a, A.K., 2019. Pha maceu ically ac i e compounds in
aqueous en i onmen : a s a us, oxici y and insigh s o emedia ion. En i on. Res.
176, 108542.
Ma ínez-Pie nas, A.B., Polo-L´
opez, M.I., Fe n´
andez-Ib´
a˜
nez, P., Agüe a, A., 2018.
Valida ion and applica ion o a mul i esidue me hod based on liquid
ch oma og aphy- andem mass spec ome y o e alua ing he plan up ake o 74
mic opollu an s in c ops i iga ed wi h ea ed municipal was ewa e .
J. Ch oma og . A 1534, 10–21.
Mille , T.H., Bu y, N.R., Owen, S.F., MacRae, J.I., Ba on, L.P., 2018. A e iew o he
pha maceu ical exposome in aqua ic auna. En i on. Pollu . 239, 129–146.
Mille , T.H., Ng, K.T., Bu y, S.T., Bu y, S.E., Bu y, N.R., Ba ona, L.P., 2019.
Biomoni o ing o pes icides, pha maceu icals and illici d ugs in a eshwa e
in e eb a e o es ima e oxic o e ec p essu e. En i on. In . 129, 595–606.
Molina, C.B., Sanz-San os, E., Boukhemkhem, A., Bedia, J., Bel e , C., Rod iguez, J.J.,
2020. Remo al o eme ging pollu an s in aqueous phase by he e ogeneous Fen on
and pho o-Fen on wi h Fe
2
O
3
-TiO
2
-clay he e os uc u es. En i on. Sci. Pollu . Res.
27, 38434–38445.
Mo eno-Gonz´
alez, R., Rod íguez-Mozaz, S., Hue a, B., Ba cel´
o, D., Le´
on, V.M., 2016. Do
pha maceu icals bioaccumula e in ma ine molluscs and ish om a coas al lagoon?
En i on. Res. 146, 282–298.
Oso io, V., La a˜
naga, A., Ace˜
na, J., P´
e ez, S., Ba cel´
o, D., 2016. Concen a ion and isk
o pha maceu icals in eshwa e sys ems a e ela ed o he popula ion densi y and
he li es ock uni s in Ibe ian Ri e s. Sci. To al En i on. 540, 267–277.
Osuna-Jim´
enez, I., Ab il, N., Vioque-Fe n´
andez, A., G´
omez-A iza, J.L., Pueyo, C., 2014.
The en i onmen al quali y o Do˜
nana su ounding a eas a ec s he immune
ansc ip ional p o ile o inhabi an c ay ish P ocamba us cla kii. Fish Shell ish
Immunol. 40 (1), 136–145.
Rigos, Geo ge, Nengas, Ioannis, Alexis, Ma ia, T oisi, Ge a M., 2004. Po en ial d ug
(oxy e acycline and oxolinic acid) pollu ion om Medi e anean spa id ish a ms.
Bio emedia ion 2, 221–248.
Rodil, R., Quin ana, J.B., Concha-G a˜
na, E., L´
opez-Mahía, P., Munia egui-Loo enzo, S.,
P ada-Rod íguez, D., 2012. Eme ging pollu an s in sewage, su ace and d inking
wa e in Galicia (NW Spain). Chemosphe e 86, 1040–1049.
Rod íguez-Mozaz, S., Hue a, B., Ba cel´
o, D., 2016. Bioaccumula ion o eme ging
pollu an s in aqua ic bio a: pa e ns o pha maceu icals in medi e anean i e
ne wo ks. Eme ging pollu an s in i e ecosys ems. The Handbook o En i onmen al
Chemis y book se ies (HEC 46, 121–141.
Rogowska, J., Cieszynska-Semenowicz, M., Ra ajczyk, W., Wolska, L., 2020.
Mic opollu an s in ea ed was ewa e . Ambio 49, 487–503.
R¨
osch, A., Anlike , S., Hollende , J., 2016. How bio ans o ma ion in luences
oxicokine ics o azole ungicides in he aqua ic in e eb a e gamma us pulex.
En i on. Sci. Technol. 50 (13), 7175–7188.
Sco , P.D., Ba kow, M., Blockwell, S.J., Coleman, H.M., Khan, S.J., Lim, R.,
McDonald, J.A., Nice, H., Nugegoda, D., Pe ig o e, V., T emblay, L.A., Wa ne, M.S.
J., Leusch, F.D.L., 2014. A na ional Su ey o ace o ganic Pollu an s in Aus alian
i e s. J. En i on. Qual. 43 (5), 1702–1712.
Szymczycha, B., Bo ecka, M., Białk-Bieli´
nska, A., Siedlewicz, G., Pazd o, K., 2020.
Subma ine g oundwa e discha ge as a sou ce o pha maceu ical and ca eine
esidues in coas al ecosys em: bay o Puck, sou he n Bal ic Sea case s udy. Sci. To al
En i on. 713, 136522.
Vazquez-Roig, P., And eu, V., Blasco, C., Pic´
o, Y., 2012. Risk assessmen on he p esence
o pha maceu icals in sedimen s, soils and wa e s o he Pego-Oli a Ma shlands
(Valencia, eas e n Spain). Sci. To al En i on. 440, 24–32.
Vioque-Fe n´
andez, A., Al es de Almeida, E., Balles e os, J., Ga cía-Ba e a, T., L´
opez-
Ba ea, J., 2007. Do˜
nana Na ional Pa k su ey using c ay ish (P ocamba us cla kii) as
bioindica o : es e ase inhibi ion and pollu an le els. Toxicol. Le . 168 (35),
260–268.
Vioque-Fe n´
andez, A., Al es de Almeida, E., L´
opez-Ba ea, J., 2009. Assessmen o
Do˜
nana Na ional Pa k con amina ion in P ocamba us cla kii: in eg a ion o
con en ional bioma ke s and p o eomic app oaches. Sci. To al En i on. 407 (515),
1784–1797.
Vys a na, Y., Huneau, F., G ynenko, V., Ve geles, Y., Celle-Jean on, H., Tapie, N.,
Budzinski, H., Le Cous ume , P., 2012. Pha maceu icals in i e s o wo egions wi h
con as ed socio-economic condi ions: occu ence, accumula ion, and compa ison
o Uk aine and F ance. Wa e Ai Soil Pollu . 223 (5), 2111–2124.
Wo ld Wildli e Fund Espa˜
na, 2009. Caudales ecol´
ogicos de la ma isma del Pa que
Nacional de Do˜
nana y su ´
a ea de in luencia. WWF Espa˜
na. June, 2009.
J. Kazako a e al.