Academic Edi o s: Jie Gao and
Zhuo an Gao
Recei ed: 15 Ma ch 2025
Re ised: 5 Ap il 2025
Accep ed: 7 Ap il 2025
Published: 11 Ap il 2025
Ci a ion: Espe anço, P.; Egi o, R.;
Oli ei a, V.; Ama al, A.L.; Rod igues,
C. In luence o E luen Quali y
Pa ame e s on Daphnia spp.
O e g ow h in an U ban Was ewa e
T ea men Plan : A Mul iyea Case
S udy Analysis. P ocesses 2025,13,
1164. h ps://doi.o g/
10.3390/p 13041164
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
In luence o E luen Quali y Pa ame e s on Daphnia spp.
O e g ow h in an U ban Was ewa e T ea men Plan :
A Mul iyea Case S udy Analysis
Ped o Espe anço 1,2 , Rômulo Egi o 3, Ve ónica Oli ei a 1,2,* , An ónio Luís Ama al 1,2,4
and Ca la Rod igues 1,2
1Poly echnic Uni e si y o Coimb a, Rua da Mise icó dia, Laga dos Co iços, S. Ma inho do Bispo,
3045-093 Coimb a, Po ugal; [email p o ec ed] (P.E.); [email p o ec ed] (A.L.A.); [email p o ec ed] (C.R.)
2Resea ch Cen e o Na u al Resou ces, En i onmen and Socie y (CERNAS), Poly echnic Uni e si y o
Coimb a, Bencan a, 3045-601 Coimb a, Po ugal
3Public Wa e s o Alen ejo, Rua D . A es a B anco, 7800-310 Beja, Po ugal; [email p o ec ed]
4CEB—Cen e o Biological Enginee ing, Uni e si y o Minho, Campus de Gual a , 4710-057 B aga, Po ugal
*Co espondence: e [email p o ec ed]
Abs ac : Was ewa e ea men plan s (WWTPs) play a c ucial ole in ea ing sewage,
which unde goes mul iple ea men s ages o ensu e a sa e ea ed e luen . Howe e ,
any in e e ence du ing hese s ages can comp omise he inal e luen quali y. Such is he
case o he o e g ow h o he mic oc us acean Daphnia spp., known o inhabi WWTPs,
bu wi h i s p esence in he decan a ion s age nega i ely impac ing e luen cla i ica ion
and u he disin ec ion. This s udy aimed o e alua e how he e luen quali y pa ame e s
in luence he occu ence o Daphnia spp. in he seconda y decan e o a WWTP. Was ewa e
moni o ing da a collec ed om 2017 o 2022 we e analyzed. Fi s ly, as he COVID-19
pandemic occu ed du ing he s udied pe iod, i was assessed whe he he quali y and
load o he aw was ewa e changed. Subsequen ly, an analysis was ca ied ou using
mul i a ia e s a is ical me hods o all he s eps o WWTP. Compa ing he pe iods be o e
and du ing he pandemic, he aw was ewa e olume dec eased by 19.58%, and he BOD,
COD, and TSS dec eased by 37.78%, 16.86%, and 35.75%, espec i ely. These we e he
pa ame e s a ec ed he mos . The s a is ical analysis e ealed co ela ions be ween he
p esence o Daphnia spp. and speci ic e luen quali y pa ame e s, including aw was ewa e
BOD alues below 500 mg L
−1
, ea ed e luen BOD alues below 13 mg L
−1
, and pH
le els exceeding 7.3. Addi ionally, BOD and pH we e highligh ed as c i ical pa ame e s
in luencing hei p esence o absence.
Keywo ds: was ewa e ea men plan ; Daphnia spp.; biochemical oxygen demand;
was ewa e moni o ing
1. In oduc ion
Was ewa e ea men plan s (WWTPs) a e acili ies wi h he pu pose o educing he
pollu an s p esen in was ewa e so ha i can be e u ned o he ecei ing en i onmen
wi hou nega i ely a ec ing i [
1
–
3
]. Fo his o happen, was ewa e unde goes ea men
p ocesses in which he pollu an s p esen a e elimina ed h ough physical, biological, and,
i necessa y, chemical ea men s [
3
,
4
]. In a gene al con ex , was ewa e ea men gene ally
comp ises ou key s ages: p e- ea men , p ima y ea men , seconda y ea men , and
e ia y ea men [
3
–
6
]. Du ing p e- ea men , mechanical me hods a e employed o e-
mo e up o 80% o solid ma e ial, including sand, plas ics, and ood was e [
7
]. Following
P ocesses 2025,13, 1164 h ps://doi.o g/10.3390/p 13041164
P ocesses 2025,13, 1164 2 o 15
his, p ima y ea men in ol es sedimen a ion, whe e suspended solids a e sepa a ed,
allowing he cla i ied was ewa e o p oceed o he seconda y ea men phase. In he
seconda y ea men phase occu s he biological ea men ha elies on ae obic mic oo -
ganisms, commonly known as ac i a ed sludge, o deg ade he o ganic ma e p esen in
he was ewa e [
1
,
8
]. Finally, he e ia y ea men , conduc ed a e he seconda y sedimen-
a ion phase, inco po a es ad anced p ocesses, such as chlo ina ion, ul a iole i adia ion,
and memb ane il a ion, ha a e designed o elimina e esidual con aminan s, such as
pa hogens and o he ha m ul mic oo ganisms [
9
]. Howe e , he p esence o Daphnia spp.,
commonly known as wa e leas, is o en signi ican in seconda y decan e s, be o e he
ea ed e luen p oceeds o he e ia y ea men , due o he al eady inc eased wa e
quali y gua an eed by he seconda y ea men , and i s p esence is easily de ec ed by hei
la ge size and apid pa henogene ic ep oduc ion [10].
Daphnia spp. p esence comes wi h bo h bene i s and disad an ages. On one hand, hey
con ibu e o con olling algae g ow h and o emo ing o ganic ma e and nu ien s om
was ewa e [
11
,
12
]. On he o he hand, hei o e g ow h in seconda y decan e s can pose
subs an ial challenges o he inal s ages o ea men , mainly when disin ec ion is needed.
Thei mo emen and eeding beha io esuspend solids, leading o inc eased u bidi y
and highe solids load, comp omising he applied ul a iole -based e ia y ea men by
blocking he adia ion om e ec i ely passing h ough he sys em o disin ec he inal
e luen , as epo ed in his case s udy. This no only challenges he sys em’s abili y o mee
legal discha ge limi s bu also dis up s disin ec ion p ocesses, as he inc eased u bidi y
and pa icula e ma e educe he e ec i eness o disin ec ion ea men s ollowing he
decan a ion s ep. Unde s anding why Daphnia spp. occu s in his speci ic ea men s ep
is c ucial o managing hei p esence and p e en ing hei uncon olled p oli e a ion,
assu ing he quali y o he inal e luen .
Daphnia spp. exhibi s ema kable adap abili y o a ious en i onmen al condi ions, includ-
ing pollu ion, empe a u e changes, pho ope iod, salini y, and nu ien le els [
13
,
14
]. When
unde highly s ess ul condi ions, Daphnia spp. popula ions swi ch o sexual ep oduc ion,
p oducing do man eggs ha acili a e coloniza ion o new habi a s and he e-es ablishmen
o hei popula ion [
13
,
15
]. These cha ac e is ics a e he p ima y eason why Daphnia spp.
has success ully colonized WWTPs and con inues o h i e by epea edly e-es ablishing
hei popula ions. Indeed, he p esence o Daphnia spp. in WWTPs is well-documen ed.
Fo example, Kuma and Ki an iden i ied Daphnia spp. and o he zooplank on in wa e
collec ed om a sewage- ed ank, demons a ing ha nu ien - ich wa e s coupled wi h
aqua ic weeds a o ed he abundance o zooplank on [
16
]. In hei s udy, wa e qual-
i y was assessed om samples aken du ing he occu ence o Daphnia spp., e ealing a
wa e empe a u e ange o 22.5 o 32
◦
C, an alkaline pH, high dissol ed oxygen le els
(2.4–4.8 mg L
−1
), low biochemical oxygen demand (BOD) le els (4.8–16.8 mg L
−1
), and nu-
ien concen a ions such as ni a es (14.6–54.4 mg L
−1
) and phospha es
(0.28–1.08 mg‘L
−1
) [
16
]. O he s udies ha e sough o explo e hei po en ial o enhance
was ewa e ea men e iciency, as hey ha e shown he capaci y o emo e pa icula e
ma e and educe bac e ia and coli o m con en s [
17
–
22
]. Some s udies, such as Pau e al.,
epo ed he Daphnia magna abili y o il e sludge pa icles om he ea ed was ewa e on
seconda y decan e s, wi h labo a o y es s epo ing a 29.4% educ ion in he suspended
pa icles and in si u es s epo ing a 30.4% educ ion [
19
]. Te ia y ea men based on
Daphnia magna il a ion has e en been conside ed by Se a e al., showing a 35% educ ion
in he concen a ion o pa icles wi h diame e s below 30
µ
m and a signi ican inac i a ion
o E. coli by 1.2 log uni s, al hough lowe han con en ional ul a iole i adia ion (inac i-
a ion be ween 1.5 and 4.0 log uni s) depending on empe a u e and hyd aulic e en ion
ime [
22
]. Howe e , no esea ch is ound on hei occu ence when hey become p oblem-
P ocesses 2025,13, 1164 3 o 15
a ic. In cases such as he o e g ow h epo ed he ein, he e is a lack o knowledge o he
ac o s d i ing hei p oli e a ion o o s a egies o p edic and e ec i ely con ol hei
p esence.
Conside ing he abo e, he main objec i e o his wo k was o s udy he occu ence o
Daphnia spp. in an u ban WWTP seconda y decan e . The speci ic objec i es consis ed o
p ocessing he moni o ing da a o he WWTP om 2017 o 2022 and iden i ying he mos
ele an physicochemical condi ions o hei p esence. To his end, s a is ical ools we e
used o iden i y pa e ns in he quali y o he e luen when Daphnia spp. appea ed. I was
also sough o iden i y possible ways o con olling his o ganism in WWTP wi hou loss o
quali y in he inal ea ed e luen .
2. Ma e ials and Me hods
2.1. Case S udy
The analyzed u ban WWTP is loca ed in Fá ima, Po ugal, and comp ises a p ima y
inle channel, which ecei es aw was ewa e ha is subsequen ly dis ibu ed ac oss wo
independen lines (Figu e 1). Each line includes an oxida ion di ch ollowed by a seconda y
decan e . Pos -decan a ion, he ea ed e luen s om bo h lines con e ge and unde go
ul a iole (UV) disin ec ion ea men p io o discha ge in o he ecei ing wa e body.
Upon analyzing he WWTP’s ea men s ages, he ollowing six da a collec ion poin s we e
iden i ied: (1) in luen o aw was ewa e , (2) seconda y sludge line 1, (3) seconda y sludge
line 2, (4) ae obic zone 1, (5) ae obic zone 2, and (6) inal e luen .
P ocesses 2025, 13, x FOR PEER REVIEW 3 o 16
become p oblema ic. In cases such as he o e g ow h epo ed he ein, he e is a lack o
knowledge o he ac o s d i ing hei p oli e a ion o o s a egies o p edic and effec-
i ely con ol hei p esence.
Conside ing he abo e, he main objec i e o his wo k was o s udy he occu ence
o Daphnia spp. in an u ban WWTP seconda y decan e . The speci ic objec i es consis ed
o p ocessing he moni o ing da a o he WWTP om 2017 o 2022 and iden i ying he
mos ele an physicochemical condi ions o hei p esence. To his end, s a is ical ools
we e used o iden i y pa e ns in he quali y o he effluen when Daphnia spp. appea ed.
I was also sough o iden i y possible ways o con olling his o ganism in WWTP wi hou
loss o quali y in he inal ea ed effluen .
2. Ma e ials and Me hods
2.1. Case S udy
The analyzed u ban WWTP is loca ed in Fá ima, Po ugal, and comp ises a p ima y
inle channel, which ecei es aw was ewa e ha is subsequen ly dis ibu ed ac oss wo
independen lines (Figu e 1). Each line includes an oxida ion di ch ollowed by a second-
a y decan e . Pos -decan a ion, he ea ed effluen s om bo h lines con e ge and un-
de go ul a iole (UV) disin ec ion ea men p io o discha ge in o he ecei ing wa e
body. Upon analyzing he WWTP’s ea men s ages, he ollowing six da a collec ion
poin s we e iden i ied: (1) in luen o aw was ewa e , (2) seconda y sludge line 1, (3) sec-
onda y sludge line 2, (4) ae obic zone 1, (5) ae obic zone 2, and (6) inal effluen .
Figu e 1. Sa elli e o e iew o he s udied WWTP (Fá ima, Po ugal) and collec ion poin s: (1) aw
was ewa e , (2) seconda y sludge line (1, 3) seconda y sludge line (2, 4) ae obic zone (1, 5) ae obic
zone 2, and (6) inal effluen .
These poin s we e chosen o p o ide a comp ehensi e assessmen o he WWTP pe -
o mance, co e ing ups eam, wi hin, and downs eam loca ions o he seconda y ea -
men sys em. Raw was ewa e (1) e lec s he cha ac e is ics o was ewa e en e ing he
plan . As he effluen subsequen ly lows h ough wo independen lines, i is necessa y
o iden i y whe he he ea men is equally efficien in bo h lines. Fo his eason, poin s
(2) o (5) we e iden i ied as impo an o his s udy. A e he seconda y decan a ion, he
ea ed effluen joins up again in jus one line, wi h his moni o ing poin conside ed he
inal effluen (6). F om hese 6 poin s i was possible o cha ac e ize he aw was ewa e ,
he WWTP ea men in luence on he imp o emen o wa e quali y, and, a e all he
ea men s ages, cha ac e ize he ea ed effluen .
Figu e 1. Sa elli e o e iew o he s udied WWTP (Fá ima, Po ugal) and collec ion poin s: (1) aw
was ewa e , (2) seconda y sludge line (1, 3) seconda y sludge line (2, 4) ae obic zone (1, 5) ae obic
zone 2, and (6) inal e luen .
These poin s we e chosen o p o ide a comp ehensi e assessmen o he WWTP
pe o mance, co e ing ups eam, wi hin, and downs eam loca ions o he seconda y
ea men sys em. Raw was ewa e (1) e lec s he cha ac e is ics o was ewa e en e ing
he plan . As he e luen subsequen ly lows h ough wo independen lines, i is necessa y
o iden i y whe he he ea men is equally e icien in bo h lines. Fo his eason, poin s
(2) o (5) we e iden i ied as impo an o his s udy. A e he seconda y decan a ion, he
ea ed e luen joins up again in jus one line, wi h his moni o ing poin conside ed he
inal e luen (6). F om hese 6 poin s i was possible o cha ac e ize he aw was ewa e ,
he WWTP ea men in luence on he imp o emen o wa e quali y, and, a e all he
ea men s ages, cha ac e ize he ea ed e luen .
P ocesses 2025,13, 1164 4 o 15
The u ban WWTP p o ided an ex ensi e da ase o analy ical pa ame e s ob ained
h ough mon hly moni o ing om 2017 o 2022. F om his da ase , 12 pa ame e s we e
selec ed o analysis due o hei a ailabili y h ough weekly moni o ing, esul ing in a
obus and comp ehensi e da abase. The selec ed pa ame e s include olume ic low,
biochemical oxygen demand (BOD), chemical oxygen demand (COD), ammonium ion
(NH
4+
), ni a e (NO
3−
), o al ni ogen (TN), pH, o al phospho us (TP), o al and ola ile
suspended solids (TSS and VSS), and o al and ola ile solids (TS and VS). In addi ion,
eco ds documen ing he occu ence o Daphnia spp. wi hin WWTP we e also inco po a ed,
enabling a de ailed assessmen o he ac o s in luencing hei p esence and po en ial
impac s on he ea men p ocess.
As he p oblem wi h Daphnia spp. was iden i ied a an unusual ime, du ing a pan-
demic pe iod, a s udy was ca ied ou o iden i y whe he his e en in luenced he load and
quali y o he aw was ewa e . Fo his analysis, he ollowing pa ame e s we e compa ed:
olume ic low, BOD, COD, pH, TN, TP, and TSS. All mon hly alues moni o ed a he
WWTP we e a e aged be ween June 2017 and Feb ua y 2020, a pe iod p io o COVID-
19. These alues we e nex compa ed wi h he pe iod du ing he COVID-19 pandemic,
be ween Ma ch 2020 and Feb ua y 2022.
S a is ical ools we e hen used o enligh en he occu ence pa e ns o Daphnia spp.
in he WWTP, ela ing o he selec ed pa ame e s (moni o ed a 6 di e en poin s in
he WWTP). A o al o 23 sample poin s we e chosen due o hei ep esen a i eness
o he WWTP ea men s ages and da a a ailabili y om 2017 o 2022, namely: Flow,
Biochemical Oxygen Demand-Raw Was ewa e (BOD-RW), Biochemical Oxygen Demand-
E luen (BOD-E), Chemical Oxygen Demand-Raw Was ewa e (COD-RW), Chemical
Oxygen Demand-E luen (COD-E), Ammonium Ion-E luen (NH4-E), Ni a e-E luen
(NO3-E), Ni ogen-Raw Was ewa e (TN-RW), Ni ogen-E luen (TN-E), pH-A, pH-E,
Phospho us-Raw Was ewa e (TP-RW), Phospho us-E luen (TP-E), To al Suspended
Solids-Raw Was ewa e (TSS-RW), To al Suspended Solids-E luen (TSS-E), To al Sus-
pended Solids-Ae obic Zone 1 (TSS-AZ1), Vola ile Suspended Solids-Ae obic Zone 1 (VSS-
AZ1), To al Solids-Seconda y Sludge 1 (TS-SS1), Vola ile Solids-Seconda y Sludge 1 (VS-
SS1), To al Suspended Solids-Ae obic Zone 2 (TSS-AZ2), Vola ile Suspended Solids-Ae obic
Zone 2 (VSS-AZ2), To al Solids-Seconda y Sludge 2 (TS-SS2), and Vola ile Solids-Seconda y
Sludge 2 (VS-SS2). The selec ed WWTP poin s include essen ial pa ame e s commonly
moni o ed in WWTPs, allowing us o ack how he aw was ewa e con en in o ganic
ma e (BOD, COD, and TSS), nu ien s (TN and TP), and pH luc ua e as hey en e he
biological ea men and ul ima ely impac he inal e luen . By including measu emen s
a di e en ea men s ages, he assessmen o he ea men pe o mance and ope a ional
s abili y can be ca ied ou . The con en on TSS and s. on bo h he anae obic zones
and decan ed sludge, 1 and 2, a e o ex eme impo ance o e alua e i bo h s eams a e
ope a ing a simila le els.
2.2. S a is ical Analysis
Pea son’s co ela ion ma ix, logis ic eg ession, and P incipal Componen Analy-
sis (PCA) we e compu ed using he R p og am (R Co e Team 4.3.1, 2023), adop ing a
signi icance le el o <0.05 o s a is ical di e ences [23].
2.2.1. Pea son’s Co ela ion Ma ix
The i s app oach consis ed o submi ing he selec ed pa ame e s o a Pea son’s
co ela ion ma ix in o de o in es iga e how hey a e co ela ed and o iden i y and
emo e collinea pa ame e s. A co ela ion coe icien abo e 0.7 was adop ed as he cu -o
alue o emo ing colinea pa ame e s [24].
P ocesses 2025,13, 1164 5 o 15
2.2.2. P incipal Componen Analysis (PCA)
The subse o he e ained pa ame e s was nex subjec ed o a s a is ical p ocedu e,
PCA [
25
–
27
], o educe he dimensionali y o he da ase and iden i y possible clus e s
ega ding he absence and p esence o Daphnia spp. This second app oach assumes ha he
occu ence o Daphnia spp. can be ela ed o a ious analy ical pa ame e s, so he scena ios
o ‘absence’ and ‘p esence’ o he mic oc us acean would o m wo dis inc clus e s wi hin a
mul i a ia e space. The analy ical pa ame e s we e i s log-no malized o s anda diza ion
pu poses and o a oid possible da a non-no mali y e ec s. In addi ion, S uden ’s - es s
we e ca ied ou o check o s a is ical di e ences be ween he absence and p esence
o Daphnia spp. (ca ego ical pa ame e s) using he i s h ee axes o he PCA ( esponse
pa ame e s) o he analy ical pa ame e s.
2.2.3. Logis ic Reg ession
The hi d app oach aimed o unde s and he ela i e con ibu ion o he analy ical
pa ame e s ha bes explain he occu ence o Daphnia spp. in he u ban WWTP. In his case,
a logis ic eg ession was applied, wi h he esponse pa ame e being bina y (0 = absence
and 1 = p esence) and he ca ego ical pa ame e s being he analy ical pa ame e s [
28
,
29
].
Once again, he analy ical pa ame e s we e log-no malized o s anda dize he pa ame e s
in e ms o uni s. The explana o y pe o mance o he logis ic model was assessed using
he Wald es (chi-squa ed) and he Akaike c i e ion (AIC) [
30
]. Fo his se o analyses, a
signi icance le el o <0.05 was adop ed o assess signi ican s a is ical di e ences.
2.2.4. Classi ica ion T ee
Finally, a classi ica ion ee analysis was pe o med using he pa ame e s iden i ied by
he logis ic eg ession as mos in luen ial o Daphnia spp. p esence. The mos p edic i e
pa ame e de e mines he i s spli (b anches), wi h subsequen b anches added un il
mee ing he s opping c i e ia. Unlike eg ession models, which i a single ela ionship
ac oss he en i e domain, classi ica ion ees adap o changing ela ionships wi hin di e en
inpu egions. Model pe o mance is e alua ed using con usion ma ices and ecei e
ope a ing cha ac e is ic (ROC) cu es ha quan i y ue/ alse posi i es and nega i es,
enabling he calcula ion o sensi i i y ( ue posi i e a e), speci ici y ( ue nega i e a e),
and accu acy (co ec classi ica ion a e). A well-balanced model exhibi s high and simila
sensi i i y and speci ici y, minimizing alse classi ica ions [31].
3. Resul s
3.1. E ec o he COVID-19 Pandemic
This i s assessmen o he quali y o he aw was ewa e is essen ial because he i s
case o he p esence o Daphnia spp. was iden i ied in Decembe 2020, 10 mon hs a e he
decla a ion o he COVID-19 global pandemic. Be ween he da e o his i s appea ance and
June 2022, app oxima ely 30 in e e ences in he ea men we e iden i ied caused by he
p esence o he mic oc us acean. The i s sign o he appea ance o mic oc us aceans was
an inc ease in u bidi y in he inal ea ed e luen . A e ha , WWTP echnicians ca ied
ou a isual inspec ion, obse ing ed clouds in he seconda y decan e and egis e ing
hei occu ence.
Table 1shows he a ia ions in he aw in low be ween he pe iod be o e
(June 2017–Feb ua y 2020) and du ing (Ma ch 2020–Feb ua y 2022) he pandemic.
P ocesses 2025,13, 1164 6 o 15
Table 1. Va ia ion in aw was ewa e quali y caused by COVID-19 (mean ±s anda d de ia ion).
Pa ame e s Be o e COVID-19
Du ing COVID-19
Va ia ion (%) p-Value
Raw was ewa e low
(m3·day−1)2640 ±278 2036 ±270 −19.6% 0.002 1
BOD (mg O2·L−1)651 ±129 405 ±101 −37.8% <0.001 1
COD (mg O2·L−1)1026 ±210 854 ±274 −16.8% 0.048 1
Ni ogen (mg N·L−1)83.5 ±28.7 90.5 ±32.3 +8.38% 0.467
Phospho us (mg P·L−1)9.40 ±3.34 10.7 ±2.01 +13.8% 0.219
TSS (mg·L−1)437 ±110 281 ±131 −35.7% 0.004 1
pH 7.28 ±0.04 7.43 ±0.11 +1.99% <0.001 1
1indica es signi ican (p< 0.01) o ma ginally signi ican (p< 0.05) di e ences.
3.2. Pea son’s Co ela ion Ma ix
The Pea son’s co ela ion ma ix shows a se ies o highly co ela ed pa ame e s
(Figu e 2). In ac , 10 o he pa ame e s analyzed p esen ed co ela ion coe icien s ( )
g ea e han 0.7, leading o he ollowing being emo ed: COD_RW, COD_E, TP_RW, TP_E,
TSS_A, TSS_E, VSS_AZ1, TS_SS1, VSS_AZ2, and TS_SS2. Such high co ela ions a e due o
he WWTP ope a ing s uc u e, wi h he aw was ewa e ollowing a single ea men line
h ough he ini ial s ages (g ading/sampling and desanding/deg easing) be o e being spli
in o wo sepa a e biological eac o lines and seconda y decan a ion s ages, as seen in
Figu e 1. This di ision esul s in non-co ela ed pa ame e s be ween bo h s eams
(VSS_AZ1, TS_SS1, VSS_AZ2, and TS_SS2) and could u he be a ec ing he co ela ion o
he COD and TP pa ame e s be ween he aw was ewa e and ea ed e luen .
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Table 1. Va ia ion in aw was ewa e quali y caused by COVID-19 (mean ± s anda d de ia ion).
Pa ame e s Be o e
COVID-19
Du ing
COVID-19 Va ia ion (%) p-Value
Raw was ewa e low
(m3·day−1) 2640 ± 278 2036 ± 270 −19.6% 0.002
1
BOD (mg O2·L–1) 651 ± 129 405 ± 101 −37.8% <0.001
1
COD (mg O2·L–1) 1026 ± 210 854 ± 274 −16.8% 0.048
1
Ni ogen (mg N·L–1) 83.5 ± 28.7 90.5 ± 32.3 +8.38% 0.467
Phospho us (mg P·L–1) 9.40 ± 3.34 10.7 ± 2.01 +13.8% 0.219
TSS (mg·L–1) 437 ± 110 281 ± 131 −35.7% 0.004
1
pH 7.28 ± 0.04 7.43 ± 0.11 +1.99% <0.001 1
1 indica es signi ican (p < 0.01) o ma ginally signi ican (p < 0.05) diffe ences.
3.2. Pea son’s Co ela ion Ma ix
The Pea son’s co ela ion ma ix shows a se ies o highly co ela ed pa ame e s (Fig-
u e 2). In ac , 10 o he pa ame e s analyzed p esen ed co ela ion coefficien s ( ) g ea e
han 0.7, leading o he ollowing being emo ed: COD_RW, COD_E, TP_RW, TP_E,
TSS_A, TSS_E, VSS_AZ1, TS_SS1, VSS_AZ2, and TS_SS2. Such high co ela ions a e due
o he WWTP ope a ing s uc u e, wi h he aw was ewa e ollowing a single ea men
line h ough he ini ial s ages (g ading/sampling and desanding/deg easing) be o e being
spli in o wo sepa a e biological eac o lines and seconda y decan a ion s ages, as seen
in Figu e 1. This di ision esul s in non-co ela ed pa ame e s be ween bo h s eams
(VSS_AZ1, TS_SS1, VSS_AZ2, and TS_SS2) and could u he be affec ing he co ela ion
o he COD and TP pa ame e s be ween he aw was ewa e and ea ed effluen .
Figu e 2. Pea son’s co ela ion ma ix wi h he measu ed analy ical pa ame e s. The ci cle’s size
co esponds o he co ela ion’s magni ude, a ying om
−
1 o 1 (ba on he igh side). Red indica es
nega i e co ela ions, while blue indica es posi i e co ela ions.
3.3. P incipal Componen Analysis
The i s h ee p incipal componen s o he PCA explained a ound 60% o he o al
a iabili y o he da a: PC 1 (27.9%), PC 2 (16.4%), and PC 3 (14.7%). A high deg ee o
P ocesses 2025,13, 1164 7 o 15
o e lap be ween he wo g oups (absence and p esence o Daphnia spp.) is appa en , wi h
only a sligh endency owa ds seg ega ion, as obse ed by he dis ance o he cen oids
(Figu e 3a–c).
P ocesses 2025, 13, x FOR PEER REVIEW 7 o 16
Figu e 2. Pea son’s co ela ion ma ix wi h he measu ed analy ical pa ame e s. The ci cle’s size
co esponds o he co ela ion’s magni ude, a ying om −1 o 1 (ba on he igh side). Red indi-
ca es nega i e co ela ions, while blue indica es posi i e co ela ions.
3.3. P incipal Componen Analysis
The i s h ee p incipal componen s o he PCA explained a ound 60% o he o al
a iabili y o he da a: PC 1 (27.9%), PC 2 (16.4%), and PC 3 (14.7%). A high deg ee o
o e lap be ween he wo g oups (absence and p esence o Daphnia spp.) is appa en , wi h
only a sligh endency owa ds seg ega ion, as obse ed by he dis ance o he cen oids
(Figu e 3a–c).
P ocesses 2025, 13, x FOR PEER REVIEW 8 o 16
Figu e 3. PCA biplo s demons a e a pa e n o high o e lap be ween he absence ( ed) and p esence
(blue) g oups o Daphnia spp., wi h a sligh endency owa ds seg ega ion (dis ance be ween cen-
oids). (a) PC 1 × PC 2; (b) PC 1 × PC 3; (c) PC 2 × PC 3.
The Daphnia spp. p esence pa e n, hough no s a is ically signi ican as nex de e -
mined, was mo e ela ed o lowe PC1 and PC3 alues. Analyzing Figu e 3b, i is also clea
ha he highe alues o PC1 and PC3 (and hence o lowe Daphnia spp. occu ence) co -
espond o he di ec ion o he BOD_RW and BOD_E (in bo h cases co obo a ed by he
logis ic eg ession) and NH4_E and a e opposi e o he di ec ion o VS_SS1 (in hese wo
la e cases jus sho ly ailing o be conside ed as signi ican d i e s by he logis ic eg es-
sion). I is also possible o e i y in Figu e 3a ha la ge alues o VS_SS1, TN_E, and
TN_RW co espond o a g ea e dis inc ion be ween he p esence and absence o Daphnia
spp. Finally, Figu e 3c sugges s ha highe alues o VS_SS1, VS_SS2, TSS_AZ1, and
TSS_AZ2 a e associa ed wi h a mo e p onounced dis inc ion be ween he p esence and
absence o Daphnia spp. Howe e , in hese wo la e igu es (Figu e 3b,c), he ac ha PC2
does no p o ide o any dis inc ion be ween Daphnia p esence o absence limi s hei use
in his analysis.
3.4. T Tes
Rega ding S uden ’s - es , i did no egis e any signi ican diffe ence in he h ee
p incipal componen s (axes) o he PCA (en i onmen al pa ame e s) be ween he “ab-
sence” and “p esence” g oups o Daphnia (Axis 1 (PC 1): = 1.439, p = 0.162; Axis 2 (PC 2):
= −0.840, p = 0.415; Axis 3 (PC 3): = 1.401, p = 0.173). These esul s co obo a e he pa e n
o high o e lap be ween he absence and p esence o Daphnia spp. ound by he PCA (Fig-
u e 4a–c).
Figu e 3. PCA biplo s demons a e a pa e n o high o e lap be ween he absence ( ed) and p esence
(blue) g oups o Daphnia spp., wi h a sligh endency owa ds seg ega ion (dis ance be ween cen oids).
(a) PC 1 ×PC 2; (b) PC 1 ×PC 3; (c) PC 2 ×PC 3.
P ocesses 2025,13, 1164 8 o 15
The Daphnia spp. p esence pa e n, hough no s a is ically signi ican as nex de e -
mined, was mo e ela ed o lowe PC1 and PC3 alues. Analyzing Figu e 3b, i is also
clea ha he highe alues o PC1 and PC3 (and hence o lowe Daphnia spp. occu ence)
co espond o he di ec ion o he BOD_RW and BOD_E (in bo h cases co obo a ed by
he logis ic eg ession) and NH4_E and a e opposi e o he di ec ion o VS_SS1 (in hese
wo la e cases jus sho ly ailing o be conside ed as signi ican d i e s by he logis ic
eg ession). I is also possible o e i y in Figu e 3a ha la ge alues o VS_SS1, TN_E, and
TN_RW co espond o a g ea e dis inc ion be ween he p esence and absence o Daphnia
spp. Finally, Figu e 3c sugges s ha highe alues o VS_SS1, VS_SS2, TSS_AZ1, and
TSS_AZ2 a e associa ed wi h a mo e p onounced dis inc ion be ween he p esence and
absence o Daphnia spp. Howe e , in hese wo la e igu es (Figu e 3b,c), he ac ha PC2
does no p o ide o any dis inc ion be ween Daphnia p esence o absence limi s hei use
in his analysis.
3.4. T Tes
Rega ding S uden ’s - es , i did no egis e any signi ican di e ence in he h ee
p incipal componen s (axes) o he PCA (en i onmen al pa ame e s) be ween he “absence”
and “p esence” g oups o Daphnia (Axis 1 (PC 1): = 1.439, p= 0.162; Axis 2 (PC 2):
=
−
0.840, p= 0.415; Axis 3 (PC 3): = 1.401, p= 0.173). These esul s co obo a e he
pa e n o high o e lap be ween he absence and p esence o Daphnia spp. ound by he
PCA (Figu e 4a–c).
P ocesses 2025, 13, x FOR PEER REVIEW 9 o 16
Figu e 4. Boxplo g aphs o he T- es esul s show no signi ican diffe ences be ween he “absence”
and “p esence” g oups o Daphnia o PC 1 (a), PC 2 (b), and PC 3 (c), used as p oxies o analy ical
pa ame e s.
3.5. Logis ic Reg ession
Logis ic eg ession iden i ied h ee pa ame e s wi h diffe en le els o in luence on
he occu ence o Daphnia spp. The biochemical oxygen demand o bo h he aw
was ewa e (BOD_RW) and he effluen (BOD_E) had a mode a e nega i e effec (i.e.,
ma ginally signi ican p- alue) on he esponse pa ame e , i.e., he p esence o Daphnia
spp. was ela ed o low le els o BOD_RW (<500 mg/L) and BOD_E (<13 mg/L) (Figu e
5a,b).
Figu e 4. Boxplo g aphs o he T- es esul s show no signi ican di e ences be ween he “absence”
and “p esence” g oups o Daphnia o PC 1 (a), PC 2 (b), and PC 3 (c), used as p oxies o analy ical
pa ame e s.
P ocesses 2025,13, 1164 9 o 15
3.5. Logis ic Reg ession
Logis ic eg ession iden i ied h ee pa ame e s wi h di e en le els o in luence on he
occu ence o Daphnia spp. The biochemical oxygen demand o bo h he aw was ewa e
(BOD_RW) and he e luen (BOD_E) had a mode a e nega i e e ec (i.e., ma ginally
signi ican p- alue) on he esponse pa ame e , i.e., he p esence o Daphnia spp. was ela ed
o low le els o BOD_RW (<500 mg/L) and BOD_E (<13 mg/L) (Figu e 5a,b).
P ocesses 2025, 13, x FOR PEER REVIEW 10 o 16
Figu e 5. G aphs o he logis ic eg ession model showing he in luence o biochemical oxygen
de iciency (a,b) and pH (c) on he occu ence o Daphnia (0 = absence/1 = p esence). (Do s ep esen
he obse a ional da a; blue line depic s he p edic mean; shadow indica es 95% con idence
in e als).
These ela ionships p oduced close AIC and Wald (chi-squa ed) alues (Table 2),
which shows he simila effec s o hese a iables.
Figu e 5. G aphs o he logis ic eg ession model showing he in luence o biochemical oxygen
de iciency (a,b) and pH (c) on he occu ence o Daphnia (0 = absence/1 = p esence). (Do s ep esen
he obse a ional da a; blue line depic s he p edic mean; shadow indica es 95% con idence in e als).
