Jou nal o En i onmen al Chemical Enginee ing 11 (2023) 111185
A ailable online 4 Oc obe 2023
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Remo al o he wa e bo ne pa asi e C yp ospo idium pa um om d inking
wa e using g anula ac i a ed ca bon
Seila Couso-P´
e ez
a
,
1
, Ma ía Jesús Abeledo-Lamei o
a
,
2
, Ana Isabel Vidal-Va ela
a
,
Hip´
oli o G´
omez-Couso
a
,
b
,
*
a
Labo a o y o Pa asi ology, Depa men o Mic obiology and Pa asi ology, Facul y o Pha macy, Uni e si y o San iago de Compos ela, Campus Vida, 15782 San iago
de Compos ela, A Co u˜
na, Spain
b
Ins i u e o Resea ch on Chemical and Biological Analysis (IAQBUS), Uni e si y o San iago de Compos ela, Campus Vida, 15782 San iago de Compos ela, A Co u˜
na,
Spain
ARTICLE INFO
Edi o : Luigi Rizzo
Keywo ds:
Ac i a ed g anula ca bon
C yp ospo idium
D inking wa e
Household wa e ea men
ABSTRACT
C yp ospo idium is a genus o apicomplexan pa asi es ha in ec he gas oin es inal ac o a wide ange o
e eb a e hos s, including humans. These en e opa hogens a e commonly de ec ed in su ace wa e s and
C yp ospo idium is esponsible o nume ous wa e bo ne ou b eaks in indus ialised coun ies. Fil e s o g anula
ac i a ed ca bon (GAC) a e mainly used o elimina e eme ging mic opollu an s o con ol unpleasan odou and
as e in d inking wa e . Recen ly, GAC has been also employed o emo al o mic oo ganisms om di e en
ypes o wa e . This wo k e alua es he capabili y o GAC in he elimina ion o he in ec i e o ms o C yp o-
spo idium pa um (oocys s) om d inking wa e . Fo his, well wa e was spiked wi h oocys s o C. pa um and
passed h ough a ch oma og aphic column illed wi h esh GAC a di e en bed heigh s (5–50 cm) and a
il a ion a e o 100 mL/min, app oxima ely. By immuno luo escence mic oscopy, he numbe o oocys s in he
wa e samples was de e mined. The loga i hmic educ ion (LR) was calcula ed by compa ing he numbe o
oocys s quan i ied in he il e ed samples and he co esponding in luen samples. High e iciencies o GAC in
emo ing C. pa um oocys s om wa e we e ob ained. Thus, wi h a bed heigh o 35 cm, emo al e iciencies >
2 LR we e achie ed. The highes LR (3.47 ±0.31) was obse ed when a GAC bed heigh o 50 cm was used.
Taking in o accoun he esul s ob ained, C. pa um oocys s we e elimina ed ema kably and, he e o e, GAC
adso p ion il e s may be conside ed addi ional ba ie s agains his wa e bo ne en e opa hogen in d inking
wa e a he household le el in de eloping coun ies.
1. In oduc ion
C yp ospo idium is a genus o apicomplexan pa asi es ha in ec he
gas oin es inal ac o a wide ange o e eb a e hos s, including
humans [1]. C yp ospo idiosis is ansmi ed by he aecal-o al ou e
and can be acqui ed bo h di ec ly, h ough con ac wi h in ec ed hos s,
and indi ec ly, by inges ion o ood and wa e con amina ed wi h oo-
cys s ( he obus in ec i e o m), being he la e he mos common
sou ce o in ec ion [1,2]. The ubiqui ous occu ence o C yp ospo idium
in he en i onmen , i s pe sis ence, and esis ance o con en ional
disin ec ion ea men s based on physical, chemical and biological
me hods ha e made his wa e bo ne pa asi e one o he c i ical pa ho-
gens o he d inking wa e indus y [3]. Thus, C yp ospo idium was
in ol ed in 76.5% o wa e bo ne ou b eaks epo ed in indus ialised
coun ies du ing he pe iod 2017–2020 [4]. In middle and low-income
coun ies, C yp ospo idium, along wi h Ro a i us and Shigella, a e he
h ee pa hogens ha mos commonly cause dia hoeal disease in chil-
d en unde 2 yea s old, wi h C yp ospo idium being esponsible o
30–50% o childhood mo ali y in hese coun ies [5,6].
Unsa e d inking wa e is s ill esponsible o mo e han hal o he
* Co esponding au ho a : Labo a o y o Pa asi ology, Depa men o Mic obiology and Pa asi ology, Facul y o Pha macy, Uni e si y o San iago de Compos ela,
Campus Vida, 15782 San iago de Compos ela, A Co u˜
na, Spain.
E-mail add ess: [email p o ec ed] (H. G´
omez-Couso).
1
Cu en add ess: Nano echnology and In eg a ed BioEnginee ing Cen e (NIBEC), School o Enginee ing, Uls e Uni e si y, Bel as Campus, 2–24 Yo k S ee ,
Bel as BT15 1AP, Uni ed Kingdom
2
Cu en add ess: Pla a o ma Sola de Alme ía–CIEMAT, Ca e e a Sen´
es, Km 4.5, 04200, Tabe nas, Alme ía, Spain
Con en s lis s a ailable a ScienceDi ec
Jou nal o En i onmen al Chemical Enginee ing
jou nal homepage: www.else ie .com/loca e/jece
h ps://doi.o g/10.1016/j.jece.2023.111185
Recei ed 26 May 2023; Recei ed in e ised o m 1 Sep embe 2023; Accep ed 4 Oc obe 2023
Jou nal o En i onmen al Chemical Enginee ing 11 (2023) 111185
2
dia hoeal diseases in he wo ld [7]. The Wo ld Heal h O ganiza ion
(WHO) es ima es ha a ound 2 billion people a e cu en ly using
con amina ed wa e sou ces [8]. Fo his eason, he Uni ed Na ions
app o ed “The 2030 Agenda o Sus ainable De elopmen ”, which includes
17 Sus ainable De elopmen Goals (SDGs). The SDG 6 pu sues “ensu ing
wa e a ailabili y and sus ainable wa e managemen and sani a ion o all”
[9,10]. To p o ec public heal h, he WHO es ablished he concep o
e e ence pa hogen, which includes he pa hogens wi hin a g oup ha
a e mos di icul o emo e o con ol and ha e he la ges associa ed
heal h bu den, bo h on a popula ion and indi idual basis [3]. Rega ding
he p o ozoan pa asi es g oup ha may be ansmi ed o humans
h ough he d inking wa e ou e (C yp ospo idium, Gia dia, En amoeba,
Toxoplasma, Balan idium and Cyclospo a), C yp ospo idium pa um was
chosen by he WHO as he e e ence pa hogen because i s oocys s a e he
mos pe sis en in he en i onmen , he mos esis an o chemical
disin ec ion, and he smalles in size. The e o e, his p o ozoan species is
he mos di icul o emo e by il a ion p ocesses [3].
The WHO es ima es ha a ound 2 billion people li e in a eas wi hou
access o sa e d inking wa e and wo- hi ds o he wo ld’s popula ion
li e in a eas o high wa e s ess [8]. Cu en ly, many households in
middle and low-income coun ies use su ace wa e , which may be
con amina ed wi h pa hogens o di e en o igins, o cooking, d inking,
ba hing and ec ea ional ac i i ies [4,11]. Con amina ion o wa e wi h
he en e opa hogen C yp ospo idium is a uni e sal challenge because i
comp omises he p o ec ion o wa e supplies [12]. Majo wa e bo ne
ou b eaks o c yp ospo idiosis associa ed wi h con amina ed d inking
wa e ha e been a ibu ed o ea men de iciencies and he e o e,
C. pa um in ec ious oocys s shall ep esen a challenge in he e alua ion
o wa e ea men sys ems in e ms o p o ozoa educ ion o inac i a-
ion pe o mance [4,13].
Ac i a ed ca bon (AC) is he name gi en o a g oup o po ous ca bons
manu ac u ed by ca bonising ca bonaceous ma e ials imp egna ed wi h
dehyd a ing chemicals o by ea ing cha coal (ca bonized wood) wi h
oxidising gases. The main ea u e o ACs is hei po ous s uc u e, as
hese ca bons exhibi a high po osi y deg ee and a la ge in e nal su ace
a ea, which makes hese ma e ials an excellen adso ben . Molecules
om he gas o liquid phase can be a ached o he su ace o he AC by
an de Waals- ype o ces, al hough chemiso p ion is also possible by
s onge alence o ces on he so-called ac i e si es o he ca bon su ace
[14,15].
Al hough he use o cha coal was desc ibed in Egyp a ound 1550
BC, i s comme cial p oduc ion began in he ea ly 20 h cen u y,
ex ending i s uses o he pu i ica ion o chemical, pha maceu ical, wa e
and ood ades. Today, AC is used in a wide ange o indus ial appli-
ca ions, including gas and ai cleaning. Howe e , i s main applica ion is
in he wa e indus y, o elimina e eme ging mic opollu an s o con ol
unpleasan odou and as e in d inking wa e [14]. Mo eo e , AC is also
employed in g oundwa e , se ice wa e and was ewa e ea men s due
o i s capabili y o adso b dissol ed o ganic impu i ies and o elimina e
subs ances a ec ing odou , as e, and colou and also se e al pollu an s
such as a oma ic compounds, hyd oca bons, de e gen s, soluble dyes,
e c. [14,15]. Thus, AC can be used in d inking wa e ea men plan s in
wo o ms: powde ed ac i a ed ca bon (PAC) o g anula ac i a ed
ca bon (GAC). PAC (pa icles size ypically be ween 10 and 100 µm in
diame e ) is no mally applied as needed o deal, o example, wi h a
apid in lux o as e and odou compounds o oxins om cyanobac e ia
[16]. The inco po a ion o PAC occu s du ing he coagulan dosing
be o e coagula ion/ loccula ion o du ing sedimen a ion p io o sand
il a ion [17]. By con as , GAC (pa icles in he ange o 0.2–5 mm in
size) is placed in il e s o con inuous applica ion as a inal ba ie o
an h opogenic mic opollu an s be o e wa e disin ec ion [17,18].
The use o GAC has been also employed o emo al o mic oo -
ganisms om di e en ypes o wa e du ing he las 20 yea s [19].
Ne e heless, he s udies ha e alua e a labo a o y and pilo scales he
capabili y o GAC il e s (columns o ca idges), o emo e C yp ospo-
idium oocys s a e sca ce [19–21]. The aim o his s udy was o e alua e
he capabili y o GAC o emo e C yp ospo idium oocys s om d inking
wa e as a me hod o household wa e ea men in middle and
low-income coun ies o poo esou ce en i onmen s.
2. Ma e ial and me hods
2.1. C yp ospo idium oocys s
Oocys s o C yp ospo idium we e ob ained om a aecal sample
collec ed om he ec um o a na u ally in ec ed neona al F ie-
sian–Hols ein cal . The aecal sample was subjec ed o a diphasic con-
cen a ion me hod and he oocys s we e pu i ied, quan i ied and
molecula ly cha ac e ized acco ding o he p o ocols p e iously
desc ibed [22]. B ie ly, aecal ma e ial was homogenized in phospha e
bu e ed saline (PBS, 0.04 M, pH 7.2) and il e ed h ough wo sie es
(mesh sizes 150 and 45 µm). Then, die hyl e he (2:1, / ) was added
and a diphasic concen a ion me hod (cen i uga ion a 2000g, o 15
min, a 4 ◦C) was applied. The supe na an was disca ded, and he
sedimen was washed wi h PBS (0.04 M, pH 7.2) by cen i uga ion a
2000g o 15 min a 4 ◦C. The pu i ica ion o he C yp ospo idium oocys s
was pe o med by discon inuous caesium chlo ide g adien s o 1.05,
1.10 and 1.40 g/mL by cen i uga ion a 2000g o 30 min a 4 ◦C [23].
The oocys s we e quan i ied using a modi ied Neubaue haemocy-
ome e chambe unde a phase con as mic oscope (BH2, Olympus
Op ical Co. L d., Tokyo, Japan). Finally, by ampli ica ion and
sequencing o agmen s o ~587 bp and ~850 bp o he small subuni
DNA (SSU DNA) and he 60 kDa glycop o ein (GP60) genes, espec-
i ely [24,25], he isola e was molecula ly iden i ied as C. pa um sub-
ype IIaA15G2R1.
2.2. Wa e ype
Well wa e (WW) collec ed in a p i a e house was used in his s udy
and was s o ed a 8 ◦C un il i s use. The pH was measu ed using a pH
me e (CRISON Ins umen s, S.A., Ba celona, Spain) [26] and a u bidi y
me e (TN-100, Eu ech Ins umen s P e L d., Singapo e) was used o
measu e he u bidi y o wa e [27]. To al dissol ed solids (TDS) we e
measu ed by weighing a il e ed sample and d ying i un il no u he
mass was los [28]. Mo eo e , wa e samples we e sen o he Depa -
men o Soil Science and Ag icul u al Chemis y o he Uni e si y o
San iago de Compos ela o de e mine he alkalini y and he ee chlo ine
o he wa e [27]. Table 1 shows he physicochemical and mic obio-
logical cha ac e is ics o he WW.
2.3. Expe imen al design
A ch oma og aphy column (ø =2.5 cm; heigh 50 cm + ese oi )
illed wi h esh GAC a di e en bed heigh s (5–50 cm) was employed in
each assay, which included h ee con amina ion s eps. The GAC used
was a comme cial p oduc (No i ® GAC 1240, 12–40 mesh, Ac os O -
ganics, Geel, Belgium) ob ained om a ege al sou ce, speci ically co-
conu shell by s eam ac i a ion, wi h pa icle size be ween 1680–0.425
mm. Thus, he column was loaded wi h he co esponding GAC bed and
washed wi h bi-dis illed wa e o bed expansion. Then, WW was spiked
wi h 5 ×10
5
C. pa um oocys s pe li e (in luen ) and passed h ough
he ch oma og aphy column a a il a ion a e o 100 mL/min,
app oxima ely (con ac ime ⁓5 min) (Fig. 1). The il e ed samples
(e luen s) we e collec ed and passed h ough ni ocellulose memb anes
(47 mm diame e ; 3 µm po e size; Me ck Millipo e L d., Ca ig wohill,
I eland), which we e placed in acuum il a ion uni s connec ed o a
pump (Fishe b and™ FB 70155, Fishe Scien i ic S.L., Mad id, Spain).
The memb anes we e inse ed in e-sealable polye hylene bags and
washed h ee imes wi h 5 mL o PBS 0.04 M pH 7.2. Then, he samples
we e cen i uged a 2000g o 15 min. Simila ly, 0.5 L o he in luen
samples we e subjec ed o he same p ocedu e. The supe na an was
disca ded and he sedimen s om he in luen and e luen samples we e
S. Couso-P´
e ez e al.
Jou nal o En i onmen al Chemical Enginee ing 11 (2023) 111185
3
esuspended in 5 and 1.5 mL o PBS 0.04 M pH 7.2, espec i ely. Each
bed GAC assay, ca ied ou in duplica e, in ol ed h ee con amina ions
and 10 washes wi h 0.5 L o clean well wa e be ween each con ami-
na ion ( o al ope a ing il e ime was app oxima ely 165 min). Finally,
he GAC was emo ed, and he column was washed wi h bi-dis illed
wa e and d ied in an o en a 65 ◦C be o e he e alua ion o ano he
bed heigh .
2.4. Quan i ica ion o C. pa um oocys s
A di ec immuno luo escence an ibody es (Aqua Glo™ G/C Di ec ,
Wa e bo ne Inc., New O leans, LA, USA) and subsequen s aining wi h
he luo ogenic dye 4
′
,6-diamino-2- enilindol (DAPI; Wa e bo ne Inc.)
we e applied on all samples o iden i y and quan i y he numbe o
C. pa um oocys s in he wa e [29]. B ie ly, aliquo s o 10
μ
L o he
in luen samples (p e iously dilu ed 1:10 wi h bi-dis illed wa e ) and
aliquo s o 100
μ
L o he e luen samples we e deposi ed on 3-well
slides (Ge ha d Menzel B.V & C. KG, B aunschweig, Ge many) ea ed
wi h a 0.1% poly-L-lysine solu ion (Sigma-Ald ich, Inc., S . Louis, MO,
USA). Once he samples we e ai -d ying a oom empe a u e, hey we e
ixed wi h 50
μ
L o absolu e me hanol (Mallinc o Bake B.V., De en e ,
The Ne he lands). Then, a o al o 40
μ
L mix con aining 10
μ
L o an i--
C yp ospo idium monoclonal an ibodies labelled wi h luo escein iso-
hiocyana e (FITC) (Aqua-Glo™ G/C Di ec , Wa e bo ne Inc.), 5
μ
L o
E ans blue coun e s ain (Wa e bo ne Inc.) and 15
μ
L o PBS 0.04 M pH
7.2 was added o e each sample. The slides wi h he samples we e
incuba ed in humid chambe s o 30 min, a 37 ◦C and in da kness and
hen hey we e washed wi h PBS 0.04 M pH 7.2. Finally, 50
μ
L o 1X
DAPI solu ion (1
μ
L o 2 mg/mL o DAPI s ock solu ion added o 5 mL o
PBS 0.04 M pH 7.2) was added o e each sample and he slides we e
again incuba ed o 2 min, a oom empe a u e and in da kness. A e
washing he samples wi h PBS 0.04 M pH 7.2 o emo e excess o DAPI
and ai -d ying, he slides wi h he samples we e moun ed using a
comme cial medium (No-Fade™ Moun ing Medium, Wa e bo ne Inc.).
The oocys s we e iden i ied by examining he samples unde an
epi luo escence mic oscope (AX70, Olympus Op ical Co., L d., Tokyo,
Japan) equipped wi h a FITC il e (exci a ion a 450–480 nm; ba ie a
515 nm) on he basis o hei shape, size and he pa e n and in ensi y o
immuno luo escence s aining ollowing he cha ac e is ics es ablished
by he Uni ed S a es En i onmen al P o ec ion Agency (USEPA) [29].
The oocys iden i ica ion was con i med by u he examina ion unde
UV il e o DAPI s aining (exci a ion a 358–461 nm; ba ie a
500 nm), again in acco dance wi h USEPA [29]. Fo each assay, he
quan i ica ion o C. pa um oocys s was pe o med in iplica e, applying
he co esponding mul iplica ion ac o on he in luen and e luen
samples (5000 and 15, espec i ely).
An USEPA analy ical quali y es was applied on he de ec ion
me hod selec ed o e i y he eco e y pe cen ages ob ained, which
accep ance c i e ia o C yp ospo idium a e mean pe cen age eco e y o
38–100% and p ecision o 46 exp essed as maximum ela i e pe cen age
di e ence [29].
2.5. Calcula ion o he emo al capabili y o GAC
The ma hema ical pa ame e o loga i hmic educ ion (LR) was used
o e alua e he capabili y o he GAC o elimina e he in ec ious o ms o
C. pa um om wa e . This pa ame e is ecommended by he WHO o
e alua e he e ec i eness o a sys em in he disin ec ion o mic obio-
logical decon amina ion used as household wa e ea men me hods
[30]. Loga i hmic educ ion is calcula ed using he geome ic mean
(GM) o he numbe o oocys s ob ained in wa e samples acco ding o
he ollowing equa ion [30]:
Loga i mic educ ion = − log10(GMe /GMin )(1)
whe e GM
in
is he GM o he C. pa um oocys numbe in in luen
samples and GM
e
is he GM o he C. pa um oocys numbe in he
e luen wa e samples.
Table 1
Physical, chemical, and mic obiological pa ame e s o well wa e (WW) used in
he e alua ion o he capabili y o g anula ac i a ed ca bon (GAC) o emo e
C yp ospo idium pa um oocys s.
Pa ame e Value Uni s
Physical
Tu bidi y
1.0 NTU
To al Dissol ed Solids (TDS) 90.0 mg/L
Conduc i i y 190.0
μ
S/cm
Chemical
pH 5.6 -
Ammonia <0.1 mg/L
Sulpha es <25.0 mg/L
Ni i es 0.2 mg/L
Ni a es 23.7 mg/L
Phospha es 0.1 mg/L
To al phospho ous 0.4 mg/L
Combined chlo ine <0.1 mg/L
F ee chlo ine <0.1 mg/L
Aluminium <0.1 mg/L
I on <0.1 mg/L
Po assium 2.2 mg/L
Magnesium 10.0 mg/L
Calcium 90.0 mg/L
Alkalini y 100.0 mg/L CaCO
3
Mic obiological
To al Coli o ms a 37 ◦C 0 CFU/100 mL
Esche ichia coli 0 CFU/100 mL
En e ococcus spp. 0 CFU/100 mL
Fig. 1. Ma e ials employed du ing he expe imen s. A, 50 cm g adua ed
ch oma og aphy column wi h ese oi ; B, esh g anula ac i a ed ca bon
(GAC); C, oocys s o C yp ospo idium pa um. Ba =10 µm.
S. Couso-P´
e ez e al.
Jou nal o En i onmen al Chemical Enginee ing 11 (2023) 111185
4
2.6. S a is ical analysis
S a is ical es s we e pe o med using S a g aphics Cen u ion
18 .18.1.12 (©1982–2018 S a g aphics Technologies, Inc., The Plains,
VA, USA). The Kolmogo o -Smi no es was used o es he da a i o a
no mal dis ibu ion. Di e ences in he GM be ween he di e en GAC
beds e alua ed we e compa ed using ANOVA and Fishe ´s leas signi i-
can di e ence (LSD) mul iple compa ison es s. Di e ences we e
conside ed s a is ically signi ican a P<0.05. The esul s p esen ed in
he g aph a e he a e age o h ee eplica es wi h he s anda d de ia ion
as he e o ba .
3. Resul s and discussion
The p esen wo k e alua es he capabili y o GAC o emo al
C. pa um oocys s om wa e as a simple, sus ainable, and e ec i e
me hod o household d inking wa e ea men . High e iciencies o
GAC o emo e oocys s o C. pa um om d inking wa e was ob ained,
and as expec ed, he emo al capabili y was inc eased as he GAC bed
heigh inc eased. Thus, emo al e iciencies >2 LR we e eached a
≥30 cm bed heigh s and >3 LR we e de e mined when bed heigh s
≥45 cm we e used. When he highes es ablished GAC bed heigh
(50 cm) was loaded, oocys s o C. pa um we e emo ed by 3.47 ±0.31
LR (Fig. 2). Signi ican s a is ical di e ences we e obse ed among he
GM o he GAC bed heigh s (F =63.67; P<0.01), de ec ing signi ican
di e ences be ween he esul s ob ained using he GAC heigh o 30 cm
in compa ison wi h ≤25 cm, and 50 cm wi h espec o ≤40 cm GAC
beds (P<0.05). The analy ical quali y es o he de ec ion me hod
e ealed an o e all eco e y pe cen age o 69.19 ±5.02. Thus, he
mean pe cen age eco e ies anged om 55.56% o 73.74% and p eci-
sion alues anged om 0 o 25.71 o he GAC bed heigh e alua ed.
These alues a e wi hin he quali y con ol accep ance c i e ia o
de ec ion me hods o C yp ospo idium in wa e es ablished by he USEPA
[29].
One o he i s s udies including GAC o emo e C. pa um oocys s
om d inking wa e was a wa e pu i ie moun ed in a auce and
consis ing o a ca idge composed o a hollow ib e memb ane il e
wi h 0.1 µm mul i-laye po es and a laye o GAC [20]. While he wa e
was unning, C. pa um oocys s (3 ×10
7
) we e injec ed in o he ube
connec ed o he wa e pu i ie and he auce . The p oposed wa e
pu i ie e ec i ely emo ed C. pa um oocys s om d inking wa e , as
oocys s we e no de ec ed in he pu i ied wa e collec ed om all he
ca idges [20].
In a p e ious s udy using i e wa e , i was obse ed ha he
capabili y o GAC il e s o elimina e C. pa um oocys s is highe in
compa ison wi h i uses and bac e ia [19]. Howe e , he educ ions
ob ained we e lowe han hose achie ed in his wo k. Thus, a he pilo
scale, wo columns (ø =0.15 m; heigh 1.35 m) illed wi h 1 m o GAC
(median g ain size 1 mm) we e supplied wi h p e- ea ed i e wa e a a
cons an low a e o 5 m/h (con ac ime o 12 min), aising 1.2 and 2.7
LR o C. pa um in loaded and esh GAC, espec i ely [19].
Ano he s udy assessed he e icacy o he GAC bio il e o educe i e
pa hogen su oga es using syn he ic g eywa e (SGW), which is
composed o chemical p oduc s o simula e o ganic and ino ganic
pollu ion o g eywa e om ba h ooms [31]. The expe imen s we e
pe o med in a cylind ical column eac o (ø =9 cm; e ec i e heigh o
60 cm). The eac o ’s e ec i e dep h was packed wi h comme cial GAC
(mesh pa icle size o 4–12). In he s udy, he highes educ ion (3.4 LR)
was achie ed wi h Saccha omyces ce e isiae, used as su oga e o
C yp ospo idium and Gia dia oo/cys s, ob aining di e en emo al e i-
ciencies agains he o he ou su oga es: 1.1 LR o S aphylococcus
epide midis; 0.9 LR o En e ococcus aecalis; 0.3 LR o E. coli and no
educ ion was obse ed o MS2 bac e iophage [31]. The LR alue
aised wi h SGW was e y simila o he co esponding ob ained in ou
wo k (3.4 s. 3.5, espec i ely), al hough he mesh size o he GAC
employed was di e en . In he p esen wo k, he mesh size o he GAC
was 12–40, while Sha a e al. [31] used GAC wi h a smalle mesh size,
speci ically 4–12, which could inc ease he emo al capabili y.
The in o ma ion ega ding he mechanisms in ol ed in he oocys s
emo al by GAC pa icles is limi ed. Hijnen e al. [19] e alua ed he
GAC adso p ion il e s as ba ie s o C yp ospo idium oocys s, among
o he pa hogens, in wa e ea men and explained he emo al o he
oocys s by s aining and a achmen . Based on he expe imen s and he
ma hema ical models applied, hey epo ed a mino con ibu ion o
s aining in emo ing C yp ospo idium oocys s. They a ibu ed he
emo al o a s ong a achmen o hese pa asi ic o ms o he GAC.
Howe e , hey concluded ha he e needs o be mo e in o ma ion abou
se e al ac o s, such as he in luence o GAC cha ac e is ics (shape and
size), he column ea u es (diame e and heigh ) and he adhesion e i-
ciency [19].
Al hough in he p esen wo k, he expe imen s we e ca ied ou wi h
esh GAC and well wa e , which p e en s he o ma ion o bio ilms,
p e ious s udies sugges ed ha he sa u a ion and age o he GAC il e
should be conside ed in he elimina ion o wa e bo ne p o ozoan pa a-
si es because o hese pa ame e s can imp o e he emo al o C yp o-
spo idium oocys s. Thus, poly inyl chlo ide (PVC) columns (ø =3.2 cm;
e ec i e heigh o 74 cm) we e loaded wi h GAC and ed du ing 18
weeks wi h i e wa e amended wi h a nu ien solu ion ( low 5 m/h)
o p omo e bio ilm g ow h in he il e . Then, anspo expe imen s
we e pe o med wi h i e wa e spiked wi h C. pa um oocys s o a
concen a ion o 6 ×10
5
oocys s/mL. The bes emo al pe o mance
was obse ed wi h aged GAC (71 ±2% o 0.54 LR) which suppo ed he
highes amoun o biomass [21]. Mo eo e , Sha a e al. [31] demon-
s a ed 2.7 and 0.7 LR o S. ce e isiae (used as su oga e o C yp ospo-
idium oocys s) in sa u a ed and unsa u a ed zones o a GAC bio il e ,
being he deg ee o bio ilm de elopmen well co ela ed wi h high e -
iciencies o emo al.
Fu he mo e, euka yo ic o ganisms p esen in su ace wa e s can
g ow in GAC il e s o wa e ea men plan s, p oli e a ing along he
dis ibu ion sys ems [32]. In addi ion, some wa e bo ne pa hogens can
su i al inside o he mic oscopical o ganisms, which ac as ca ie s
h ough wa e sys ems [32–36]. The ole o zooplank on, such as o i-
e s, copepods and cladoce ans, as a empo a y ehicle o pa hogens
h ough d inking wa e is s ill no well known. Howe e , he impac o
he p eda ion by zooplank on on he anspo and des ina ion o
C yp ospo idium oocys s in GAC il e s was s udied [37,38]. Thus, Bichai
e al. [37] pe o med a s udy wi h wo pa allel GAC il e columns (ø =
15 cm; bed heigh o 1 m) in a pilo plan , which ope a ed unde
ull-scale condi ions ( il a ion a e 5 m/h; con ac ime o 12 min; and
no backwashing). A e wo weeks, hey obse ed a mean mass educ-
ion o C. pa um oocys s o 32.1% and 66.2% in he lowe (50–95 cm)
Fig. 2. Loga i hmic educ ions o each g anula ac i a ed ca bon (GAC) bed
heigh ob ained du ing he expe imen s ca ied ou o e alua e he capabili y o
GAC on he emo al o C yp ospo idium pa um oocys s om well wa e (WW).
S. Couso-P´
e ez e al.
Jou nal o En i onmen al Chemical Enginee ing 11 (2023) 111185
5
and he uppe (0–30 cm) zones o he GAC il e beds, espec i ely [37].
Then, he zooplank on was isola ed om he GAC il e bed and e luen
wa e , iden i ying mainly o i e s, which a e p eda o s o oocys s [36,
37]. The au ho s concluded ha he emobilisa ion o C yp ospo idium
oocys s e ained on GAC il e s and he ansmission o hese pa asi ic
o ms o d inking wa e may be in luenced by zooplank on [37].
Finally, C. pa um is conside ed by he WHO as he e e ence pa h-
ogen o wa e bo ne p o ozoan pa asi es [3]. The In e na ional Scheme
o E alua e Household Wa e T ea men Technologies es ablishes a
minimum equi ed LR o ≥2 o p o ec i e me hods (2-s a ) and ≥4 o
highly p o ec i e me hods (3-s a ) when C. pa um oocys s concen a-
ions o ≥5×10
4
oocys s/mL a e subjec ed wi h an accep able educ-
ion de ia ion o 1 loga i hmic a iance [13,30]. In he p esen wo k,
>2 LR we e achie ed wi h a bed heigh o 35 cm, speci ically, 2.93
±0.53 LR. The e o e, he educ ions aised by GAC il a ion a e
consis en wi h a 2-s a p o ec ion me hod acco ding o he WHO, o -
e ing comp ehensi e p o ec ion agains c yp ospo idiosis.
4. Conclusions
S udies e alua ing he e icacy o GAC il e s o he emo al o he
in ec ious o ms o he wa e bo ne p o ozoan pa asi e C yp ospo idium
a e sca ce. Conside ing he esul s ob ained in he p esen wo k and he
il a ion condi ions, he oocys s o C. pa um we e emo ed signi i-
can ly om WW. The e o e, unlike bac e ia and i uses, GAC il e s can
ep esen impo an addi ional ba ie s o C yp ospo idium oocys s in
household wa e ea men s in middle and low-income coun ies o poo
esou ce en i onmen s, p o iding ex a p o ec ion agains
c yp ospo idiosis.
5. Recommenda ions o u u e s udies
Fu he s udies a e needed o e alua e he e ec s o GAC il e s in he
oocys su i al and he in luence o GAC ype and backwashing on he
emo al capabili y a ull-scale. Mo eo e , i is well known ha C yp-
ospo idium oocys s a e sensi i e o UV adia ion and sola wa e disin-
ec ion (SODIS) me hod imp o es he mic obiological quali y o
d inking wa e in medium and low-income coun ies, al hough C yp o-
spo idium oocys s a e esis an o his p ocedu e. In his way, he use o
GAC il e s may cons i u e one impo an ba ie o p o ozoan pa asi es
om d inking wa e , e en can educe i s u bidi y. The e o e, he
implemen a ion o hese il e s as p e ious s ep o SODIS would inc ease
he p o ec ion agains wa e bo ne p o ozoan pa asi es.
CRediT au ho ship con ibu ion s a emen
Seila Couso-P´
e ez: In es iga ion, Fo mal analysis, W i ing – o ig-
inal d a , W i ing – e iew & edi ing. Ma ía Jesus Abeledo-Lamei o:
In es iga ion, W i ing – e iew & edi ing. Ana Isabel Vidal-Va ela:
In es iga ion. Hip´
oli o G´
omez-Couso: In es iga ion, Fo mal analysis,
Supe ision, W i ing – e iew & edi ing, P ojec adminis a ion, Fund-
ing 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
This s udy is pa o he esea ch p ojec en i led “Pho o-i adia ion
and Adso p ion based No el Inno a ions o Wa e - ea men (PANI-
WATER)” (g an numbe 820718), which was join ly unded by he
Eu opean Union’s Ho izon 2020 Resea ch and Inno a ion P og amme o
he Eu opean Commission and he Depa men o Science and Tech-
nology o India. SC-P is he bene icia y o a Ma ga i a Salas con ac
unded by he P og amme o equali ica ion, in e na ional mobili y,
and alen a ac ion in he Spanish uni e si y sys em, Minis y o Uni-
e si ies (g an UP2021–042).
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