Co esponding au ho : Ugochukwu Romanus Onyeukwu
Copy igh © 2025 Au ho (s) e ain he copy igh o his a icle. This a icle is published unde he e ms o he C ea i e Commons A ibu ion License 4.0.
An agonis ic and Addi i e Toxici y Assessmen o Quina y Mix u es o Me al and 2,4-
D Combina ions on Algal Phospha ase Func ion
Ugochukwu Romanus Onyeukwu 1, *, Emeka Sabas ine Asiwe 2, Ma yRose Ogechi Eche a 1, Chizoba P ecious
Tony-Nze 1 and Japhe E asmus Aisoni 3
1 Depa men o Mic obiology, Facul y o Science and Compu ing, Uni e si y o Ag icul u e and En i onmen al Sciences
Umuagwo, Imo S a e, Nige ia.
2 Depa men o Biochemis y, Facul y o Science and Compu ing, Uni e si y o Ag icul u e and En i onmen al Sciences
Umuagwo, Imo S a e, Nige ia.
3 Depa men o Mic obiology, Facul y o li e science, Baye o Uni e si y, Kano, Kano S a e, Nige ia.
GSC Biological and Pha maceu ical Sciences, 2025, 33(01), 146-163
Publica ion his o y: Recei ed on 02 Se embe 2025; e ised on 08 Oc obe 2025; accep ed on 11 Oc obe 2025
A icle DOI: h ps://doi.o g/10.30574/gscbps.2025.33.1.0388
Abs ac
The p esen s udy comp ehensi ely examined he impac o combined me al and pes icide mix u es on he ac i i y o
he hyd oly ic phospha ase enzyme in he eshwa e alga Chlo ella ulga is. Toxicological assessmen s en ailed
e alua ing inhibi ion o phospha ase-media ed con e sion o p-ni ophenylphospha e o p-ni ophenol, moni o ed
spec opho ome ically a 410 nm. Bo h una y and quina y mix u es o Coppe (Cu²⁺), Zinc (Zn²⁺), Lead (Pb²⁺),
Ch omium (C ²⁺), Cadmium (Cd²⁺), and 2,4-dichlo ophenoxyace ic acid (2,4-D) we e in es iga ed a p ede e mined
ixed mola a ios. Expe imen al and model-p edic ed dose- esponse analyses e ealed ha all es ed hea y me als
elici ed concen a ion-dependen inhibi o y e ec s on phospha ase ac i i y. No ably, C ²⁺ and Cu²⁺ demons a ed he
highes oxici ies wi h IC50 alues o 0.19 ± 0.01 mM and 0.28 ± 0.00 mM, espec i ely, whe eas 2,4-D mani es ed
compa a i ely low inhibi ion, inducing app oxima ely 20% enzyma ic supp ession. Quina y mix u es o mula ed a
ixed a ios o 20:20:20:20:20 and 16:21:21:21:21 exhibi ed di e en ial oxicological p o iles whe ein he 2,4-
D/Pb²⁺/Zn²⁺/C ²⁺/Cd²⁺ mix u e consis en ly p esen ed he highes oxici y. Ac oss he di e en mix u es, a gene al
decline in oxici y was obse ed, ye oxici y sa u a ion was eached, indica ing uni o m oxic e ec s a highe
concen a ions. Toxici y index e alua ions p edominan ly signalled an agonis ic in e ac ions (TI >>1), aside om one
mix u e in he 20:20:20:20:20 a io displaying addi i e in e ac ions. The modula ion o oxici y by 2,4-D wi hin hese
cock ails no ably a enua ed me al oxici y ye concu en ly ampli ied i s own oxic e ec s. This dualis ic modula ion
likely a ises om di e en ial modes o ac ion, empo al exposu e a iances, and ecep o si e in e ac ions among he
oxican s. Collec i ely, he indings unde sco e he ecological isk posed by me al and pes icide co-exposu es o
eshwa e mic oalgae, media ed h ough complex an agonis ic and addi i e enzyma ic inhibi ion mechanisms.
Keywo ds: Toxicological assessmen ; Enzyma ic inhibi ion mechanisms; Dose- esponse analysis; IC50 alues; Me al-
pes icide in e ac ion; Ecological isk o mic oalgae
1. In oduc ion
Chemical mix u es a e combina ions o wo o mo e chemicals ha main ain hei indi idual chemical iden i ies wi hou
al e a ion (Eu opean Commission, 2012). Despi e mos oxici y da a used o chemical sa e y e alua ions ocusing on
single compounds, eal-wo ld aqua ic en i onmen s a e ypically exposed o mix u es a he han indi idual chemicals
(Bel ield e al., 2023 in Yang e al., 1998). The e o e, exposu e o chemical mix u es is he e o e he no m in
en i onmen al oxicology (Jongwoon e al., 2022). Fu he mo e, adi ional oxici y assessmen s may ei he e alua e
GSC Biological and Pha maceu ical Sciences, 2025, 33(01), 146-163
147
he mix u e as a whole ( op-down app oach) o analyze he indi idual componen s and hei combined e ec s (bo om-
up app oach). Gene ally, chemical oxici y e alua ions use he bo om-up me hod, whe e he oxici ies o he sepa a e
componen s a e modelled o p edic he o e all mix u e e ec (He nandez, Gil, and Lacasana, 2017). A key challenge in
assessing mix u es a ises when indi idual chemicals a e p esen a concen a ions below hei no-obse ed-e ec le el
(NOEL), bu in combina ion exhibi unexpec ed oxic e ec s (Jongwoon e al., 2022; Eu opean Commission, 2012b).
P edic i e oxici y me hods o en ely on assump ions like concen a ion addi ion (CA) o chemicals wi h simila modes
o ac ion o independen ac ion (IA) o hose wi h di e en modes. In many aqua ic ecosys ems, o ganisms such as
mic oalgae occupy an impo an posi ion in ene gy ophic le els and a e exposed o o ganic and ino ganic chemical
mix u es a he han single pollu an s (A onzon, Peluso, and Coll, 2020; Ko alako a e al., 2020; Topaz e al., 2020;
Zhang e al., 2021). The inhibi ion o phospha ase ac i i y in hese mic oalgae se es as a use ul indica o o e alua ing
he e ec s o en i onmen al oxican s (Nguyen, Moon, and Lee, 2020; Nunes e al., 2018; Göbölös e al., 2024; Onyeukwu
and Edna, 2022a; 2022b; Wenq e al., 2021). The e ec i e concen a ions o chemical mix u es can be quan i ied using
he combina ion index (CI) me hod, and da a analysis is o en acili a ed by sigma s a is ical plo s. The esponses o
bo h indi idual compounds and mix u es a e ypically e alua ed by me ics such as he median inhibi o y concen a ion
(MIC50) o e ec i e concen a ion (EC50). Aside combina ion index me hod, quan i a i e s uc u e p ope y ela ionship
(QSPR) model can be used o de elop ma hema ical ela ionships ha p edic chemical’s physical o chemical p ope y
based on i s molecula desc ip o s. Fo example, QSPR models can used o p edic he modynamic p ope ies such as
he mal ene gy, en opy, and hea capaci y o camp o hecin d ug de i a i es, using molecula desc ip o s which is
op imized by gene ic algo i hms and mul iple linea eg ession me hods. The model's p edic i e abili y is said o be
alida ed by c oss- alida ion echniques o ensu e accu acy in p ope y p edic ion (Yue and Li, 2022; Ahmadinejad, e
al., 2018). Addi ionally, is he use o a i icial neu al ne wo ks combined wi h QSPR modelling o p edic p ope ies o
chemical mix u es mo e accu a ely han expe imen al me hods alone. These ad anced QSPR models can help in
p edic ing p ope ies ela ed o new biomass uels, pollu ion analyses, o oxicological isks (Mu and Li, s2022).
Quan i a i e s uc u e-p ope y ela ionship (QSPR) models se e as p edic i e ools o es ima e he physicochemical
p ope ies o pu e compounds and mix u es. To add ess gaps in QSPR p edic ions, new o mula ions and models ha e
been de eloped o add ess some inconsis ences in QSPR usage. Molecula desc ip o s used in hese models can be
cons i u ional, opological, geome ic, o quan um chemical in na u e. QSPR and QSAR models a e inc easingly applied
in oxicology, eco oxicology, pha maceu icals, pha macodynamics, pha macokine ics, chemome ics, and physical-
chemical p ope y p edic ions (Hassold e al., 2021; Eu opean Commission, 2020, 2012b; He nandez e al., 2017; WHO,
2017). A majo di e ence be ween QSAR modelling o single chemicals and mix u es in ol es he ypes o molecula
desc ip o s used, which a e c i ical o accu a ely p edic ing oxici y, especially when dealing wi h nonlinea p ope ies.
Common ma hema ical echniques in QSAR/QSPR include Mul iple Linea Reg ession (MLR), Pa ial Leas Squa es
(PLS), Neu al Ne wo ks (NN), and Suppo Vec o Machines (SVM), which con inue o be e ined wi h ad anced
algo i hms and hyb id me hods. In his s udy, he ocus is o e alua e bo h indi idual and combined e ec s o single and
quina y mix u es o me al ions Coppe (Cu2+), Zinc (Zn2+), Lead (Pb2+), Ch omium (C 2+), Cadmium (Cd2+) alongside 2,4-
dichlo ophenoxyace ic acid on he alkaline phospha ase ac i i y o Chlo ella ulga is, using a oxic index model. The
in e ac ion e ec s o mix u es based on concen a ion we e assessed using he oxic index (TI), which sums he oxic
uni s o each componen (Boillo and Pe odin, 2008). The TU alues we e calcula ed using he exp ession:
I
i
iIC
Cmix
TU
50
=
=
=
n
1i
i
TUTI
, While TI is he summa ion o TU o n oxican s in he mix u e, whe e Cmixi is he
concen a ion o he i h oxican in he mix u e and EC50i is he EC50 o he same oxican when es ed singly. TI=1 implies
addi i e in e ac ion, TI > 1 implies an agonis ic in e ac ion and TI < 1 implies a syne gis ic in e ac ion (Boillo and
Pe odin, 2008). The syne gis ic, addi i e, and an agonis ic e ec s be ween he hea y chemicals and 2,4-
dichlo ophenoxyace ic acid in a ious mix u es a di e en e ec i e concen a ions we e de e mined using he
combina ion index (CI) me hod, aside o he me hods (Kaha agahawa e and Ha a-Yamamu a, 2020).
2. Ma e ial and me hods
2.1. Sample a ea
Ihiagwa is a own si ua ed wi hin he Owe i Wes Local Go e nmen A ea in Imo S a e, loca ed in sou heas e n Nige ia.
I lies app oxima ely 12 kilome e s sou h o Owe i, he s a e capi al. The O ami i Ri e lows h ough Ihiagwa, wi h
geog aphic coo dina es a la i ude 4°54'14.00" N and longi ude 7°08'30.00" E. The i e 's wa e shed ex ends o e
oughly 10,000 squa e kilome e s (3,900 squa e miles) and ecei es an annual ain all anging be ween 2,250 and 2,500
millime e s. The a ea is p edominan ly co e ed by deple ed ain o es ege a ion, wi h a e age empe a u es ho e ing
a ound 27°C (81°F) yea - ound. Un o una ely, he O ami i Ri e is subjec o pollu ion om o ganic was e and
chemicals, due o ine icien was e managemen sys em in Owe i which exace ba es he i e 's con amina ion. This
pollu ion p ima ily esul s om in ensi e human ac i i ies, including household was e disposal, ag icul u al uno ,
GSC Biological and Pha maceu ical Sciences, 2025, 33(01), 146-163
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ailing d ainage in as uc u e, ac o y discha ges, and a ious indus ial ope a ions. This has led o inc eased
concen a ions o pollu an s such as phospha es, ni a es, me als, pes icides, and he bicides in he O ami i Ri e . Despi e
hese challenges, he i e se es as an impo an al e na i e sou ce o d inking wa e when he public wa e supply is
una ailable. To be e unde s and he ex en o pollu ion, he cu en s udy ocuses on a la ge segmen o he O ami i
Ri e along he Nekede o Ihiagwa s e ch. The geog aphical map o he s udy a ea is p esen ed in Figu e 1.
Figu e 1 Digi al Geog aphical map o O ami i Ri e along he Nekede -. Ihiagwa s e ch
2.2. Sampling
2.2.1. Sampling Ma e ial
S e ile glass bo les, wi h capaci y o 200 ml was used o wa e sampling collec ion. They we e i ed wi h g ound glass
o sc ew caps. The s oppe o cap and neck o he bo le we e p o ec ed om con amina ion by sui able co e o
s e ilized hin aluminum oil. Silicon ubbe lines, ha could wi hs and epea ed s e iliza ion a 1600C, was used inside
he sc ew caps. A e being s e ilized, he bo le was asep ically s o ed wi hou opening he s e ilized bo le. The
samples we e collec ed a a ime in e al o en (10) minu es o each sampling poin s. The iming was egula ed by he
use o a s op wa ch by one o he ield assis an s. The Imo S a e Wa e co-ope a ion shows wa e o ea men in he
ups eam o his s e ch and many ac i i ies such as ishing and sand mining ha go on downs eam.
2.3. Collec ing Sample
2.3.1. Sample collec ion and anspo a ion:
Samples o wa e we e collec ed in s e ile bo les. Ca e was ensu ed o p e en acciden al con amina ion o wa e
du ing collec ion. The Ri e wa e was collec ed om O ami i Ri e in Ihiagwa, Imo S a e sou h-eas e n Nige ia. Wa e
samples we e collec ed mids eam along he cou se o he i e a h ee spo s (uppe , middle and lowe -cou se) wi h
di e en coo dina es (a): (5◦24.25 ״0.32 ׳ N, 7◦0.36 ״ 0.036 ׳E; (b): 5◦24.28 ״ 0.55 ׳N, 7◦0.38 ״0.36 ׳E and (c): 5◦23.55 ״0.20
׳N, 6◦59.46 ״ 0.39 ׳ E) om a dep h o 30 cm and pooled in 1-li e s e ile plas ic bo le o 200 ml s e ile glass bo le.
Immedia ely a e collec ion, samples we e placed in an insula ed cold box o coole o anspo o a wa e es ing
labo a o y. Wa e samples we e examined upon a i al, 6 hou s o collec ion o ensu e con inual iabili y o cells.
2.4. Isola ion
The pooled sample we e s o ed in a coole and aken o he labo a o y. The algal load o he sample was de e mined by
washing o cen i uga ion me hod and aga -pla ed wi hin six hou s o collec ion. Wi hin he pe iod he wa e sample
was collec ed, pu e cul u es o he es o ganism we e isola ed and 2-3weeks he ea e o axenic cul u es, ha was
s o ed unde s anda d mic obiological condi ions be o e oxici y assay. The algal load o he wa e sample was
es ima ed a colony o ming uni (CFU/ml).
2.5. Algological analysis
2.5.1. P epa a ion o aga pla e
Aga pla es we e p epa ed by dissol ing 2% aga (w/ ) in BBM. The pla es we e au ocla ed a 1260C o 15 minu es.
The pla es we e allowed o cool down and 5 ml wa m aga medium was pu in he pla es. The pla es we e allowed o
cool, kep in in e ed posi ion o comple e emo al o s eam and d ying a leas 72 h s be o e s eaking in bold basal
medium (modi ied wi h highly en iched ace me al solu ion and F/2 i amin solu ion), (S ein, 1973). Modi ied bold
basal media pla es con ained (g/ml); KH2PO: 48.75g/500(10ml), CaCl2•2H2O: 12.5g/500ml (1ml), MgSO4•7H2O:
37.5g/500ml (1ml), NaNO3: 125g/500ml (1ml), K2HPO: 4.37.5g/500ml (1ml), NaCl: 12.5g/500ml (1ml),
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Na2EDTA•2H2O: 10g/L (1ml), KOH: 6.2g/L, FeSO4•7H2O: 4.98g/L (1ml), H2SO4 (concen a ed): 1ml/L, T ace Me al
Solu ion: 1ml, H3BO3 5.75g/500ml: (0.7), F/2 Vi amin Solu ion: (op ional: cyanocobalamin, bio in, hiamine) (Nichols
and Bold, 1965).
2.5.2. Cul u e o isola e
An aliquo o 12ml o washed and cen i uged algal sample was aken om anspo ed pooled samples in o he s e ile
ubes. The ubes we e cen i uge a 3000 pm o 15 minu es. The supe na an was emo ed. The cells we e suspended
in a esh s e ile wa e in each ube using o ex mixe ( o a ed a 1000-1500 pm up o homogeneous suspension).
Cen i uga ion and washing o algal cells we e epea ed o six imes o expel con aminan s and mos mic oo ganisms
p esen in he algal sample (Pa in and Habib, 2007). A loop ul o he labeled isola e was s eaked on o bold basal
medium aga , supplemen ed wi h an ibac e ial and an i- ungal d ugs. Cul u e pla es we e kep unde luo escen ligh .
Subsequen ly, he Cul u e pla es we e incuba ed o 2 weeks a oom empe a u e (28±20C). The ea e , he cul u e was
ans e ed in o a b o h inocula ed wi h bold basal medium con ained in 250 ml conical lask. Con inuous cul u e was
main ained o sus ain algal g ow h a exponen ial phase.
2.5.3. Subcul u e o isola e
Axenic cul u e o es o ganism was sub-cul u ed in an ino ganic liquid medium p epa ed as ecommended by OECD
(1981).
2.5.4. S o age o pu e s ock cul u e
Fo op imal yield o es o ganism(s), axenic b o h cul u e was incuba ed a empe a u e (20±20C), unde whi e
luo escen ligh (3000-4000) lux, on a o a y shake . New s ock cul u e was ini ia ed a 40C in he da k, in e e y 40-60
days, by inocula ing app oxima ely 5×104 cells ml-1, (Jonsson and Aoyama, 2007).
2.5.5. P epa a ion and s anda diza ion o inoculum
A loop ul o he isola es, s o ed in bold basal medium slan in he e ige a o was inocula ed in o 200ml o bold basal
medium b o h con ained in 500 ml conical lask and incuba ed on a o a y shake a 150 pm a oom empe a u e
(28±20C), o 24h ou s. A e incuba ion, he cells we e ha es ed by cen i uga ion a 3000 pm o 10minu es, he
supe na an s we e disca ded and he sedimen which con ained he g een pigmen cells was ha es ed. The ha es ed
cells we e washed wice in s e ile dis illed wa e . The cell ex ac s we e s anda dized in a spec opho ome e o a
densi y o 1.8 a 600nm.
2.6. Mo phological Iden i ica ion o Isola ed o ganism
The mo phological ai s e alua ed, comp ised o colony mo phology, g een pigmen and chlo ophyll a/b p oduc ion.
Mo phological analyses we e based on ype, elas ici y and appea ance, while colony mo phology pa ame e was based
on colou , o m, anspa ency and diame e . The o he es s ca ied ou o iden i ica ion o he isola e included;
chlo ophyll p oduc ion, ca alase, phospha ase, s a ch and lipase (S ein, 1973).
2.7. Molecula (Genome) Iden i ica ion o he Isola ed o ganism
The ollowing molecula iden i ica ion we e ca ied ou : DNA ex ac ion, DNA quan i ica ion, 18S sequencing
Ampli ica ion, Assembly and Anno a ion and Phylogene ic analysis.
2.7.1. DNA ex ac ion
Ex ac ion was done using a ZR ungal/algal/bac e ial DNA mini p ep ex ac ion ki supplied by Inqaba Sou h A ica. A
hea y g ow h o pu e cul u e o he suspec ed isola es was suspended in 200 mic oli e o iso onic bu e in o a ZR
Bashing Bead Lysis ubes, 750 mic oli e o lysis solu ion was added o he ube. The ubes we e secu ed in a bead bea e
i ed wi h a 2ml ube holde assembly and p ocessed a maximum speed o 5 minu es. The ZR bashing bead lysis ube
was cen i uged a 10,000xg o 1 minu e. Fou hund ed (4s00) mic oli e o supe na an was ans e ed o a Zymo
Spin IV spin Fil e (o ange op) in a collec ion ube and cen i uged a 7000xg o 1 minu e. One housand wo hund ed
(1200) mic oli e o algal/ ungal/bac e ial DNA binding bu e was added o he il a e in he collec ion ubes b inging
he inal olume o 1600 mic oli e, 800 mic oli e was hen ans e ed o a Zymo-Spin IIC column in a collec ion ube
and cen i uged a 10,000xg o 1 minu e, he low h ough was be disca ded om he collec ion ube. The emaining
olume was ans e ed o he same Zymo-spin and spun. Two hund ed (200) mic oli e o he DNA P e-Wash bu e
was added o he Zymo-spin IIC in a new collec ion ube and spun a 10,000xg o 1 minu e ollowed by he addi ion o
500 mic oli e o algal/ ungal/bac e ial DNA Wash Bu e and cen i uged a 10,000xg o 1 minu e. The Zymo-spin IIC
GSC Biological and Pha maceu ical Sciences, 2025, 33(01), 146-163
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column was ans e ed o a clean 1.5 mic oli e cen i uge ube, 100 mic oli e o DNA elu ion bu e was added o he
column ma ix and cen i uged a 10,000xg mic oli e o 30 seconds o elu e he DNA. The ul a-pu e DNA was hen
s o ed a -20 deg ee o o he downs eam eac ion.
2.7.2. DNA quan i ica ion
The ex ac ed genomic DNA was quan i ied using he Nano d op 1000 spec opho ome e . The so wa e o he
equipmen was launched by double clicking on he Nanod op icon. The equipmen was ini ialized wi h 2ul o s e ile
dis illed wa e and blanked using no mal saline. Two mic oli e o he ex ac ed DNA was loaded on o he lowe
pedes al; he uppe pedes al was b ough down o con ac he ex ac ed DNA on he lowe pedes al. The DNA
concen a ion was measu ed by clicking on he “measu e” bu on.
2.7.3. 18S sequence Ampli ica ion
The 18S egions o he isola es was ampli ied using he 18S C-2 b: 5‘- ATTGGAGGGCAAGTCTGGT-3‘' and 18S D-2 b: 5'-
ACTAAGAACGGCCATGCAC-3, P ime s on a ABI 9700 Applied Bio sys ems he mal cycle a a inal olume o 30 mic o
li es o 35 cycles. The PCR mix included: The X2 D eam aq Mas e mix supplied by Inqaba, Sou h A ica ( aq
polyme ase, DNTPs, MgCl), he p ime s a a concen a ion o 0.4M and he ex ac ed DNA as empla e. The PCR
condi ions was main ained as ollows: Ini ial dena u a ion, 950C o 5 minu es; dena u a ion, 950C o 30 seconds;
annealing, 530C o 30 seconds; ex ension, 720C o 30 seconds o 35 cycles and inal ex ension, 720C o 5 minu es. The
p oduc was esol ed on a 1% aga ose gel a 120V o 15 minu es and isualized on a blue ligh ans illumina o .
2.7.4. Sequencing
Sequencing was done using he BigDye Te mina o ki on a 3510 ABI sequence by Inqaba Bio echnological, P e o ia
Sou h A ica. The sequencing was done a a inal olume o 10ul; he componen s includes: 0.25ul BigDye® e mina o
1.1/ 3.1, 2.25ul o 5 x BigDye sequencing bu e , 10uM P ime PCR p ime , and 2-10ng PCR empla e pe 100bp. The
sequencing condi ion was main ained as ollows 32 cycles o 960C o 10s, 550C o 5s and 600C o 4min.
2.7.5. Phylogene ic Analysis
Ob ained sequences was edi ed using he bioin o ma ics algo i hm T ace edi , simila sequences we e downloaded om
he Na ional Cen e o Bio echnology In o ma ion (NCBI) da a base using BLASTN. These sequences we e aligned using
MAFFT. The e olu iona y his o y was in e ed using he Neighbou -Joining me hod in MEGA 6.0 (Sai ou and Nei, 1987).
The boo s ap consensus ee in e ed om 500 eplica es (Felsens ein, 1985) was aken o ep esen he e olu iona y
his o y o he axa analysed. The e olu iona y dis ances we e compu ed using he Jukes-Can o me hod (Jukes and
Can o , 1969; Sai ou and Nei, 1987; and Felsens ein,1985).
2.7.6. Phospha ase Ex ac ion
Phospha ase ex ac ion ollowed he me hod desc ibed by Jonsson and Aoyama (2007). Algal pelle s iden i ied
molecula ly we e ozen in liquid ni ogen and mace a ed in a mo a wi h ace a e bu e . The mix u e was subjec ed
o eeze- haw cycles by s o ing a −20°C and hawing a oom empe a u e o p oduce a 1:4 (w/ ) suspension. Cell
dis up ion was achie ed by p obe sonica ion on ice (0°C) wi h 50 seconds sonica ion ollowed by 20 seconds in e als
(1 cycle) a 70% ampli ude; epea ed wice. The lysa e was cen i uged a 10,000 pm o 20 min. A e wa ds, he ex ac
om supe na an was assayed o he con e sion o colou less p-ni ophenylphospha e o yellow, p-ni ophenol. The
supe na an con aining he enzyme ex ac was iden i ied as alkaline phospha e using li mus indica o and s o ed o be
used o phospha ase assays.
2.8. Toxici y o Una y-Quina y mix u es o me als and pes icide o Chlo ella ulga is alkaline phospha ase
ac i i y
The indi idual o single dose o g aded ionic concen a ions o coppe , zinc, ch omium, lead, cadmium and 2,4-
dichlo ophenoxyace ic acid was assessed. The me als we e each p epa ed in 10mM s ock concen a ion. The
concen a ion anges om Coppe (Cu2+), Zinc (Zn2+) and Ch omium (C 2+) was (0-0.5mM), Lead (Pb2+) and Cadmium
(Cd2+) ions (0-7.0mM) and 2,4-dichlo ophenoxyace ic acid (2,4-D) (0-25mM). Quina y mix u es o pes icide/me al we e
amalgama ed in a simple pe cen age a io o 20:20:20: 20:20 and 16:21:21: 21:21 o he quina y combina ions’ a ios.
Thei oxici ies we e de e mined a concen a ions om 0-9.0mM. Inhibi o y s udy on he alkaline phospha e (ALP)
ac i i y was de e mined in 3ml eac ion inal olume consis ing o he g aded concen a ion o single o quina y
oxican s mix u e, dis illed wa e , bu e ed enzyme, and subs a e (p-NPP), con ained in 15ml s e ilized cul u e ubes.
The se -up was conduc ed in iplica es. The con ol consis s o se -ups de oid o oxican s. The se -up con ained g aded
concen a ions o oxican s amended in equisi e olume o dis illed wa e , 0.5ml bu e ed subs a e p-NPP (pH 8.0)
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and 0.5ml c ude enzyme was added and he cul u e ubes we e incuba ed o 30-40 minu es a oom empe a u e
(370C). A 0.1ml o sodium hyd oxide was added o s op he eac ion. The ea e , he ubes we e cen i uged and 2ml o
he supe na an s was spec opho ome ically measu ed a 410nm.
2.9. S a is ical Analysis
2.9.1. Es ima ion o ela i e esponse, Median Inhibi o y Concen a ion o Una y- Quina y Combina ions o Me als and
Pes icide and Modeling o Da a om he Inhibi ion analysis.
The ela i e esponses we e e alua ed as pe cen age ela i e es o con ol, due o inhibi ion o he oxican s and i ed
model equa ions (Figu e 2),
Figu e 2 Rela i e esponses
In equa ion: (1), RC is he esponse o he con ol and RT is he esponse in he es s (a di e en concen a ions o he
oxican ). The da a gene a ed om ela i e inhibi ion esponses o una y-quina y o me als and pes icide we e i ed
in o dose- esponse models (Cede g een, e .al., 2005) using sigma plo ( e sion 10). The indi idual median inhibi o y
concen a ion (EC50) o he indi idual oxican s and oxican s mix u e o phospha ase enzyma ic ac i i y in Chlo ella
ulga is was gene a ed om he models. The i ed models a e also elabo a ed wi h hei equa ions below (Figu e 3).
Figu e 3 Ppe cen age ela i e con ol inhibi ion and i ed model equa ions
Whe e:
• y is he ela i e esponse.
• y0 is he esponse a in ini e x.
• b pa ame e de e mining he slope o he ho me ic inc ease,
• a is he maximum esponse.
• is he pa ame e desc ibing he deg ee o ho me ic inc ease.
• x is he concen a ion o phenol.
• x0 is EC50.
2.10. Analysis o oxici y using oxic index model
The oxici y in e ac ion o he mix u es was assessed using he oxic index (TI), which sum he componen s oxic uni
(Boillo and Pe odin, 2008). The TU alues we e calcula ed using he exp ession as shown in igu e ou :
GSC Biological and Pha maceu ical Sciences, 2025, 33(01), 146-163
152
𝑇𝑈𝑖 = 𝐶𝑚𝑖𝑥𝑖
𝐼𝐶50𝐼
....................................................................(6)
While TI is he summa ion o TU o n oxican s in he mix u e
Figu e 4 Toxici y exponen model equa ions
Whe e Cmixi is he concen a ion o he i h oxican in he mix u e and IC50I o EC50i is he EC50 o he same oxican when
es ed singly. TI=1 implies addi i e in e ac ion, TI > 1 implies an agonis ic in e ac ion and TI < 1 implies a syne gis ic
in e ac ion (Boillo and Pe odin, 2008). The expe imen al da a was i ed and he median inhibi o y concen a ions
(EC50) o indi idual and mix u es o pes icides and me als we e e alua ed using Sigma plo so wa e (10.0). S a is ical
analysis o EC50 alues was compu ed using one-way analysis o a iance (p<0.05) in s a is ical package o he social
sciences (IBM, SPSS so wa e 22).
3. Resul s
3.1. Mo phological and biochemical iden i ica ion o he isola e R1(AY591506).
The mo phological ai s o isola e R1(AY591506) unde he elec on mic oscope comp ises o a sphe ical mic oscopic
cell wi h 2-10μm diame e . In mo phome ic obse a ions, cell diame e was 3-12 μm. Chlo oplas was pa ie al and
cup-shaped wi h a single py enoid he isola e showed many s uc u al elemen s simila o plan s. I has a unilamina
cell wall wi h subsphe ical o ellipsoidal shape wi hou lagella. The isola e R1(AY591506) also comp ises o a single
chlo oplas wi h a double en eloping memb ane composed o phospholipids. The singly, s anded-shaped chlo oplas
wi h py enoids, con ains a clus e o used hylakoids o g een chlo ophyll pigmen . I con ains a gel-like subs ance
con ined wi hin he ba ie o he cell memb ane, a double-laye memb ane ha esembles he mi ochond ion and a
dense ci cula pa ch ( ig 5). Unde he ligh mic oscopy i appea ed g een, unicellula , and sphe ical (coccoid) o
subsphe ical. Chlo oplas was pa ie al and cup-shaped wi h a single py enoid. All mo phological and phylogene ic
acing wi h desc ip ions o R1(AY591506 ( ig 6), was en a i ely iden i ied as Chlo ella sp ollowing he wo ks o
Tomaselli, (2004); Bo owi zka, (2018); Sa i, e al., (2014); K ieni z, e al., (2015); Yamamo o, e al., (2005); Champenois,
e al., (2015); Ga cia, (2012); Beije inck, e al., (1890); Yamamo o, e al., (2004). All mo phological and biochemical
sc eening a e p esen ed in able 1
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Table 1 Mo phological and biochemical sc eening assay o Chlo ella ulga is
Pa ame e s
Resul s
Shape
Sphe ical/cup-shaped py enoid
Fo m
Ellipsoidal
Chlo oplas
(Single anded) p esen
Colou
G een hylakoids pigmen
Type
Unilamina
Cell diame e
2-12 um
Phospha ase es
++
Ni ogenase es
++
Chlo ophyll analysis
++
Ni iles es
++
Lipoxygenases
++
Amylase
+
Lipase es
++
U ease es
++
Thiolase/gela inase es
++
Ca alase
++
Hyd ogenases
++
Ca bonic anhyd ase
++
Species: Chlo ella ulga is
Figu e 5 Schema ic Mic oscopic s uc u e o Chlo ella ulga is
3.2. Molecula (Genome) Iden i ica ion o he Isola e R1(AY591506).
F om he molecula iden i ica ion o specie le el using he p ocess o DNA ex ac ion, DNA quan i ica ion,18S
sequencing Ampli ica ion, Assembly, Anno a ion and Phylogene ic analysis, he ob ained 18S sequence om he isola es
(R1(AY591506) p oduced an exac ma ch du ing he megablas sea ch o highly simila sequences om he NCBI non-
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edundan nucleo ide (n /n ) da abase. The 18S o he isola es showed a pe cen age simila i y o o he species a 99-
100%. The e olu iona y dis ances compu ed using he Jukes-Can o me hod we e in ag eemen wi h he phylogene ic
placemen o 18S o he isola es wi hin he Chlo ella sp and e ealed a closely ela edness o Chlo ella ulga is
espec i ely
Figu e 6 Phylogene ic ee showing he isola e Chlo ella ulga is
3.3. Toxici y o Indi idual Me als and 2,4-D on Chlo ella ulga is Phospha ase Ac i i y
Resul p esen ed in igu e 4 shows he expe imen al (da a poin s) and model-p edic ed dose- esponse da a cu e o
una y oxici y o hea y me als: Coppe , Lead, Ch omium, Cadmium, Zinc ions and 2, 4-D o Chlo ella ulga is
phospha ase ac i i y. The esul s indica e ha he inhibi ion o phospha ase ac i i y closely i ed in o a Sigmoidal 3
Pa ame e model; wi h R2 alues anging om 0.98-0.99. Th eshold inhibi o y concen a ions (EC50) o he single
compounds a e p esen ed in able 2.0.
Table 2 Median inhibi o y concen a ion (IC50) o he e ec o Una y oxici y o (me al/pes icide) o phospha ase
enzyme ac i i y om Chlo ella ulga is.
Toxican s
IC50 (mM)
Toxici y index
In e ac ion
Una y (100%)
Cu2+
Zn2+
Pb2+
C 2+
Cd2+
2,4-D
0.28 ±0.01c
0.31±0.02c
1.86±0.76c
0.19±0.01c
2.59±0.32c
16.82±1.47c
-
-
-
-
-
-
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
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