Acta agriculturae Slovenica, 117/4, 1–12, Ljubljana 2021
doi:10.14720/aas.2021.117.4.2111 Original research article / izvirni znanstveni članek
Evaluation of biochemical treatments applied in polluted soils irrigated 
with low quality water for long periods of time through the CO
2
 efflux
Mohamed SABER
 1
, Alaa ZAGHLOUL
 2, 3
Received February 21, 2021; accepted November 26, 2021.
Delo je prispelo 21. februarja 2021, sprejeto 26. novembra 2021
1 Department of Agricultural Soil Microbiology, National Research Centre, Dokki, Cairo, Egypt
2 Soils and Water Use Department, National Research Centre, Dokki, Cairo, Egypt
3 Corresponding author, e-mail: alaazaghloul2008@gmail.com
Evaluation of biochemical treatments applied in polluted 
soils irrigated with low quality water for long periods of time 
through the CO
2
 efflux
Abstract: To sightsee the bearings of the certain reme-
diation amendments, usually applied in the bioremediation of 
soils irrigated with low quality water for extended periods on 
the indigenous microbial population, a greenhouse experiment 
was conducted at National Research Centre (NRC) where the 
soil ecosystem was supplied with varied mineral remediation 
amendments and the carbon dioxide (CO
2
) refluxes were fol-
lowed up. In this study, microbial activity through CO
2
 efflux 
was taken as an indicator to evaluate the effectiveness of eight 
soil amendments in minimizing the hazards of inorganic pol-
lutants in soil ecosystem irrigated with low quality water s for 
more than 40 years. Results showed that Ni and Zn were the 
most dominant contaminants that adversely influenced indig-
enous microbial activities in untreated soil, while Cu was the 
most persuasive. All trailed remediation amendments signifi-
cantly minimized the hazards of inorganic pollutants in treated 
soil ecosystems. In addition, modified bentonite (Probentonite) 
was the best persuasive one. Mechanisms take place between 
trailed remediation amendments and inorganic pollutants in 
the studied soil ecosystems were discussed. In conclusion ap-
plication of certain raw or modified clay minerals especially 
Probentonite could be a good tool in decreasing the rate of the 
studied inorganic pollutants in a contaminated soil ecosystem 
irrigated with low quality water for extended periods. 
Key words: soil; low quality water; bioremediation; po-
tential toxic elements; soil indigenous microbial Activities; 
modified clay minerals
Ovrednotenje biokemičnih obravnavanj onesnaženih tal, ki 
so bila dalj časa namakana z vodo slabe kakovosti z meritvijo 
sproščanja CO
2
 
Izvleček: Za prepoznavanje obremenitev, ki jih povzroča-
jo nekateri remediacijski dodatki, ki se navadno uporabljajo pri 
bioremediaciji tal namakanih dalj časa z vodo slabe kakovosti 
na samoniklo mikrobno populacijo, je bil izveden poskus v ra-
stlinjaku v nacionalnem raziskovalnem centru (NRC). Tlem so 
dodajali različne mineralne remediacijske dodatke in spremlja-
li sproščanje ogljikovega dioksida (CO
2
). Mikrobna aktivnost, 
izražena kot iztok CO
2,
 je služila kot indikator ovrednotenja 
učinkovitosti osmih talnih dodatkov, ki naj bi zmanjšali škodo, 
ki jo v talnem ekosistemu povzročajo anorganska onesnaže-
vala iz vode za namakanje slabe kakovosti v obdobju več kot 
40 let. Rezultati so pokazali, da sta bila Ni in Zn dominantna 
kontaminanta, ki sta negativno vplivala na aktivnost samoni-
klih mikrobov v obravnavanih tleh medtem, ko je bil učinek 
Cu največji. Vsi poskusi dodatkov v remediaciji so značilno 
zmanjšali tveganja poškodbe tal zaradi anorganskih polutantov 
v obravnavanih talnih ekosistemih. Pri tem je bil spremenjeni 
bentonit (Probentonite) najučinkovitejši. V raziskavi so inter-
pretirani mehanizmi, ki potekajo med dodatki v remediaciji in 
anorganskimi onesnaževali v preučevanih talnih ekosistemih. 
Zaključek je, da je dodatek nekaterih osnovnih ali spremenje-
nih glinenih mineralov, še posebej probentonita lahko dobro 
sredstvo za zmanševanje onesnaženja z nekaterimi anorganski-
mi onesnaževali v onesnaženih ekosistemih, ki so bili namakani 
z vodo slabe kakovosti v daljšem obdobju. 
Ključne besede: tla; voda slabe kakovosti; bioremediacija; 
potencialno toksični elementi; aktivnost samoniklih talnih mi-
krobov; spremenjeni glineni minerali 

Acta agriculturae Slovenica, 117/4 – 2021 2
M. SABER and A. ZAGHLOUL 
1 INTRODUCTION
Through low quality water farming varied organic 
and inorganic contaminates reach the soil together with 
enteric pathogens, and cause vital adverse agricultural, 
environmental and public health troubles. Biological 
pollutants include bacteria, viruses, and parasites that 
are responsible for waterborne diseases, such as typhoid 
fever, cholera, dysentery, polio, hepatitis, and schistoso-
miasis. The presence of coliform bacteria is indicator of 
recent fecal pollution, this type of contamination is ex-
clusively attributed to human and animal waste. Also, in 
these materials inorganic pollutants include cations and 
anions, most of them naturally occurring in soils, sedi-
ments, and rocks. Cations include heavy metals, such as 
cadmium (Cd), chromium (Cr VI), lead (Pb), manganese 
(Mn), mercury (Hg), and nickel (Ni). These highly toxic 
chemicals may reach soil after mineral dissolution with 
low quality waters applied or from industrial activities or 
after industrial emissions (Saber et al., 2015).Although 
low quality water farming was always associated with po-
tential benefits as well as with problems, yet appropriate 
practices to ensure its safe and effective sustainable use 
are not well developed (Doaa Ali et al, 2020). The role of 
microorganism on the health of soil ecosystem is incon-
testable. Since 1930 huge amounts of agrochemicals were 
applied to soil ecosystems and adversely impacted their 
biome activities, Khan and Scullion (2000) recorded a 
shift in bacterial to fungal population in soil ecosystems 
as a result of contamination with inorganic pollutants. 
Recently, one of the narrative concepts of soil health is bi-
oremediation of contaminated soil ecosystems using var-
ied remediation amendments, some of which with nega-
tive impacts on soil microbial activities (Kelly and Tate 
III, 1998). It is worthy to state that the influence of the 
inorganic pollutants on the respiration intensity in a soil 
ecosystem irrigated with low quality water for extended 
periods was somewhat inconsistent, Yonebayashi and 
Hattori (1989) and Doelman and Haanstra (1984) 
verified significant decreases in CO
2
 evolution from 
such soils, while Bardgett and Saggar (1994) and W elp 
(1999), on the other hand, recorded high increases. Such 
phenomenon is linked with varied interferences. 
The current study aimed at evaluating the delayed 
effect of a natural modified clay mineral fortified with 
certain remediation amendments on a soil irrigated with 
low quality water s for extended periods through their 
microbial activities represented by CO
2
 evolution. 
2 MATERIAL AND METHODS
2.1 STUDY SITE
A surface soil sample (0-30 cm), irrigated with low 
quality water for 40 years was collected from El-Rahawy 
farm, Giza governorate. The chemical characterization of 
the soil showed it to have pH, 6.83; EC, 0.2 dSm
-1
; OM 
(organic matter), 0.2 %; clay content, 4.4 % and with a 
sandy texture. Determined inorganic pollutants in the 
tested soil were 18 ppm Ni; 35.65 ppm Cu; 400.6 ppm Zn; 
596 ppm Fe; 45.19 Mn 57.7ppm Pb and the Zn equivalent 
parameter was 633.9. 
2.2 EXPERIMENTAL DESIGN
In a completely randomized pot experiment with 
four replicates, single and combined mixtures of varied 
remediation inorganic amendments were trailed to re-
tain PTE’s from a contaminated soil ecosystem irrigated 
with low quality water for extended periods.  The soil was 
treated with either 2 % probentonite (T1), 2 % kaolinite 
(T2), 1 % probentonite + 1 % Kaolinite (T3), 1 % proben-
tonite + 1 % rock phosphate (RP) (T4), 1 % kaolinite + 
1 % RP (T5), 1 % Bentonite + 0.5 % kaolinite + 0.5 % RP 
(T6), 2 % iron oxide (T7) and 1 % iron oxide + 1 % RP 
(T8). Treated and untreated control soils were moistened 
to 60 % of the soil water holding capacity and incubated 
for 60 days at 25 
o
C. At the end of incubation time (60 
days), a kinetic study was carried out on treated and un-
treated soil ecosystems followed by a distribution study 
of inorganic pollutants studied. 
2.3 INORGANIC POLLUTANTS INSTRUMENTA-
TION AND ANALYSIS
A Perkin–Elmer flame atomic absorption spectrom-
eter (FAAS) and HACH DR890 colorimeter was used 
in inorganic pollutants instrumentation and analyses. 
Atomic absorption measurements were carried out using 
air: acetylene flame while HACH colorimeter measure-
ment with the provided test kits. The operating param-
eters for working elements were set of as recommended 
by the manufacturer.
2.4 POTENTIAL TOXIC ELEMENTS DISTRIBU-
TION ANALYSIS
Inorganic pollutants were fractionated to wa-
ter soluble, exchangeable, (readily available form RA), 

Acta agriculturae Slovenica, 117/4 – 2021 3
Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water ...
carbonate-bound, Fe-Mn oxides-bound ad organic-
bound which was considered to be the residual fraction 
(Zaghloul, 2002). The soil quality criterion index (Zn 
equivalent model) was numerically expressed for the lev-
els of PTE’s toxicity as described by Saber et al. (2012). A 
quality criterion index for zinc equivalent over 250 units 
indicated a risky situation. Kinetic studies were carried 
out using the Electrical Stirred Flow Unit (ESFU) meth-
od. 
2.5 CO
2
 EFFLUX AND KINETIC EQUATION
CO
2
 evolved during the incubation was trapped in 
1 M NaOH, and the excess NaOH was titrated with 0.1 
M HCl after the addition of BaCl
2
. Total CO
2
 mineral-
ized was calculated as cumulative CO
2
 evolution (Leifeld 
et al., 2002). The specific respiration activity (qCO
2
) was 
expressed as the production of CO
2
-C per unit biomass 
C and time. 
Kinetic equations: The following four kinetic equa-
tions representing both empirical and theoretical equa-
tions were used to test the conformity of the CO
2
 release 
data to each of them.
2.5.1 First order equation
 Log (q
j
 - q
t
 ) = Log q
0
 - k
1
 t
where:
q
j 
= the maximum amount of CO
2
 release
q
0
 = the initial amount of CO
2
 at the time of added 
the resin.
q
t
 = the  amount of  CO
2
 release at time t.
t = time in minute
k
1 
 = the rate constant of reaction n in sec
-1
2.5.2 Elovich équation
q= (1/b) ln ( ab ) + (1/b) ln t
 
where:
q = amount of CO
2
 desorbed at time t
a = constant in ppm CO
2
 min 
-1
b = constant in (ppm CO
2
)
 -1
t =  time in minute
2.5.3 Modified Freundlich equation
q = k
d
 t
b\
where:
q = amount of CO
2
 desorbed in time t
k
d
 = desorption rate coefficient in mg CO
2
 kg
-1
 soil 
min
-1
b
\
 = constant in mg CO
2
 kg
-1 
soil
2.6 SOIL MICROBIAL BIOMASS
Soil microbial biomas was measured by the fumi-
gation–extraction method after 24, 72, 96, 168, 240 and 
336 hours. Three replicates of each treatment were fumi-
gated with ethanol-free chloroform for 24 h at 25 
⁰
C. The 
soil samples were then extracted with 0.5 M K
2
SO
4
 for 30 
min. Three replicates of non-fumigated soil samples were 
extracted similarly. The extracted PTE’s were determined 
by dichromate oxidation at 100 ⁰C (2 ml of extract, 1.5 
ml of 15 M H
2
SO
4
 and ‏1.5 ml of saturated aqueous solu-
tion of K
2
Cr
2
O
7
). The residual K
2
Cr
2
O
7
 was determined 
by photometrical at 565 nm (Kuzyakov, 1997). The cali-
bration of the extracted C (carbon) measurements was 
carried using glucose. CO
2
 evolved during incubation 
was trapped in 1 M NaOH, and the excess NaOH was 
titrated with 0.1 M HCl after addition of BaCl
2
 (USEPA. 
2001). Total mineralized C was calculated as cumulative 
CO
2
 evolution (Leifeld et al., 2002). The specific respira-
tion activity (qCO
2
) was expressed as the production of 
CO
2
-C per unit biomass C and time (Anvar and Oliver 
Dilly, 2002).
2.7 STATISTICAL ANALYSIS
Multiple linear regressions, discriminant analysis 
and the fitting of curves to the data were performed using 
separate two-way ANOVAs. The data of biomass inor-
ganic pollutants and soil organic C were analyzed by dis-
criminate analysis. Statistical analyses aimed to examine 
the succession of the applied remediation amendments 
in returning the studied contaminated soil ecosystem to 
its normal settings. SAS software was used to evaluate the 
kinetic models that describe CO
2
 efflux under the action 
of the used different remediation amendments. 
3 RESULTS AND DISCUSSION 
3.1 KINETICS OF CO
2
 EFFLUX IN BIO-REMEDI-
ATED SOIL ECOSYSTEM
Results drawn in Figure 1 demonstrate the kinetics 
of CO
2
 efflux from both contaminated and bio-remediat-
ed soil ecosystems. All trailed remediation amendments 

Acta agriculturae Slovenica, 117/4 – 2021 4
M. SABER and A. ZAGHLOUL 
led to marked decreases in the rate of CO
2
 efflux com-
pared to control contaminated soil ecosystem. In con-
trol contaminated soil ecosystem, maximum CO
2
 efflux 
reached 14.11 mg kg
-1
 soil; while being 10.45 mg kg
-1
 soil 
in T3 (soil fortified with a mixture of bentonite + kaolin-
ite) and decreased to 8.2 in T8 (iron oxide + RP). Other 
trailed remediation amendments, thereafter decreased 
CO
2
 efflux with values higher than in abovementioned 
treatments was recorded. 
Chander and Brookes (1993), stated that when Zn 
and Cu are present together in the soil ecosystem the in-
crease in Zn and Cu bioavailability above 123 or 3.0 ppm 
causes marked decreases in the intensity of soil microbial 
biomass. 
Results given in Figure 2 show that the impacts of 
the trailed remediation amendments in minimizing the 
hazards of PTE’s varied according to their type. Modified 
bentonite, iron oxide, rock phosphate as well as the mix-
ture of these remediation amendments decreased signifi-
cantly the evolution of CO
2
 or restored the soil ecosystem 
to its normal conditions. Of all treatments T8, T3 and 
T7 were the best that condensed normal conditions, in 
some cases; however, certain remediation amendments 
increased the CO
2
 to a non-significant level compared to 
control especially when fortified with kaolinite (T5) even 
in the absence of RP .
3.2 RATE CONSTANTS OF BEST FITTED MODELS 
DESCRIBE CO
2
 EFFLUX AS AFFECTED BY 
REMEDIATION AMENDMENTS APPLIED TO 
SOIL ECOSYSTEM
Kinetic approach was used to evaluate the effec-
tiveness of the trailed remediation amendments on CO
2
 
efflux that express the biomass activity as well as the 
depressing action of PTE’s associated with the trailed re-
mediation amendments. 
As given in Tables 1-3, the rate constants of CO
2
 
efflux from the soil irrigated with low quality water for 
extended periods, as an indicator for microbial activ-
ity, varied according to type of the trailed remediation 
amendment. For MFE, the best fitted model, the appli-
cation of kaolinite and RP (T5) decrease CO
2
 efflux to 
0.71 mg g
-1
 soil, in control the value was 0.77 mg g
-1
 min
-
1
. Fortification of the soil ecosystem irrigated with low 
quality water with iron oxide also decreased CO
2
 efflux to 
0.68 mg g
-1
 min
-1
, and hence might be
 
used to cure such 
contaminated soil ecosystem. Also, using bentonite in 
T1 decreased the CO
2
 flux to 0.68 mg g
-1
 min
-1
. All other 
trailed remediation amendments significantly decreased 
CO
2
 efflux compared to control. The negative values re-
corded for the capacity factor in the same model means 
returning the soil ecosystem to optimum conditions for 
microbial activity.
3.3 MICROBIAL BIOMASS IN PTE’S CONTAMI-
NATED AND BIO-REMEDIATED SOIL ECO-
SYSTEMS
Results given in Table 4 show the amount of con-
taminants absorbed by the microbial biomass in the soil 
ecosystem. In control microorganism’s absorbed signifi-
cant amounts of inorganic pollutants reached 0.46 ppm 
Zn, 0.03 Cu and 0.10 Ni. The existence and uptake of 
all inorganic pollutants directly led to an increase in the 
microbial activity in the soil ecosystem. For example, in 
T1 only Ni was absorbed by microorganisms, meanwhile 
other studied inorganic pollutants did not presented in 
side microorganisms. 
The same trend was re-exhibited in T3, T5 and T7 
treatments. Bentonite + kaolinite + rock phosphate (RP) 
treatment (T6) and iron oxide + RP treatment (T8) also 
increased the microbial activity through increasing CO
2 
efflux of rate constants of Elovich kinetic model (Table 
3). Results in the same table point to that bentonite as 
remediation amendment decreased inorganic pollutants 
uptake at different degrees with some exception observed 
in case of Ni. 
Although the application of oxides as remediation 
amendment was extensively mentioned in literature, the 
application of iron oxides in this study enhanced Zn and 
Ni by microorganisms found in the soil ecosystem. It 
is worth to state that Cu was the only PTE that was de-
pressed by the trailed remediation amendments though 
the increasing of non fumigated values compared to fu-
migated ones. 
3.4 CONTRIBUTION OF TRAILED REMEDIATION 
AMENDMENTS IN SOIL ECOSYSTEMS ON 
MINIMIZING THE HAZARDS OF PTE’S
The effect of trailed remediation amendments on 
minimizing the hazards of Ni, Cu and Zn is drawn in 
Figure 3 that show all trailed treatments significantly 
decreased the available forms of the studied inorganic 
pollutants compared to control. The comparison be-
tween the different treatments indicated that bentonite 
significantly decreased the available form of PTE’s. For 
instances, the application of modified bentonite to soil 
(T4) led to a decrease reaching 34 % of available of Cu, 
79 % of available Zn and 77 % of available Ni.
The mixture of bentonite and kaolinite decreased 
68 % of available Cu, 66 % of available Zn and 59 % of 

Acta agriculturae Slovenica, 117/4 – 2021 5
Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water ...
Figure 1: Kinetics of CO
2
 efflux from contaminated soil as affected by remediation materials compared to control treatment. Where: (T1) 2 % probentonite, (T2) 2 % kaolinite, 
(T3) 1 % probentonite + 1 % Kaolinite, (T4) 1 % probentonite + 1 % rock phosphate (RP), (T5) 1 %kaolinite + 1 % RP , (T6) 1 % bentonite + 0.5 % kaolinite + 0.5 % RP , (T7) 2 % 
iron oxide and (T8) 1 % iron oxide + 1 % R 

Acta agriculturae Slovenica, 117/4 – 2021 6
M. SABER and A. ZAGHLOUL 
available Ni, this trend perhaps represents the selectivity 
of used clay minerals in retain PTE’s. Results pointed to 
that increasing of Cu and Zn was retained by kaolinite 
over bentonite, meanwhile a reverse trend was observed 
in Ni. The modification of kaolinite with RP increased the 
retention of Ni by 64 % in the soil ecosystem; meanwhile 
it did not exceed 18 % under sole kaolinite application. 
The same treatment, however, did not influence the 
retention level of Cu and Zn in the soil ecosystem. In 
contrast, fortification of the soil ecosystem with sole iron 
oxide decreased the retention of inorganic pollutants to 
65, 72 and 45 % of Cu Zn and Ni respectively, while the 
mixture of PR with iron oxide decreased these values re-
spectively to 50, 30 and 36 % of total form in soils. Results 
drawn in the same figure indicated that the mixture of all 
treatment (T6) did not exhibit the predicted trend since 
the decreasing orders of Inorganic pollutants did not ex-
ceed 34, 62 and 55 % of total Cu, Zn and Ni compared to 
control in order to be a valued treatment but not the best 
3.5 DISTRIBUTION OF THE STUDIED PTE’S IN 
REMEDIATED SOIL ECOSYSTEM
Results drawn in Figure 4 exhibit the distribution of 
Ni, Cu and Zn in soil ecosystem irrigated with low qual-
Figure 2: Map showing the experimental plots and location of the experiment at the Institute of Agricultural Research and Train-
ing in Ibadan, Oyo state, Nigeria. (T1) 2 % probentonite, (T2) 2 % kaolinite, (T3) 1 % probentonite + 1 % kaolinite, (T4) 1 % 
probentonite + 1 % rock phosphate (RP), (T5) 1 % kaolinite + 1 % RP , (T6) 1 % bentonite + 0.5 % kaolinite + 0.5 % RP , (T7) 2 % 
iron oxide and (T8) 1 % iron oxide + 1 % RP
Treatments a*10^4 b R
2
SE
cont -5.51 11.58 0.99** 0.21
T1 -5.96 10.95 0.99** 0.40
T2 -5.12 9.62 0.99** 0.35
T3 -7.71 12.53 0.97** 0.81
T4 -8.95 14.36 0.99** 0.80
T5 -6.43 11.33 0.96** 0.73
T6 -5.94 11.00 0.99** 0.43
T7 -3.88 7.40 0.99** 0.36
T8 -5.51 11.58 0.98** 0.21
Table 1: Rate constants of 1
st
 order model describe CO
2
 efflux 
from contaminated soil as affected by remediation treatments 
Treatments a b R
2
SE
cont 0.77 2.29 0.99** 0.05
T1 0.68 -1.86 0.99** 0.04
T2 0.74 -2.17 0.99** 0.04
T3 0.72 -2.39 0.99** 0.05
T4 0.68 -2.56 0.99** 0.05
T5 0.71 -1.99 0.98** 0.07
T6 0.75 -2.29 0.99** 0.05
T7 0.68 -1.86 0.99** 0.04
T8 0.74 -2.17 0.99** 0.04
Table 2: Rate constants of MFE describe CO
2
 efflux from con-
taminated soil as affected by remediation treatments 

Acta agriculturae Slovenica, 117/4 – 2021 7
Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water ...
ity water for long periods as affected by the trailed re-
mediation amendments applied to minimize the hazards 
of PTE’s and to optimize microbial activities through re-
mediation of soil ecosystem. Generally, as shown in the 
Figure 4, trailing the different remediation amendments 
decreased the readily available form and increased the 
residual one with rates varied according to amendment 
used. Three main categories of the trailed remediation 
amendments are distinguished, the 1
st
 category included 
the best ones, i.e., pro-bentonite (T4), pro-kaolinite (T5) 
and iron oxide (T7), which minimized the readily avail-
able form to zero in Ni and from 98 to 100 % for Cu and 
Treatments a b R2 SE
cont 3.85 -28.08 0.92** 1.77
T1 3.82 -27.09 0.95** 1.40
T2 3.34 -24.00 0.94** 1.25
T3 4.57 -33.12 0.95** 1.54
T4 5.24 -38.31 0.95** 1.76
T5 3.97 -28.46 0.94** 1.59
T6 3.82 -27.77 0.94** 1.44
T7 2.54 -18.10 0.93** 1.04
T8 3.85 -28.08 0.92** 1.77
Table 3: Rate constants of Elovich equation describe CO
2
 
efflux from contaminated soil as affected by remediation treat-
ments 
Zn, i.e., increased the residual form in these treatments. 
The 2
nd
 category included the remediation amendments 
able to minimize the available form of one of the test-
ed inorganic pollutants to zero such as T3 (the mixture 
between bentonite and kaolinite) in case of Cu. The 3
rd
 
category included the rest of treatments that significantly 
minimized inorganic pollutants at different rates accord-
ing to the studied inorganic pollutants.
It should be mention that all applied remediation 
amendments are locally available in Egypt and are con-
sidered with economic low coast. Various methods such 
as hydrometallurgical technologies, ion exchange, electro 
dialysis, reverse osmosis, precipitation and adsorption 
had been trailed to remove inorganic pollutants from 
aqueous solution phase in aquatic ecosystems (La Grega 
et al., 1994). It is well known that the reduction in the 
readily available forms of inorganic pollutants’ in con-
taminated soil ecosystems is a commonly technique used 
to reduce the negative impacts of inorganic pollutants on 
environment and improve the quality of contaminated 
soil ecosystems (Zaghloul, 2006). It is well known that 
clay minerals interact with almost all soil contaminants 
(Prost and Yaron, 2001). The adsorption of Ni, Cd, Zn, 
and Pb by the clay mineral montmorillonite was reported 
by Schulthess and Huang (1990). Sorption technique is 
one of the most efficient methods of cleaning the envi-
ronment from Inorganic pollutants. 
In this study microbial activity through CO
2
 efflux 
was used as an indicator to evaluate the effectiveness of 
eight remediation amendments in minimizing the haz-
ards of PTE’ s in soil ecosystem. Bentonite is a well-known 
as one of the most effective remediation amendments 
Treatments
NF
Zn
Fum.
Zn
NF
Cu
Fum
Cu
NF
Ni
Fum
Ni
ppm
Control 2.65 3.11 1.24 1.27 1.80 10.9
T1 0.86 0.42 1.69 1.34 1.89 2.36
T2 2.00 0.99 0.82 0.71 2.3 1.61
T3 0.54 0.31 0.58 0.41 1.88 2.23
T4 6.53 4.85 1.21 1.08 2.04 2.22
T5 3.13 2.71 0.85 0.77 1.44 2.02
T6 0.3 0.44 0.96 0.52 1.66 2.36
T7 1.21 0.78 0.89 0.89 1.75 2.05
T8 3.29 4.39 0.88 0.78 1.89 2.71
Table 4: Microbial biomass for Ni, Cu and Zn in both contaminated and remediated soils
NF: non-fumigated, Fum: Fumigated

Acta agriculturae Slovenica, 117/4 – 2021 8
M. SABER and A. ZAGHLOUL 
Figure 3: Potential toxic elements availability in contaminated as affected by remediation materials compared to control treat-
ments 

Acta agriculturae Slovenica, 117/4 – 2021 9
Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water ...
Figure 4: Distribution of PTE’s in the studied low quality water soils as affcted by certain remediative amendments. (T1) 2 % 
probentonite, (T2) 2 % kaolinite, (T3) 1 % probentonite + 1 % kaolinite, (T4) 1 % probentonite + 1 %  rock phosphate (RP), (T5) 
1 % kaolinite + 1 % RP , (T6) 1 % bentonite + 0.5 % kaolinite + 0.5 % RP , (T7) 2 % iron oxide and (T8) 1 % iron oxide + 1 % RP

Acta agriculturae Slovenica, 117/4 – 2021 10
M. SABER and A. ZAGHLOUL 
used in curing soil ecosystems contaminated with certain 
inorganic pollutants such as Ni, Cu, Zn and others (An-
dini et al., 2006). In addition, bentonite has been shown 
to improve the overall soil quality (Phillips, 1998). Appli-
cation of bentonite to soils irrigated with low quality wa-
ter s significantly retained PTE’s in different mechanisms 
such as sorption and ion exchange mechanisms. The op-
timum conditions of soil ecosystem to decrease the avail-
ability of PTE’s might obviously monitored through the 
microbial activity of decreasing the CO
2
 efflux.
Although both bentonite and pro-bentonite showed 
priority in minimizing the hazards of PTE’s in the stud-
ied soil ecosystem, yet, kaolinite exhibited the least ex-
changeability among bentonite clay mineral group, as 
several studies confirmed the potential of natural kaolin-
ite in metal ion adsorption from solution. O’Day et al. 
(1994) mentioned that CO
2
 is always binding to kaolinite 
as co-complexes at both inner and outer sphere complex-
es using X-ray absorption spectroscopy (XAS). Boron 
adsorption on kaolinite was studied by Singh and Mat-
tigod (1992) using phenomenological equations and sur-
face complication reactions. Samaneh and Jalali (2016) 
evidenced strong preference for the ion exchanged form 
of kaolinite for Cu ions. Exchange capacity of both cation 
as well as anion of kaolinite and their relation with homo 
ionic counterparts with Na+ was critically examined by 
Ferris and Jepson (1975). 
Results indicated that the succession of more than 
one model in describing the kinetic results having high 
R
2
 values ranged between 96-99** in used models. This 
means that the different mechanisms that took place in 
the sorption of studied PTE’s by the trailed remediation 
amendments improved CO
2
 efflux by decreasing the 
available form of PTE’s.
The outer groups were situated along the unshared 
plane of the alumina hydroxyl sheet, while the inner 
groups were located along the plane that is shared with 
and borders on the silica oxide sheet. The movement of 
the inner hydroxyl plane is restricted as a result of chemi-
cal bonding between the silica and alumina sheets. The 
pro- clay mineral treatment in all cases directly increased 
the retention of PTE’s, this trend decreased the microbial 
biomass of these treatment. This result might be due to 
the mode of phosphate reaction with all PTE’s in having 
complex compounds (Ma and Harris, 1997). Worth to 
mention that iron oxide exhibited the same trend when 
RP was applied to the soil ecosystem.
Sorption and immobilization of inorganic pollut-
ants in soil ecosystem is an effective detoxification pro-
cess and thus it is an essential part of the buffering capac-
ity of the soil ecosystem (Welp 1999). 
Immobilization of inorganic pollutants caused an 
increase in basal respiration rate, litter decomposition 
and microbial activity. There are several methods for 
immobilization of inorganic pollutants in soil, through 
either adding natural and synthetic chemical additives 
such as alkaline materials, phosphate minerals, iron and 
manganese oxides, alumino-silicates or coal fly ashes 
(Mench et al., 1998). Clay minerals are among the major 
materials that interact with almost all soil contaminants 
(Prost and Yaron, 2001). The adsorption of Ni, Cd, Zn, 
and Pb by montmorillonite was reported by Schulthess 
and Huang (1990). Immobilization of inorganic pollut-
ants by natural zeolite (clinoptilolite) and six synthetic 
zeolites was studied by Oste et al. (2002), who found that 
the synthetic zeolites had an effect on immobilization of 
Cd and Zn. The improvement of the quality of the mi-
crobe’s media through is mainly due to ameliorative ac-
tion of PTE’s in soil ecosystem irrigated with low quality 
water for extended periods.
4 CONCLUSIONS AND RECOMMENDA-
TIONS
The application of microbial activity through CO2 
flux in evaluation soil remediation technology(ies) can 
be a viable and innovative best way. In this work, the 
use of some clay minerals, crude or modified with some 
microbes, significantly reduced the content of harmful 
heavy metals in a soil ecosystem irrigated with low-qual-
ity water for long periods. The treatments used led to a 
significant decrease in the available forms of the studied 
heavy elements in parallel with the increase in the resid-
ual unavailable forms of pollutants. According to the ob-
tained results, heavy metal ions showed a tendency to ac-
cumulate on clay minerals, which shows the importance 
of this method of treatment and suitability for improving 
soil quality by restoring appropriate ecosystem condi-
tions and flourishing microbial activity. This method is 
also characterized by low costs and an economical way 
that encourage the farmers to use it for having safe food. 
It is worth noting the importance and necessity of using 
this technique more in other studies with other polluted 
metals that did not fall within the scope of this research.
5 ABBREVIATIONS
NRC: National Research Centre
PR: Rock phosphate
CO2: Carbon dioxide
Cu: copper
Ni: Nickel
Zn: Zinc
PR: Rock phosphate

Acta agriculturae Slovenica, 117/4 – 2021 11
Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water ...
K
2
Cr
2
O
7
: Potassium dichromate 
ANOV A: Analysis of variance
R2: Coefficient of determination
SE: Standard Error
NF: Non-fumigated                    
Fum: Fumigated
6 ACKNOWLEDGMENT
The authors would like to express their appreciation 
and gratitude to the Science, Technology & Innovation 
Funding authority (STDF) for financing the present work 
through the project number 41523 contracted with the 
National Research Center and extended till present.
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