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Use of the land snail Helix aspersa for monitoring heavy metal
soil contamination in Northeast Algeria
R. Larba &N. Soltani
Received: 22 June 2013 /Accepted: 21 March 2014 /Published online: 2 April 2014
#Springer International Publishing Switzerland 2014
Abstract The objective of this study was to investigate
the impact of anthropogenic activities on soil quality
using the land snail Helix aspersa as a bioindicator. Soil
samples and snails were collected from several sites in
Northeast Algeria during the summer and winter of
2010. All of the sites were chosen due to their proximity
to industrial factories—a potential source of soil pollu-
tion via heavy metal contamination. The concentration
of heavy metals (Pb, Cd, Mn, and Fe) in soil samples
was analyzed using atomic absorption spectrophotome-
try. Activity levels of glutathione S-transferase (GST)
and acetylcholinesterase (AChE), indicators of oxida-
tive stress and neurotoxicity, respectively, were mea-
sured in snails collected from each site. GSTand AChE
activity were found to vary between sites and by season.
The highest levels of GST activity were registered dur-
ing the summer at sites closest to potential sources of
pollution. AChE activity levels also peaked during the
summer with the highest values recorded at the site in El
Hadjar. These increased levels of bioindicative stress
response correlated with increasing metal concentration
in soil samples collected at each site.
Keywords Pollution .Soils .Heavy metals .Helix
aspersa .Biomarkers
Introduction
The city of Annaba, a major economic hub and tourism
center located in East Algeria, has observed markedly
increased levels of pollution in recent decades (Semadi
and Deruelle 1978; Abdenour et al. 2000,2004), most
notably heavy metal contamination (Beldi et al. 2006;
Maas et al. 2010). In an effort to understand the envi-
ronmental impact of this contamination, several recent
studies have examined the degradation of
diflubenzuron, a commonly used insecticide, in saltwa-
ter (Soltani and Morsli 2003) and freshwater (Zaidi et al.
2013) and evaluated its ecotoxicological risks on non-
target species such as shrimp (Morsli and Soltani 2003)
and fish (Zaidi and Soltani 2010,2011).
The regional coastal environments have been sub-
jected to various forms of degradation, including
chemical contaminants associated with densely popu-
lated urban areas via harbors and other industrial
complexes (Semadi and Deruelle 1978; Abdenour
et al. 2000; Beldi et al. 2006). In one study demon-
strating the extent of this pollution, elevated levels of
heavy metals (Cu, Zn, Pb, Cd) were detected in a
locally prevalent edible mollusk Donax trunculus
(Beldi et al. 2006). This species has been successfully
used as a bioindicator of marine pollution (Verlecar
et al. 2006;Sifietal.2007; Amira et al. 2011; Soltani
et al. 2012) through the direct measurement of several
biomarkers, such as malondialdehyde (MDA), lactate
dehydrogenase (LDH), acetylcholinesterase (AChE),
glutathione S-transferase (GST), and catalase (CAT)
(Soltani et al. 2012).
Land snails have also been widely used as a sentinel
species for the assessment of metallic pollution in
Environ Monit Assess (2014) 186:4987–4995
DOI 10.1007/s10661-014-3753-2
R. Larba :N. Soltani (*)
Laboratory of Applied Animal Biology,
Department of Biology, Faculty of Sciences,
Badji Mokhtar University of Annaba,
23000 Annaba, Algeria
e-mail: noureddine.soltani@univ-annaba.org
terrestrial ecosystems (Gomot de Vaufleury and Pihan
2000;Regolietal.2006;Jordaensetal.2006;
Notten et al. 2006). Helix aspersa is a good
bioindicator of metal and organic soil contamination
(Scheifler et al. 2002; Gimbert et al. 2006) and is the
most abundant and widespread gastropod species in
Northeast Algeria (Larba and Soltani 2013). This
pollution causes oxidative stresses which can induce
the activity of antioxidants in local organisms
(Kaloyianni et al. 2009; Tlili et al. 2010). Aerobic
organisms have a variety of enzymatic and non-
enzymatic antioxidant defenses that maintain endog-
enous reactive oxygen species (ROS) at relatively
low levels and attenuate the damage related to their
high reactivity. The enzymatic and non-enzymatic
antioxidants are essential for the conversion of ROS
to harmless substances and for maintenance of cellu-
lar metabolism and function (Mates 2000;Lietal.
2003; Zhang et al. 2008).
Enzymatic antioxidants such as superoxide dismut-
ase (SOD), CAT, and GST as well as several low
molecular weight antioxidants such as vitamins B, C,
E, and glutathione (GSH) (Sussarellu et al. 2013)
protect against oxidative damage by inhibiting reac-
tive oxygen species formation. The activity of AChE
has been used as a biomarker of exposure to various
types of chemicals such as organophosphates, carba-
mate insecticides (Coeurdassier et al. 2001;
Neuberger-Cywiak et al. 2007; Oliveira et al. 2007),
metals, synthetic detergents, fuel oil components, and
algal toxins (Amiard-Triquet et al. 1998; Guilhermino
et al. 1998; Tim-Tim et al. 2009).
For human and ecological risk assessment, a
growing body of evidence has shown the importance
of determining the spatial distribution of pollutants
(Maas et al. 2010). The main objective of the pres-
ent study was to assess the utility of H. aspersa in
environmental monitoring as a bioindicator of heavy
metal contamination (Pb, Cd, Mn, and Fe) in North-
east Algeria by measuring selected biomarkers
(AChE and GST). Samples were collected during
the summer and winter of 2010 from various sites
located along a terrestrial soil pollution gradient ac-
cording to their proximity to factories and other
potential sources of pollution. The data collected
allowed for the determination of a spatial mapping
of contaminated soils and consequently to identify
the location where remediation efforts should be
focused.
Materials and methods
Study area and sampling sites
Sampling sites used in this study were uncultivated and
located in Northeast Algeria between the east and west
of the Annaba area. Sampling sites include El Hadjar,
one of the most populated areas of this region, in addi-
tion to Ben M'Hidi, Sidi Kaci, Bouteldja, and El Tarf.
Each of these sites was chosen along a terrestrial soil
pollution gradient according to its proximity to several
types of factories, including those involved in the pro-
duction of phosphoric fertilizers (Fertial), pesticides
(Asmidal), steel products (ArcelorMittal), and metallic
construction (Ferovial). The sampling site at El Kala,
located in a protected nature reserve, the National Park
of El Kala, was used as a control site due to its location
far from motorized traffic and other anthropogenic
sources of metal contamination. The geographical posi-
tions of each site are listed in Fig. 1and Table 1.
Animal biomarker assay
In this study, five adult specimens of H. aspersa (weight
12.4± 0.5 g, shell diameter 29.09± 0.5 mm) were col-
lected during the summer and winter of 2010 at each
sampling site (including the control site), transferred to
the laboratory, and dissected the same day. The animals
were sacrificed and the head and hepatopancreas were
sampled. AChE and GST were individually analyzed as
previously described (Zaidi and Soltani 2010,2011).
AChE activity was measured in the brain (Ellman
et al. 1961). The heads were homogenized in a 1-ml
solution composed of 38 mg ethylene glycol tetraacetic
acid (EGTA), 1 ml Triton X-100 %, 5.845 g NaCl, and
80 ml Tris buffer (0.01 M, pH 7). After centrifugation
(5,000 rpm, 5 min), AChE activity was measured in
aliquots (100 μl) of supernatant added to 100 μlof
5,5′-dithiobis-2-nitrobenzoic acid (DNTB) and 1 ml of
Tris buffer (0.1 M, pH 7). After 5 min, 100 μlof
acetylthiocholine was added. Measurements were con-
ducted at 412 nm every 4 min for a period of 20 min.
GST activity was determined in hepatopancreas ac-
cording to Habig et al. (1974) with the use of GSH
(5 mM) and 1-chloro-2-4-dinitrobenzoic acid (CDNB;
1 mM). Hepatopancreas were individually homoge-
nized in 1 ml of buffer phosphate (0.1 M, pH 6). The
homogenate was centrifuged (1,300 rpm for 30 min),
and the supernatant was used for the enzymatic assay.
4988 Environ Monit Assess (2014) 186:4987–4995
Hereto, 200 μl of the supernatant was added to
1.2 ml of the mixture GSH-CDNB in phosphate
buffer (0.1 M, pH 7). Changes in absorbance were
measured at 340 nm every minute for a period of
5 min. The protein concentrations in the total homog-
enate were also quantified according to Bradford
(1976) with Coomassie brilliant blue G250 as a re-
agent and bovine serum albumin (BSA) as a standard.
The absorbance was read in a spectrophotometer at
595 nm.
Soils sampling and heavy metal extraction and analysis
Three subsamples of soil (~100 g) were randomly taken
at a depth of 10 cm from each site, placed in acid-
washed polyethylene bags, and homogenized. Soil anal-
yses were performed by the Laboratory of Pharmacolo-
gy and Phytochemistry (courtesy of Pr. E. Leghouchi,
Jijel University, Algeria) according to the procedure of
Laib and Leghouchi (2011). In brief, the extraction was
made with the following steps. All reagents were
obtained from Merck and were of analytical grade. Each
sample was dried at 105 °C for 24 h to a constant dry
weight and sieved to 150 μm. Then, three 2.0-g repli-
cates of each dried sample were digested in concentrated
HCl and HNO
3
(Merck) solution at a 3:1 ratio. The
mixture was heated (180 °C) in glass flasks for 30 min
and then cooled for 30 min at an ambient temperature
(25 °C). Each sample was filtered using filter paper
(Whatman No. 1) and diluted with double deionized
water in the approximate range of standard concentra-
tions prepared from stock standard solution of each
metal (Merck). Concentrations of Cd, Fe, Pb, and Mn
in the extracts were evaluated using a flameless atomic
absorption spectrophotometer (Shimadzu model
AA6200) with air-acetylene flame equipped with a deu-
terium background corrector (Laib and Leghouchi
2011). The values are expressed by the mean ± standard
deviation (m ± SD) in the analysis of three subsamples
for each soil sample. All metal samples were analyzed in
duplicate, and concentration was expressed in milli-
grams per kilogram of dry mass.
Statistical analysis
The normality of data was verified using the
Kolmogorov-Smirnov test, and the homogeneity of var-
iances was checked by Levene’s test. Data are expressed
as mean ± standard deviation (m ± SD) and were sub-
jected to two-way analysis of variance (ANOVA). Dif-
ferences between sites were determined by Tukey’stest.
All statistical analyses were performed using Minitab
Software (Version 15, Penn State College, PA, USA)
Fig. 1 Geographical location of
sampling sites (El Hadjar, Ben
M'hidi, Sidi Kaci, Bouteldja, El
Tar f, El K ala )
Tabl e 1 Geographic position of sampling sites
Sites North East
El Hadjar 36° 48′00.36″7° 44′00.00″
Ben M’hidi 36° 46′02.06″7° 54′11.64″
Sidi Kaçi 36° 45′34.32″7° 58′22.59″
Bouteldja 36° 46′57.16″8° 12′00.08″
El Tarf 36° 46′01.58″8° 19′01.84″
El Kala 36° 53′48.55″8° 26′36.80″
Environ Monit Assess (2014) 186:4987–4995 4989
with p<0.05 considered as a statistically significant
difference.
Results
Glutathione S-transferase and acetylcholinesterase
activities
Data on AChE and GST activity in snails collected from
the six study sites are presented in Figs. 2and 3.Atsites
located near pollution sources, snails showed higher
GST activity compared to snails from the control refer-
ence site El Kala (14.18±1.78 nM/min/mg). The highest
GST activity was found in El Hadjar (30.57±1.30 nM/
min/mg). In addition, higher levels of GST activity were
found in samples taken during the summer months
compared to winter at all study sites. Two-way ANOVA
revealed significant effects of both seasons (F
1,24
=
411.63; p<0.001) and site (F
5,24
=138.67; p<0.001),
and no significant season-site interaction (F
5,24
=1.82;
p=0.231).
AChE activity was significantly lower in snails col-
lected from all sampling sites compared to snails from
the reference site. During the winter season, the lowest
AChE activity was observed in El Hadjar (25.57±
1.39 nM/min/mg) and the highest activity was observed
in El Kala (40.472±1.236 nM/min/mg protein). At all
study sites, AChE activity levels were found to be lower
during the summer compared to the winter. Two-way
ANOVA revealed significant effects of both season
(F
1,24
=2,332.79; p<0.001) and site (F
5,24
=277.18;
p<0.001), and season-site interaction (F
5,24
=5.62; p=
0.001).
Soil heavy metal concentrations
In order to determine the level of correlation between
land snail biomarker responses and heavy metal con-
tamination of soils, the concentrations of the most im-
portant heavy metals (Fe, Mn, Pb, and Cd) were mea-
sured in soil samples at each site (Tables 2and 3).
Globally, the average concentration of each metal re-
corded exhibited the following decreasing order: Fe,
Mn, Pb, and Cd. At all sites, higher values were mea-
sured in samples collected in the summer compared to
samples collected in the winter. Moreover, the concen-
trations of each heavy metal varied between sites. No-
tably, Sidi Kaci, Bouteldja, and El Tarf, sites which
showed increased Pb levels during sampling, are all
located near major road networks that are potential
sources of Pb pollution exposure from highway traffic.
The lowest heavy metal concentrations were registered
in the site of El Kala, which validates its selection as a
control site.
For each heavy metal, two-way ANOVA (season,
site) indicated a number of effects. Fe levels obtained
during sampling showed a significant effect due to
season (F
1,24
=5.5×10
4
;p<0.001), site (F
5,24
=3.1×
10
4
;p<0.001), and season-site interaction (F
5,24
=
1,131.86; p<0.001); for Mn, a significant effect due to
season (F
1,24
=5,311.60; p<0.001), site (F
5,24
=660.65;
p<0.001), and season-site interaction (F
5,24
=33.54;
p<0.001); for Pb, a significant effect due to season
(F
1,24
=547.98; p<0.001), site (F
5,24
=191.07;
p<0.001), and season-site interaction (F
5,24
=5.52; p=
0.002); and for Cd, a significant effect due to season
(F
1,24
=422.57; p<0.001) and site (F
5,24
=140.02;
p<0.001) and no significant effect due to season-site
interaction (F
5,24
=1.42; p=0.231).
Fig. 2 Specific activity of GST
(nM/mn/mg proteins) in
hepatopancreas of H. aspersa
collected from the different sites
in summer and winter of 2010
(m ± SD; n=5; for each site, mean
values followed by the same letter
are not significantly different at
p>0.05)
4990 Environ Monit Assess (2014) 186:4987–4995
Discussion
In recent years, considerable concern has mounted over
the problem of soil contamination with heavy metals
due to industrialization and urbanization. Environmen-
tal concentrations of heavy metals depend on both nat-
ural and anthropogenic factors. Anthropogenic process-
es recognized as potential sources of heavy metal soil
contamination predominately consist of agricultural uti-
lization of metal-containing fertilizers and pesticides,
vehicle traffic, combustion of petroleum fuels contain-
ing metal additives (Reichman 2010), and surface runoff
produced by atmospheric pollutants (Calvet and
Barriuso 1994; Fernández et al. 2006). Heavy metals
can cause serious threats to environmental health due to
their bioaccumulation in terrestrial ecosystems and af-
fect food quality and safety (Agarwal 2009; Ryu et al.
2010).
Biomarkers are now becoming an integral part of
ecosystem health assessment and management in addi-
tion to more traditional water chemical analysis (Lam
2009; Boyd 2010). The antioxidant defense system is
being increasingly studied because of its potential utility
to provide biochemical biomarkers that can be used in
environmental monitoring systems (Ballesteros et al.
2009). They serve as important biological defense
against environmental oxidative stress at a cellular level
(Van der Oost et al. 2003; Pandey et al. 2003). The
antioxidant defense system consists of both enzymatic
and non-enzymatic systems. Enzymatic system includes
enzymes such as SOD, glutathione peroxidase
(GSHPx), catalase, and GST.
Many studies confirm that the antioxidant defense
system can be markedly induced under certain levels of
stress at a given time. However, with increasing expo-
sure time and concentration of pollutants, they show a
decrease tendency (Hao and Chen 2012). GSTs, a family
of dimeric multifunctional enzymes, have been shown to
be involved in detoxification of xenobiotics, protection
from oxidative damage, and the intracellular transport of
hormones, endogenous metabolites, and exogenous
chemicals in diverse organisms (Zhou et al. 2009). Pol-
lution by heavy metals plays an important role in in-
creasing the rate of GST activity (Hamed et al. 2003).
In this study, GST activity was measured in hepato-
pancreas samples of H. aspersa. The hepatopancreas
Fig. 3 Specific activity of AChE
(nM/mn/mg proteins) in head of
H. aspersa collected from the
different sites in summer and
winter of 2010 (m ± SD; n=5; for
each site, mean values followed
by the same letter are not
significantly different at p>0.05)
Tabl e 2 Soil concentrations of
heavy metals (at a depth of 10 cm
in mg/kg dry mass) in the differ-
ent sites during the summer of
2010 (m ± SD; n=3; for each
metal, mean values followed by
different lowercase letters are
significantly different at p<0.05)
Site Fe Mn Pb Cd
El Hadjar 886.99±2.96 a 20.55±0.38 a 1.63±0.04 a 0.15± 0.00 a
Ben M’hidi 683.44±5.89 b 15.76±0.46 b 1.17± 0.08 b 0.12 ±0.00 b
Sidi Kaci 620.42±1.45 b 13.66±0.08 c 1.26±0.07 b 0.11± 0.00 b
Bouteldja 493.52± 1.46 c 14.08± 0.25 c 1.89± 0.05 c 0.08± 0.00 c
El Tarf 412.67±52.67c 13.98± 0.27 c 1.95± 0.03 d 0.07±0.00 c
El Kala 330.70±0.65 c 10.76±0.41 d 1.06±0.06 e 0.06±0.00 c
Environ Monit Assess (2014) 186:4987–4995 4991
was chosen as a target organ for biochemical assessment
because it plays an integral role in detoxification pro-
cesses resulting in heavy metal deposition
(Nowakowska et al. 2012). GST activity was found to
be significantly higher in the snails from sample sites
adjacent to potential sources of pollution (factories,
harbors, etc.) compared to samples taken from the ref-
erence control site, El Kala. The induction of GST
activity indicates an adaptation of the organism to en-
hanced pollution stress (Astani et al. 2012). In addition,
increased GST activity suggests that the detoxification
process against pro-oxidation forces, which are mediat-
ed by this enzyme, is induced (Elia et al. 2007; Radwan
et al. 2010). GST activity was also found to vary sea-
sonally with higher induction of GST activity in the
summer compared to winter. This may be due to winter
rains which promote leaching of soil pollutants. Indeed,
the climate of the study areas is Mediterranean, with an
average annual temperature of 18 °C and an annual
rainfall ranging from 650 to 1,000 mm with peak rainfall
in winter and deficits occurring typically during summer
(Debieche 2002). Variation in GST levels between sites
may be due to increasing or decreasing proximity to
pollution sources (ArcelorMittal, Fertial).
AChE activity has commonly been used as a bio-
marker of exposure to several chemicals such as neuro-
toxic pesticides (Oliveira et al. 2007) and heavy metals
(Amiard-Triquet et al. 1998; Lam 2009). Our study
revealed decreased levels of AChE activity in snails
collected in summer compared to winter. This decrease
is potentially due to the effect of heavy metal pollutant
leaching. As with GST, variation in AChE levels be-
tween sites may be due to increasing or decreasing
proximity to pollution sources (ArcelorMittal, Fertial).
Flame atomic absorption spectrometry is one of the
most reliable techniques for the evaluation of metal ion
content but has some limitations such as low concentra-
tions of metal ions in environmental samples and com-
plicated matrix interferences (Ghaedi et al. 2013c).
Solid-phase extraction has currently become the most
common technique for environmental sample pretreat-
ment of trace metals from matrices because of advan-
tages such as high recovery, short extraction time, high
enrichment factor, and lower cost and consumption of
organic solvents over liquid-liquid extraction. Thus, the
efficiency and utility of novel sorbents for removal and
recoveries of heavy metal ions by solid-phase extraction
were recently investigated (Ghaedi et al. 2013a,b,d).
In the present study, soil samples from six sites were
collected in 2010 and analyzed by a method commonly
used for several years and applied for determination of
heavy metal concentrations in various media
(Leghouchi et al. 2009; Laib and Leghouchi 2011).
The lowest heavy metal concentrations were registered
in El Kala, a protected area used as a control site. In
contrast, the soil collected from the other five sample
sites showed higher concentrations of Fe, Cd, Pb, and
Mn, indicating the presence of metallic pollution. The
highest levels of Fe, Mn, and Cd were found in soil
samples adjacent to the waste dumpsite of El Hadjar,
indicating that the waste produced by this industrial area
is a potential major source of soil contamination. Pb
levels were significantly higher at Sidi Kaci, Bouteldja,
and El Tarf. This may be linked to the increased traffic
congestion in these sites in accordance with previous
studies (Ho and Tai 1988; Garcia and Millan 1998).
Tabl e 3 Soil concentrations of
heavy metals (at a depth of 10 cm
in mg/kg dry mass) in the differ-
ent sites during the winter of 2010
(m ± SD; n=3; for each metal,
mean values followed by different
lowercase letters are significantly
different at p<0.05)
Site Fe Mn Pb Cd
El Hadjar 586.94± 3.92 a 11.39± 0.39 a 1.14±0.05 a 0.09±0.00 a
Ben M’hidi 490.29± 0.53 b 8.67± 0.21 b 0.85±0.05 b 0.07± 0.00 b
Sidi Kaci 412.55± 0.52 b 8.23± 0.10 b 1.24±0.04 c 0.06±0.00 b
Bouteldja 332.46±1.52 c 8.35± 0.05 b 1.24±0.04 c 0.04±0.00 c
El Tarf 295.87± 1.16 c 10.56±0.26 c 1.39±0.05 d 0.03±0.00 c
El Kala 192.75± 0.78 d 3.20± 0.07 d 0.38±0.00 e 0.01±0.00 c
Tabl e 4 Regulatory
limits of heavy metal
concentrations (g/kg) in
soils according to
AFNOR U44-041
na not available
Heavy metals Concentrations
Cd 2
Cr 150
Cu 100
Pb 100
Zn 300
Fe na
Mn na
4992 Environ Monit Assess (2014) 186:4987–4995
Average metal concentrations across the six sites varied
in a similar manner with Fe being the most significant
pollutant, followed by Mn, Pb, and Cd in decreasing
order. Metal concentration in soil samples seems to
decrease with increasing distance from highways and
other pollution sources such as the industrial area of El
Hadjar and the ArcelorMittal steel complex. The disper-
sion of contaminants may also be influenced by meteo-
rological conditions such as wind (Piron-Frenet et al.
1994), rainfall, or motorized traffic intensity (Garcia and
Millan 1998). According to Debieche (2002), the dom-
inant wind in the studied area comes from the North and
Northeast and, to a lesser extent, from the North and the
West. Recently, the concentrations of some heavy
metals were determined in soils from Annaba and its
surroundings (Maas et al. 2010). The average concen-
trations reported for Cd, Cr, Cu, Pb, Zn, Fe, and Mn
were 0.30, 28.3, 23.8, 42.3, 64.7, 25,240, and
405.9 mg/kg, respectively. Moreover, the spatial distri-
bution of heavy metals observed in the Annaba area was
explained by various anthropogenic activities including
urban traffic (Pb and Zn), local industrial contamination
(Cd and Cr), and agriculture (Cu). In the present study,
the concentrations of heavy metals determined in soils
from six sites located in Northeast Algeria were below
the standard limits (Table 4)(AFNOR1996) and are in
agreement with natural background values (Möller et al.
2005). Moreover, Alloway (1995) indicated that Cd
concentrations do not exceed 1.0 mg/kg for most of
surface soils worldwide.
Conclusion
In conclusion, the results show a decreasing gradient of
pollution from west to east correlating to the proximity
of the pollution sources. The highest levels of contam-
ination were observed at El Hadjar near the
ArcelorMittal steel complex. H. aspersa, a common
species of land snail present in the area, was tested as a
bioindicator of metal contamination. GST and AChE
activities measured from each animal served as sensitive
parameters measuring exposure to pollution and the
induction of an environmentally mediated biochemical
stress response. Overall results show that H. aspersa is a
useful bioindicator for assessing heavy metals from
atmospheric pollution, several industrial factories, and
vehicle traffic. Moreover, AChE and GST are reliable
biomarkers of environmental stress.
Acknowledgments The authors thank Pr. E. Leghouchi
(Laboratory of Pharmacology and Phytochemistry, Faculty of
Sciences,Jijel University, Algeria) and Pr. M. Lahouel (Laboratory
of Molecular Toxicology, Faculty of Sciences, Jijel University,
Algeria) for heavy metal analysis. This study was financed by
the Algerian Fund for Scientific Research and by the Ministry of
High Education and Scientific Research of Algeria (CNEPRU and
PNR projects, Pr. N. Soltani).
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