Chemistry and Ecology

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Impact of heavy metals of industrial plant

wastewater on benthic communities of Bizerte
Lagoon (Tunisia)

Ibtihel Saidi, Olfa Ben Said, Jamel Ben Abdelmalek, Soufiane Jouili, Luis
Chicharo & Hamouda Beyrem

To cite this article: Ibtihel Saidi, Olfa Ben Said, Jamel Ben Abdelmalek, Soufiane Jouili, Luis
Chicharo & Hamouda Beyrem (2019) Impact of heavy metals of industrial plant wastewater on
benthic communities of Bizerte Lagoon (Tunisia), Chemistry and Ecology, 35:8, 746-774, DOI:
10.1080/02757540.2019.1644324

To link to this article: https://doi.org/10.1080/02757540.2019.1644324

Published online: 05 Aug 2019.

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CHEMISTRY AND ECOLOGY
2019, VOL. 35, NO. 8, 746–774
https://doi.org/10.1080/02757540.2019.1644324

RESEARCH ARTICLE

Impact of heavy metals of industrial plant wastewater on

benthic communities of Bizerte Lagoon (Tunisia)
Ibtihel Saidia, Olfa Ben Saida, Jamel Ben Abdelmalekc, Soufiane Jouilia, Luis Chicharob
and Hamouda Beyrema
a
Laboratory of Environment Biomonitoring, Faculty of Sciences of Bizerte (FSB), University of Carthage,
Bizerte, Tunisia; bMarine and Environmental Research Centre (CIMA), Faculty of Sciences and Technology,
University of Algarve, Faro, Portugal; cLaboratory Quality Control, Cement Bizerta Bay Sabra, Bizerte, Tunisia

ABSTRACT ARTICLE HISTORY

The aim of this study was to assess the consequences of human Received 15 April 2018
impact on the characteristics of sediments heavy metal Final Version Received 12
concentration, grain size and its influence on the structure of the July 2019
microbial and meiofaunal community assemblages. A survey was
KEYWORDS
carried out in July 2013 within six sites located in the Bizerte Metals; resistant bacteria;
Lagoon (Tunisia), both downstream and upstream of industrial meiofauna; marine
effluents. The highest total sediment metal concentrations were sediments; industrial
detected in stations located close to the industrial sewage effluents; Bizerte Lagoon
discharge points. In these stations, the lowest densities of the
total meiofauna (33 ± 13 ind/10 cm−2) and conversely the highest
densities of cultivable bacteria that are heavy metal resistant have
been reported (16 ± 80.34 CFU g−1). Univariate (ANOVA) and
multivariate (MDS/CCA) analyses demonstrate high dissimilarity
(0.06) in meiofaunal and bacterial community structures between
downstream and upstream industrial sewages. Furthermore,
canonical correspondence analysis CCA results indicated that
heavy metal sediment contamination promoted bacteria that are
resistant to heavy metals, while heterotrophic bacteria supported
the development of meiofauna taxa. The results highlight the
importance of bacteria/meiofauna interactions, as both
meiofaunal and microbial communities give indications of the
ecological impact of heavy metal contamination in sediment.

Introduction
Heavy metals are micro-pollutants, which can affect the health of the marine environ-
ment since they are not subjected to biological or chemical degradation. They can
accumulate in different links of trophic chains, generating toxic concentrations in
marine organisms [1]. These inorganic compounds, essential for biological activities
[2], originate from both natural processes and anthropogenic activities [3,4]. Thus, an
index of environmental pollution may be created that shows the environmental concen-
trations that exceed the geochemical background, the background level being the level
that was determined by natural geological and geochemical processes [5]. At higher

CONTACT Ibtihel Saidi saidi_ibtihel@hotmail.fr Laboratory of Environment Biomonitoring, Faculty of Sciences of

Bizerte (FSB), University of Carthage, 7021 Zarzouna, Bizerte, Tunisia
© 2019 Informa UK Limited, trading as Taylor & Francis Group
 CHEMISTRY AND ECOLOGY 747

concentrations, specific elements, including copper, zinc or lead, can be toxic to the
marine environment [6]. The inputs of heavy metals in marine environments are primar-
ily due to industrial activities. Coastal lagoons are generally considered to be a sink for
trace metals [7]. Metals enter the coastal lagoon through atmospheric deposition of
metal-containing dust from burning of petroleum fuels (such as petroleum coke
(Petcoke)), surface runoff, as well as industrial and domestic sewage discharges [8]
with the later being the most important [9].
The contamination of aquatic systems by heavy metals, especially in sediments, has
become one of the most challenging pollution issues. Sediments are increasingly
recognised as the most important pollutant sink and as a carrier and possible future
source of heavy metals in estuarine systems [10] because heavy metals that are
fixed to sediments may be recycled back to water column via chemical and biological
processes [11]. Particulate metals eventually settle to the bottom and may be directly
available to benthic fauna [12] or may be released to the water column through sedi-
ment resuspension, adsorption/desorption reactions, reduction/oxidation reactions, or
the decomposition of organisms. These processes could enhance the dissolved con-
centrations of trace metals in the environment and threaten the ecosystem. Metal
uptake by benthic organisms depends largely on metal mobility, total concentration
and chemical forms [13]. Since benthic metazoans of small size often associated
with sediment particles [14], meiofaunal community structure can be used as a sensi-
tive bioindicator of pollution in marine environments [14,15]. Knowledge of the
relationship between benthic fauna and sediment is a prerequisite for understanding
the structure and dynamics of benthic associations. Several others found positive
relationships between organisms and sediments in marine and estuarine environments
[16,17].
Meiofauna are diverse and abundant organisms in the marine and estuarine environ-
ment, show a wide range of tolerances to environmental stress, and interact directly
with interstitial water contaminants [18]. For these reasons, meiofauna have several
potential advantages over macrofauna as an indicator, which have traditionally been
the component of the benthos examined in pollution monitoring surveys [19]. In
addition, meiofauna are easy to maintain in the laboratory which facilitates their use
in biomonitoring experimental studies [20,21]. Moreover, meiofauna facilitate mineralis-
ation of organic matter, enhance nutrient regeneration and serve as food for a variety of
higher trophic levels [15]. Several authors have reported the adverse effects of trace
metals on meiofauna [22–24]. The same is true for benthic bacterial communities, as
many studies suggested that functional microbial variables are highly sensitive to
heavy metal contamination and could be used as bioindicators of stress conditions in
coastal sediments [25,26].
In the marine environment, structure and function of the benthic components play
a very important role in the biochemical and metabolic processes of the sediment
with important implications for the carbon cycle and the food web [27]. A large
part of these processes is carried out through bacterial metabolism, which utilise
the carbon in the detritus food chain [28]. The activities of benthic organisms
(meiofauna and macrofauna) and the fluctuations of environmental parameters are
important drivers for microbial organisation and activities [29]. The meiofauna–micro-
organisms interactions, including trophic and non-trophic interactions [30,31] play a
748 I. SAIDI ET AL.

major role in both the overall carbon fluxes [32,33] and organic matter mineralisation
[34,35]. Bioturbating meiofauna exert a top-down impact on bacterial community
structure by changing the physicochemical conditions of sediment thus enhancing
nutrient and carbon supplies [36,37]. Nevertheless, meiofauna and bacteria can also
be in competition for food sources depending on the carbon supply conditions [38].
Therefore, meiofauna have both positive and negative impacts on bacteria, which com-
plicates the interactions between these two essential compartments of the benthic food
web [14]. Thus, the presence of a contaminant in the marine environment can induce a
direct or indirect effect on the benthic communities. To investigate the toxicity of con-
taminant, it is better to examine more than one trophic echelon, especially related
echelons such as meiofauna and bacteria, as these organisms inhabit different micro-
niches within the sediment where the bioavailability of the toxins may be different.
Therefore, investigating the impact of contamination on these two communities will
allow more information to be gained. The majority of metal ecotoxicological studies
are restricted to meiofaunal communities, have been achieved mainly at an experimen-
tal scale, and showed that toxic metals have either lethal or sublethal effects (reductions
in fecundity, increased developmental time which tend to increase with increased metal
concentrations) [19]. Thus, a decrease in diversity is an expected respons of a meiofau-
nal community to metal pollution [39]. A previous pilot study indicated that there was
some commonality between meiofaunal and microbial approaches for ecological assess-
ment of contaminated sites [40]. Studies of microbial communities in sediment are
becoming more common for assessing biodiversity, bioremediation potential or the
impact of pollutants [29]. Nevertheless, at an ecosystem scale, the effect of metals on
benthic communities could be different as a result of biotic and abiotic interactions.
As is the case for many Mediterranean coastal lagoons, Bizerte Lagoon is a polluted
ecosystem subject to both urbanisation and industrialisation (cement works, metallur-
gical industry, boatyard, tyre production factories) [41]. Industry generates an average
daily flow of wastewater of approximately 31,600 m3/day into the Bizerte Lagoon and
urban sources generate 20,000 m3/day in [42]. Thus, direct and indirect discharge of
waste and runoff has resulted in the chemical contamination of the lagoon by
various toxic compounds such as heavy metals [43], organo-chlorinated pesticides
[44], halogenated aromatic compounds [45] and polycyclic aromatic hydrocarbons
[46]. DGEQV [47] and Yoshida [48] reported that metallic contamination of Bizerte
Lagoon is mainly of industrial origin (Figure 1). Trace metals originate from the activi-
ties of nine industrial plants classified as major sources of pollution in the Bizerte
Lagoon [47]. According to Ben Mna [49] based on enrichment factors and geo-accumu-
lation indexes biota from Bizerte Lagoon are affected by heavy metals. In this complex
environmental context, it is urgent to estimate the degree of pollution in the sediment
by means of the geo-accumulation index and the enrichment factor, along with heavy
metal bioavailability assessment of metals so as to evaluate their potential risk to the
biota.
The aim of the field study was to assess the chronic effects of heavy metals
on meiofaunal and bacterial community structures, in contaminated and non-contami-
nated sediment from Bizerte Lagoon, both upstream and downstream of the dis-
charge points of industrial effluents and to contribute to the management of the
system.
 CHEMISTRY AND ECOLOGY 749

Figure 1. The Bizerte Lagoon and the six selected sampling stations: Industrial plant waste-
water (WWPT): water current direction. shellfish farm. S1, S2, S3 and S4: station upstream
of the discharge; S5: downstream station of the discharge; S6: station away from discharge point
(Châara).
750 I. SAIDI ET AL.

Materials and methods

Characteristics of the study sites
Study site
Sediment and fauna were collected at the Bizerte Lagoon, a coastal Mediterranean lagoon
located in the northern part of Tunisia (latitude: 37°8’37°14′ N, longitude: 9°46′ –9°56′ E)
(Figure 1). This lagoon is of great ecological, cultural and economic importance and
covers an area of 128 km2 [41]. The lagoon has a mean depth of 7 m. and is connected
to the Mediterranean Sea through a 6-km long inlet and to the Ichkeul Lake through
Wadi Tinja (Figure 1) [7]. The lagoon lies in the vicinity of several towns (Bizerte, Zarzouna,
Menzel Abderrahmen, Menzel Jemil, and Menzel Bourguiba) and various industrial sites
(Figure 1). This ecosystem is affected by a large set of anthropogenic activities, including
domestic sewage input, industrial waste, atmospheric pollution, agriculture and bivalve
aquaculture [41] and is subject to contamination from two industrial zones, one with an
oil refinery and one with cement plants [46]. The lagoon hydrodynamics are mainly
driven by wind forces that exhibit a defined seasonal pattern with a dominance of
winds from the east (5 m s−1 in summer) and northwest (8 m s−1 in winter) [50]. The stron-
gest tidal currents are located on the northern border of the lagoon and near the inlet [51].
In contrast, currents are considerably reduced inside the lagoon [52].

Sampling locations
Six sampling sites were selected based upon on cement and steel industrial plant waste-
water discharge points into Bizerte Lagoon (Figure 1). The sites are located in two distinct
areas. Sites 2 through 4 are located in the Northeastern corner of Sabra Bay, while Site 1 is
near the artificial channel connecting Sabra Bay to the Mediterranean. Site 5 is located
near to the point where Bizerte Lagoon flows into the Sabra Bay through a natural
channel. Site 6 is located within Bizerte Lagoon.
The upstream zone (stations S1, S2, S3 and S4) was located in Sabra bay, a semi closed
bay where the steel and cement industries are located. S1, S2, S3 and S4, were 0–1000, 35,
30 m and 190–270 m from industrial effluent sources. The downstream zone (S5 and S6)
was located far from the steel and cement industry. S5 and S6 were 1500 and 4630 m away
from industrial effluent (Figure 1), and were considered as control areas, as the impacts
from the upstream contamination are not detectable. Water and natural sediment
samples including the meiobenthos were collected in July 2013 during low tide.

Sampling and processing

Triplicate cores were taken manually using hand-cores (10 cm−² surface area) adjacent to
one another at each station, to a depth of 10 cm, were gathered in sterile containers and
kept at 4°C while transported to the laboratory. On return to the laboratory, the core for
each replicate was mixed in a polythene bag and then subsampled into three subsamples,
one for microbiological analysis, one for meiofaunal analysis and one for metal analysis
(Figure 2) [29].
Sediment to be analysed for metal concentrations was frozen at −20°C pending analy-
sis. Sediment fractions of less than 63 μm were used for the determination of trace metals
and samples were kept in the refrigerator at 4°C until analysis [53].
 CHEMISTRY AND ECOLOGY 751

Figure 2. Sampling methods.

Water analysis
A Microprocessor Conductivity Meter (WTW-LF-197, Brives B.V., Germany) was used to
measure in situ temperature and salinity (using the Practical Salinity Scale). pH was deter-
mined using a pH meter (Accumet Basic AB15, Fisher Scientific) and the level of dissolved
oxygen was determined with a multi parameter probe (YSI GRANT 3800).
Samples for nutrients (500 mL) were filtered through Whatman GF/F filters. The filtrates
were collected in acid-washed vials and kept frozen (−20°C) until analysis (within two
weeks) [54]. Nutrient concentrations were determined by spectrophotometric methods
3− +
NO−3 and NO2 , PO4 : [55], 1962; NH 4 : [56]; Si (OH) 4: [57].
−

Sediment analysis
Granulometry
Sediment granulometry was determined for all three replicate samples. The particle size ana-
lyzes were performed at Malvern granulometer Masttersizer 2000. The results were then
compiled with GRADISTAT software [58], using the parameters initially developed by Folk
and Ward [59]. The size classes correspond to the international standards set by [60,61].
752 I. SAIDI ET AL.

Metals analysis
Ten metals were selected for analyses arsenic (As), barium (Ba), boron (B), chromium (Cr),
copper (Cu), lead (Pb), manganese (Mn), nickel (Ni), vanadium (V), zinc (Zn). These metals
were chosen because they present significant concentrations in Bizerte Lagoon waters
near harbours and industrial areas [41]. The fraction of sediment less than 63 μm was
used for the determination of the heavy metal concentration using atomic emission spec-
trometry with inductively coupled ICP_AES plasma. To analyse the concentration of these
metals, sediment samples were frozen and lyophilised. Samples were homogenised and
mineralised by aqua regia prior to analysis. Acid digestion was performed from original
wet material after its mineralisation by strong acids according to the method of Loring
et al. [62]. All samples were analysed in triplicate, and the data shown herein represent
the average of the triplicates [63].

Assessment of sediment contamination

Different indices were used to assess the degree of heavy metal contamination in Bizerte
Lagoon sediment.
Contamination factor
Cmetal
CF =
Cbakground (Bn)
.

The CF is the ratio obtained by dividing the concentration of each metal in the sediment
by baseline or background value [64]. For background values (Bn) we have used the
average shale value of Turekian and Wedepohl [65]. due to the absence of trace
element data in Bizerte Lagoon sediments [7]. CF values were interpreted according to
Hakanson [66]. CF was classified into four groups: CF < = low contamination factor, 1 <
CF < 3 = moderate contamination factor, 3 < CF < 6 = considerable contamination factor
and CF > 6 = very high.

Geo-accumulation index
Cn
Igeo = log 2
1.5 Bn
.

Cn is the measured concentration of the element in soil dust, Bn is the geochemical

background value. The constant 1.5 allows us to analyse natural fluctuations in the
content of a given substance in the environment and to detect very small anthropogenic
influences. The geoaccumulation index consists of 7 grades or classes [67] (Table 4). Class 6
is an open class and comprises all values of the index higher than class 5. The elemental
concentrations in class 6 may be a hundredfold greater than the geochemical background
value [68].

Bacterial and meiofaunal abundances

Bacterial enumeration was performed by 3-tubes Most Probable Number MPN method
and CFU (Colony Forming Units) method in triplicate [69]. Eosin Methylene Blue (EMB)
and Tryptone Soya agar were used, respectively, for faecal coliforms, faecal streptococci
 CHEMISTRY AND ECOLOGY 753

and pathogenic staphylococci. Tubes and plates have been incubated during 18–24 h at
37°C and 44°C.

Bacterial densities
Cultivable Heterotrophic Bacteria (CHB)
Viable heterotrophic marine bacteria in sediment samples were recovered using the
spread plate method. Sediments were homogenised by gentle hand stirring with a
large spatula, then for each collection station, the sample of 1 mg of was suspended in
9 mL of physiological saline solution [29], vortexed in order to detach bacteria from the
sediment particles, and serially diluted to 10−5. From the serial dilutions, 0.1 mL of the
10−3, 10−4 and the 10−5 dilutions was inoculated into Marine Agar and plated using
the pour plate method. All plates were prepared in triplicate [70]. Inoculated plates
were incubated at 28 °C for 48 h. The colonies were counted and the densities were
expressed as colony-forming units per gram of sediment (CFU g−1).

Cultivable Heavy Metal-Resistant Bacteria (CHMRB)

Enumeration of bacteria resistant to heavy metals was carried out for sediment samples
from each station by counting the CFU (colony forming units). Five-fold serial dilutions
were made from 10 to 10−5 on an R2A agar medium [71]. First, a bacteria was obtained
by shaking 1 g of collected sediment in 9 mL volume of Ringer’s solution at 30°C and
150 rpm [72,73]. A total of six heavy metals were tested. The salts used were 0.002 g/L
of CdCl2, 0.072 g/L of CuCl2, 0.0568 g/L of NiCl2, 0.180 g/L of PbCl2, 0.688 g/L of ZnCl2,
and 0.160 g/L of CrCl3 (Sigma) were prepared by dissolving it in distilled water, sterilised
by filtration through a membrane filter (0.22 m) and then added to the medium [74].

Meiofaunal analyses
Sediment samples were rinsed with a gentle jet of freshwater over a 1 mm sieve to exclude
macrofauna and decanted over a 40 μm sieve following the resuspension–decantation
methodology [75]. Meiofauna samples were fixed in 4% neutralised formalin [76] and
stained with Rose-Bengal. Meiofauna (M) were counted and identified at the higher
taxon level, nematodes (N), copepods (C), polychaetes (P), Oligochaetes (O) and tartigrade
(Tar), using a stereo-dissecting microscope (magnification ×40).

Statistical analysis
Univariate analyses of the meiofauna taxa for each site were calculated, including the
number of taxa (S), total abundance (N), the Shannon–Wiener diversity indices (H′ ) (calcu-
lated using Loge), Margalef’s species richness (d) and evenness (Pielou’s J) [77]. The signifi-
cance of differences in univariate measures between sites was tested using a two-way
ANOVA. Statistical analysis was performed using a two-way Analysis of Covariance
(ANCOVA) testing differences among sites in the distribution of meiofauna and bacteria.
Two-way ANCOVA was utilised to determine differences among sites in the distribution
of meiofauna and bacteria and whether there is an interaction effect between two inde-
pendent (Site and Position) variables in terms of a continuous dependent variable.
754 I. SAIDI ET AL.

Statistical analysis was carried out using a two-way ANOVA (or two-way ANCOVA) since
they clearly have two nested factors ‘site’ and ‘position’ (upstream and downstream). Pos-
teriori paired multiple comparisons were performed using Tukey HSD test. Statistically sig-
nificant differences were assumed when P < .05. Cluster analysis derived from Bray–Curtis
similarity matrices (square-root transformed) was used to view spatial differences in the
meiofaunal and nematode community structures. Furthermore, the formal significance
of all of the multivariate analyses was generated with the PRIMER v6 software [78]. The
similarity of meiofauna and bacteria among sites was determined by cluster and non-
metric multidimensional scaling ordinations (MDS) on fourth-root transformed data,
using the Bray–Curtis similarity index. Grain-size data analysis wasperformed using the
software GRADISTAT [58]. Canonical correspondence analysis CCA based on meiofaunal
and bacterial community structures, sediment heavy metal contamination in each
sampling stations and abiotic variables was performed with MVSP v3.12d software
(Kovach Computing Service, Anglesey Wales). Data were analysed using two-way
ANCOVA in SPSS software version 18.

Results
Characteristics of the study sites
The values of physico-chemical parameters measured in the Bizerte Lagoon at different
sites are given in Table 1. The overall physicochemical parameters of water did not
change significantly between the different stations sampled. Water temperature was
characterised by a minimum of 20.4 °C in S5 and a maximum of 22 °C in S1. The
minimum value for salinity (33.65 PSU) was measured at S6 located 4630 m away from
the cement industry discharge (Table 1) and the maximum value for salinity (35 PSU)
was found at station S3 located in front of the cement industry. The pH of the water
was slightly basic, with ranges from 9.02–9.20. S3 is located 10 m away from effluents
and had the lowest pH 9.03. S2 which is located close to S3, had relativity higher dissolved
oxygen (7.55) than other sites (Table 1). The range of dissolved oxygen in different sites
showed that water lagoon is well saturated. Dissolved oxygen content in surface waters
was, generally on the order of 6.32–7.55 mg L−1. Dissolved oxygen in aquatic ecosystems
is a function of many physicochemical parameters, which govern their solubility. The
highest NO3 concentrations were detected in downstream sewages discharge points
(0.1 μmol L−1) and the lowest in upstream industrial effluents (0.0053 μmol L−1), NO2
was always detectable, with concentrations commonly in the range of 0.004–
0.32 μmol L−1. NH+4 was generally 0.8–3 μmol L−1, with the highest level (3 μmol L−1)
recorded in upstream industrial sewage water. The highest Si(OH)4 concentration was
9.2 μmol L−1 in the upstream effluent, while the lowest was in downstream effluent far
away discharge points with 3 μmol L−1, PO4 was commonly (0.01–0.8 μmol L−1). The sig-
nificance of differences in univariate measures between sites was tested using a two-
way ANCOVA. None of the environmental variables (temperature, salinity, pH, oxygen
and nutrients) exhibited significant differences among the stations (P > .05). The granulo-
metric analysis showed that the particle size of the sediment at the sampling stations it is
composed principally of mud and sand (Table 2). Sand dominated the areas, reflecting the
effects of hydrodynamic and urban activity. The highest average percentage distribution
Table 1. GPS coordinates and environmental parameters measured at each sampling station.
Station S1 S2 S3 S4 S5 S6
Longitude 37°15.796N 37°15.663N 37°15.663N 37°15.655N 37°15.564N 37°15.663N
Latitude 009°51.120E 009°51.082E 009°51.105E 009°51.136E 009°51. 136 E 009°50.065E
pH 9.18 9.2 9.03 9.18 9.04 9.14
T (°C) 22 21.4 21 20.5 20.4 21.5
O2 (mg/L) 6.33 7.55 6.32 7.5 7.45 7.14
Sal (PSU) 34.2 34.16 35 34.75 34.4 33.65
NO2 (µmol/L) 0.32 0.097 0.004 0.32 0.153 0.162
NO3 (µmol) 0.032 0.0097 0.0053 0.0276 0.0565 0.1089
NH4 (µmol/L) 0.833 3 1.333 3 0.833 2.833
PO4 (µmol/L) 0.6272 0.0789 0.9781 0.0 0.7588 0.5175
Si (µmol/L) 4.314 5 9.216 0.441 3.922 3.431
Total metals (mg/kg−1) 872.13 ± 4.37 664.19 ± 4.09 682.6 ± 20.39 1089.96 ± 28.5 230.39 ± 26.82 253.04 ± 3.58

CHEMISTRY AND ECOLOGY

755
756 I. SAIDI ET AL.

Table 2. Percentage of grain size distribution in coastal superficial sediments of the Bizerte Lagoon.
S1 S2 S3 S4 S5 S6
GRAVEL 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
SAND 76.6% 88.3% 89.4% 99.3% 99.8% 99.1%
MUD 23.4% 11.7% 10.6% 0.7% 0.2% 0.9%

for sand presence was downstream of the effluents near sites S5 and S6. These percen-
tages were 99.8% and 99.1%, respectively. The lowest values (76.6%) were detected at
S1 locted upstream of the effluent discharge. The highest mud values were observed at
S1 station (23.4%), followed by S2 (11.7%), S3 (10.6%), while the lowest ones were
found at S5 (0.2%). Results of Two-way ANOVA (Table 3) indicate that there is a statistically
significant difference in the in grain size (% of MUD) among sites (P = .00).

Metals concentrations in surface sediments

Total heavy metal contents in surface sediment varied from 230.39 ± 26.82 mg kg of dry
weight (mg/kg of dry wt), in S5 to 1089.96 ± 28.5 mg kg−1 of dry wt in S4 (Figure 3). S1
located close to steel industry 872.13 ± 4.37 mg kg−1 of dry wt, showed the second
highest score for total metals contents, with the principle contaminants being Zn and Mn
(365, 114 mg kg−1 of dry wt, respectively). Low heavy metal content was recorded at S5
and S6 (230.39 mg kg−1 of dry wt, 254.90 mg kg−1 of dry wt). The total amount of As
varied from 5 to 16 mg/kg of dry wt, from 34 to 128 mg kg−1 of dry wt for Ba, from 23 to 73
mg kg−1 of dry wt for B, from 10 to 61 mg kg−1 of dry wt for Cr, from 8 to 44 mg kg−1 of dry
wt for Cu, from 15 to 50 mg kg−1 of dry wt for Pb, from 59 to 688 mg kg−1 for Mn, from 5 to
26 mg kg−1 of dry wt for Ni, from 14 to mg kg−1 of dry wt for V and from 45 to 365 mg kg−1
of dry wt for Zn across the sites (Figures 4 and 5). The metal concentrations in surface sedi-
ments collected from all sites in the Bizerte Lagoon were ranked in decreasing order, as
follow: Mn > Zn > Ba > B> V > Cr > Pb > Cu > Ni (Figure 5). A Two-way analysis of covariance
ANCOVA (Table 4) was conducted to examine the effect of % mud on heavy metals, notice
that the F-test value for ‘mud’ is no longer significant. This tells us that once we take into
account the effects of position and site there is no longer a significant effect of % mud on
average heavy metals concentration. ANCOVA, P < .05 this significant value says that the
‘Station’ is a significant predictor of the metals.
The distributions of ten heavy metals are shown in Figure 5. Higher concentrations of
As, Zn, B and Pb were found at S1 northeast of the Sabra, near the steel industry. The

Table 3. Results of the 2-way ANOVA for the % mud relationships among
stations (S) and side.
Source df Mean square F P
Corrected model 5 259.514 650.592 .000
Intercept 1 826.217 2071.295 .000
S 4 200.441 502.499 .000
SIDE 0 .
S * SIDE 0 .
Error 12 .399
Total 18
Corrected total 17
Note: Significant differences (P < .05) are in italics.
 CHEMISTRY AND ECOLOGY 757

Figure 3. Comparison of total heavy metals concentrations (average ± standard deviation) in sedi-
ments of different station in the Bizerte Lagoon.

concentrations decreased in the northwest (S2, S3 and S4) to southwest (S5) direction. At
all sampling stations, concentrations of Hg were below detection limits. Regardless of
which station was investigated, Mn and Zn were dominant. Station S1, located upstream
of the raw discharge, showed the second highest score for metal content, mainly contami-
nated with Mn, Zn and Cr (respectively 114, 364 and 55 mg/kg of dry wt). One of the most
detected metals in these samples is Zn, especially at stations S1, S2 and S3 (Figures 4 and
5). The highest zinc content (364.7 mg kg−1 of dry wt) was recorded at S1 while the lowest
content was recorded nearby at the S2 station, with 168.7 mg kg−1 of dry wt. High levels of
Mn, Ba and V were found in the southwest (S4) of the Sabra Bay respectively, 688 mg kg−1
of dry wt, 129 mg kg−1 of dry wt and 57 mg kg−1 of dry wt, nearby the cement industry.

Indices of sediment contamination

The contamination factors (CFs)
Low contamination factors of Cr and Ni were observed in almost all six stations (Table 5).
Cd reaches moderate contamination for the three sediment samples from S1, S2 and S3.
The Zn reaches moderate contamination for the sediment samples from station S2 and S3
and relatively high contamination in the sample from S1. Low contamination factors exist
in the sediment of sites S5 and S6 for all metals.

Geo-accumulation index
The geo-accumulation indexes varied from metal to metal for the different sampled
stations (Table 6). The Igeo for Cr, Cu, Pb and Ni indicated unpolluted to moderately pol-
luted sediment quality (class 1) for all study sites. Zn showed slight spatial variation in
station close to steel industry S1 (classes 1–2) moderately polluted. Mn revealed moder-
ately to strongly polluted (classes 2–3) at S4.
Cd remains at low levels in the sediment from sampling stations S4, S5 and S6,
suggesting that their concentration in the sediments is at background levels. Sediments
were unpolluted to moderately polluted with Cd (sediment quality class 1) for sites S1,
758 I. SAIDI ET AL.

Figure 4. Averaged Concentrations of each metals (mg/kg) of As, Cu, Ba, B, Pb, Mn, V, Ni, Cr and Zn in
sediments from Bizerte Lagoon stations and error bars are also shown. The error bars are the standard
deviations.

S2 and S3. The present study showed low contamination exists in sediments at sites 5 and
6 for all metals.

Meiofaunal abundance
Meiofauna were counted and sorted into different taxonomic groups in order to evaluate
the effect of pollution on meiofauna abundance and diversity. Total meiofaunal density in
Bizerte Lagoon revealed significant differences between stations upstream and down-
stream of industrial effluents, with higher average densities at S5 (867 ± 116
ind.10 cm−2) than at S1 and S2 Figure 6 (706 and 539 ind.10 cm−2, respectively),
ANCOVA, P < .05 (Table 7). A two-way analysis of covariance (ANCOVA) was conducted
for this study to examine the effect of % mud on meiofauna, P = .3 < .5, Notice that the
 CHEMISTRY AND ECOLOGY 759

Figure 5. Percentage levels of Arsenic (As), Barium (Ba), Boron (B), Chromium (Cr), Copper (Cu), Lead
(Pb), Manganese (Mn), Nickel (Ni), Vanaduim (V), Zinc (Zn) in the in the sediment of different locations.

Table 4. Results of the 2-way ANCOVA for the total metals relationships
among stations(S) and side.
Source df Mean square F P
Corrected model 6 287,640.600 18.947 .000
Intercept 1 19,791.182 1.304 .278
MUD 1 1729.684 .114 .742
S 4 76,791.657 5.058 .015
SIDE 0
S * SIDE 0
Error 11 15,181.147
Total 18
Corrected total 17
Note: Significant differences (P < .05) are in italics.

Table 5. Metal contamination factors (CFs) and for sediment of all sites studied in Bizerte Lagoon.
Cd Cr Cu Pb Ni Zn Mn
S1 2.91 0.66 1.62 2.93 0.45 5.13 1.95
S2 2.73 0.73 1.76 1.92 0.59 2.37 2.02
S3 2.9 0.61 1.56 2.54 0.58 2.23 2.47
S4 1 0.35 0.92 0.91 0.31 1.0 11.76
S5 1 0.12 0.32 0.94 0.1 0.63 1.14
S6 1 0.13 0.48 0.86 0.11 0.82 1.02

Table 6. Geo-accumulation indices (Igeo) of heavy metals for soils of all sites studied in the Bizerte
Lagoon.
Cd Cr Cu Pb Ni Zn Mn
S1 0.58 0.13 0.32 0.58 0.09 1.03 0.39
S2 0.54 0.14 0.35 0.38 0.11 0.47 0.40
S3 0.59 0.12 0.31 0.50 0.11 0.44 0.50
S4 0.0 0.07 0.18 0.18 0.06 0.19 2.36
S5 0.0 0.02 0.06 0.19 0.02 0.12 0.23
S6 0.0 0.02 0.09 0.17 0.02 0.16 0.20
760 I. SAIDI ET AL.

Figure 6. Abundance of the total meiofaunal taxa in the study area.

F-test value for ‘% mud’ is no longer significant. This tells us that once we take into account
the effects of position and site, there is no longer a significant effect of % mud on average
meiofauna. Stations located close to the industrial sewage discharge points revealed the
lowest abundance (33 ± 12.48 ind.10 cm−2) of meiofauna. However, the assemblage struc-
ture was similar across stations, with a clear dominance of nematodes. Univariate
measures of the meiofauna taxa for each site were calculated, including a number of
taxa (S), total abundance (N ), the Shannon–Wiener diversity indices (H′ ) (calculated

Figure 7. Comparison of meiofauna community structures. Relative abundance of the major meiofauna
taxonomic groups in the different stations of Bizerte Lagoon. Clustering analysis based on Bray–Curtis
similarity.
 CHEMISTRY AND ECOLOGY 761

Table 7. Results of the 2-way ANCOVA for the total mieofauna relationships
among stations (S) and side.
Source df Mean square F P
Corrected Model 6 290296.115 50.477 .000
Intercept 1 48776.290 8.481 .014
MUD 1 6281.524 1.092 .318
S 4 194114.577 33.753 .000
SIDE 0
S * SIDE 0
Error 11 5751.074
Total 18
Corrected Total 17
Note: Significant differences (P < .05) are in italics.

using Loge) and evenness (Pielou’s J ) [77]. S is lowest at station 3, H′ and J′ are lowest at
the station located in upstream effluents closer to the industrial sewage discharge points
than the other stations. Meiofaunal richness was highest at S6, distant from the steel and
cement factories (eight taxa), and lowest at S3 far from cement industry (two taxa)
(Table 8). The dominant taxon was represented by nematodes, which represented on
average 58% (Figure 7) of the total meiofauna. The next most abundant taxa were cope-
pods and polychaeta (both at 4% of the total meiofauna), followed by tardigrade (3%), oli-
gochaete (2%), gnathostomulida and turbellaria (1%). The contribution of the remaining
taxa was less than 1% and therefore these taxa were considered as the ‘unclassified
group’. However, the three biodiversity indices (H′ , J′ , and S) were negatively correlated
with the concentrations of total heavy metals (P < .05). Major meiofaunal groups, including
tardigrade, oligochaete, gnathostomulida and turbellaria, disappeared with the dominant
taxon represented by nematodes. This impact might be due to a direct effect of heavy
metals or lack of food since bacteria were affected by the heavy metal contamination.
The Shannon-diversity measure was particularly lower at stations S1, S2, S3 and S4 corre-
lated to a negative relation between biodiversity and the geo-accumulation index. Surface
sediments in stations located upstream of the effluent are unpolluted to moderately pol-
luted with Cd, Cr, Pb, Ni. S1 was also moderately polluted by Zn, with station 4 moderately
to strongly polluted by Mn. Shannon-diversity H′ was particularly higher at stations S5 and
S6 downstream of the industrial effluent with the lower geo-accumulation index (Table 6).
The comparison of meiofauna community structures by clustering analysis showed a clear
difference between upstream (S1, S2, S3, S4) and downstream (S5 and S6) cement and
steel factories wastewaters stations (Figure 7). The cluster classified the coastal lagoon
stations into two groups according to their meiofaunal composition and sediment
heavy metals contents.

Table 8. Means for univariate measures of the meiofauna community structure at different sites of
Bizerte Lagoon for number of taxa (S), abundance (N ), Shannon–Wiener diversity (H′ ) and evenness
(Pielou’s J ).
S N J′ H′ (loge)
S1 7.67 703.33 0.21 0.42
S2 4.33 539.00 0.06 0.08
S3 2.33 66.00 0.35 0.26
S4 3.67 33.00 0.61 0.81
S5 7.67 800.00 0.55 1.12
S6 8 333.67 0.75 1.32
762 I. SAIDI ET AL.

Table 9. Results of the 2-way ANCOVA for the total bacteria relationships
among stations (S) and side.
Source df Mean square F P
Corrected Model 6 1.003E8 49.332 .000
Intercept 1 2,741,968.102 1.348 .270
MUD 1 3,605,533.171 1.773 .210
S 4 6.136E7 30.173 .000
SIDE 0
S * SIDE 0
Error 11 2,033,564.621
Total 18
Corrected Total 17
Note: Significant differences (P < .05) are in italics.

Sediment cultivable bacterial abundance

We counted the abundance of Cultured Heterotrophic Bacteria (CHB) and Heavy Metal-
Resistant Bacteria (CHMRB) in sediments. CHB abundance in sediment varied among
sampled stations, mainly upstream of the industrial effluent station. The maximum
value was found at S2 (16.104 ± 29.27 CFU g−1). The lowest value was measured at S4
(3.102 CFU g−1) (Figure 7).
For Heavy metal-resistant bacteria, the higher abundance value was reported at S4
(16.104 ± 80.34 CFU g−1). The lowest abundance of CHMRB value was downstream of the
effluents at S5 and S6 (1.102 and 2.102 CFU g−1) which had the lowest content of heavy
metals (230 and 255 mg/kg of dry wt). ANCOVA test (Table 9) ‘% mud’ is no longer significant
(ANCOVA, P = .2). This tells us that once we take into account the effects of position, there is
no longer a significant effect of % mud on average bacteria. P = .00 this significant P-value
says that the ‘station’ is a significant predictor of distribution of Bacteria.

Relationship between bacterial, meiofauna and sediment metals

The MDS ordination analysis classified the coastal lagoon stations into two groups accord-
ing to their faunal composition and heavy metals similarities. This was also revealed by the
cluster analysis (Figure 8). One group was composed of S5 and S6 stations. The other

Figure 8. Bacterial abundance (average ± standard deviation) in the sediment of different locations
Cultured Heterotrophic bacteria (CH bacteria), Cultivable heavy metal-resistant bacterial (CHMR
bacteria).
 CHEMISTRY AND ECOLOGY 763

Figure 9. Non-metric MDS transformed of study area based on the sidimentological variables. (meio-
faunal bacterial, metals).

group consisted of stations S1, S2, S3 and S4. MDS (Figure 9) illustrates a clear relationship
between heavy metals and biota distribution into two groups, sites with high metal con-
centrations were less diverse than sites of low metal concentrations, with a stress factor
0.06. Canonical correspondence analysis (CCA) of the biotic data correlated with some
physical and chemical parameters (Figure 10) showing that the station S4 was highly
influenced mainly by Mn as well as Ba, V, Ni and Cu. S2 and S3 were influenced by As,
Zn and Pb. The CCA showed differences according to the presence of heavy metals at
stations (Figure 10) as S5 and S6 were not influenced by heavy metals but correlated
with the heterotrophic bacterial. Interestingly, heterotrophic bacteria was correlated
with the most abundant meiofauna group (nematodes, copepods, oligochaetes, poly-
chaetes, and other) and correlated with temperature (T ).

Discussion
In this Mediterranean lagoon, environmental variables strongly fluctuated with seasonal
variations related to precipitation, temperature and currents [79]. Water temperature at
all sampled sites exhibited comparable temporal fluctuation. Temperature is a key
factor influencing the benthic fluxes of nutrients and promotes a temporal variability in
sediment–water nutrient exchanges in coastal environments with marked seasons. The
salinity values of monitored waters varied slightly from 33.65 to 35 PUS. The low salinity
values at the study sites in Sabra Bay can be attributed to industrial and domestic
sewage released by Bizerte city [80]. We noted that the salinity values recorded in the
Sabra Bay in Bizerte Lagoon (present work) were similar to those reported by Béjaoui
et al. [41] and Ben Othman et al. [81]. The pH values from different locations indicate an
alkaline nature of water. The pH values recorded in the Bizerte Lagoon in July 2013
were similar to those reported by Ben Garali et al. [82]. Nutrient concentrations were
764 I. SAIDI ET AL.

Figure 10. Canonical correspondence analysis (CCA) based on meiofaunal bacterial, metals assem-
blages in each sampling stations and abiotic variables. Tar: abundance of tardigrada; O: abundance
of Oligochaeta; C: abundance of Copepods; P: abundance of Polychaeta; CHB: heterotrophic bacterial;
CHMRB: Heavy Metal-Resistant Bacteria; N: Nematode; Tur: abundance of turbellarians, G:Abundance of
gnathostomulida, Arsenic (As), Barium (Ba), Boron (B), Chromium(Cr), Copper (Cu), Lead (Pb), Manga-
nese (Mn), Nickel (Ni), Vanadium (V), Zinc (Zn), Nutrient ((NO2 + NO), NO3, Si, NH4), O2 Station biotic
variable.

within the range of those found by Sakka Hlaili et al. [50] When nutrients are abundant, a
bloom of living organisms typically occurs at various links in the food chain. The increase in
living organisms leads to excessive consumption of oxygen and to its scarcity both in the
water column and the sediment [83].
The grain size distribution in sampled stations was primarily characterised by fine sedi-
ments containing only mud and sand. This finding corresponds with Ben Mna et al. [84]
consequently the fine sediments accumulated more pollutants and organic matter [85].
It has long been recognised that sediment characteristics greatly influence the structure
and diversity of benthic faunal communities [86]. The results reveal narrow variation in tex-
tural composition observed in the sediments of all sites, the sediments were predomi-
nantly composed of sand and silty clay at all locations. Horowitz Elrick [87] reported
that, fine to very fine-grained sediments tend to have relatively high metal concentrations

Table 10. Standard guideline applicable for heavy metals in marine sediment
(After Pazi (2011)).
Sediment quality (μg/g)
Metal Average crustal Non polluted Moderately polluted Heavily polluted
As 1.8 <3 3–8 >8
Cu 55 <25 25–50 >50
Cr 100 <25 25–75 >75
Ni 75 <20 20–50 >50
Pb 12.5 <40 40–60 >60
Zn 70 <90 90–200 >200
 CHEMISTRY AND ECOLOGY 765

due in part to the high specific surface area of the smaller particles. This enrichment is
mainly due to surface adsorption and ionic attraction. The speciation and toxicity of
metals in the sediments are greatly dependent on the partitioning of metals between
the sediment particles and the interstitial waters [88]. Dissolved metals present in the
interstitial waters are more bioavailable and toxic than particulate metals [89]. Physical
processes (water currents, anthropogenic disturbance) and the activity of benthic organ-
isms (burrowing, irrigation) can cause sediment resuspension and mixing of previously
redox-stratified sediments with oxygenated overlying waters. This resuspension and
mixing alter metal sediment–water partitioning and metal speciation in the dissolved
phase, interstitial waters and overlying waters [90–92]. These physico-chemical changes
alter the bioavailability of metals in the pore waters [93,94] and the release rate (flux) of
metals from the sediments [88]. The fate (speciation) of metals released from sediments
is affected by overlying water conditions, in particular, the pH, salinity, dissolved oxygen
concentration and amount of suspended solids [95,96].
The highest accumulation of the total metals was found at S1, S2, S3 and S4, reflecting
the increase in anthropogenic inputs of those metals in recent decades. These results are
in accordance with Ben Said et al. [46], which indicate that the station located in the
channel, showed the second highest score for metal contents, mainly contaminated
with Mn, Zn, and Cr, suggesting that Cr and Zn are derived from industrial activities
such as cement production [97–99]. Low heavy metals contents recorded at S5 and S6
may be explained by strong hydrodynamism at this station located at the exchange
point between the lagoon of Bizerte and the Mediterranean Sea [100]. The distribution
of these heavy metals showed variations among the stations as a result of variability in
the extent of pollution inputs [101,102]. Pollution inputs are due to municipal effluents
coming from the bordering cities [103], from the naval port and the metallurgic factory
and several other industries such as the iron and steel plant, cement factory and
refinery [104], with these pollutants adsorbed by the sediments [7]. Since sampling
stations S1, S2, S3 and S4 are more influenced by the tide, the sediments can be sus-
pended due to tidal currents and the metallic content of the sediments can be mobilised
more easily to the water column [98,105]. Moreover, heavy industrial activities are located
close to stations S1, S2, S3 and S4. Higher concentrations of Pb and Zn were found in the
S1, this high Pb sediment content could have come from the synergistic deposition effects
from cement production operations and traffic activities. Pb is known to be part of heavy
metals released during cement production [106,107]. Whatever in the station investigated,
Mn and Zn were dominant. S1 is heavily polluted by Zn and Mn compared to the standard
guideline applicable for heavy metals in marine sediment (Table 10) [108]. The concen-
tration of Mn may not be derived from industrial activities but from geological processes
[109]. Manganese is one of three toxic essential trace elements, but it is toxic at high con-
centrations [110]. Manganese compounds exist naturally in the environment as solids in
the soils and small particles in the water. The distribution of Cd and Ni at the study site
is similar to values measured in Ghar El Melh Lagoon [110]. Comparison of the level of
Cu and As to the standard guideline applicable for heavy metals in marine sediment
showed that concentrations of As and Cu, in the sediment were heavily polluted by As
and moderate polluted by Cu Pazi [107]. A high level of V was found in (S4) of the
Sabra Bay. Vanadium is an abundant element in crude oils and considered to be a toxic
element in the environment. It can have an inhibitory effect on some organisms and
766 I. SAIDI ET AL.

has been used as a proxy tracer of oil pollution along the Caribbean coast of Panama [111].
Comparison of Igeo results from this study and previous works suggest that there is a pro-
gressive increase in metal contamination in surface sediments of Bizerte Lagoon. Bar-
houmi et al. [100] revealed Igeo values for Cd, Pb, Ni and Zn above 1, suggesting
widespread pollution and showed that these heavy metals originate from anthropogenic
sources such as urban runoffs and industrial effluent discharges. Zaaboub et al. [7] men-
tioned that the most accumulated trace elements in surface sediments are Zn and Pb in
Bizerte Lagoon. Ben Mna et al. [84] revealed that sediment pollution in Bizerte Lagoon
was moderate to high for Pb, while moderate for Zn and unpolluted to moderately pol-
luted sediments for Cr. In the hydrochemical cycle that governs the dynamics of the pres-
ence of metallic elements within the water systems, the sediments play an essential role as
they constitute not only very important vectors but also the principal reservoirs of a large
number of metals [112]. Therefore, sediments are considered as integrators and tracers of
the metallic pollution [112]. The studies revealed a clear inverse relationship between
metal concentrations and meiofauna diversity, the higher concentrations, the lower diver-
sity. Several studies have shown a decrease in meiofaunal diversity due to heavy metal pol-
lution in coastal systems [113]. In this study, the contamination resulted in a reduction in
both the density and diversity of the meiofauna community in sediment. We discovered
that the station S5 has the lowest heavy metal concentration 230 mg/kg−1and is the
most diversified and richest (8taxa) in total meiofauna abundance 867 ind.10 cm−2.
There are well-documented reports that nematodes show particular abilities to accumu-
late metals present in their biotope [114,115] confirming that differences between nema-
tode communities in different stations are linked to levels of heavy metals in the
sediments. The sediment granulometry may contribute only a minor role in controlling
benthic community structure within the survey area. No relationship between a number
of individuals and sediments was apparent within the survey area. In the surface sedi-
ments of the station located upstream of the effluents, the dominant taxon was rep-
resented by nematodes These surface sediments are moderately polluted sediment
with Cd, Cr, Pb, Ni and Zn. Van der Wurff et al. [116] showed that nematodes can accumu-
late and tolerate higher levels of copper, cadmium, zinc and lead than other meiofaunal
organisms. However, the relationship between sediment metals and copepod abundance
is less clear. This is supported by the findings of Warwick [117] who suggested that cope-
pods are in generally more sensitive to the effects of pollution than nematodes. The dis-
tribution of meiofauna in the station close to the industrial discharge points is distinct from
those in all other locations by a general reduction of nematode sand the presence of rela-
tively high numbers of copepods, as copepods can be tolerant to higher levels of heavy
metals [118]. These results suggest that metal contamination significantly alters the com-
position of meiofauna assemblages. Past studies have shown that the high concentrations
of Cr, Zn, As, and Cd are clearly correlated with low diversity of meiofauna [119] and may
indicate anthropogenic impact at locations where these metals are present. Some species
(Tardigrade, oligochaeta, Gnathostomulida and turbellaria) disappear; others can become
dominant [120]. The high abundance of heavy metal-resistant bacteria was correlated with
high heavy metal levels. Under the stress of combined heavy metal pollution, the shifts in
bacterial community structures were significant between the polluted and the reference
sediment samples (P < .5). The increases found in heavy metal polluted sediments could
be caused by an acquired tolerance by adaptation, and a shift in species composition.
 CHEMISTRY AND ECOLOGY 767

We found that these results, similar to other investigators, further support the hypothesis
that bacteria in heavy metal contaminated sediments are increased in number [121,122].
Microbial communities have evolved to adapt their metabolism to the presence of multi-
contaminants [123–126]. Heavy metals can change the bacterial densities and diversity
[124]. In marine sediments, bacteria play a key role in inorganic nutrient recycling, in
early organic matter diagenesis and in the transfer of energy and material to higher
trophic levels [127,128]. Therefore, the impact of heavy metals on benthic bacteria might
have important consequences both on biogeochemical cycles and on benthic trophody-
namics [25]. Some studies reported that intensive grazing by meiofauna may induce inten-
sification in bacterial production and thus an increase of microorganism abundance [129].
However, S1, S2, S3 and S4 are influenced by heavy metals and correlated with CHMR bac-
teria, pH, salinity and dissolved O2. Interestingly, nematodes were also correlated with the
CH bacterial showing the positive effect of heterotrophic bacteria for meiofauna survival
that may probably be due to bacteria-feeding meiofauna. There were significant differences
in the meiofaunal community between locations. The CCA showed that the sediment meio-
fauna community of stations located upstream of effluent were influenced by heavy metals
showing the negative effect of heavy metals for meiofauna and for heterotrophic bacterial
survival. Spatial variation of meiofauna communities at Bizerte Lagoon was observed
bamong the location sites. Most authors underline the significant effects of meiofauna on
bacteria [130], we observed inverse relationships between CHMRB and meiofauna. The
abundance of CHMRB correlate with low numerical values of meiofauna and vice-versa. It
was observed that heavy metals concentration had a clear influence on heavy metal-resist-
ant bacteria in the sediment. Both bacterial and nematode communities were sensitive to
increasing concentrations of metals [131]. The high abundance of Heavy Metal-Resistant
Bacteria correlated with low abundance of nematode, the low abundance of CHB and the
high levels of heavy metals. It appears, however, that the total nematode and total hetero-
trophic bacteria are highly correlated. Bacteria are the first step in the transfer of toxic com-
pounds to higher trophic levels of the food chain [132]. This indicates that metals will affect
the microbial component of the sediment as well as the bacterial community structure and
activities are under the influence of meiofaunal groups such as nematodes [133,134] directly
as main bacterial predators and indirectly modifying the environmental conditions. Based
on the two way analysis of covariance (ANCOVA), the results show that there are no relation-
ships between the grain size distribution, the abundance and diversity of meiobenthos in
the area. In addition, there was no statistically significant interaction between position
and site on Bacteria whilst controlling for mud. These results thus suggest that the meiofau-
nal community at Bizerte Lagoon was not influenced by sediment grain size.

Conclusion
The results from our study revealed the highest concentrations of heavy metals were
found in Sabra bay near urban areas with high anthropogenic activities. The contami-
nation factor and the geo-accumulation index show that currently, the lagoon of Bizerte
is unpolluted to moderately polluted. The majority of superficial sediments of Sabra Bay
of Bizerte Lagoon are moderately contaminated, mainly by manganese, lead, zinc, chro-
mium, barium and arsenic at least degree by nickel. The sites S2, S3 and S4 located
upstream of industrial effluents with high metal sediment contents were significantly
768 I. SAIDI ET AL.

less diverse than sites of low metal concentrations. The meiofauna and particularly its
dominant group, the nematode, respond well to the toxic effects of pollutants and can
be viewed as relevant biological indicators of stress in the marine environment. We
showed the correlation of heavy metals, mainly manganese and barium, on bacterial
and meiofaunal abundance. The heterotrophic bacteria was correlated positively with
the other meiofauna taxa (Nematods, copepods, oligochaetes, polychaetes, and other).
The metal resistant bacteria directly correlated with heavy metal concentration. In con-
clusion, this study has demonstrated that the composition of biota in Sabra Bay is more
highly correlated with heavy metals than to grain size % MUD. A suite of metals consisting
of As, Ba, Cu, Cr, Pb, Ni and Mn were identified as the dominate influence on the meiofau-
nal and bacterial communities. This finding supports the suggestion that changes in meio-
faunal and bacterial communities could be used as biomonitors of heavy metals in coastal
sediments.

Acknowledgements
Researches works and innovation are carried out within the framework of the device MOBIDOC
financed by the European Union under the PASRI programme and administrated by the ANPR.
Also we thank the ministry of Higher Education and Scientific Researches, University of Carthage,
Faculty of Sciences Bizerte Tunisaia and the cement campanie Bizerte for their support. Thanks to
Joanna Wlodarczyk and the group of the LAQ – Laboratorio de Analises Quimicas for their help
with the heavy metals analyses.

Disclosure statement
No potential conflict of interest was reported by the authors.

Notes on contributors
Ibtihel Saidi is PhD in Biology in the Department of Biology, Laboratory of Environment Biomonitor-
ing, Faculty of Sciences of Bizerte, Tunisia. Her research focuses on Environmental pollution and
Environmental Impact Assessment.
Olfa Ben Said is Postdoctoral in the Department of Biology. She obtained her PhD in microbiology
from Faculty of Sciences of Bizerte Her research focuses on Environmental Impact Assessment.
Jamel Ben Abdelmalek is an expert in cement industry in Tunisia. Engineer’s degree GEochimie. PhD
student in geology.
Soufiane Jouili is Postdoctoral in the Department of Biology his PhD in Biology from Faculty of
Sciences of Bizerte. Her research focuses on Environment.
Luis Chicharo is Associate Professor of Faculty of Sciences and Technology, University of Algarve,
Portugal, he is Director of International Centre for Coastal Ecohydrology, under the auspices of
UNESCO.
Hamouda Beyrem is Professor in the Department of Biology, Faculty of Sciences of Bizerte university
of Carthage Tunisia. His research focuses on ecology.

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