barhoumi2014

Marine Pollution Bulletin xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul
Baseline
Assessment of pollution in the Bizerte lagoon (Tunisia)

by the combined use of chemical and biochemical markers
in mussels, Mytilus galloprovincialis
Badreddine Barhoumi a,b, Karyn Le Menach b, Christelle Clérandeau b, Walid Ben Ameur a,
Hélène Budzinski b, Mohamed Ridha Driss a, Jérôme Cachot b,⇑
a
University of Carthage, Faculty of Sciences of Bizerte, Laboratory of Environmental Analytical Chemistry (05/UR/12-03), 7021 Zarzouna, Bizerte, Tunisia
b
University of Bordeaux, CNRS, UMR EPOC 5805, avenue des Facultés, 33405 Talence Cedex, France
a r t i c l e i n f o a b s t r a c t
Keywords: In order to assess the environmental quality of the Bizerte lagoon (Tunisia), biomarker and contaminant
Pollution monitoring levels were measured in Mediterranean mussels (Mytilus galloprovincialis) from five selected sites. Persis-
Persistent organic pollutants tent organic pollutants (POPs) were quantified in whole body and enzyme activities such as acetylcholin-
Bioaccumulation esterase (AChE), catalase (CAT) and glutathione S-transferase (GST) in gills. Despite the relatively low
Biomarkers
levels of organic contaminants, the selected biomarkers responded differently according to the pollution
Mediterranean mussel
Coastal lagoon
level at the different sites. GST and AChE activities were correlated with the amount of DDTs in mussel
tissues. These two enzymatic activities were also correlated to temperature and pH. No significant differ-
ence was observed for CAT activity. Principal component analysis showed a clear separation of sampling
sites in three different assemblages which is consistent with POP body burden in mussels. Our results
confirmed the usefulness of combining biomarker and chemical analyses in mussels to assess chemical
pollution in the Bizerte lagoon.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction has led to a decrease in bivalve and fish production over the last
few decades (ANPE, 1990). To put this into perspective, only
Marine ecosystems are of high ecological and economic impor- 61.06 tons of mussels were produced in 2002 (CRDA, 2002) com-
tance, because they support vital habitats for organisms and sus- pared with 116.500 tons in 1998 (CRDA, 1998). This sharp drop
tain several anthropogenic pressures. Highly productive areas, is one of the main reasons that the Tunisian authorities have begun
such as estuaries and coastal lagoons, are among the most exten- to focus on maintaining the chemical and biological quality of
sively modified and threatened ecosystems, mainly due to urban mussels in the area.
development, industrialization, and tourism (Clark, 2001). As a Biomonitoring based on the quantification of contaminant con-
result, complex mixtures of contaminants are continuously centrations in bioindicator organisms has been widely used in
released into these ecosystems, reducing water quality and impos- environmental surveys over the past few decades. Mussel watch-
ing severe restrictions on organisms. This can ultimately lead to a like programs have been developed successfully in many countries
decrease in natural resources (Monserrat et al., 2007; Cravo et al., (Goldberg, 1975). Bivalves, and especially mussels, have been
2009). One example of such an area is the Bizerte lagoon, a major widely used as indicators of marine and estuarine pollution. Such
coastal lagoon on the north coast of Tunisia. It is a particularly molluscs are sessile filter feeders, able to accumulate and tolerate
important fishing area which has also been used in shellfish pro- high concentrations of pollutants, present across a very wide geo-
duction since 1964 (Beji, 2000). Meanwhile, the lagoon is located graphical area, easy to collect, and particularly abundant in coastal
close to areas of heavy industrial and agricultural activities. This and estuarine waters (Goldberg, 1986). In addition to chemical
area is chronically polluted with industrial wastes, pesticides and analyses, the use of different biomarkers has been introduced in
chemical fertilizers, through soil erosion and water runoff. This monitoring programs to evaluate not only contaminant concentra-
tions in sediments, water column and organisms but also the
effects of pollutants on the organisms (Bayne, 1989).
⇑ Corresponding author. Tel.: +33 (0)540 003 830; fax: +33 (0)540 002 267. Contaminants usually appear in the environment as complex
E-mail address: j.cachot@epoc.u-bordeaux1.fr (J. Cachot). mixtures that can cause interactive effects on the biota that are
http://dx.doi.org/10.1016/j.marpolbul.2014.05.002
0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Barhoumi, B., et al. Assessment of pollution in the Bizerte lagoon (Tunisia) by the combined use of chemical and bio-
chemical markers in mussels, Mytilus galloprovincialis. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.05.002
2 B. Barhoumi et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
impossible to evaluate through chemical analyses alone. In this To assess the general health of mussels, 10 animals were used at
sense, biomarkers offer an integrated assessment of exposure lev- each station for the condition index measurement (CI) (the ratio
els and effects of pollutants in wildlife. There are several biomark- between whole soft tissues weight and shell weight). To determine
ers that are more specific for certain types of xenobiotics and, thus, contaminants (OCs, PAHs and PBDE), whole body tissues of 60
the use of a battery of biomarkers together with chemical analyses specimens from each station were dissected, pooled in 3 samples
has been strongly recommended to be included in monitoring pro- (each with tissues of 20 specimens), and stored at 20 °C, before
grams (OSPAR, 2000; ICES, 2008). Three biomarkers were selected being freeze-dried and homogenized with a blender. For biochem-
for this study. Acetylcholinesterase (AChE) activity is an indicator ical parameters, 10 organisms were dissected, and the individual
of synaptic neurotoxic effects (Bocquené and Galgani, 1998); gluta- gills were immediately stored at 80 °C until required for biomarker
thione S-transferase (GST) is a phase II enzyme involved in the analysis.
detoxification of organic xenobiotics (Habig et al., 1974); and cat- Lipid content was determined using microwave assisted extrac-
alase (CAT) involved in oxidative stress response caused by exces- tion (MAE) (Bodin et al., 2009). A few milligrams of freeze-dried tis-
sive oxyradical formation in the metabolism of various compounds sue were weighed and then extracted with dichloromethane
(Claiborne, 1985). (20 mL, 30 W, 10 min). Organic extract was then filtered and evap-
The first objective of our study was to determine concentrations orated to dryness using a RapidVap evaporator (Labconco, Kansas
of persistent organic pollutants, POPs (PCBs, Pesticides and PBDEs) City, MO, USA). The residue was weighed to gravimetrically esti-
and PAHs in Mytilus galloprovincialis specimens. Mussel samples mate the lipid content of the samples. The lipid content was
were collected from five sites around the Bizerte lagoon, each expressed in percentage of dry-basis lipid content (LC%) as follows:
effected by POPs (Trabelsi and Driss, 2005; Barhoumi et al., LC% = (m1/mdm) 100, where m1 is the mass of extracted lipid (g),
2014a,b) and heavy metals (Ben Garali et al., 2010) to different mdm is the mass of dry matter (g).
degrees. In this human impacted aquatic area, several previous All reagents used for biomarker analyses (bovine serum albumin
studies have investigated the application of biochemical tools for (BSA); hydrogen peroxide (H2O2); acetyl-thiocholine (AcTCh);
pollution biomonitoring (Dellali et al., 2001b; Khessiba et al., dithiobis-nitrobenzoate (DTNB); 1-chloro,2,4, dinitrobenzene
2001, 2005; Roméo et al., 2006; Mahmoud et al., 2010). However, (CDNB) and 5,50 , reduced glutathione (GSH) were purchased from
no studies have assessed the levels of POPs in Bivalves from the Sigma (France). The pesticide grade dichloromethane was pur-
Bizerte lagoon. The second purpose was to evaluate selected bio- chased from Acros Organics (Noisy le Grand, France). Pentane was
markers in mussels, in order to (i) investigate biological responses obtained from Atlantic Labo (Bruges, France), sulphuric acid Sharlau
of organisms from sites with different history of pollution and (ii) (95–98% extra pure) and isooctane Sharlau were purchased from ICS
tentatively correlate pollutant body burden and induced effects in (Gradignan, France). The copper (40 mesh, 99.5% purity, Aldrich,
mussels. In addition, environmental (temperature, pH and salinity) Strasbourg) was activated with hydrochloric acid (1 N) and then
and biological (CI) factors were quantified in water samples and washed with water, acetone, and CH2Cl2. The alumina (150 basic,
mussels from each site, in order to investigate the possible effects type T particle size 0.0630.2 mm) and the silica (Silica gel, particle
of these variables on pollutants concentration and biomarker size 0.063–0.2 mm) (Merck, Darmstadt, Germany) were washed
responses. with CH2Cl2 and activated for 14 h at 150 °C. The acid silica gel
Five sampling stations were chosen within the area of the Bizerte was prepared by mixing 600 g of silica gel and 400 g of sulphuric
lagoon used for our study (Fig. 1). They were selected based on pos- acid over night at 200 °C (Müller et al., 2001). Standard reference
sible differences in contamination levels and availability of bivalves: material SRM 2262 (chlorinated biphenyl congeners in isooctane)
two non-urbanized areas considered to be relatively uncontami- and SRM 2261 (pesticides in isooctane) was provided by the
nated (S2 and S5), and three zones clearly exposed to anthropogenic National Institute of Standards and Technology (NIST, Gaithersburg,
pressure (S1, S3 and S4). S1 (37°15.480 N, 9°51.180 E), is located in the MD, USA). Individual solutions of PBDEs (BDE47, BDE 99, BDE119,
Bizerte channel near the cement plant, and is influenced by intense BDE153) were purchased from Cambridge Isotope Laboratories
fishing, passage of ships, and urban effluents from the city of Bizerte. (CIL USA, purity 99%). CB30, CB103, CB155 and CB198 were deliv-
S2 (37°13.480 N, 9°48.260 E), is an exchange point between the lagoon ered as neat crystals by Promochem (Molsheim, France, Purity
of Bizerte and the Mediterranean Coast. It is subjected to complex 99%). Octachloronaphthalene (OCN) was used as syringe standard
hydrodynamic conditions, with relatively low levels of contamina- and purchased from Ultra Scientific (North Kingstown, USA, Purity
tion (Khessiba et al., 2001; Barhoumi et al., 2014a,b). S3 95%). The PAHs studied range from diaromatics (Naphthalene) to
(37°12.490 N, 9°47.900 E), is located close to the mouth of Haima River hexa-aromatics (Benzo(ghi) perylene). The Standard Reference
in the northwestern part of the lagoon, in a region of intensive agri- Material, SRM 2260, aromatic hydrocarbons in toluene (Nominal
cultural activity, including extensive use of pesticides. This station is Concentration 60 mg mL1), a standard compound solution of 24
also influenced by the passage of commercial cargo vessels between aromatic hydrocarbons was provided by the National Institute of
the cities of Bizerte and Menzel Bourguiba. S4 (37°13.330 N, Standards and Technology (NIST, Gaithersburg, MD, USA). The com-
9°51.500 E), is located near a town of 10,000 inhabitants, surrounded pounds used as internal standards for the quantification of PAHs
by industrial units (Textile fabrics, electronic industries and metal- were 9 perdeuterated PAHs. Naphthalene-d8, dibenzothiophene-
lurgy). The sampling site receives a constant influx of untreated d8, phenanthrene-d10, anthracene-d10, benzo(e)pyrene-d12, ben-
waste water. S5 (37°8.380 N, 9°52.350 E), is located far from urban zo(a)pyrene-d12 and benzo(ghi)perylene-d12 were from Cambridge
and industrial sources of pollution, but remains influenced by agri- Isotope Laboratories (CIL, Andover, MD, USA), fluoranthene-d10 and
cultural inputs. Physic-chemical parameters (temperature, salinity chrysene-d12 from MSD isotopes (Division of Merck Frost Canada
and pH) were measured in situ at all sampling sites using a WTW- INC, Montreal, CND). Benzo(b)fluoranthene-d12 and pyrene-d10 were
197i multimeter. used as syringe standard and purchased from MSD isotopes (Division
Approximately 80 mussels (M. galloprovincialis) of similarly- of Merck Frost Canada INC, Montreal, CND).
sized (4.4–5.2 cm shell length) wild Mediterranean mussels were Mussel samples were freeze-dried and homogenized with a blen-
collected from each sampling site in February 2012. This work der. The combined extraction protocol for OCs, PBDEs and PAHs was
was carried out by a scuba diver, operating at a depth of around adapted from Baumard et al. (1997) and Budzinski et al. (2000),
3–5 m. The specimens were then immediately taken to the labora- Thompson et al. (2002) and Tapie et al. (2008). The appropriate volume
tory in ice boxes. Mussels were sacrificed 3 h after collection to of the internal standard solutions (PCB congeners 30, 103, 155 and 198
ensure uniform sampling and transport conditions across all sites. for PCBs, OCPs and PBDEs, perdeuterated PAHs for PAHs) were
B. Barhoumi et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx 3
Fig. 1. The Bizerte lagoon and the five selected sampling stations.
gravimetrically added to about 0.5 g of lyophilized mussel tissues. capture detector (GC/ECD). Separation and detection of OCs and
Organic pollutants were extracted by accelerated solvent extraction PBDEs was performed by high-resolution gas chromatography
on a ASE 200 system (Dionex, Voisins le Bretonneux, France) with (GC; Agilent 6890 Series gas chromatography system; Agilent
dichloromethane (Tapie et al., 2008). The parameters used during Technologies, Philadelphia, PA, USA) equipped with a 63Ni electron
the extraction procedure were as follows: temperature (100 °C), sta- capture detector, and both PTE-5 (30 m 0.32 mm i.d., 0.32 lm film
tic time (8 min), pressure (130 bars), heating time (5 min), flush vol- thickness) and HP1 (30 m 0.32 mm i.d., 0.25 lm film thickness)
ume (60%), and purge time (60 s). After extraction, the extract was capillary columns were used. The operating conditions were as fol-
concentrated using a RapidVap evaporator (Labconco, Kansas City, lows: injector temperature, 270 °C, detector temperature, 300 °C.
MO, USA) near 1 mL and first purified on alumina micro-column. The GC temperature program was 70 °C (1 min hold) to 140 °C at a
The compounds were eluted with 3 5 mL dichloromethane. The rate of 25 °C min1, 179 °C at 2 °C min1, 210 °C at 1 °C min1 and
extract was reconcentrated under a gentle flow of nitrogen, the sol- then to 300 °C (10 min hold) at 5 °C min1. The carrier gas was
vent exchanged with isooctane and evaporated again under nitro- hydrogen (H2), set at a flow rate of 1.5 mL min1. The detector
gen flow up to 1 mL. The extract was further purified on silica gel make-up gas was nitrogen, set at a flow rate of 60 mL min1. Sample
micro-column, previously conditioned with n-pentane. The alkane injection volume: 1 lL. Injection mode: splitless (1 min). The data
fraction was discarded by rinsing the column with n-pentane and presented in this paper were obtained using the PTE-5 column,
the compounds were eluted with 3 5 mL n-pentane/dichloro- and a HP1 capillary column was used as second column to confirm
methane (65/35; v/v). The extract was concentrated under nitrogen the identification of OCs and PBDEs compounds in mussels. PCBs
flow and transferred into GC vials containing a 300 lL glass insert. (SRM 2262), OCPs (SRM 2261) and PBDEs (BDE47, 99, 119 and
The deuterated PAH internal standard solution (pyrene-d10 and 153) were quantified in relation to internal standards (CB30,
benzofluoranthene-d12) was gravimetrically added to the final CB103, CB155, CB198). The relative response factors of the different
extract. The latter extract was divided into two fractions, the first compounds were determined by injecting standard solutions (SRM
one being used for the analysis of PAHs by gas chromatography– 2262, SRM 2261 and a standard solution prepared from pure crystals
mass spectrometry (GC/MS), and the second purified on an acid for PBDEs) spiked with the same solution of internal standards as
silica gel column containing a bed of activated copper. OCs and that used for spiking the samples. Sample concentrations are
PBDEs were eluted with 3 5 mL n-pentane/dichloromethane expressed as nanograms per gram dry weight (ng g1 dw). The
(90/10; v/v). The extract was then concentrated, transferred to aromatic fraction was analysed by gas chromatography–mass
isooctane and the syringe standard (Octachloronaphthalene) was spectrometry in SIM mode (GC–MS; HP 5890 Series II, MSD 5972,
added before analysis by gas chromatography coupled to electron Agilent Technologies, Palo Alto, CA, USA) using an HP5-MS capillary
column (5% diphenylsiloxane–95% dimethylsiloxane; 30 m and 30 mM H2O2 (Claiborne, 1985). Catalase activity was calcu-
0.25 mm 0.25 lm; Agilent). The injector temperature was main- lated as lmol H2O2 consumed min1 mg1 of proteins. The GST
tained at 280 °C. The GC temperature program was from 50 °C assay was performed using a modified version of the method
(2 min) to 300 °C (8 min) at 10 °C min1. The carrier gas was helium developed by Habig et al. (1974). GST activity towards 1-chloro-
at a constant flow rate of 1 mL min1. The GC was coupled to a HP 2,4-dinitrobenzene (CDNB) was measured spectrophotometrically
5972 mass selective detector (MSD) (electronic impact: 70 eV, volt- (UVIKON 933 spectrophotometer, BioTek) in a reaction mixture
age: 2000 V) operating in single ion monitoring mode (SIM) using containing 100 mM phosphate buffer (pH 7.0), 20 mM CDNB and
the molecular ion of each compound at 1.23 scan/s. The interface 20 mM GSH. The activity rate of GST was measured as the change
temperature was 290 °C. The PAHs were quantified relative to per- in OD/min at 340 nm (extinction coefficient, e = 9.6 mM1 cm1),
deuterated PAHs added to the samples prior to the extraction. The and expressed as lmol min1 mg1 protein.
response factors of the different compounds were measured by Statistical analyses were performed using the software STATIS-
injecting SRM 2260 solution spiked with the same solution contain- TICA 7.1 from StatSoft (Maison Alfort, France). The results of chem-
ing the perdeuterated PAHs as the one used for spiking the mussels. ical (PAH, PCB, DDT and PBDE), biological (CI and LC) and
The PAHs studied ranged from di-aromatic (Naphthalene) to hexa- biochemical (GST, CAT and AChE enzymatic activities) parameters
aromatic (benzo(ghi)perylene) and included: Naphthalene (Nap); were reported as mean ± SD. To identify differences of chemical
Acenaphthylene (Acy); Acenaphtene (Ace); Fluorene (Fl); Dibenzo- and biochemical parameters between sites (level of significance
thiophene (DBT); Phenanthrene (Phe); Anthracene (Ant); Fluo- at p < 0.05), results were initially tested for normality (Shapiro–
ranthene (Flu); Pyrene (Pyr); 2,1 Benzonaphtothiophene (2,1BNT); Wilk’s test on residues with 1% risk) and equality of variance
Benz(a)anthracene (BaA); Chrysene (Chry); Triphenylene (Triph); (Levene’s test, 5% risk). Afterwards, significant differences between
Chry plus Triph (Ch); Benzo(b)fluoranthene (BbF); Benzo(j)fluo- treatments were tested with one way analysis of variance (ANOVA)
ranthene (BjF); Benzo(k)fluoranthene (BkF); BbF plus BjF plus BkF followed by a post-hoc Tukey’s test (p < 0.05). If conditions for
(BF); Benzo(e)pyrene (BeP); Benzo(a)pyrene (BaP); Perylene (Per); ANOVA were not fulfilled, non-parametric Kruskall–Wallis’s test
Dibenz(a,h)anthracene (DahA); Dibenz(a,c)anthracene (DacA); DahA and Bonferroni–Dunn’s post-hoc test were used (p < 0.05). The
plus DacA (DA); Indeno(1,2,3-cd)pyrene (IP); benzo(ghi)perylene existence and strength of relationships between parameters were
(BP). PAH concentrations are given in ng g1 dw. To ensure quality determined by parametric Pearson’s bivariate correlation analysis.
assurance, procedural blanks were regularly performed during the Principal component analysis (PCA) was performed to discriminate
extraction process. No pesticides, PCBs, or PBDEs were found in the different sites using the mean values of the entire dataset
the blanks. Naphthalene, the most volatile PAH, was detected, albeit (Chemical, biological, biochemical and abiotic parameters).
at low concentration, in the blanks. Because of this, samples were Physicochemical minimum and maximum parameters of water
corrected for blanks only for that particular compound. Samples samples including temperature, salinity, and pH levels varied little
and blanks were spiked with internal recovery standards (CB30, across sites changing between 12.0–16.5 °C, 33–35.2 psu and 7.1–
CB103, CB155 and CB198 for OCs and PBDEs, 9 perdeuterated PAHs 7.8, respectively (data not shown).
for PAHs) prior to extraction procedures and by syringe standard Condition index of the sampled mussels did not show any sig-
(OCN for OCs and PBDEs, Benzo(b)fluoranthene-d12 and pyrene- nificant difference between stations with the exception of stations
d10 for PAHs) before injection to monitor methodological analyte S1 and S2 (p < 0.05). The lowest value, indicating poor physiologi-
losses. Recoveries over 80% were accepted. Blank injections of isooc- cal condition, was measured at station S1. The highest CI value was
tane were performed between each sample injection to ensure the measured at station S2 (Table 1). The lipid content of the sampled
cleanliness of the GC/ECD and GC/MS injector. Limits of detection mussels varied from 13.7% at S3 to 20.9% at S2 but no significant
(LODs) and limits of quantification (LOQs) were calculated by the variation was observed between sites (Table 1). No significant cor-
signal-to noise ratio (3:1 and 10:1, respectively) for each chemical relation was found between OCPs, PCBs, PAHs or PBDE concentra-
class and were respectively 0.10 and 0.40 ng g1 dw for DDTs, 0.12 tions and total lipid content.
and 0.22 ng g1 dw for HCB, 0.02 and 0.10 ng g1 dw for PCBs, 0.10 Content of organochlorinated compounds (RPCBs, RDDTs and
and 0.30 ng g1 dw for BDE 47 and 0.10 and 0.25 ng g1 dw for PAHs. HCB) in mussel tissues from the Bizerte lagoon is presented in
For biochemical analyses, gill samples were removed from Table 1. PCB contamination was homogeneous within the lagoon,
80 °C, thawed on ice and homogenized with an Ultra-TurraxÒ since no concentration difference was found between the five sam-
tissue homogenizer in 1:3 (v:v) 100 mM Tris–HCl buffer contain- pling sites. More specifically, station S3 (150.6 ± 3.6 ng g1 dw),
ing 0.1% Triton X-100, pH 7. Homogenates were centrifuged at located close to the mouth of Haima River, was the most contam-
10,000 g for 20 min. All preparation procedures were carried inated site, showing values twice those of S2 (89.1 ± 14.3 ng g1
out at 4 °C. After centrifugation, the supernatant (fraction S9) dw). Overall, CB153 was the most abundant congener accumulated
was retained; divided into several aliquots and kept at 80 °C in bivalve soft tissues, accounting for 36% of the RPCBs, followed
for subsequent analyses of biomarkers (AChE, CAT and GST). All by CB138 (17%) and CB126 (9%) (Fig. 2A). Grouping PCB congeners
assays were performed in triplicate. The protein concentration according to their grade of chlorination, the hexa-CBs were the
in the S9 fraction was measured using Bradford’s (1976) colori- most representative of the contamination pattern (53%), followed
metric method with bovine serum albumin (BSA) as a standard. by penta-CBs (20.7%), tetra-CBs (14.4%) and hepta-CBs (5.9%),
All protein measurements were carried out using a spectropho- while octa-CBs (2.8%) and trichlorobiphenyls (3.3%) made up a
tometer microplate reader (Synergy HT, BioTek), and expressed small percentage of the RPCBs (Fig. 2B). For all DDTs (RDDTs),
in mg mL1. Measurements of AChE activity were performed the spatial pattern was similar to that exhibited by PCBs (Table 1).
using the colorimetric method of Ellman et al. (1961). Absorbance The highest concentrations of residues were observed in S3
at 412 nm was recorded for samples and blanks by using a micro- (32.3 ± 11.3 ng g1 dw) and S1 (28.3 ± 5.7 ng g1 dw), while the
plate-reading UV-spectrophotometer (Synergy HT, BioTek). AChE lowest in S2 (13.1 ± 2.3 ng g1 dw). No significant difference was
activity was expressed in nmol min1 mg1 protein using a molar found among sampling sites. The 4,40 -homologues were the most
extinction coefficient of 13.6 mM1 cm1. representative of the contamination pattern for each sampling site:
CAT activity was measured on a UVIKON 933 spectrophotome- 4,40 -DDD was the most abundant compound, accounting on aver-
ter (BioTek) by the decrease in absorbance at 240 nm due to H2O2 age for the 36% of the RDDTs, followed by the 4,40 -DDE (29%)
consumption (extinction coefficient, e = 0.04 mM1 cm1), in a and 4,40 -DDT (4%) (Fig. 2C). In contrast, 2,40 -homologues were less
reaction solution containing 100 mM phosphate buffer, pH 7.4 abundant than 4,40 ones, with the exception of 2,40 -DDT, whose
Table 1
Mussels characteristics and concentrations of organo-halogenated compounds (ng g1 dw, mean ± SD), in their soft tissue.
Site S1 S2 S3 S4 S5
CI 1.2 ± 0.3 (b) 1.6 ± 0.3 (a) 1.4 ± 0.2 (ab) 1.3 ± 0.3 (ab) 1.5 ± 0.3 (ab)
LC (%) 14.1 ± 1.6 (a) 20.9 ± 1.2 (a) 13.7 ± 7.5 (a) 16.4 ± 0.3 (a) 14.0 ± 3.0 (a)
Pesticides
HCB 0.5 ± 0.1 (b) 0.7 ± 0.2 (b) 0.6 ± 0.1 (b) 0.1 ± 0.1 (a) 0.9 ± 0 (b)
Lindane <lod <lod <lod <lod <lod
cis chlordane <lod <lod <lod <lod <lod
trans chlordane <lod <lod <lod <lod <lod
Mirex <lod <lod <lod <lod <lod
2,40 DDE 2.0 ± 0.8 1.4 ± 1.2 1.5 ± 1.3 1.2 ± 0.8 0.7 ± 0
2,40 DDD nq nq nq nq nq
2,40 DDT 8.3 ± 0.9 3.3 ± 0.4 7.1 ± 4.3 5.1 ± 0.8 5.6 ± 4.1
4,40 DDE 6.7 ± 1.0 5.2 ± 1.7 10.9 ± 2.9 4.5 ± 0.2 5.5 ± 1.1
4,40 DDD 9.3 ± 3.9 3.3 ± 0.3 9.5 ± 2.3 12.3 ± 2.6 8.3 ± 4.0
4,40 DDT 2.0 ± 0.4 <lod 3.4 ± 0.9 <lod <lod
RDDTs 28.3 ± 5.7 (a) 13.1 ± 2.3 (a) 32.3 ± 11.3 (a) 23.1 ± 1.7 (a) 20.1 ± 9.0 (a)
ROCPs 28.8 ± 5.8 13.8 ± 2.4 33.0 ± 11.2 23.2 ± 1.7 21.0 ± 9.0
PCBs
CB-28 <lod <lod <lod <lod <lod
CB-52 5.1 ± 1.2 5.3 ± 0.6 7.4 ± 6.3 0.7 ± 0.6 1.3 ± 1.2
CB-101 10.8 ± 7.1 6.5 ± 2.3 12.9 ± 3.6 8.0 ± 1.3 10.7 ± 1.6
CB-118 4.5 ± 0.5 <lod 5.3 ± 3.2 2.3 ± 0.2 2.0 ± 0.8
CB-138 18.1 ± 11.1 11.7 ± 4.3 25.7 ± 2.4 17.8 ± 1.5 14.7 ± 1.8
CB-153 32.9 ± 20.4 32.0 ± 4.5 53.8 ± 1.0 35.3 ± 1.8 34.3 ± 2.0
CB-180 5.7 ± 4.5 3.8 ± 0.7 9.1 ± 1.2 7.4 ± 6.2 4.8 ± 0.6
CB-18 4.5 ± 0.5 3.8 ± 1.1 4.5 ± 0.7 2.3 ± 0.6 2.0 ± 0.2
CB-44 1.8 ± 0.6 2.4 ± 0.8 2.7 ± 0.4 1.3 ± 0.2 2.0 ± 0.9
CB-50 3.4 ± 1.2 3.5 ± 0.5 4.8 ± 0.3 2.2 ± 2.2 4.3 ± 1.7
CB-66 5.6 ± 3.2 4.9 ± 0.7 7.0 ± 2.4 5.4 ± 0.9 4.1 ± 0.5
CB-77 + 154 nq nq nq nq nq
CB-126 10.6 ± 3.7 9.6 ± 1.9 9.8 ± 0.8 7.8 ± 0.8 6.6 ± 1.7
CB-128 + 187 2.5 ± 1.2 <lod 4.5 ± 0.7 2.2 ± 0.7 2.0 ± 0.8
CB-195 1.4 ± 0.5 0.4 ± 0.2 2.3 ± 0.1 1.3 ± 0.3 1.1 ± 0.1
CB-201 1.4 ± 0.6 5.1 ± 0.4 0.8 ± 0.2 0.5 ± 0.2 <lod
RPCBs 108.4 ± 52.7 (a) 89.1 ± 14.3 (a) 150.6 ± 3.6 (a) 94.6 ± 5.8 (a) 89.7 ± 3.5 (a)
PBDEs
BDE-47 1.7 ± 0.4 0.8 ± 0.1 4.9 ± 0.1 3.4 ± 0.4 3.0 ± 0.3
BDE-99 <lod <lod <lod <lod <lod
nq: Not quantifiable.

<lod: below the limit of detection.
Values in the same row marked with different letters indicate significant differences at p < 0.05.
levels were higher than those of the parental compound 4,40 -DDT varied from 47.5 ± 3.2 ng g1 dw to 390.0 ± 57.3 ng g1 dw, with
in each station. HCB concentrations in mussels were absolutely an average concentration of 169.3 ng g1 dw. Maximum PAHs lev-
negligible, with average values of 0.56 ± 0.25 ng g1 dw (range els were found in site S3 (390.0 ± 57.3 ng g1 dw), the next highest
0.12 ± 0.08 – 0.87 ± 0.03 ng g1 dw) (Table 1). However significant in site S1 (141.1 ± 13.6 ng g1 dw) and site S4 (139.2 ± 6.9 ng g1
differences were observed between S4 and the remaining stations dw). Significant differences were observed between stations S1
(p < 0.05). Lindane, cis chlordane, trans chlordane and Mirex were and S2, and between S3 and the remaining stations (p < 0.05). Sig-
below the limit of detection. Accumulation of PBDEs was much nificant differences in PAH distribution can be seen in Table 2. Low
lower than that of PCBs and DDTs, with the highest value recorded molecular weight PAHs (2 + 3 rings) were predominant (53.5–
at the station S3 (4.9 ± 0.1 ng g1 dw). The only congener measured 69.3%) at sites S1, S2, S4 and S5, while 4 ring PAHs (>60%) were
above the detection limit was BDE47 (Table 1). Concentrations of mainly present at station S3. For all sites, there were less high
individual PAHs in soft tissues of mussels from the Bizerte lagoon molecular weight PAHs with 5 + 6 rings. Percentages ranged from
are shown in Table 2. Some PAH compounds, such as BeP, BaP, Per, 1.1% to 17.8%. The Phenanthrene/Anthracene (Phe/Ant) and Fluo-
and DA were absent in the studied samples, while some others ranthene/Pyrene (Flu/Pyr) ratios currently used to identify the ori-
such as Phe and Pyr were found at high concentrations. Phe, Pyr, gin of PAHs, are different according to the sampling site location
Ch, and Flu reached high concentrations at S3. Elevated levels of (Table 2). At site S5, PAHs are mainly petrogenic in origin (Phe/
Phe were also observed at S1 and S5. Total PAH concentrations Ant ratio > 10 and Flu/Pyr ratio 6 1), while the origins of those
A 100%
CB201
activity was positively correlated to pH and negatively correlated
90% CB195 to temperature. RPAHs and RPCBs were inversely correlated to
80% CB180 salinity. Lastly, RPCBs was positively correlated to the levels of
CB153 RPAHs. The PCA of biological (LC, CI), biochemical (AChE, CAT,
Relative amount
70%
CB138
60% GST), chemical (HCB, RPAHs, RPCBs, RDDTs) and environmental
CB126
50% CB118 (temperature, salinity, pH) data produced three principal compo-
40% CB101 nents that accounted for 98.7% of the total variance. The plot of
CB66 variable vectors for the first two principal components PC1 and
30%
CB52 PC2 that explained 91.9% of the total variance is shown in
20% CB50
CB44
Fig. 4A. The third component took into account only 6.7% and
10%
CB18 was therefore excluded, because it did not provide any significant
0%
S1 S2 S3 S4 S5 additional information. PC1 accounted for 73% of the total variance
and was driven on one side (positive part) by CAT, GST, PAHs, PCBs,
DDTs, and temperature and on the other side (negative part) by LC,
B 100%
90%
CI, AChE, Sal and pH. PC2 explained 19% of the total variance and
was correlated only with HCB on the negative part. The plot of
80%
Octa-Cl scores for different sites for the two principal components PC1
70%
Relative amount
Hepta-Cl and PC2 separated three groups of sites (Fig. 4B). S2 and S5 (First
60%
group), S1 and S4 (Second group) and the third group (S3).
50% Hexa-Cl
This study is (to our knowledge) the first time that chemical and
40% Penta-Cl
biological measurements have been combined in this way to mon-
30% Tetra-Cl
itor pollution in the Bizerte lagoon, using wild Mediterranean mus-
20% Tri-Cl sels (M. galloprovincialis) as an indicator organism. Statistical
10% comparisons of PAH body burden in mussel tissues showed signif-
0% icant differences between sites. More specifically, the level of con-
S1 S2 S3 S4 S5
tamination at S3 was higher and significantly different from all the
other sampling stations, suggesting a possible higher exposure of
C 100%
native bivalves to PAHs, while limited differences were found
90%
between the other sites. For all other investigated POPs (Table 1),
80% no significant difference was detected between locations. Stations
70% 2,4'-DDT
Relative amount
S1 and S3, where the highest PAH, PCB and DDT concentrations
60% 2,4'-DDE were measured, are located in the channel of Bizerte or close to
50% 4,4'-DDT the Haima River, respectively. These two highly populated areas
40% see heavy boat traffic, and are characterized by agricultural and
4,4'-DDD
30% industrial activities, including oil refineries and cement manufac-
4,4'-DDE
20% turing. Station S2 is also located in the channel, but PAH, PCB
10% and DDT concentrations in mussels were lower than those mea-
0% sured at stations S1 and S3. This can be explained by the sediment
S1 S2 S3 S4 S5 re-suspension phenomena, since sediments from the three stations
had different concentrations of POPs. PCBs and DDTs concentra-
Fig. 2. Profile of contamination of mussels from the Bizerte lagoon by (A) PCBs and
(B) different Cl-substituted PCBs and (C) DDTs. tions at stations S1, S2 and S3 were respectively 11.5 and
14.6 ng g1 dw, 0.4 and 1.8 ng g1 dw, 10.6 and 5.2 ng g1 dw
(Barhoumi et al., 2014a). For PAHs, the significant difference in
detected at the other stations are both petrogenic and pyrolitic concentrations between station S1 (141.1 ± 13.6 ng g1 dw) and
(Phen/Ant and Flu/Pyr ratios < 10 and >1, respectively). S3 (390.0 ± 57.3 ng g1 dw) can be explained by the position of sta-
Spatial variations of GST, CAT and AChE activities in gills of mus- tion S1 close to the sea, where there are significant water
sels are presented in Fig. 3. The GST activity in S3 exchanges (Barhoumi et al., 2014b). It should be noted that the
(372.7 nmol min1 mg1 protein) was significantly higher than the high values of total DDT residue levels at site S1 and S3 could be
activity registered at S2 and S5 sites (p < 0.05). No significant change a result of the discharge of untreated effluents from Bizerte City
was observed in GST activity in mussels from sites S1, S2, S4 and S5 and agricultural areas located upstream in the Haima River catch-
(277.2 to 348.7 nmol min1 mg1 protein) (Fig. 3A). AChE activity ment, transported and deposited in the downstream sediment.
ranged from 8.5 to 9.8 nmol min1 mg1 protein at sites influenced Historically, chemical analyses of POPs in mussels have not been
by anthropogenic activities (S1, S3 and S4) and from 12.6 to regularly monitored in the Bizerte lagoon, making it impossible
16 nmol min1 mg1 protein at S2 and S5 which are considered as to establish temporal trends for those contaminants. The levels of
reference sites (Fig. 3B). Significantly lower AChE activity was PCBs and DDTs measured in our study are within the same range
found in mussels at sites S1, S3, and S4 compared to the reference of concentrations determined in M. galloprovincialis collected from
site S2 (p < 0.01). No difference was observed in CAT activity Ría, NW Spain (Suárez et al., 2013). PAH levels are similar to those
between the five stations, with values ranging from 25.0 to found by other authors along the NW Mediterranean coast
28.1 lmol min1 mg1 protein (Fig. 3C). (Villeneuve et al., 1999), along the North coast of Tunisia
Relationships between chemical and biochemical parameters (Mzoughi and Chouba, 2012) and the NW Portuguese coast (Lima
are shown in Table 3. With regard to organic contaminants, GST et al., 2007). However, these PAH levels were lower than reported
and AChE displayed positive and negative correlations with RDDT, in mussels from areas affected by oil spills or tanker accidents,
respectively. CAT activity was inversely correlated to AChE activity such as the Prestige (Soriano et al., 2006), the Aegean Sea, at La
and CI levels. Also, AChE activity was inversely correlated to GST Coruña (Porte et al., 2000), and the Erika, in the Bay of Biscay
activity. RDDTs and GST activity were negatively correlated to (Tronczynski et al., 2004). HCB concentrations were also similar
pH and positively correlated to temperature. In contrast, AChE to those reported by Deudero et al. (2007) in several invertebrate
Table 2
PAH concentrations (ng g1 dw, mean ± SD) in the soft tissues of mussels (Mytilus galloprovincialis) collected at five sites from the Bizerte lagoon.
Site S1 S2 S3 S4 S5
Nap 5.7 ± 1.7 1.8 ± 1.4 7.3 ± 3.0 5.6 ± 1.7 3.0 ± 1.2
Acy <lod 2.0 ± 0.1 4.9 ± 0.1 <lod <lod
Ace <lod <lod 8.2 ± 1.0 <lod 8.0 ± 1.0
Fl 15.1 ± 2.9 5.5 ± 0.5 12.8 ± 3.7 14.2 ± 1.4 9.0 ± 7.9
DBT 10.4 ± 1.7 2.8 ± 0.8 11.6 ± 1.8 13.0 ± 0.9 6.6 ± 5.0
Phe 61.3 ± 8.1 18.9 ± 2.7 66.9 ± 6.2 57.5 ± 4.3 40.4 ± 29.7
Ant <lod 2.0 ± 0.1 <lod <lod 1.9 ± 0.1
Flu 5.8 ± 0.9 3.6 ± 0.5 73.7 ± 26.4 6.1 ± 4.3 12.3 ± 5.4
Pyr 12.9 ± 1.5 7.5 ± 1.9 102.9 ± 35.5 14.0 ± 3.7 11.9 ± 3.4
2,1BNT 1.6 ± 0.6 <lod 6.0 ± 2.7 1.8 ± 0.2 1.8 ± 0.2
BaA <lod <lod 13.9 ± 2.1 2.4 ± 0.4 2.0 ± 0.1
Ch 14.7 ± 2.0 2.5 ± 0.8 56.2 ± 2.0 13.5 ± 0.8 14.7 ± 1.3
BF 12.6 ± 1.8 0.6 ± 0.4 21.2 ± 4.4 9.1 ± 1.9 15.1 ± 2.0
BeP <lod <lod <lod <lod <lod
BaP <lod <lod <lod <lod <lod
Per <lod <lod <lod <lod <lod
DA <lod <lod <lod <lod <lod
IP <lod <lod 1.7 ± 0.7 0.6 ± 0.1 0.9 ± 0.1
BP 1.0 ± 0.1 0.3 ± 0.0 2.4 ± 0.8 1.4 ± 0.1 1.2 ± 0.1
RPAHs 141.1 ± 13.6 (b) 47.5 ± 3.2 (c) 390.0 ± 57.3 (a) 139.2 ± 6.9 (bc) 128.9 ± 37.7 (bc)
Flu/(Flu + Pyr) 0.31 ± 0.01 0.33 ± 0.04 0.42 ± 0.00 0.28 ± 0.08 0.50 ± 0.05
Ant/(Ant + Phe) nd 0.10 ± 0.01 nd nd 0.06 ± 0.03
Phe/Ant nd 9.57 ± 1.17 nd nd 20.94 ± 15.90
Flu/Pyr 0.45 ± 0.02 0.50 ± 0.09 0.71 ± 0.01 0.41 ± 0.18 1.00 ± 0.17
2 + 3 rings (%) 65.5 69.3 28.7 64.9 53.5
4 rings (%) 24.8 28.7 64.8 27.1 33.1
5 + 6 rings (%) 9.6 2.0 6.5 8.0 13.4
Values in the same row marked with different letters indicate significant differences at p < 0.05.
<lod: below the limit of detection.
BF = BbF + BjF + BkF.
Ch = Chry + Triph.
DA = DahA + DacA.
species from the Balearic Islands. Considering the toxicity of POPs approximate, and must be done with caution, depending on the
to animals, it seems important to rank the sampling sites according metabolisation of individual PAHs that are affected by species
to the concentrations of these compounds. Therefore, based on POP and trophic states.
concentrations determined in the whole body of mussels, the rank- PCB congener accumulation pattern found in mussels from the
ing is: S3 (high contamination) > S1 and S4 (moderate contamina- Bizerte lagoon are consistent with those reported by Suárez et al.
tion) > S2 and S5 (low contamination). According to the number of (2013). These authors found that mussels (M. galloprovincialis)
rings in the molecule, the predominant PAH compounds in mussels sampled in the Ría of Vigo accumulate mainly hexa and pentachlo-
collected from the Bizerte lagoon (exception of station S3) were: robiphenyls (71.3% and 18.6% respectively of the total PCBs). Sim-
2 + 3 ring PAHs > 4 ring PAHs > 5 + 6 ring PAHs. One reason for ilar percentages were also reported in clams (Binelli and Provini,
the greater number of 2 + 3 ring PAHs in mussels could be related 2003) and can be explained by slow biotransformation and excre-
to their uptake pathway. Indeed, the fewer number of rings PAH tion kinetics of congeners with higher degree of chlorination (Van
molecules haven, the more water soluble they are, meaning that den Brink and Bosveld, 2001). The most abundant PCBs were hexa-
they can be easily ingested through food consumption or through chlorobiphenyls PCB 153 and PCB 138. This indicates that mussels
the gill membrane. It could also be caused by the different of from the Bizerte lagoon were exposed predominantly to higher
decontamination kinetics of each individual PAH (Rantamaki, chlorinated PCB formulations. Congeners 153 and 138 are the main
1997). Fleming et al. (2004) and Rocher et al. (2006) similarly constituents of Aroclor 1254 and 1260 which were the most largely
found that low weight compounds (3–4 rings PAHs) are more bio- used commercial PCB mixtures in several countries (Ivanov and
accumulated in Mytilus sp. than 5 and 6 ring PAHs. In mussels from Sandell, 1992; Barakat et al., 2002). While the use of PCBs in Tuni-
station S3, 4 ring PAHs represented about 65% of total PAHs. The sia is not well established, PCBs in transformers, electrical equip-
significant differences in the pattern of PAHs between S3 and the ment and other industries is fairly common. Since the 1950s,
remaining stations could be explained by variability of sources, around 100 industrial sites have opened in the immediate vicinity
hydrodynamic conditions, turbidity, or the presence of organic of the lagoon, the biggest being the cement factory (1950) and ‘‘El
matter (river inputs) at different sampling sites. Molecular ratio Fouledh’’ steelworks (1965). From 1970 to 1985, SACEM company
calculation revealed that in the following stations S1, S2, S3 and settled in the Menzel Bourguiba area, manufactured approximately
S4, PAHs originated from both pyrolitic and petrogenic sources. 908 electrical transformers and imported 900 tonnes of PCBs
These stations are located near harbours (Bizerte and Menzel (APEK, 2005). The relative concentration of DDT and its metabo-
Abderahmen Harbours) and receive PAHs from petrogenic sources. lites, DDD and DDE, can be used as indicators of possible sources
Since these stations are located close to the cities of Bizerte and of pollution. Since DDT can be biodegraded in aerobic conditions
Menzel Abderahmen, urban discharges may also contribute to to DDE and in anaerobic conditions to DDD, ratio of
PAH inputs (mainly pyrolitic PAHs). Unlike other stations, S5 is less (DDE + DDD)/RDDT > 0.5 can be indicative of long-term weather-
urbanized and located far from any cities. This station probably ing (Zhang et al., 1999). In our study, ratios of (DDE + DDD)/RDDT
receives PAHs from petrogenic sources by atmospheric deposition ranged between 0.64 and 0.78, indicating that the degraded
and water current. The identification of PAH sources still remains metabolites formed a significant proportion of total DDT
A 450 ab a ab
was no recent input of technical DDT from the agricultural areas
into the Bizerte lagoon following their ban in 1984. Ameur et al.
400 (2013) reached the same conclusion for the aged input of DDTs
b
(nmol min-1 mg-1 prot)
350 b based on DDT concentration level in fish muscles. Environmental

300 conditions, such as salinity, temperature, pH and the amount of
250 organic particles may affect the concentration and distribution of
GST
200 chemical contaminants in the water column, sediment and biota

(Eggleton and Thomas, 2004). Organism physiology and tissue
150
composition, particularly lipid content, can also affect the concen-
100
tration and distribution of organic contaminants (Baumard et al.,
50
1999). In Mytilus sp., lipid content varies depending on exogenous
0 and endogenous factors such as food availability, energy reserves,
S1 S2 S3 S4 S5
and stage of reproduction (Ross et al., 2000). Suárez et al. (2013)
showed that the highest lipid content was observed in late winter,
B 25
a
prior to the main spawning period and in late spring and early
summer, coinciding with sexual rest and with an abundance of
(nmol min-1 mg-1 prot)
20 ab nutrients in the environment. This observation is concordant with

our study showing higher levels of lipids in late winter (February).
15
b No correlation between lipid content and organic contaminant lev-
AChE
b b
els in mussel tissues was observed. The differences in mussel con-
10
tamination levels between sampling sites cannot, therefore, be
explained by an increase in contaminant bioaccumulation due to
5 a high tissue lipid content. Baumard et al. (1999) and Thompson
et al. (1999) did not observe any relationship between lipid content
0 and concentrations of PAHs, PCBs or DDTs in several bivalve spe-
S1 S2 S3 S4 S5
cies collected from the Western Baltic Sea and the Arcachon Bay,
France. In this study, a significant negative correlation was
C 35 a
a
a a
observed between PCBs, PAHs and salinity, in a manner similar
a
30 to that previously described by other authors (Duursma et al.,
(µmol min-1 mg-1 prot)
1986; Suárez et al., 2013). This inverse relationship with salinity

25
could suggest that river inputs (Haima, Tinja, Gueniche, Garek
20 and Guenine Rivers) are the main entry of these pollutants into
CAT
15
the Bizerte lagoon. In addition, RDDTs was negatively correlated
to pH. This result can be explained by pH affecting the structure
10 of OCPs causing their biodegradation (Wenzel et al., 2002).
5 Eggleton and Thomas (2004) have shown that pH changes can
accelerate the desorption, partitioning, bacterial degradation and
0 oxidation of organic pollutants.
S1 S2 S3 S4 S5
While chemical analyses give a qualitative and quantitative dis-
Fig. 3. GST (A), AChE (B) and CAT (C) activities (mean ± SD) in the gills of mussels crimination of pollution between sampled sites, they do not provide
Mytilus galloprovincialis collected from the Bizerte lagoon. Different letters indicate any information about the possible effects on aquatic organisms.
significant differences between stations (Anova, N = 10, p < 0.05). Furthermore, measurements from only a few classes of contami-
nants are likely not representative of the global chemical stress
aquatic organisms are facing. Instead, the application of a suite of
compounds. Also, most values of DDD/DDE ratio were greater than sensitive and integrative biomarkers could clearly differentiate
unity, indicating that soft tissue in mussels was dominated by 4,40 - between healthy and stressed organisms (Brooks et al., 2011),
DDD, the product of anaerobic degradation of 4,40 -DDT. These allowing a more precise differentiation of pollution hazard and pos-
results clearly indicate an old DDT pollution and suggest that there sible health risk for exposed organisms. Considering the
Table 3
Pearson correlation coefficients among the different biological (CI, LC), biochemical (AChE, CAT, GST), chemical (HCB, RPAHs, RPCBs, RDDTs) and environmental (T °C, salinity,
pH) variables tested across sites (n = 5).
CI AChE CAT GST HCB RPAHs RPCBs RDDTs T (°C) Sal pH

LC 0.583 0.801 0.652 0.531 0.043 0.625 0.513 0.818 0.801 0.541 0.804
CI 1 0.820 0.948* 0.709 0.639 0.241 0.266 0.711 0.652 0.190 0.797
AChE 1 0.937* 0.884* 0.494 0.728 0.675 0.951* 0.880* 0.636 0.993***
CAT 1 0.811 0.701 0.461 0.413 0.815 0.722 0.363 0.876
GST 1 0.497 0.779 0.839 0.919* 0.890* 0.777 0.904*
HCB 1 0.018 0.033 0.268 0.124 0.068 0.413
RPAHs 1 0.959* 0.839 0.829 0.969** 0.768
RPCBs 1 0.833 0.850 0.992*** 0.733
RDDTs 1 0.982** 0.808 0.980**
Numbers in bold indicate significant correlations.

Asterisks indicate the signification of the correlations
*
p < 0.05.
**
p < 0.01.
***
p < 0.001.
1,5 3,0
A B
2,5
S4
1,0
2,0
Sal CAT
1,5
0,5 S1
1,0
PC2 (18.97%)
LC
PC2 (18.97%)
GST 0,5
0,0 pH DDTs
AChE T 0,0
PAHs
PCBs S2
-0,5
-0,5 S5
CI
-1,0
HCB
-1,0 -1,5 S3
-2,0
-1,5 -2,5
-1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 -5 -4 -3 -2 -1 0 1 2 3 4 5
PC1 (72.98%) PC1 (72.98%)
Fig. 4. Results of the PCA for the two principal components produced by biological (CI), biochemical (AChE, CAT, GST), chemical (HCB, RPAHs, RPCBs, RDDTs) and
environmental (T °C, salinity, pH) variables in mussels collected at five stations in the Bizerte lagoon. (A) Plot of variable vectors. (B) Plot of scores of different sites.
widespread presence of pro-oxidants in environmental mixtures, RDDTs. At stations S4 and S1, the inhibition of AChE activity in
and the well-known toxicity of reactive oxygen species (ROS), the mussels may be related to complex pollution inputs from both
application of oxidative stress biomarkers is recommended by industrial and domestic activities. In station S5, which is located
OSPAR in biomonitoring programs. Overall, mussel health seems far from urban and industrial sources of pollution, high AChE activ-
to be particularly affected by the whole contaminant mixture of ity was recorded, which could indicate a lower level of contamina-
the Bizerte lagoon. This is particularly evident for bivalves from tion of this site by POPs. The highest levels of AChE activity were
the most polluted site (S3), which showed significant increase of found at station S2. This high AChE activity level was similar to
GST activity compared to control (S2 and S5). GST enzyme activity those obtained by Mora et al. (1999) on M. galloprovincialis collected
has been suggested as a useful indicator of mussel exposure to from a low polluted site at the entrance of the Arcachon bay (south-
organic pollutants (Fitzpatrick and Sheehan, 1993). Although some west France). When compared with data from literature, the values
field studies have reported a significant induction of GST activity in from our study are in the same range as those recorded by Serafim
mussels exposed to organic compounds (Moreira and Guilhermino, et al. (2011) in mussels collected from the South coast of Portugal
2005; Rocher et al., 2006), others have detected little or no GST and by Dellali et al. (2001b) in mussels collected from the Bizerte
response (Fitzpatrick et al., 1997; De Luca-Abbott et al., 2005). lagoon. No significant difference in CAT activity was observed
The activation of GST activity in bivalves from S3 may be due to between stations. Lionetto et al. (2003) also found no difference
the high levels of DDTs accumulated in their soft tissue as con- in CAT activity levels between mussels collected from ‘‘clean’’ and
firmed by the significant positive correlation between RDDTs and polluted stations on the Salento Peninsula (Italy). The lack of differ-
GST. Our data was consistent with the findings of Parolini et al. ence between sampled stations may be caused by exposure to low
(2013) who noticed a significant correlation between induction of or moderate oxidative stress and/or by biochemical adaptation of
GST and RDDTs in soft tissues from Zebra mussel (Dreissena mussels to environmental conditions. Previous field (Regoli, 1998)
polymorpha) exposed to an organochlorine mixture. In this regard, and laboratory studies (Cajarville et al., 1997) reported ‘‘biochemi-
GST has been found to play an important role in the conjugation cal adaptation’’ in the CAT response of M. galloprovincialis chroni-
of electrophilic compounds with glutathione, and these reactions cally exposed to pollutants. Other enzymes such as superoxide
are of vital importance for the detoxification of xenobiotics dismutases, glutathione peroxidase, and glutathione reductase
(Kaaya et al., 1999). AChE is an enzyme involved in recycling of ace- were also shown to be involved in the anti-oxidant response of pol-
tylcholine, a neuromediator of cholinergic nerves. Numerous stud- lutant-exposed bivalves (Di Salvatore et al., 2013; Parolini et al.,
ies reported AChE inhibition by various neurotoxic compounds 2013). Such enzymatic activities were not recorded in our study.
such as organophosphate and carbamate pesticides (Bocquené Compared with data from the same region and from other parts
and Galgani, 1998). The inhibition of this enzyme was also related of the world, values of catalase activity obtained in this study are
to exposure to many other chemical groups, e.g. metals, hydrocar- similar to those reported by Dellali et al. (2001a) in mussels from
bons and detergents (Guilhermino et al., 1998; Elumalai et al., the Bizerte lagoon (station S2) and by Bocchetti and Regoli (2006)
2002). AChE inhibition has thus been suggested as indicative of in mussels from the Adriatic Sea. Biological responses are influ-
general neurotoxic stress (Lehtonen et al., 2006). Our results enced by natural environmental factors. Previous studies using bio-
showed decreased AChE activity not only at sites in areas influenced markers pointed out the need to incorporate the effects of abiotic
by agricultural practices where pesticide contamination could be (temperature, salinity, diet, etc.) and biotic factors (reproduction
expected (S3 and S5) but also at sites in areas receiving urban and cycle, growth, age, sex, etc.) to correctly interpret biomarker
industrial waste (S1 and S4), where a wide variety of pollutants responses (Smolders et al., 2002; Damiens et al., 2004). Mussel pop-
are found (Dellali et al., 2001b; Trabelsi and Driss, 2005; ulations in the study area were subjected to different water temper-
Barhoumi et al., 2014a,b). In fact, negative effects of organochlori- atures, salinity, pH, and food availability. Temperature and salinity
nated pesticides on AChE activity can be hypothesized in our study patterns along the Bizerte lagoon are influenced by two main cur-
for mussels from station S3, as confirmed by the significant nega- rents, marine currents from the Mediterranean Sea, which enter
tive correlation between AChE activity and the concentration of the lagoon through the Bizerte channel and increase water salinity
from 33 psu (Zaouali, 1974) to 36 psu (Aïssa, 1991), and fresh water Acknowledgment
currents from the Ichkeul Lake, which enter the lagoon through the
Tinja River. The condition index (CI) of mussels collected in the Biz- The authors gratefully acknowledge the Ministry of Higher
erte lagoon was high in all stations, with the exception of S2, con- Education and Scientific Research, Tunisia for its financial support.
firming that nutrient availability in the Bizerte lagoon is high. It
References
was shown that CI is strongly linked to the availability of food
resources (Mourgaud et al., 2002). In addition, a significant negative Aïssa, P., 1991. Ecologie des nématodes libres de la lagune de Bizerte: écologie et
relationship was observed between CI and CAT. Similarly, CAT biocénotique. Thèse de Doctorat, University de Tunis, Tunisie.
activity was shown to be significantly decreased in the clam R. Ameur, W.B., Trabelsi, S., El Megdiche, Y., Ben Hassine, S., Barhoumi, B., Hammami,
B., Eljarrat, E., Barceló, D., Driss, M.R., 2013. Concentration of polychlorinated
decussatus when food was available (Roméo et al., 2003). When
biphenyls and organochlorine pesticides in mullet (Mugil cephalus) and sea
food is regularly available, animals have better physiological condi- bass (Dicentrarchus labrax) from Bizerte Lagoon (Northern Tunisia).
tions and their background level of anti-oxidant defences may be Chemosphere 90, 2372–2380.
ANPE, 1990. Diagnostic préliminaire pour l’Etude de l’Equilibre Ecologique du lac de
lower. Due to their sessile condition and filter feeding habits,
Bizerte. GIC-NNEA-TECI, ANPE, Tunisie.
bivalves might also be particularly sensitive to changes in environ- Barakat, A.O., Kim, M., Qian, Y., Wade, T.L., 2002. Organochlorine pesticides and
mental conditions, such as temperature, pH and salinity. It has been PCBs residues in sediments of Alexandria Harbour, Egypt. Mar. Pollut. Bull. 44,
suggested that salinity fluctuation may influence the capacity of 1421–1434.
Barhoumi, B., Le Menach, K., Dévier, M.-H., El megdiche, Y., Hammami, B., Ameur,
mussels to handle oxidative stress (Prevodnik et al., 2007). Fluctu- W.B., Hassine, S.B., Cachot, J., Budzinski, H., Driss, M.R., 2014a. Distribution and
ations in biochemical responses have also been reported under ecological risk of polychlorinated biphenyls (PCBs) and organochlorine
hypo saline stress in M. galloprovincialis (Hamer et al., 2008). Never- pesticides (OCPs) in surface sediments from the Bizerte lagoon Tunisia.
Environ. Sci. Poll. Res. 21, 6290–6302.
theless, the range of salinity variations detected in our study area Barhoumi, B., Le Menach, K., Devier, M.-H., Ben Ameur, W., Etcheber, H., Budzinski,
seems to be insufficient to elicit any disturbance in the biochemical H., Cachot, J., Driss, M.R., 2014b. Polycyclic aromatic hydrocarbons (PAHs) in
responses. Temperature was positively and negatively correlated surface sediments from the Bizerte Lagoon, Tunisia: levels, sources, and
toxicological significance. Environ. Monit. Assess. 186, 2653–2669.
with GST, and AChE activity, respectively. The influence of temper- Baumard, P., Budzinski, H., Garrigues, P., 1997. Analytical procedure for the analysis
ature on enzyme activities has been reported in different bivalve of PAHs in biological tissues by gas chromatography coupled to mass
species (Dellali et al., 2001b; Pfeifer et al., 2005). Although the range spectrometry: application to mussels. Fresenius J. Anal. Chem. 359, 502–509.
Baumard, P., Budzinski, H., Garrigues, P., Dizer, H., Hansen, P.D., 1999. Polycyclic
of temperatures recorded in this study could not be regarded as aromatic hydrocarbons in recent sediments and mussels (Mytilus edulis) from
representative of high thermal stress (very little temperature differ- the Western Baltic Sea: occurrence, bioavailability and seasonal variations. Mar.
ence observed between sites), they can be considered to be an addi- Environ. Res. 47, 17–47.
Bayne, B.L., 1989. Measuring the biological effects of pollution: the mussel watch
tional stress to pollutant exposure. In addition, it is important to
approach. Water Sci. Technol. 21, 1089–1100.
take into account that gills are in direct contact with water, mean- Beji, O., 2000. Les ressources vivantes exportables du lac de Bizerte: Etat actuel et
ing that epithelium cells can be directly affected by water temper- potentialités (première partie). Bull. Inst. Nat. Sci. Technol. Mer Salammbô 27,
ature fluctuations. Indeed, Regoli and Principato (1995) have shown 45–60.
Ben Garali, A., Ouakad, M., Gueddari, M., 2010. Contamination of superficial
that gills are more susceptible to changes in environmental param- sediments by heavy metals and iron in the Bizerte lagoon, northern Tunisia.
eters and more prone to oxidative stress than other tissues. GST and Arab. J. Geosci. 3, 295–306.
AChE activities were also shown to be correlated to pH. Ben Ben Khedher, S., Jebali, J., Kamel, N., Banni, M., Rameh, M., Jrad, A., Boussetta, H.,
2012. Biochemical effects in crabs (Carcinus maenas) and contamination levels
Khedher et al. (2012) have also documented a significant negative in the Bizerta Lagoon: an integrated approach in biomonitoring of marine
correlation between pH and GST activity in gills and digestive gland complex pollution. Environ. Sci. Pollut. Res. 20, 2616–2631.
of crabs (Carcinus maenas), collected from the Bizerte lagoon. Binelli, A., Provini, A., 2003. POPs in edible clams from different Italian and
European markets and possible human health risk. Mar. Pollut. Bull. 46, 879–
PCA allowed the identification of variables that had a major 886.
contribution to the total variance of the data set. Out of all the Bocchetti, R., Regoli, F., 2006. Seasonal variability of oxidative biomarkers,
studied parameters, POP concentrations appeared as driving fac- lysosomal parameters, metallothioneins and peroxisomal enzymes in the
Mediterranean mussel Mytilus galloprovincialis from Adriatic Sea.
tors for spatial variations, revealing three groups of stations. The Chemosphere 65, 913–921.
first one includes mussels from stations S2 and S5 with low PAHs, Bocquené, G., Galgani, F., 1998. Biological effects of contaminants: cholinesterase
PCBs, and DDTs body burden, and high CI and AChE activity. The inhibition by organophosphate and carbamate compounds. Proc. ICES Tech.
Mar. Environ. Sci. 22, 12.
second encompasses stations S1 and S4, with moderate POP levels
Bodin, N., Budzinski, H., LeMénach, K., Tapie, N., 2009. ASE extraction method for
and CI, high CAT activity, and low AChE activity. Lastly, mussels simultaneous carbon and nitrogen stable isotope analysis in soft tissues of
from station S3 are characterized by high PCBs, PAHs, and DDTs aquatic organisms. Anal. Chim. Acta 643, 54–60.
body burden, moderate CI, and induction of GST and AChE Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal.
inhibition. Biochem. 772, 248–264.
To conclude, this study provides concordant information on the Brooks, S., Harman, C., Zaldibar, B., Izagirre, U., Glette, T., Marigómez, I., 2011.
presence of contaminants and the resulting biological responses on Integrated biomarker assessment of the effects exerted by treated produced
water from an onshore natural gas processing plant in the North Sea on the
mussels in the Bizerte lagoon. The battery of parameters applied in mussel Mytilus edulis. Mar. Pollut. Bull. 62, 327–339.
this study included GST, CAT, and AChE activities in gills and POPs, Budzinski, H., Letellier, M., Thompson, S., LeMenach, K., Garrigues, P., 2000.
and lipid quantification in the whole body of mussels. This study Combined protocol for the analysis of polycyclic aromatic hydrocarbons
(PAHs) and polychlorobiphenyls (PCBs) from sediments using focussed
allowed three groups of stations within the Bizerte lagoon to be microwave assisted (FMW) extraction at atmospheric pressure. Fresenius J.
identified according to the chemical contamination levels and bio- Anal. Chem. 367, 165–171.
logical responses in mussels. The inter-station variations of bio- Cajarville, M.P., Orbea, A., Marigomez, I., Cancio, I., 1997. Peroxisome proliferation in
the digestive epithelium of mussels exposed to the water accommodated
marker responses were associated with chemical contamination fraction of three oils. Comp. Biochem. Phys. C 117, 233–242.
and fluctuations in abiotic (pH, salinity and temperature) and bio- Claiborne, A., 1985. Catalase activity. In: Greenwald, R.A. (Ed.), Handbook of
tic (condition index) parameters. These results demonstrate that Methods for Oxygen Radical Research. CRC Press, Boca Raton, pp. 283–284.
Clark, R.B., 2001. Marine Pollution. Oxford University Press, Oxford.
M. galloprovincialis constitutes a suitable bioindicator species for
Cravo, A., Lopes, B., Serafim, A., Company, R., Barreira, L., Gomes, T., Bebianno, M.J.,
pollution biomonitoring in the Bizerte lagoon and in other coastal 2009. A multibiomarker approach in Mytilus galloprovincialis to assess
marine areas of the Mediterranean sea. This work represents the environmental quality. J. Environ. Monit. 11, 1673–1686.
first step towards a new monitoring program that is being devel- CRDA, 1998. Commissariat régional au développement agricole de Bizerte.
Statistiques annuelles de l’arrondissement de la pêche. Bizerte.
oped for M. galloprovincialis in the Bizerte lagoon to evaluate both CRDA, 2002. Commissariat régional au développement agricole de Bizerte.
the contamination and environmental risks of POPs. Statistiques annuelles de l’arrondissement de la pêche. Bizerte.
Damiens, G., His, E., Gnassia-Barelli, M., Quiniou, F., Roméo, M., 2004. Evaluation of Mahmoud, N., Dellali, M., Bour, M.E., Aissa, P., Mahmoudi, E., 2010. The use of Fulvia
biomarkers in oyster larvae in natural and polluted conditions. Comp. Biochem. fragilis (Mollusca: Cardiidae) in the biomonitoring of Bizerta lagoon: a
Phys. C 138, 121–128. mutimarkers approach. Ecol. Indic. 10, 696–702.
Dellali, M., Romeo, M., Aissa, P., 2001a. Suivi annuel de l’activité catalase chez des Monserrat, J.M., Martínez, P.E., Geracitano, L.A., Amado, L.L., Martins, C.M.G., Pinho,
moules et des palourdes originaires de la lagune de Bizerte. Oceanol. Acta 24, G.L.L., Chaves, I.S., Ferreira-Cravo, M., Ventura-Lima, J., Bianchini, A., 2007.
263–271. Pollution biomarkers in estuarine animals: critical review and new
Dellali, M., Gnassia Barelli, M., Romeo, M., Aissa, P., 2001b. The use of perspectives. Comp. Biochem. Phys. C 146, 221–234.
acetylcholinesterase activity in Ruditapes decussatus and Mytilus Mora, P., Michel, X., Narbonne, J.-F., 1999. Cholinesterase activity as potential
galloprovincialis in the biomonitoring of Bizerta lagoon. Comp. Biochem. Phys. biomarker in two bivalves. Environ. Toxicol. Pharmacol. 7, 253–260.
C 130, 227–235. Moreira, S.M., Guilhermino, L., 2005. The use of Mytilus galloprovincialis
De Luca-Abbott, S.B., Richardson, B.J., McClellan, K.E., Zheng, G.J., Martin, M., Lam, acetylcholinesterase and glutathione S-transferases activities as biomarkers of
P.K.S., 2005. Field validation of antioxidant enzyme biomarkers in mussels environmental contamination along the Northwest Portuguese Coast. Environ.
(Perna viridis) and clams (Ruditapes philippinarum) transplanted in Hong Kong Monit. Assess. 105, 309–325.
coastal waters. Mar. Pollut. Bull. 51, 694–707. Mourgaud, Y., Martinez, E., Geffard, A., Andral, B., Stanisiere, J.Y., Amiard, J.C., 2002.
Deudero, S., Box, A., March, D., Valencia, J.M., Grau, A.M., Tintore, J., Calvo, M., Metallothionein concentration in the mussel Mytilus galloprovincialis as a
Caixach, J., 2007. Organic compounds temporal trends at some invertebrate biomarker of response to metal contamination: validation in the field.
species from the Balearics, Western Mediterranean. Chemosphere 68, 1650– Biomarkers 7, 479–490.
1659. Müller, A., Björklund, E., Von Holst, C., 2001. On-line clean-up of pressurized liquid
Di Salvatore, P., Calcagno, J.A., Ortíz, N., Ríos de Molina, M.D.C., Sabatini, S.E., 2013. extracts for the determination of polychlorinated biphenyls in feedingstuffs and
Effect of seasonality on oxidative stress responses and metal accumulation in food matrices using gas chromatography–mass spectrometry. J. Chromatogr. A
soft tissues of Aulacomya atra, a mussel from the South Atlantic Patagonian 925, 197–205.
coast. Mar. Environ. Res. 92, 244–252. Mzoughi, N., Chouba, L., 2012. Heavy metals and PAH assessment based on mussel
Duursma, E.K., Nieuwenhuize, J., Van Liere, J.M., 1986. Partitioning of caging in the North Coast of Tunisia (Mediterranean Sea). Int. J. Environ. Res. 6,
organochlorines between water, particulate matter and some organisms in 109–118.
estuarine and marine systems of the Netherlands. Neth. J. Sea Res. 20, 239–251. OSPAR, 2000. Quality Status Report: Region IV – Bay of Biscay and Iberian Coast.
Eggleton, J., Thomas, K.V., 2004. A review of factors affecting the release and OSPAR Commission, London.
bioavailability of contaminants during sediment disturbance events. Environ. Parolini, M., Pedriali, A., Binelli, A., 2013. Chemical and biomarker responses for site-
Int. 30, 973–980. specific quality assessment of the Lake Maggiore (Northern Italy). Environ. Sci.
Ellman, G.L., Courtney, K.O., Anders, V., Featherstone, R.M., 1961. A new and rapid Pollut. Res. 20, 5545–5557.
colorimetric determination of acetylcholinesterase activity. Biochem. Pfeifer, S., Schiedek, D., Dippner, J.W., 2005. Effect of temperature and salinity on
Pharmacol. 7, 88–95. acetylcholinesterase activity, a common pollution biomarker, in Mytilus sp.
Elumalai, M., Antunes, C., Guilhermino, L., 2002. Single metals and their mixtures on from the south-western Baltic Sea. J. Exp. Mar. Biol. Ecol. 320, 93–103.
selected enzymes of Carcinus maenas. Water Air Soil Pollut. 141, 273–280. Porte, C., Biosca, X., Pastor, D., Solé, M., Albaigés, J., 2000. The Aegean Sea oil spill. 2.
Fitzpatrick, P.J., O’Halloran, J., Sheehan, D., Walsh, A.R., 1997. Assessment of a Temporal study of the hydrocarbons accumulation in Bivalves. Environ. Sci.
glutathione S-transferase and related proteins in the gill and digestive gland of Technol. 34, 5067–5075.
Mytilus edulis (L.), as potential organic pollution biomarkers. Biomarkers 2, 51– Prevodnik, A., Gardeström, J., Lilja, K., Elfwing, T., McDonagh, B., Petrovic, N.,
56. Tedengren, M., Sheehan, D., Bollner, T., 2007. Oxidative stress in response
Fitzpatrick, P.J., Sheehan, D., 1993. Separation of multiple forms of glutathione S- to xenobiotics in the blue mussel Mytilus edulis L.: evidence for variation
transferase from the blue mussel, Mytilus edulis. Xenobiotica 23, 851–861. along a natural salinity gradient of the Baltic Sea. Aquat. Toxicol. 82,
Fleming, H.P., Adalberto, J., Asencio, P., Gutierrez, E., 2004. Polycyclic aromatic 63–71.
hydrocarbons in sediments and mussels of Corral Bay, south central Chile. J. Rantamaki, P., 1997. Release and Retention of selected polycyclic aromatic
Environ. Monit. 6, 229–233. hydrocarbons (PAH) and their methylated derivatives by the common mussel
Goldberg, E.D., 1975. The mussel watch. A first step in global marine monitoring. (Mytilus edulis) in the brackish water of the Baltic sea. Chemosphere 35,
Mar. Pollut. Bull. 6, 111. 487–502.
Goldberg, E.D., 1986. The mussel watch concept. Environ. Monit. Assess. 7, 91–103. Regoli, F., 1998. Trace metals and antioxidant enzymes in gills and digestive gland
Guilhermino, L., Barros, P., Silva, M.C., Soares, A.M.V.M., 1998. Should the use of of Mediterranean mussel Mytilus galloprovincialis. Arch. Environ. Contam.
cholinesterases as a specific biomarker for organophosphate and carbamate Toxicol. 34, 48–63.
pesticides be questioned? Biomarkers 3, 157–163. Regoli, F., Principato, G., 1995. Glutathione, glutathione-dependent and antioxidant
Habig, W.H., Pabst, M.J., Jakoby, B., 1974. Glutathione-S-transferase. The first enzymes in mussel, Mytilus galloprovincialis, exposed to metals under field and
enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. laboratory conditions: implications for the use of biochemical biomarkers.
Hamer, B., Jaksic, Z., Pavicic-Hamer, D., Perik, L., Medakovic, D., Ivankovic, D., Aquat. Toxicol. 31, 143–164.
Pavicic, J., Zilberberg, C., Schröred, H.C., Müller, W.E.G., Smodlaka, N., Batel, R., Rocher, B., Le Goff, J., Peluhet, L., Briand, M., Manduzio, H., Gallois, J., Devier, M.H.,
2008. Effect of hypoosmotic stress by low salinity acclimation of Mediterranean Geffard, O., Gricourt, L., Augagneur, S., Budzinski, H., Pottier, D., Andre, V.,
mussels Mytilus galloprovincialis on biological parameters used for pollution Lebailly, P., Cachot, J., 2006. Genotoxicant accumulation and cellular defence
assessment. Aquat. Toxicol. 89, 137–151. activation in bivalves chronically exposed to waterborne contaminants from
ICES, 2008. Report of the Fourth ICES/OSPAR Workshop on Integrated Monitoring of the Seine River. Aquat. Toxicol. 79, 65–77.
Contaminants and their Effects in Coastal and Open Sea Areas (WKIMON IV), 5– Roméo, M., Gharbi-Bouraoui, S., Gnassia-Barelli, M., Dellali, M., Aissa, P., 2006.
7 February 2008. ICES, Copenhagen, Denmark. ICES CM 2008/ACOM: 49, pp. 71. Responses of Hexaplex (Murex) trunculus to selected pollutants. Sci. Total
Ivanov, V., Sandell, E., 1992. Characterization of polychlorinated biphenyl isomers in Environ. 359, 135–144.
sovol and thrichlorodiphenyl formulations by high-resolution gas Roméo, M., Hoarau, P., Garello, G., Gnassia-Barelli, M., Girard, J.P., 2003. Mussel
chromatography with electron capture detection and high-resolution gas transplantation and biomarkers as useful tools for assessing water quality in the
chromatography-mass spectrometry techniques. Environ. Sci. Technol. 26, NW Mediterranean. Environ. Pollut. 122, 369–378.
2012–2017. Ross, P.S., Ellis, G.M., Ikonomou, M.G., Barret-Lennards, L.G., Addison, R.F., 2000.
Kaaya, A., Najimi, S., Ribera, D., Narbonne, J.F., Moukrim, A., 1999. Characterization Genetic diversity and metal tolerance of two marine species: a comparison
of glutathione S-transferases (GST) activities in Perna perna and Mytilus between populations from contaminated and reference sites. Mar. Pollut. Bull.
galloprovincialis used as a biomarker of pollution in the Agadir Marine Bay 44, 671–679.
(South of Morocco). Bull. Environ. Contam. Toxicol. 62, 623–629. Serafim, A., Lopes, B., Company, R., Cravo, A., Gomes, T., Sousa, V., Bebianno, M.J.,
Khessiba, A., Hoarau, P., Gnassia-Barelli, M., Aissa, P., Roméo, M., 2001. Biochemical 2011. A multi-biomarker approach in cross-transplanted mussels Mytilus
Response of the mussel Mytilus galloprovincialis from Bizerta (Tunisia) to galloprovincialis. Ecotoxicology 20, 1959–1974.
chemical pollutant exposure. Arch. Environ. Contam. Toxicol. 40, 222–229. Smolders, R., Bervoets, L., De Boeck, G., Burst, R., 2002. Integrated condition indices
Khessiba, A., Roméo, M., Aissa, P., 2005. Effects of some environmental parameters as a measure of whole effluent toxicity in zebrafish (Danio rerio). Environ.
on catalase activity measured in the mussel (Mytilus galloprovincialis) exposed Toxicol. Chem. 21, 87–93.
to lindane. Environ. Pollut. 133, 275–281. Soriano, J.A., Viñas, L., Franco, M.A., Gonzalez, J.J., Ortiz, L., Bayona, J.M., Albaiges, J.,
Lehtonen, K.K., Schiedek, D., Köhler, A., Lang, T., Vuorinen, P.J., Förlin, L., Barŝiené, J., 2006. Spatial and temporal trends of petroleum hydrocarbons in wild mussels
Pempkowiak, J., Gercken, J., 2006. The BEEP project in the Baltic Sea: overview from the Galician coast (NW Spain) affected by the Prestige oil spill. Sci. Total
of results and outline for a regional biological effects monitoring strategy. Mar. Environ. 370, 80–90.
Pollut. Bull. 53, 523–537. Suárez, P., Ruiz, Y., Alonso, A., San Juan, F., 2013. Organochlorine compounds in
Lima, I., Moreira, S.M., Osten, J.R.-V., Soares, A.M.V.M., Guilhermino, L., 2007. mussels cultured in the Ría of Vigo: accumulation and origin. Chemosphere 90,
Biochemical responses of the marine mussel Mytilus galloprovincialis to 7–19.
petrochemical environmental contamination along the North-western coast of Tapie, N., Budzinski, H., Le Menach, K., 2008. Fast and efficient extraction methods
Portugal. Chemosphere 66, 1230–1242. for the analysis of polychlorinated biphenyls and polybrominated diphenyl
Lionetto, M.G., Caricato, R., Giordano, M.E., Pascariello, M.F., Marinosci, L., Schettino, ethers in biological matrices. Anal. Bioanal. Chem. 391, 2169–2177.
T., 2003. Integrated use of biomarkers (acetylcholinesterase and antioxidant Thompson, S., Budzinski, H., Garrigues, P., Narbonne, J.F., 1999. Comparison of PCB
enzymes activities) in Mytilus galloprovincialis and Mullus barbatus in an Italian and DDT distribution between water-column and sediment-dwelling bivalves
coastal marine area. Mar. Pollut. Bull. 46, 324–330. in Arcachon Bay, France. Mar. Pollut. Bull. 38, 655–662.
Thompson, S., Budzinski, H., LeMenach, K., Letellier, M., Garrigues, P., 2002. Multi- Villeneuve, J.-P., Carvalho, F.P., Fowler, S.W., Cattini, C., 1999. Levels and trends of
residue analysis of polycyclic aromatic hydrocarbons, polychlorobiphenyls, and PCBs, chlorinated pesticides and petroleum hydrocarbons in mussels from the
organochlorine pesticides in marine sediments. Anal. Bioanal. Chem. 372, NW Mediterranean coast: comparison of concentrations in 1973/1974 and
196–204. 1988/1989. Sci. Total Environ. 237, 57–65.
Trabelsi, S., Driss, M.R., 2005. Polycyclic aromatic hydrocarbons in superficial Wenzel, K., Manz, M., Hubert, A., Schuurmann, G., 2002. Fate of POPs (DDX,
coastal sediments from Bizerte Lagoon, Tunisia. Mar. Pollut. Bull. 50, 344–359. HCHs, PCBs) in upper soil layers of pine forests. Sci. Total Environ. 286,
Tronczynski, J., Munschy, C., Héas-Moisan, K., Guiot, N., Truquet, I., Olivier, N., Men, 143–154.
S., Furaut, A., 2004. Contamination of the Bay of Biscay by polycyclic aromatic Zaouali, J., 1974. Les peuplements dans les biocoenoses lagunaires tunisiennes.
hydrocarbons (PAHs) following the T/V ‘‘Erika’’ oil spill. Aquat. Living Resour. Etude de la biologie de l’espèce pionnière Cerastoderma glaucum Poiret. Thèse
17, 243–259. de Doctorat, University de Caen, France.
Tunisia Country Situation Report Association for Environmental Protection in Zhang, G., Min, Y.S., Mai, B.X., Sheng, G.Y., Fu, J.M., Wang, Z.S., 1999. Time trend of
Kerkennah (APEK) Tunisia, October 2005. <http://www.ipen.org>. BHCs and DDTs in a sedimentary core in Macao estuary, southern China. Mar.
Van den Brink, N.W., Bosveld, B.T.C., 2001. PCB concentrations and metabolism Pollut. Bull. 39, 326–330.
patterns in common terns (Sterna hirundo) from different breeding colonies in
The Netherlands. Mar. Pollut. Bull. 42, 280–285.

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Marine Pollution Bulletin xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Marine Pollution Bulletin

Assessment of pollution in the Bizerte lagoon (Tunisia)

nq: Not quantiﬁable.

350 b based on DDT concentration level in ﬁsh muscles. Environmental

200 chemical contaminants in the water column, sediment and biota

20 ab nutrients in the environment. This observation is concordant with

1986; Suárez et al., 2013). This inverse relationship with salinity

CI AChE CAT GST HCB RPAHs RPCBs RDDTs T (°C) Sal pH

Numbers in bold indicate signiﬁcant correlations.

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