Marine Pollution Bulletin 74 (2013) 477–489

Contents lists available at SciVerse ScienceDirect

Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Baseline

Factors contributing to heavy metal accumulation in sediments

and in the intertidal mussel Perna perna in the Gulf of Annaba (Algeria)
Bourhane-Eddine Belabed a, Xavier Laffray b, Amel Dhib c, Mouna Fertouna-Belakhal d, Souad Turki c,
Lotfi Aleya b,⇑
a
Département des Sciences de la Mer, Faculté des Sciences, Université Badji Mokhtar d’Annaba, BP 12, Algeria
b
Université de Franche-Comté, Laboratoire de Chrono-Environnement, UMR CNRS 6249, 1, Place Leclerc, F-25030 Besançon cedex, France
c
Institut National des Sciences et Technologies de la Mer (INSTM), La Goulette Port de Pêche 2060, Tunisia
d
Institute of Fisheries and Aquaculture, Errimel, 7080 Bizerta, Tunisia

a r t i c l e i n f o a b s t r a c t

Keywords: This paper presents the results of a seasonal survey of heavy metals accumulated in sediments and in the
Mussels soft parts of the body of the mussel Perna perna at four stations in the Gulf of Annaba (Algeria). Pooled soft
Perna perna tissues from 10 mussels representing the entire range of sizes were digested in nitric acid. Statistical anal-
Heavy metals ysis reveals a significant seasonal effect on all the measured metals, the highest values being recorded in
Sediment
winter. With the exception of Cr, the levels for all metals were significantly higher in the east, at the out-
Annaba Gulf
Algeria
let of the Seybouse River, than at all other monitoring stations. The study also shows that north-western
waters are subject to a significantly lower degree of heavy metal pollution than elsewhere in the gulf.
Levels were nevertheless within the limits of public health standards.
The results confirm the usefulness of P. perna as a bioindicator for heavy metal pollution.
Ó 2013 Elsevier Ltd. All rights reserved.

In many coastal and estuarine ecosystems bivalves, especially
mussels, are useful biological indicators of organic and inorganic
pollutants, including heavy metals, since they exhibit a broad set
of major criteria for successful biomonitoring (Phillips, 1976a,
1976b; Fowler and Oregioni, 1976). In addition to their abundance
and wide geographical distribution in all temperate marine re-
gions, mussels offer interesting traits such as a sessile nature in
the adult stage (e.g. reflecting site-specific conditions), their mode
of feeding (filter-feeders), or consumption by humans (risk assess-
ment of seafood chain transfer and population health (Casas et al.,
2008). Extremely tolerant of fluctuations in salinity, oxygen con-
centration and temperature (Rainbow, 1995), the majority of mus-
sels also present a broad range of tolerance to toxicity, associated
with a capacity to accumulate contaminants in tissues in relation
to surrounding water levels without major metabolic transforma-
tions (Goldberg, 1975; Chase et al., 2001). These advantageous
traits had led to the use of bivalve molluscs, particularly mussels,
as sentinel organisms for marine pollution studies in many areas
of the world (Luten et al., 1986; Stronkhorst, 1992; Boening,
1999; Neuberger-Cywiak et al., 2003) and noticeably in major gov-
ernment marine monitoring programmes (World Mussel Watch,
WMW) which began in the 1970s (Goldberg, 1975, 1986; O’Connor
et al., 1994; Cantillo, 2003; Melwani et al., 2013). Although high
⇑ Corresponding author. Tel.: +33 03 81 66 57 64; fax: +33 03 81 66 57 97.
E-mail address: lotfi.aleya@univ-fcomte.fr (L. Aleya).
levels of heavy metals occur naturally (e.g. deep oceanic water
upwelling, run-off from mineralised areas in estuarine environ-
ments), anthropogenic pollution may also be related to industrial
production, agriculture or mining processing, fossil fuel and urban
waste burning, all of which are capable of significant contamina-
tion of coastal waters throughout many parts of the world (Usero
et al., 2005). Marine ecosystems are highly vulnerable to pollution
due to their recipient position from continents, but their size and
complexity often makes it difficult to evaluate the real impact of
anthropogenic interference. For instance, heavy metal pollution is
a serious worldwide problem for wildlife conservation due to the
metals’ toxic effect on the biota (Islam and Tanaka, 2004). They in-
duce potential sub-lethal population altering dynamics for most of
the species in contact with their components (e.g. alteration of
fecundity or survival, teratogenic effects on offspring). Risk of
exposure to the heavy metals of aquatic ecosystems is particularly
high and a great deal of attention is still paid to their mechanisms
in toxicology and their pernicious effects on deep ecosystem func-
tioning (Grosell and Brix, 2005). The brown mussel Perna perna is
widely distributed in Algerian coastal waters and therefore its
use as a biomonitoring tool is of major scientific interest. However,
as indicated by Rajagopal et al. (2006), this mussel is characterised
by relatively narrow incipient thermal limits, with limited temper-
ature adaptation ability and a significantly lower combined tem-
perature–salinity tolerance than that of Perna viridis. In addition,
since many factors such as season and physiological state or

0025-326X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.marpolbul.2013.06.004
478 B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489
animal size may affect heavy metal bioaccumulation in molluscs
(Roméo and Gnassia-Barelli, 1988), the sampling strategy of mon-
itoring schemes must be carefully designed in order to take into ac-
count P. perna size and sex so as to minimise the effects of these
confounding factors. We therefore conducted a seasonal survey
of nine heavy metals (Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn) accu-
mulated in sediments and in the soft parts of P. perna at four sta-
tions in the Gulf of Annaba (eastern Algeria) and evaluated their
potential impact on the biota.
Located in the extreme eastern section of the Algerian coast,
600 km from Algiers and 100 km from the Tunisian border, the
Annaba Gulf is a wide-mouthed bay, open to the Mediterranean
Sea on the north, extending from Cape Rosa (8°150 E and 36°580 N)
in the East, to Cape de Garde (7°470 E and 36°580 N) in the West,
with a distance of 40 km in between. The Seybouse River in the
south-east, the second longest river in Algeria (with a catchment
basin of about 6470 km2), and the Mafrag River in the east drain
into the gulf (Belabed et al., 2013). The two rivers receive agricul-
tural water discharges from cereal farming, market gardening and
arboriculture, and domestic releases from important conurbations
(Khelifi-Touhami et al., 2006) and untreated sewage (Abdennour
et al., 2000) contributing to sediment contamination by heavy met-
als. Moreover, discharges from industries, which settle in the lower
part of the Meboudja River, the final tributary of the Seybouse Riv-
er, are important source points of heavy metals measured in the
Annaba Gulf superficial sediments. The industry on the Seybouse/
Meboudja plain is represented by the largest integrated steel pro-
duction site of the Maghreb region (Arcelor Mittal, El Hadjar), and
three other major industrial complexes: Chaïba (mechanical pro-
duction site, cement works, glass factory, paper mills), Pont Bou-
chet (plastics and biomedical manufacturing, mechanical repair
and battery recycling sites) and Allélick (milk products (ORELait)
and metallic equipment (Ferrovial) manufacturing plants (Belabed
et al., 2013)).
The location has a Mediterranean climate, with a mean annual
temperature of 18 °C and rainfall ranging from 650 to 1000 mm,
mainly distributed in winter (60–70% of total precipitation). Sum-
mers are fairly hot and dry, and winters cold and wet (Debièche,
2002). The gulf is subjected to a dominant sea-breeze with a speed
Fig. 1. Map of the four sampling stations and of the main processes governing the marine dynamics inside the Annaba Gulf (Algeria).
of up to 6 m s1 from the north–north-east (30%), and, to a lesser
extent, from the north (13%) and the west (10%) (Debièche,
2002). The maximal depth of the Gulf is 65 m. The main processes
governing the Annaba Gulf marine dynamics are described in
Fig. 1. The principal water circulation along the Annaba Gulf coast
consists of a permanent current (speed varying from 0.50 to
1 m s1) flowing eastward along the Algerian coast, which derives
from the Atlantic Ocean waters (AW) flowing into the Mediterra-
nean Sea through the narrow Strait of Gibraltar. Operating as a sur-
face costal jet, the Algerian coastal current, is associated with
important upwelling processes along the North African coast
(Arnone et al., 1990). This permanent current enters the Annaba
Gulf near Cap de Garde (NW Annaba coast) and flows eastward to-
wards the north-eastern part of the Bay (Rosa Cape). East of Cap de
Garde, the current splits into strong residual currents flowing east-
ward along the Annaba coast, inducing effective dispersion of Oued
Seybousse freshwater inflows into the Annaba Gulf coastal waters.
Moreover, near the shoreline, a major water current flows in a cir-
cular pattern, favouring the mixing of fresh and marine waters.
The samplings were conducted over 1 year (2006–2007) during
both the dry (05-28-06, 08-27-06, 10-29-06) and the wet period
(11-26-06 and 01-28-07) at four stations located in the western
part of the gulf (Fig. 1):
– Sidi Salem Beach (36°520 02.000 N and 007°460 26.600 E), located in
the eastern part of the study area, close to the outlet of the Sey-
bouse River which receives agricultural, industrial (from iron,
steel and fertiliser plants) and urban waste from the west valley
of the city of Annaba (Abdennour et al., 2000) which is Algeria’s
most industrialised region.
– Sunrise Beach (36°540 33.900 N and 007°460 20.900 E), located near
Annaba Harbour, also receives urban waste.
– Saint Cloud Beach (36°550 22.200 N and 007°450 50.200 E) receives
urban waste.
– La Kyenne Beach (36°580 02.000 N and 007°460 24.000 E), located on
the north-western coast of the Gulf of Annaba, about 7 km north
of the Annaba district. This last station was chosen as it repre-
sents an area relatively free of urban interference and its water
movements are different from those of the other three stations.
 B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489
Sediment samples were taken near the mussel collecting points.
For heavy metal analysis, samples (1000 g) were collected from 5
cores, 4 cm deep and 5 cm wide, extracted with an Eckmann grab
from the top 15 cm layer to a depth of over 10 m and stored at
4 °C until analysis. Sampling tools were washed and dried with
deionised water before the next sample was taken. Mature adult
mussels were scraped by hands from depths ranging between
the surface and 10 m, placed in plastic bags, properly packed and
immediately put into an insulated box with ice cubes to be trans-
ported to the laboratory for further preparation and analysis. In or-
der to minimise natural variability, for both sexes, a mussel sample
was prepared from 25 or more individuals representing the avail-
able size range (35–40 mm) existing in the study area (equal to
0.5–1.5 g weight range). In the laboratory, the samples were imme-
diately frozen to 20 °C before heavy metal analysis.
Sediment and mussel samples were prepared using analytical
grade reagents (Carlo Erba). For Annaba sediments: a representa-
tive portion of sediment samples collected in the Annaba Gulf sta-
tions were treated with an excess of 10% HCl for carbonate removal
before the determination of the Total Organic Matter content
according to Parker’s method (1983). Triplicate sediment samples
were oven-dried at 80 °C for 24 h and transferred into a pre-
weighed container and weighed (A) using an analytical balance.
Samples were then heated in a Thermolyne 1400 muffle furnace
at 450 °C for 4 h to remove organic matter. After cooling in a dry
box, the burn residue was weighed (B) and organic matter percent-
ages were determined as: %TOM = 100  (A  B)/A  100. The water
content of the sediment was determined by the weight lost during
drying. For analysis, sediment samples were air-dried in the labo-
ratory at room temperature, homogenised and ground into fine
powder using a mortar and pestle and then passed through a poly-
propylene sieve (200 lm pore-size mesh). A small amount of sed-
iment material was then dry-sieved into a 63 lm pore-size mesh
to separate the clay/silt size fraction. Grain size fraction <63 lm
was determined and sediment samples were stored in a polythene
container before digestion and analysis. After each sediment sam-
ple, the mortar, pestle and sieve were cleaned with 10% redistilled
HNO3 and rinsed with reagent grade water. Each sediment sample
(0.5 g DW) was then digested with aqua regia at 95 °C in a micro-
processor-controlled digestion box for 2 h (DIN ISO 11466 1997).
Prior to analysis the solution was diluted 1:10 with deionised
water. For Annaba mussel tissues: 20-selected mussels (10 males
and 10 females) from each sampling station were defrosted at
room temperature and subsequently analysed for heavy metals.
Mussels were rinsed with distilled water and dissected at room
temperature to remove the byssus thread and to shuck tissues
from the shell (Chiu et al., 2000). Before tissue preparation and
analysis, mussel shell length (mean size of 10-mussels:
38.6 ± 1.4 mm) was measured with a pair of vernier calipers to
0.1 mm. The wet weight (mean weight of 10-mussels:
10.4 ± 1.2 g) was individually determined to 0.01 g, using an elec-
tronic balance. As significant differences in heavy metal amounts
could be found with maturation stage evolution, only specimens
in stage III of gonadal maturation were selected for analysis
(Lunetta, 1969). Pooled-mussel tissues were minced using sharp
stainless steel scissors pre-rinsed in hexane, then homogenised
using a plastic spatula and freeze-dried. An aliquot of the freeze-
dried soft tissues from the different collected mussels was with-
drawn and oven-dried at 105 °C for 24 h to calculate, at constant
dry weight, its water percentage (Mo and Neilson, 1994). To
achieve homogenised soft tissue samples, the freeze-dried mussels
were homogenised once again with a mixer mill before storage in
acid-washed glass vials at room temperature until analysis. Freeze-
dried tissue (approximately 0.5 g DW) was digested in sealed Tef-
lon vessels with 10 ml of HNO3 for 2 h at room temperature and
479
subsequently at 95 °C in a microwave digestion system (CEM Cor-
poration, MDS-2100) according to standard procedures (Robisch
and Clark, 1993). After cooling, volumes were adjusted by bi-dis-
tilled water to 100 ml.
Heavy metals in the digested samples were determined by Atom-
ic Absorption Spectrophotometry (AAS 680 Shimadzu model) with
either an air–acetylene flame (Zn, Cu, Ni, Cr, Mn, Fe), a graphite fur-
nace (Cd, Pb) or GF-AAS (also known as Electrothermal Atomic
Absorption Spectrometry (ETAAS)), depending on the elements to
be analysed (UNEP/FAO/IAEA/IOC, 1984b). The operational parame-
ters and matrix modifiers were those recommended by the manu-
facturer. Total mercury was determined according to UNEP/FAO/
IAEA/IOC, 1984a by the cold vapour technique associated with atom-
ic fluorescence with a Perkin–Elmer FIMS-400 system (SnCl2 as
reducing agent) (Chiffoleau et al., 2003). All elements were subse-
quently expressed in mg kg1 dry weight. Detection limits for heavy
metals in sediment and mussel samples for cadmium (Cd), copper
(Cu), chromium (Cr), iron (Fe), mercury (Hg) manganese (Mn), nickel
(Ni), lead (Pb) and zinc (Zn) were 0.02, 0.03, 0.02, 0.04, 0.01, 0.04,
0.06, 0.06, and 0.05 mg kg1 respectively. For quality assurance
and quality control, blanks and Standard Reference Materials
(SRM) were analysed after every 10 sample injections, or at 2-h
intervals. The accuracy of the method was checked by several mea-
surements on the standard reference materials of the International
Agency for Atomic Energy (IAAE): ERM-CE 278 (mussel tissue: Myti-
lus edulis), SRM-1566b (Oyster tissue: Crassostrea virginica), NBS
1646 (Estuarine river sediment), SL-1 (Lake sediment) and Soil-5
(Soil). Recoveries were above 90% for all the heavy metals measured.
Results are presented in Table 1.
Basic hydrological parameters of surface coastal waters – tem-
perature, salinity, concentration of dissolved oxygen, turbidity (as
contents of suspended particulate matter (SPM)) – were recorded
simultaneously at the same sampling stations as the molluscs.
Temperature, salinity, and dissolved oxygen were measured in
the field with a WTW Universal Pocket Meter MultiLine P4, while
water turbidity was measured in laboratory according to stan-
dard methods recommended for marine waters (Kramer et al.,
1994).
The gametogenic cycle of P. perna mussels inside the Annaba
Gulf was determined in the present study. At each sampling, about
10 molluscs were randomly chosen and fixed with 4% formalde-
hyde solution for gonad development study. The appearance of
the gonad and reproductive cells was observed under microscope
in order to determine the reproductive status of the sampled mus-
sels. According to Keck et al. (1975), gonad conditions were classi-
fied into five main categories: Stage I – immature (ranking
stage = 1), Stage II – developing (ranking stage = 2), Stage III – ripe
(ranking stage = 3), Stage IV – spawning (ranking stage = 4), and
Stage V – spawning/resting (ranking stage = 0). Gonad index (GI)
was then determined according to Chipperfield (1953). Sex ratio
was also determined for each sampling station.
Data were expressed as mean ± standard deviation (SD). For sta-
tistical analysis, one-way analysis of variance (ANOVA) with a Tu-
key post-test was used to evaluate statistically significant
differences in heavy metal concentrations in sediment and in mus-
sel tissues between stations for each sampling season. Then, two-
way analysis of variance using stations and season as factors was
also used to determine their effects on heavy metal concentration
in sediments as well as in P. perna soft body tissues. A Pearson cor-
relation coefficient analysis was performed considering all samples
and elements to obtain a general overview of the relationship be-
tween heavy metal concentration in sediment and in P. perna tis-
sues. In all tests, the significance level for differences in critical
values was set to p-value <0.05. Statistical analysis of results was
performed with R 2.15.0 (R Development Core Team, 2011).
 480 B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489

Water characteristics of each station in the Annaba Gulf are pre-

Concentrations of heavy metals in ERM-CE 278 (mussel tissue: Mytilus edulis), SRM-1566b (American Eastern Oyster tissue: Crassostrea virginica), in SRM-1646a (Estuary sediment), IAEA-SL-1 (Lake sediment) and IAEA-SL-5 (Agricultural

867.24 ± 46.18

126.42 ± 37.79
0.834 ± 0.092
sented in Table 2. Hydrological conditions of surface water in the

29.76 ± 3.42
79.81 ± 5.97

17.19 ± 9.76

376.54 ± 9.88
4.62 ± 0.26
1.54 ± 0.08
coastal zone showed considerable seasonal variations for tempera-
Measured

ture and dissolved oxygen, while salinity, pH and turbidity varied

significantly but to a lesser extent. On the basis of the temporal dis-
tribution patterns of the environmental variables measured, two
Agricultural soil

main periods of different hydrographical characteristics can be dis-

tinguished: from late spring through late summer (May through
4.45 ± 0.19
IAEA-SL-5

28.9 ± 2.8
77.1 ± 4.7

368 ± 8.2
852 ± 37

129 ± 26
Certified

September) and from early autumn through early spring (October

0.79a

through April). Water temperature showed considerable seasonal

1.5a

13a

variations (Table 2) with maximal values during late spring/late

summer months (25.35 ± 0.38 °C in station 1, in August). Water
temperature decreased to 15–16 °C at the end of October to rela-
107.09 ± 11.17

352.28 ± 18.47

228.04 ± 14.08
0.132 ± 0.065
34.03 ± 6.39

49.13 ± 7.47
37.29 ± 8.95
6.54 ± 0.27
0.26 ± 0.06

tively cool values in autumn and winter (ranging from

Measured

11.05 ± 0.28 to 15.00 ± 0.31 °C in November and January according

to stations). As water temperature increased salinity varied greatly
between seasons (between 35.39 ± 0.69 and 39 ± 1.95), increasing
in summer (maximum values observed during the spring/summer
periods (39.00 ± 1.9 in May)), and then decreasing during the wet
Lake sediment

0.260 ± 0.050

season from October through January. Similarly, dissolved oxygen

6.74 ± 0.17
IAEA-SL-1

37.7 ± 7.4
44.9 ± 8.0
30.0 ± 5.6

concentrations and pH varied during the four seasons, with the

346 ± 16
Certified

223 ± 10
104 ± 9

lowest concentration of dissolved oxygen recorded in the warmest

0.13a

waters in May and August (26 ± 1.30% and 37 ± 1.92% respectively

at Sidi Salem station). The pH increased during the dry seasons
(from early spring to early autumn), and decreased during the
235.12 ± 3.24

rainy winter season. Furthermore, no significant spatial differences

41.23 ± 1.73

11.91 ± 1.54
49.42 ± 1.94
10.08 ± 0.29
0.15 ± 0.01

2.01 ± 0.05
Measured

were observed for salinity, whilst important variations were ob-

served for dissolved oxygen concentration and turbidity among
n.r.

n.r.

stations. Moreover, water temperature showed significant spatial

variations with a light decrease along the gulf shore from east to
Estuary sediment

west, i.e. from station 1 to 4.

2.008 ± 0.039
0.148 ± 0.007

The available size of the P. perna mussels collected at the four

10.01 ± 0.34
SRM-1646a

234.5 ± 2.8

stations ranged in length from 35 to 40 mm and in weight from

11.7 ± 1.2
48.9 ± 1.6
40.9 ± 1.9
Certified

0.5 to 1.5 g. Gonad development followed an annual cycle with a

n.r.

n.r.

spring/summer peak activity (from May to August), and a period

of inactivity from autumn to late-winter (October to January).
The major spawning occurred in summer (50% of the population
1435.16 ± 39.24

were classified stage IV). Gametogenesis then diminished consid-

207.34 ± 7.17
0.038 ± 0.002

erably from autumn to late-winter. 20% of the mussels remained

71.52 ± 1.86

18.71 ± 0.34
2.54 ± 0.09

1.08 ± 0.11
0.31 ± 0.01
Measured

in Stage III (ripe stage) during the study. The percentage of contri-
Non-certified values for concentrations of certain elements in IAEA-SL1 and IAEA-SL2.

bution of the sexes appeared to be skewed in favour of males,

n.r.

which represented 50–60% of the samples, while females made

up 30–40%. 5–10% were not identifiable for the sex ratio determi-
nation (Fig. 2).
0.0371 ± 0.0013

The fine-grained material (<63 lm) represented about 30% of

0.308 ± 0.009
Oyster tissue
SRM-1566b

the sediment samples collected inside the gulf (excepted for La Ky-
205.8 ± 6.8
2.48 ± 0.08

1.04 ± 0.09

1424 ± 46
71.6 ± 1.6

18.5 ± 0.2
Certified

enne Beach station; 51.9%). La Kyenne Beach station excepted

(8.5%), the Total Organic Matter content of sediment (%TOM) col-
n.r.
soil); average ± standard deviation, values in mg/kg dry weight.

lected ranged from 18.0% to 19.5%. The same pattern was observed
for water content with 32.4% mean value for stations 1–3
(68.6 ± 1.7 for station 4). Spatial and seasonal variations of toxic
0.198 ± 0.005

83.33 ± 1.91

element (Cd, Cr, Cu Fe, Hg, Mn, Ni, Pb, and Zn) concentrations mea-
9.52 ± 0.28

7.73 ± 0.27

2.01 ± 0.11
0.35 ± 0.02
0.81 ± 0.07
Measured

sured in the <63 lm grain size fraction of the upper layer of the
sediments of the gulf’s four sampled stations are summarised in
n.r.

n.r.

Table 3. As demonstrated by the results of the 2-way ANOVA per-

formed to investigate the effects of season and station on heavy
metal concentrations in the sediment, sampling locality had signif-
Mussel tissue

0.348 ± 0.007

0.196 ± 0.009
ERM-CE278

icant effects on all of the studied elements (Table 3). Considering

9.45 ± 0.13

7.69 ± 0.23
0.78 ± 0.06

2.00 ± 0.04
83.1 ± 1.7
Certified

toxic metal levels, a concentration decrease was generally ob-

served from station 1 (highest concentration) to station 4 (lowest
n.r.

n.r.

concentrations, outside of the gulf and far from the urbanised

zone,) for all seasons. When all heavy metals are considered, sta-
tion comparison showed that the lowest values were recorded in
Element

the sediments of station 4. In contrast, Cr concentrations in the

Table 1

Mn

sediments of stations 2 and 3 (415 and 390 mg kg1 DW) were

Hg
Cu
Cd

Zn
Pb
Ni
Fe
Cr

higher (p < 0.01) than those of stations 1 (120 mg kg1 DW) and
 B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489 481

Table 2
Seasonal and spatial physical water quality variations for the four sites within the Annaba Gulf (N = 3).

Seasons Station 1 Station 2 Station 3 Station 4 2-Way ANOVA

Temperature (°C)
Spring 24.85 ± 0.37a 23.50 ± 0.46b 23.40 ± 0.39b 22.30 ± 0.43c Station 27.43***
Summer 25.35 ± 0.38a 24.40 ± 0.41ab 24.70 ± 0.42ab 24.20 ± 0.45b Season 2866***
Fall 16.00 ± 0.45a 15.70 ± 0.35a 15.70 ± 0.30a 15.75 ± 0.36a Interaction 3.090**
Winter (early) 12.50 ± 0.35a 11.75 ± 0.25ab 11.68 ± 0.26b 11.05 ± 0.28b
Winter (late) 15.00 ± 0.31a 14.85 ± 0.36a 14.55 ± 0.35a 14.35 ± 0.34a
Salinity
Spring 36.50 ± 0.83a 38.20 ± 0.91ab 38.00 ± 0.90ab 39.00 ± 0.95b Station 1.518 ns
Summer 38.58 ± 0.96a 38.52 ± 0.94a 38.57 ± 0.97a 37.99 ± 0.92a Season 23.32***
Fall 35.90 ± 0.84a 36.75 ± 0.76a 36.55 ± 0.83a 37.97 ± 0.91a Interaction 1.804 ns
Winter (Early) 36.40 ± 0.79a 35.90 ± 0.83a 35.89 ± 0.76a 35.80 ± 0.75a
Winter (Late) 36.05 ± 0.85a 35.40 ± 0.71a 35.39 ± 0.69a 35.90 ± 0.84a
pH
Spring 8.80 ± 0.22a 8.43 ± 0.21ab 8.37 ± 0.28ab 7.91 ± 0.18b Station 41.23***
Summer 8.64 ± 0.23a 8.00 ± 0.21b 7.88 ± 0.19b 7.86 ± 0.18b Season 88.69***
Fall 7.75 ± 0.21a 7.34 ± 0.20ab 7.38 ± 0.18ab 7.05 ± 0.17b Interaction 0.83 ns
Winter (early) 7.80 ± 0.17a 7.29 ± 0.18b 7.15 ± 0.16b 6.93 ± 0.18b
Winter (late) 7.55 ± 0.16a 7.22 ± 0.16ab 7.25 ± 0.17ab 6.96 ± 0.15b
Dissolved oxygen (%)
Spring 26.00 ± 1.30a 39.00 ± 1.90b 36.00 ± 1.80b 60.00 ± 1.90c Station 194.4***
Summer 37.00 ± 1.90a 40.00 ± 2.00a 40.50 ± 2.10a 41.00 ± 2.10a Season 638.3***
Fall 48.20 ± 2.40a 47.00 ± 2.30a 48.50 ± 2.40a 92.00 ± 2.20b Interaction 24.64***
Winter (early) 75.00 ± 3.80a 86.00 ± 4.00b 88.00 ± 4.10b 102.00 ± 4.80c
Winter (late) 70.00 ± 3.10a 77.00 ± 3.90ab 76.00 ± 3.60ab 85.00 ± 4.10b
Turbidity
Spring 2.20 ± 0.11a 2.40 ± 0.12a 2.70 ± 0.14a 8.00 ± 0.40b Station 2228***
Summer 1.80 ± 0.11a 2.00 ± 0.13ab 2.30 ± 0.16b 6.00 ± 0.29c Season 65.20***
Fall 1.70 ± 0.09a 2.00 ± 0.11ab 2.20 ± 0.13b 5.20 ± 0.23c Interaction 17.47***
Winter (early) 1.50 ± 0.15a 1.80 ± 0.11a 1.90 ± 0.09a 6.20 ± 0.27b
Winter (late) 1.80 ± 0.11a 1.90 ± 0.08a 2.00 ± 0.12a 6.50 ± 0.25b
**
P<0.01.
***
P<0.001.

dates are shown in Table 3. If concentrations were enhanced for all

Fig. 2. Sex ratio of mussels collected at the four sites of the Annaba Gulf during the
study.
4 (90 mg kg1 DW). Cu concentrations were also slightly higher at
Sunrise (station 2) compared to Sidi Salem (station 1) (p < 0.05).
Concerning seasonal trends, the mean values of the five collecting
elements during winter (early and late-winter), no statistically sig-
nificant variations were observed between the five sampling dates
(2-way ANOVA, Table 3). Contrast in seasonal concentrations was
also determined by calculating the ratio (E) of heavy metal levels
measured during the wet and dry season (Table 4). The results of
the 2-way ANOVA considering the wet/dry season contrast shows
that both season and station had significant effects on heavy metal
concentrations (except for Cr and Cu) (Table 4). For each element,
toxic metal values peaked during the wet season, likely associated
with increased inputs via run-off from the catchment basin, as
indicated by E-ratio values of up to 1. Thus, sediments at stations
1 (Sidi Salem), 2 (Sunrise) and 3 (Saint Cloud) were relatively en-
riched in toxic metals during the wet season with greater varia-
tions in the vicinity of the Seybouse River inlet and Annaba
Harbour even though dilution processes may have occurred. As ex-
pressed by the E-ratio, a slight decrease in the dry/wet season hea-
vy metal concentration variations in sediments was observed from
station 1 to station 3. In contrast, higher toxic metal concentrations

Table 3
Metal concentrations in sediment samples (mg kg1 DW) and results of 2-way ANOVA testing the effects of site, season and interaction between the two factors on heavy metal
concentration in sediment.

Cd Cr Cu Fe Hg Mn Ni Pb Zn
Station 1 2.62 ± 0.44a 120.09 ± 14.36a 60.32 ± 4.81a 4590.11 ± 370.33a 0.10 ± 0.01a 425.16 ± 49.13a 51.63 ± 2.86a 186.19 ± 17.08a 301.17 ± 35.24a
Station 2 1.52 ± 0.23b 415.16 ± 50.19b 90.24 ± 4.13b 1750.04 ± 65.97b <DL b 220.26 ± 23.12b 44.62 ± 1.63b 175.26 ± 13.27a 90.22 ± 9.84b
Station 3 0.9 ± 0.1c 390.04 ± 36.22b 15.01 ± 0.64c 1420.23 ± 46.14c <DL b 175.42 ± 11.31c 42.14 ± 0.83b 98.19 ± 6.03b 75.26 ± 5.29c
Station 4 <DL d 90.27 ± 2.07c <DL d 1300.16 ± 27.06d <DL b 101.28 ± 4.12d 17.03 ± 0.04c 10.04 ± 0.02c 190.31 ± 2.03d
Station 239.7*** 200.8*** 2152*** 700.8*** 616.4*** 343.1*** 936.9*** 599.9*** 407.0***
Season 2.476 ns 0.4447 ns 1.463 ns 1.656 ns 0.7610 ns 1.930 ns 1.879 ns 1.489 ns 2.123 ns
Interaction 1.366 ns 0.2317 ns 1.660 ns 0.8927 ns 0.7610 ns 1.826 ns 1.255 ns 1.244 ns 1.450 ns
***
P<0.001.
482 B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489

during the dry season, induced by important summer anthropo-
genic source contributions, implied values as low as 1 for the E-ra-
tio, as for Cu and Pb at station 2 or for Cd, Mn, and Ni at station 3
(Ni at station 4). However, the small annual variations measured in
toxic metal concentrations in the Annaba Gulf sediments are
underscored by the E-ratio values near 1.
Chemical contamination in the sediments was evaluated by
comparison with the sediment quality guideline proposed by the
United States Environmental Protection Agency (USEPA) whose
criteria are shown in Table 5. The sediments of all stations under
investigation are considered unpolluted by Cd, but heavily polluted
by Cr. Only stations 1 and 2 are evaluated as heavily polluted by Cu,
while the others belong to the non-polluted sediment class. Only
station 1 sediments are classified as moderately polluted by Mn
(other stations classified as non-polluted). The same pattern was
observed for Ni with sediments defined as moderately to heavily

Table 4
Ratio (E) of sediment metal concentrations in wet/dry season and results of 2-way ANOVA testing the effects of site, season and interaction between the two factors on heavy
metal concentration in sediment.

Cd Cr Cu Fe Hg Mn Ni Pb Zn
Station 1 1.300 1.219 1.140 1.107 1.154 1.208 1.083 1.158 1.166
Station 2 1.105 1.022 0.986 1.055 / 1.002 1.019 0.999 1.026
Station 3 0.999 1.026 1.021 1.019 / 0.998 0.997 1.009 1.050
Station 4 / 1.002 / 1.017 / 1.014 0.999 1.001 0.999
Station 294.3*** 264.0*** 2440*** 951.8*** 777.6*** 433.5*** 1114*** 718.3*** 441.9***
Season 10.63** 1.006 ns 4.130 ns 9.061** 9.600** 9.564** 7.094* 5.280* 6.850*
Interaction 6.639** 0.2000 ns 5.995** 4.403* 9.600*** 9.106*** 4.798** 4.888** 4.967**
*
P<0.05.
**
P<0.01.
***
P<0.001.

Table 5
EPA guidelines for sediments with maximum acceptable limits (lg g1) of some trace heavy metals.

USEPA guidelines Annaba Gulf stations

Not polluted Moderately polluted Heavily polluted Station 1 Station 2 Station 3 Station 4
Cd – – >6 2.2–3.1 1.3–1.7 0.8–1.0 <DL
Cr <25 25–75 >75 103–135 356–466 345–432 87–92
Cu <25 25–50 >50 54.9–66.2 85.7–94.8 14.3–15.6 14.3–15.6
Mn <300 300–500 >500 366–476 192–243 161–188 96–105
Ni <20 20–50 >50 48.6–54.4 42.6–46.1 41.1–42.8 41.1–42.7
Pb <40 40–60 >60 164–204 158–189 89–104 9.5–10.5

Table 6
Trace metal concentrations in P. perna tissues for the studied stations and seasons.

Mean ± SD Cd Cr Cu Fe Hg Mn Ni Pb Zn
All seasons taken into account
Station1 0.51 ± 0.11a 1.85 ± 0.11a 26.74 ± 2.47a 932.04 ± 12.95a 0.159 ± 0.031a 22.28 ± 1.34a 8.99 ± 2.27a 1.68 ± 0.15a 358.96 ± 5.4a
Male 0.46 ± 0.10 1.80 ± 0.08 25.61 ± 2.02 926.05 ± 10.78 0.143 ± 0.03 21.60 ± 1.11 7.93 ± 1.92 1.61 ± 0.11 356.24 ± 4.68
Female 0.56 ± 0.10 1.90 ± 0.11 27.86 ± 2.41 938.02 ± 12.36 0.175 ± 0.025 22.95 ± 1.23 10.04 ± 2.14 1.75 ± 0.15 361.67 ± 4.73
Station 2 0.22 ± 0.02b 2.25 ± 0.09b 17.91 ± 1.71b 421.99 ± 13.05b 0.02 ± 0.008b 16.08 ± 0.24b 9.03 ± 2.35a 0.79 ± 0.07b 158.03 ± 8.35b
Male 0.22 ± 0.02 2.20 ± 0.07 16.94 ± 1.46 415.18 ± 11.02 0.016 ± 0.008 15.95 ± 0.18 7.69 ± 1.69 0.75 ± 0.05 153.34 ± 7.92
Female 0.22 ± 0.02 2.30 ± 0.09 18.89 ± 1.37 428.79 ± 11.42 0.025 ± 0.005 16.22 ± 0.22 10.36 ± 2.17 0.82 ± 0.07 162.72 ± 5.85
Station 3 0.22 ± 0.02b 2.44 ± 0.03c 20.92 ± 1.12c 454.95 ± 4.97c 0.01 ± 0.001c 14.39 ± 0.42c 5.73 ± 0.79b 0.58 ± 0.08c 183.95 ± 4.16c
Male 0.21 ± 0.02 2.43 ± 0.03 20.70 ± 1.17 454.05 ± 3.55 0.01 ± 0.001 14.29 ± 0.43 5.58 ± 0.80 0.53 ± 0.07 183.09 ± 3.68
Female 0.22 ± 0.03 2.44 ± 0.04 21.15 ± 1.05 455.85 ± 6.04 0.01 ± 0.001 14.49 ± 0.40 5.87 ± 0.77 0.63 ± 0.06 184.81 ± 4.51
Station 4 <DL c 0.72 ± 0.02d 6.09 ± 0.69d 371.99 ± 4.47d 0.02 ± 0.001b 5.41 ± 0.43d 3.49 ± 0.31c <DL d 130.97 ± 7.36d
Male <DL 0.71 ± 0.02 5.71 ± 0.60 369.44 ± 3.59 0.019 ± 0.001 5.17 ± 0.37 3.31 ± 0.28 <DL 126.73 ± 4.80
Female <DL 0.73 ± 0.02 6.46 ± 0.56 374.54 ± 3.8 0.02 ± 0.001 5.66 ± 0.35 3.67 ± 0.24 <DL 135.20 ± 7.11

Table 7
Results of 2-way ANOVA testing the effects of station, season and interaction between the two factors on heavy metal concentration in P. perna soft body tissues.

Cd Cr Cu Fe Hg Mn Ni Pb Zn
Male
Station 3134*** 53,920*** 15,620*** 242,700*** 4351*** 34,910*** 565.7*** 3073*** 138,200***
Season 87.70*** 203.7*** 340.0*** 165.4*** 90.56*** 183.3*** 148.3*** 12.51*** 323.5***
Interaction 39.60*** 20.43*** 18.34*** 15;32*** 43.10*** 27.53*** 15.68*** 2.050* 10.74***
Female
Station 12,520*** 24,690*** 11,130*** 221,800*** 17,010*** 35,500*** 1997*** 10,190*** 120,100***
Season 280.4*** 153.5*** 227.9*** 198.9*** 163.1*** 190.9*** 301.1*** 74.39*** 315.0***
Interaction 99.32*** 15.31*** 19.70*** 14.54*** 82.78*** 34.12*** 41.35*** 22.25*** 5.692***
*
P<0.05.
***
P<0.001.
 B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489
polluted. With the exception of station 4 (non-polluted sediments),
all studied station sediments belong to the heavily polluted class
according to the EPA guidelines.
Spatial and seasonal variations of mean heavy concentrations
(mg kg1 DW) in P. perna soft body tissues for the four stations
are summarised in Table 6. Table 7 shows the results of a 2-way
ANOVA performed to investigate the effects of station and season
on heavy metal concentrations, also in P. perna soft body tissues.
Both station and season had significant effects on all the elements
studied. However, though there were seasonal effects at the differ-
ent stations, sampling locality had more significant effects on the
heavy metal concentrations (Table 7). The ANOVA results are con-
sistent with trends observed in Fig. 3 showing relatively higher
heavy metal concentrations in the soft body tissues of mussels col-
lected during the colder months with a peak in the early winter
months. When comparing the four stations, station 1 was consis-
tently higher (p < 0.001) than the reference station, the latter al-
ways being the less-polluted one. The greatest differences were
observed for Cd which were always above 200-fold higher during
all the sampling periods. The spatial differences in heavy metal
concentrations are highly significant for all heavy metals, in both
sexes (Table 6). The concentrations decreased from the east to
the west, i.e. from station 1 to 4 (Fig. 3). Their levels showed wide
variations among stations, which directly depended on exposure to
pollutants. With the exception of Cr and Ni (1.85 and
0.90 mg kg1 DW, respectively), the mean concentrations of ele-
ments in tissues collected from station 1 (Sidi Salem) were higher
(p < 0.001) than those of other stations. In contrast, Cr concentra-
tions were higher (p < 0.001) in tissues of both male and female
mussels collected at station 2 (Sunrise) and 3 (Saint Cloud) (2.25
and 2.44 mg kg1 DW, respectively) than in those collected at sta-
tion 1, and the lowest was measured at station 4 (0.72 mg kg1 -
DW). Ni concentrations in mussel bodies were similar at stations
Fig. 3. Heavy metal concentrations in P. perna soft body tissues (for male and female) for the studied stations and seasons.
483
1 and 2 whatever the season. The mean concentrations of Zn, Cu,
Cr and Fe in tissues collected from station 3 (18.40, 2.09, 2.44
and 45.50 mg kg1, respectively) were higher (p < 0.001) than
those of station 2 (15.80, 1.79, 2.25 and 42.20 mg kg1, respec-
tively). The mean concentrations of Hg in tissues collected from
station 1 (0.16 mg kg1) were higher (p < 0.001) than those of sta-
tions 2, 3 or 4 which were close to the detection limits of the AAS
(0.2, 0.1 and 0.2 mg kg1, respectively). Fig. 3 presents the seasonal
variations of toxic metal concentrations in P. perna male and fe-
male tissues from the four stations. The highest concentrations
were measured during the winter season for both sexes. Significant
seasonal variations of heavy metal content were recorded, peaking
in early winter for all elements except Zn (which peaked in au-
tumn). This pattern was most pronounced for Pb, Hg, Cd, Cu and
Ni, and to a lesser extent for Cr, Mn and Fe in molluscs at stations
1, 2 and 3. During the summer, all metal concentrations were com-
paratively low. The temporal pattern in heavy metal concentra-
tions in mussel soft tissues at station 1 may be separated into
two heavy metal groups: first, metals with high temporal stability
(i.e. Cr, Fe, Zn), second, heavy metal with temporal variability in
mussel soft body tissues (i.e. Cd, Cr, Hg, Ni, Mn, Pb) (Fig. 3). The
highest variations recorded at station 1 occurred for Cd, Hg, and
Ni with up to 15% of variation for both sexes. Station 2 exhibited
the same temporal patterns with a high temporal variability in
Cd, Cu, Hg, Ni and Pb, and high temporal stability for the other met-
als. The highest variations occurred for Hg, and Ni, up to 20% for
both sexes. The heavy metal concentrations accumulated in male
and female soft body tissues of station 3 show a very low temporal
variability except for Hg and Ni where it is higher (variation up to
10%) (Fig. 3). Station 4 appears to have very stable concentrations
for all the heavy metals analysed in P. perna soft body tissues, for
either sex. While the data showed that all metals varied signifi-
cantly with season, concentrations were relatively stable at station
484 B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489

Table 8 ment varied between the wet and dry seasons with enhanced con-
Biota-sediment accumulation factors (BSAF = concentration in male and female soft centrations in wet season samples at stations 1–3 located inside
body tissues/concentration in sediment) in the Annaba Gulf.
the Annaba Gulf, though no significant seasonal variation between
BSAF Cd Cr Cu Fe Hg Mn Ni Pb Zn the five sampling dates was found. Heavy metal in marine sedi-
Station 1 ments, originating from both natural and anthropogenic sources,
Male 0.18 0.42 0.20 1.43 0.05 0.15 0.01 1.18 0.18 accumulated simultaneously which makes it difficult to determine
Female 0.21 0.46 0.20 1.75 0.05 0.19 0.01 1.20 0.21 the ratio of each source (Loring and Rantala, 1992). The lack of sig-
Station 2 nificant variations in concentration in superficial sediment be-
Male 0.14 0.19 0.24 / 0.07 0.17 0.00 1.70 0.14 tween the sampling dates may be explained by deposition (of
Female 0.14 0.21 0.25 / 0.07 0.23 0.00 1.80 0.14
natural and/or anthropic elements) and post-depositional remobil-
Station 3 isation processes of elements, with modification of diagenetic
Male 0.23 1.38 0.32 / 0.08 0.13 0.01 2.43 0.23
Female 0.24 1.41 0.32 / 0.08 0.14 0.01 2.46 0.24
activity, which are likely to influence the concentrations (with or
without seasonal cycle) (Ridgway and Price, 1987). However,
Station 4
Male / / 0.28 / 0.05 0.19 / 0.67 /
numerous factors related to natural variability over the seasons
Female / / 0.29 / 0.06 0.22 / 0.71 / in the physical and chemical sediment characteristics (notably be-
tween the wet and dry seasons), may also strongly affect the heavy
metal retention capacity and thus the metal concentration in
superficial sediment.
4 compared to the others. Cr, Fe, Hg and Pb are the most stable ele- The capacity of sediment to retain heavy metals is controlled by
ments with season at station 4 showing less than 10% variation for numerous factors related to sediment characteristics that are
both sexes. determinant in heavy metal accumulation processes. One of the
Defined as the ratio between heavy metal levels in molluscs and most decisive factors acting on heavy metal accumulation pro-
sediment respectively (Szefer et al., 1999), the biota-sediment cesses is grain size (Goldberg, 1954; Hirst, 1962; Stoffers et al.,
accumulation factor (BASF) was calculated in order to evaluate 1977). According to the literature, decreased heavy metal concen-
the rate of heavy metal accumulation in male and female mollusc trations in sediment are strongly and positively correlated to in-
tissues. Except for station 3, Cu was poorly bioaccumulated in mus- creased grain size. Thus, a high level of heavy metal
sel bodies. High Zn values were found all along the gulf shore, with concentration is generally associated with the fine-grained mate-
the exception of station 4, the highest mean BSAF values recorded rial (<63 lm) when compared with the >63 lm fractions, as their
at station 3 (Table 8). Hg is also accumulated by P. perna as shown large surface areas influence the collection and transport of inor-
by the high BSAF values in the vicinity of the Seybouse River inlet. ganic constituents (Krauskopf, 1956; Romankevich, 1984). As re-
The other heavy metals under study did not achieve bioaccumula- ported in many studies, heavy metal accumulation is also related
tion levels in the mussel tissues above those found in the sur- to the organic matter content of the sediment (Goldberg, 1954;
rounding sediments of station 4, since the biota-sediment Krauskopf, 1956; Stoffers et al., 1977; Gonçalves and Boaventura,
accumulation factors were <1 (Table 8). 1991). Thus, coatings of organic matter prevalent in the fine
Relations between heavy metal concentrations (Pearson’s corre- grained sediments lead to relatively higher concentrations of heavy
lation coefficient) in P. perna soft body tissues (both sexes) and metal accumulation (Wangersky, 1986; Zhong et al., 2006). In
those measured in the sediments are presented in Table 9. The addition, the silt and clay composition variations recorded in the
strength of these relations varies according to the different heavy estuarine environment may lead to higher heavy metal concentra-
metals studied and to a lesser extent among seasons. A significant tions (Green et al., 2001).
relation (p < 0.05) is observed for concentrations of Cd, Fe, and Hg Besides variations in heavy metal retention capacity of superfi-
in P. perna relative to their concentration in surface sediments cial sediment, natural and/or anthropic input variations related to
(r > 0.952). The Pearson’s correlation coefficient obtained for Cr, climatic conditions and human activities (agriculture, urban and
Mn, Ni, Pb and Zn is positive with up to 0.681 but with a low level industrial activities) respectively might also affect the heavy metal
of confidence (p > 0.05). The lower coefficient was obtained for Cu deposition and thus the concentrations in sediments. If associated
(r < 0.602). with the increased pluviometry in winter, this may be linked to
There is now a baseline of information on heavy metal contam- storm-water run-off from the Annaba catchment basin, and mainly
ination of superficial sediment and of P. perna soft body tissues for from agricultural sources, i.e. from field fertilisation (phosphates
the Annaba Gulf. Heavy metal concentrations in superficial sedi- can be enriched in Cd) and pesticide use (mostly organochlorines).

Table 9
Pearson’s correlation coefficient analysis for heavy metal concentrations in P. perna with sediment for male and female from the different seasons studied.

Cd Cr Cu Fe Hg Mn Ni Pb Zn
Male
Spring 0.956* 0.807 ns 0.434 ns 0.988* 0.992** 0.927 ns 0.960* 0.780 ns 0.681 ns
Summer 0.977* 0.835 ns 0.461 ns 0.988* 0.997** 0.920 ns 0.932 ns 0.854 ns 0.721 ns
Autumn 0.971* 0.851 ns 0.527 ns 0.987* 0.998** 0.897 ns 0.947 ns 0.896 ns 0.741 ns
Winter (early) 0.967* 0.822 ns 0.554 ns 0.992** 0.998** 0.913 ns 0.925 ns 0.918 ns 0.797 ns
Winter (late) 0.965* 0.835 ns 0.531 ns 0.988* 0.998** 0.910 ns 0.953* 0.891 ns 0.792 ns
Female
Spring 0.964* 0.810 ns 0.483 ns 0.989* 0.997** 0.952* 0.889 ns 0.797 ns 0.685 ns
Summer 0.980* 0.831 ns 0.538 ns 0.991** 0.997** 0.935 ns 0.837 ns 0.866 ns 0.725 ns
Autumn 0.981* 0.837 ns 0.602 ns 0.991** 0.997** 0.919 ns 0.834 ns 0.907 ns 0.755 ns
Winter (early) 0.971* 0.811 ns 0.595 ns 0.992** 0.996** 0.936 ns 0.867 ns 0.902 ns 0.812 ns
Winter (late) 0.968* 0.816 ns 0.586 ns 0.991** 0.996** 0.940 ns 0.879 ns 0.879 ns 0.812 ns
*
P<0.05.
**
P<0.01.
 B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489
However, if agricultural sources account for such an increase in the
ratio, the relatively high heavy metal concentrations measured in
the Annaba Gulf sediments may be explained by significant indus-
trial facilities as well as main domestic waste effluents discharged
directly into the Seybouse River. Industrial and domestic waste-
waters containing Cd (and other metals such as Cu and Zn) dis-
charged into the river may furnish a dominant and constant input
in sediments in the gulf, thus masking natural seasonal patterns.
All these influences were manifested differently according to
station conditions and the specific heavy metal under
consideration.
According to the literature, accumulation processes and heavy
metal concentration variation in mollusc soft body tissues are
influenced by a host of biotic and abiotic factors (Phillips and
Rainbow, 1994). Surrounding environmental factor variations re-
lated to spatial (Blackmore and Wang, 2003) and/or seasonal influ-
ence (Regoli, 1998) may influence accumulation of heavy metals.
The effects of factors such as water temperature (Phillips, 1976b),
heavy metal bioavailibility, hydrodynamics, salinity (Chong and
Wang, 2001) or organic matter content (Pan and Wang, 2004) have
been reported. Biological parameters such as food acquisition
capability (Saavedra et al., 2004), sex (Sokolowski et al., 2004)
and related factors such as size/weight (Phillips, 1976a, 1976b), tis-
sue composition modification and the reproductive cycle (stage of
gonadal development) (Boyden and Phillips, 1981) also play an
important role in the accumulation processes of heavy metals in
molluscs. In the results obtained at the three stations (in compar-
ison to the reference station of La Kyenne Beach), heavy metal con-
taminations in P. perna soft body tissues were mainly related to
local sources and local environmental conditions in the Annaba
Gulf, which might have affected bioavailablity, and therefore the
uptake of heavy metals in mussels, differently from station to sta-
tion. These observations are corroborated by the results of 2-way
ANOVA for the combined effects of spatial and seasonal factors
on P. perna, showing that ‘‘station’’ rather than ‘‘season’’ influenced
the accumulation of heavy metals in P. perna soft body tissues. In-
deed, the beaches located inside the Annaba Gulf (stations 1–3) are
subjected to a higher degree of heavy metal pollution than the La
Kyenne Beach station, probably in relation to discharges from the
Sidi Seybouse River and from the Annaba urban centre, harbour
and industrial areas (Abdennour et al., 2000, 2004; Belabed et al.,
2013). A significant gradient concentration in Annaba Gulf waters
has been demonstrated for all of the heavy metals analysed. How-
ever, the origin of heavy metals accumulated in the gulf’s sediment
and transferred to marine mussels cannot be easily established due
to the diversity of sources. Cr and Ni excepted, the concentration
level of heavy metals in P. perna soft body tissues, related to in-
creased metal bioavailability, were significantly higher at Sidi
Salem (station 1) which is located near the outlet of the Seybouse
River. Indeed, agricultural and urban wastewater, along with un-
treated discharge from sewers and other industrial activities (iron,
steel or fertiliser industries) in the catchment area of the west val-
ley of Annaba city probably contribute to the heavy metals in the
gulf as shown by Abdennour et al. (2000, 2004). In addition, this
contamination may originate in the harbour activity and urbanised
area of Annaba, located near Sunrise station, with its sewage out-
falls, storm water outlet effluent and road runoff discharging di-
rectly into Annaba Gulf. With the exception of Sidi Salem Beach,
similar levels of Hg were measured in stations without a clear spa-
tial pattern. Generally, heavy metal concentrations in mussel soft
body tissues at station 4 (La Kyenne Beach) could be considered ba-
sic natural levels. The data reflect the usual fluctuations of heavy
metals in tissues and their availability for the biota incorporation
on the northern coast of Annaba district. Significant temporal vari-
ations (p < 0.001) for all heavy metal concentrations were recorded
in the Annaba Gulf, with similar patterns among stations. Despite
485
significant variations among stations, concentrations of all studied
heavy metals were higher during early winter (November), possi-
bly due to a common source. However, there was also some inter-
action between station and season (2-way ANOVA), meaning that
though the patterns were similar, the strength of the influence of
season was relatively different at each of the four stations. Such
observations can be attributed to differences in seasonal changes
in heavy metal exposure and bioavailability between the more
and the less-polluted stations. A good example is given with Cd
which showed a greater difference between wet and dry season
values at station 1 than at the other three stations, notably in com-
parison to the reference station. Furthermore, temporal fluctua-
tions of factors such as salinity, temperature and pH are due to
the specific hydrological conditions in the Annaba Gulf. During
the spring/summer months, a marked increase in the temperature
of surface waters (though less than for salinity and pH) was ob-
served for all stations on the gulf, coinciding with an important de-
crease in the concentration of dissolved oxygen (%), probably as a
result of significant wind-induced coastal upwelling along the
Algerian coast in summer (Bakun and Agostini, 2001). Moreover,
at the end of autumn and during winter, important changes oc-
curred in the hydrological conditions in the gulf (drop in salinity
and temperature), as the Algerian coast is an area of coastal down-
welling. Seasonal rainfall variations are also likely to induce mod-
ification of heavy metal concentrations in marine waters and
therefore in sediment and in mussels too, although to a lesser ex-
tent. As demonstrated by the lower water salinity levels recorded
for the different stations, increased rains in winter led to an impor-
tant inflow of fresh water to the coastal zone from the wadi sys-
tem. The results from studies of differing marine mollusc species
reported by various authors (Phillips, 1977; Blust et al., 1992;
Blackmore and Wang, 2003), have shown that heavy metal uptake
and accumulation in soft body tissues is affected by element bioav-
ailibility which generally tends to increase with decreasing salin-
ity, according to the concentration of free heavy metal ions in
the surrounding seawater (Jackim et al., 1977). Lower salinity leads
to a reduction in the chloro-complexation of heavy metals, result-
ing in increased bioavailability of the free ions. This is enhanced by
the combination of decreased salinity and pH induced by torrential
rainfall during the winter months. As this also affects the extract-
ability of metals, it may partially explain the higher concentrations
measured in the sediment and the greater accumulation in mollusc
tissues in the lower salinity conditions of the cold season. More-
over, increased mobilisation of differing sources of heavy metals
may contribute to higher concentration in marine waters and to
greater availability to marine organisms during winter. Thus, in
addition to regular wastewaters and untreated sewage discharge,
suspended particulate matter containing heavy metals, from agri-
cultural as well as urban and industrial areas, may also be collected
by the Seybouse River from superficial runoffs and released into
the gulf near Sidi Salem Beach (station 1). Similarly, the winter
peaks of heavy metal concentrations recorded at stations 2 and 3
may be related to increased runoff from the impervious surface
of urbanised areas and from Annaba Harbour, due to greater rain-
fall from October to March. The effects of surface water tempera-
ture variations on absorption and accumulation of heavy metals
such as As, Cd, Co, Cu, Pb, Ni and Zn, by both filter-feeding and de-
posit-feeding marine bivalves, have been reported by several
authors (Phillips, 1976a; Jackim et al., 1977; Mubiana and Blust,
2007). In seawater, temperature influences numerous physio-
chemical factors, with changes in pH and the chemical speciation
of metals figuring among the most important (Byrne et al., 1988).
However, according to the literature, heavy metal speciation is
poorly affected by temperature variations, particularly the free me-
tal ion activity which is the form most bioavailable to molluscs
(Campbell, 1995).
486 B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489
Seasonal trends at the most polluted station are very similar to
those of the reference station, which also suggests that seasonal
changes in heavy metal concentrations in P. perna soft body tissues
probably depend more on changes in animal physiology than on
conditions of exposure. However, these fluctuations do not neces-
sarily depend on seasonal changes in metal bioavailability but may
be related to seasonal changes in flesh weight or in the relative tis-
sue composition of sentinel organisms (Marigomez and Ireland,
1990; Regoli and Orlando, 1993). Mussels are known to show
strong seasonal changes in physiological energetics, growth, ani-
mal body weight and other related biochemical changes (Hawkins
and Bayne, 1992) due to modifications in food availability, water
temperature or reproduction, often in correlation with variations
in heavy metal concentration (lg g1 tissue fresh weight) while
metal body-burden (lg individual1) remains unchanged (Phillips,
1977; Wang and Fisher, 1997). In particular, the importance of the
mussel reproductive cycle to heavy metal concentration variations
in soft body tissues, related to seasonal changes in flesh weight
during gonadal development (notably prior to the spawning peri-
od), has been highlighted by several authors (Latouche and Mix,
1982; Páez-Osuna et al., 1995; Joiris et al., 1998; Otchere, 2003)
for different mollusc species such as Anadara senilis, Mytilus gallo-
provincialis, Mytilus edulis, P. perna, P. ciridis, Crassostrea gigas, and
Crassostrea tulipa. Thus, a summertime decrease in the heavy metal
concentrations in soft body tissue may be influenced by the
spawning process (which, for many mollusc species, is itself in-
duced by changes in surface water temperature), while heavy me-
tal accumulation often occurs simultaneously to gonadal
development during winter (Regoli, 1998). Since the 1970s, sea-
sonal variations in heavy metal concentrations recorded in marine
molluscs have been demonstrated with maximum values recorded
in winter and minimum in summer, as reported by many authors
for numerous marine bivalve species such as C. gigas (Boyden
and Phillips, 1981), M. edulis (Phillips, 1976a), M. galloprovincialis
(Regoli and Orlando, 1993), P. perna (Sokołowski et al., 2004),
and P. viridis (Chu et al., 1990). Therefore, significant changes in
these biological parameters may greatly influence heavy metal
concentrations in wild mussel populations, thus explaining wide
seasonal variations. In addition, as demonstrated by Páez-Osuna
et al., 1995 for Crassostrea iridescens (Oyster), the increase in heavy
metal concentrations in soft body tissues during the winter months
may depend on gonadal development, which in turn may be influ-
enced by the presence in the surrounding environment of metals
such as Zn. Several authors have also highlighted the importance
of the specimen’s sex on heavy metal accumulation. In accordance
with previous observations, significant variations between the P.
perna male and female, with higher heavy metal concentrations
in the latter, were recorded at two of the four stations. No signifi-
cant differences were observed between the sexes at station 3 for
Cd, Cu and Fe in spring and summer, or for Hg whatever the season.
Corroborating the Gomes Ferreira et al. (2004) observations of soft
body tissues in P. perna from the coast of Rio de Janeiro State
(Brazil), the lack of significant differences between the sexes at sta-
tion 3 may be linked to the sampling strategy which was limited by
the available size range (35–40 mm) found in this area. Moreover, as
the weight of animals collected in the Annaba Gulf was restricted to
the 0.5–1.5 g range, no significant differences in metal quantities in
specimens of differing weight were observed. Additional studies
must be undertaken in order to evaluate heavy metal fluctuations
between the sexes at station 3 in organisms up to 40 mm. Copper,
which is an important and essential microelement acting as a respi-
ratory pigment of marine invertebrates and which may be metabol-
ically regulated by molluscs (Förstner and Wittmann, 1983),
exhibited low variations in soft body tissues among seasons (less
than 11% for both sexes). Similar findings were observed by Gomes
Ferreira et al. (2004) in P. perna in Rio de Janeiro (Brazil).
Comparisons of the male/female mean heavy metal concentra-
tions measured during the present study to those published in
worldwide marine environmental studies using P. perna as a bioin-
dicator are presented in Table 10. However, even though it is the
same species (P. perna), great caution must be exercised in compar-
ative analyses since differences in environmental (temperature,
pH, salinity) and biological (size, sex, reproduction) parameters
may be responsible for variability in heavy metal accumulation.
Except for Zn, La Kyenne Beach, located on the north-western coast
of the Gulf of Annaba, can be considered the natural background le-
vel along the Algerian coast, as world data comparison suggests re-
duced contamination by heavy metals in the mussels collected at
this station. Concentrations in soft body tissues are either in the
same range as (Cu for example) or significantly lower than values
cited for low-pollution areas reported by the World Mussel Watch
Programme (Cantillo, 2003) or other worldwide studies. Only Zn
showed concentrations significantly higher than those reported
for non-impacted areas (Table 10). Since this station is located in
a relatively unaffected area, heavy metal variations there may sim-
ply reflect the usual fluctuations and natural availability of these
metals for biota incorporation in the Annaba Gulf. Moreover, the
concentrations found in mussel soft body tissues were lower than
the maximum concentration permitted for human consumption
specified by legislation in Brazil, Luxemburg and Spain (Zn ex-
cepted). The heavy metal concentrations in P. perna collected on
the Annaba Gulf beaches are expressed in dry weight (water con-
tent in P. perna mussels is about 80%), while the values cited in
the legislation are expressed in wet weight. For beaches located in-
side the Annaba Gulf (i.e. station 1–3), heavy metal concentrations
measured in P. perna soft body tissues are significantly lower than
values from the World Mussel Watch environmental studies for Cd
and Pb, but significantly higher for Cr, Cu, Ni and Zn (Cantillo,
2003) (no comparative data available for Fe, Hg and Mn). The range
of concentrations of Cd, Cr, Ni and Pb recorded at stations located
inside the gulf were close to those reported in soft body tissues of
P. perna collected from non-impacted areas by Baraj et al. (2003),
Gomes Ferreira et al. (2004) or Saenz et al. (2010). Higher values
were reported for Cd and Pb in the impacted sites in Brazil
(Carvalho et al., 1991; Avelar et al., 2000) and the Gulf of Aden
(Szefer et al., 1997). Higher values were also recorded in Maurita-
nia and Ghana by Roméo et al. (2000) and Otchere (2003). Con-
cerning Cu specifically, the results obtained in the present study
show higher concentrations than the maximum recorded in both
impacted and non-impacted areas. Similar results were observed
by Szefer et al. (1997) in P. perna in the Aden Gulf. Thus, the
Annaba Gulf may be considered as under high impact pollution.
Except for the station located at the mouth of the Seybouse River,
the concentration of Fe in soft body tissues of mussels collected in-
side the Annaba Gulf are lower than those reported by Carvalho
et al. (1998) and by Gomes Ferreira et al. (2004) for non-impacted
Brazilian sites. Concentrations recorded at station 1 are near those
recorded in the Aden Gulf by Szefer et al. (1997) or in the more im-
pacted sites of the Rio Grande do Sul State (Brazil) by Baraj et al.
(2003). However, the highest concentrations of Fe were found in
samples from the beaches of Rio de Janeiro State (Gomes Ferreira
et al., 2004) or in Sakumo Lagoon in Ghana (Otchere, 2003).
Previous studies have produced results showing levels of Mn and
Ni in the same range as those found in the present study. Analyses
performed on mussels collected from beaches affected by both
industrial pollution and untreated urban waste release have been
reported for the Rio de Janeiro State coast by Carvalho et al.
(1991), for Guanabara Bay by Carvalho and Lacerda (1992) and
for Santana Island by Carvalho et al. (1998). Similar values were
also found by Otchere (2003) in Ghana lagoons. The concentrations
of Zn observed in the present study are higher than those detected
in non-impacted areas by Brazilian Atlantic coast monitoring
Table 10
Heavy metal concentrations in P. perna from impacted (I) and non-impacted areas (NI) from different countries. M: Mean value in mg kg1 dry weight; MM: minimum–maximum values in mg kg1 dry weight; RM: Range of mean from
different sites in mg kg1 dry weight; MSD: Mean ± standard deviation in mg kg1 dry weight.

Sampling site Cd Cr Cu Fe Hg Mn Ni Pb Zn Reference

Algeria
St1 0.51 ± 0.11 1.85 ± 0.11 26.74 ± 2.47 932.04 ± 12.95 0.159 ± 0.031 22.28 ± 1.34 8.99 ± 2.27 1.68 ± 0.15 358.96 ± 5.4 Present work 2013
St2 0.22 ± 0.02 2.25 ± 0.09 17.91 ± 1.71 421.99 ± 13.05 0.02 ± 0.008 16.08 ± 0.24 9.03 ± 2.35 0.79 ± 0.07 158.03 ± 8.35
St3 0.22 ± 0.02 2.44 ± 0.03 20.92 ± 1.12 454.95 ± 4.97 0.01 ± 0.001 14.39 ± 0.42 5.73 ± 0.79 0.58 ± 0.08 183.95 ± 4.16
St4 <DL 0.72 ± 0.02 6.09 ± 0.69 371.99 ± 4.47 0.02 ± 0.001 5.41 ± 0.43 3.49 ± 0.31 <DL 130.97 ± 7.36
Brazil
RJ Statea
I 1.2–1.6 (RM) / 8–9 (RM) / / 10–20 (RM) 7–10 (RM) 2.0–13 (RM) 180–250 (RM) Carvalho et al. (1991)
NI 0.1 (M) / 10 (M) / / 17 (M) 6 (M) 2 (M) 150 (M) Carvalho and Lacerda
(1992)
NI 0.3 (M) 1.3 (M) 5.1 (M) 675 (M) / 9.3 (M) 8.6 (M) 1.8 (M) 79 (M) Carvalho et al. (1998)
NI 0.3 (RM) 1.7–2.5 (RM) 5.7–6.7 (RM) 1000–1200 (RM) / 20–26 (RM) 4–12 (RM) 0.1–0.5 (RM) 40–51 (RM) Gomes Ferreira et al.
(2004)
SC Statea
I 0.66 (M) 0.86 (M) 6.08 (M) / / / 8.49 (M) 0.53 (M) 90.5 (M) Saenz et al. (2010)
NI 0.48–0.87 (RM) 0.63–1.10 (RM) 4.52–5.29 / 0.03 (RM) / 2.87–6.75 0.24–0.47 (RM) 51.9–81.1 (RM)
(RM) (RM)
SP Statea
I 0.98–4.57 (MM) 0.23–48.48 1.63–8.90 / / / / 3.94–60.00 43.18–132.7 Avelar et al. (2000)
(MM) (MM) (MM) (MM)
RGS Statea 0.086–1.0 (MM) 0.53–2.96 (MM) 3.77–20.6 254.8–970.6 0.051–0.220 13.7–61.7 / 0.35–1.23 (MM) 41.1–109.2 (MM) Baraj et al. (2003)
(MM) (MM) (MM) (MM)
Yemen 0.6 (M) / 27 (M) 900 (M) / 60 (M) / 5.2 (M) 40 (M) Szefer et al. (1997)
Mauritania 5.54 ± 2.12 / 6.2 ± 3.2 / / / / / 34 ± 5 (MSD) Roméo et al. (2000)
(MSD) (MSD)
Ghana 1.4–1.9 (RM) / 15–16 (RM) 900–1130 (RM) 0.33–0.37 (RM) 12–15 (RM) / / 12–16 (RM) Otchere (2003)
Morocco 0.41 ± 0.17 / 7.0 ± 2.3 / 0.036–2.6 (MM) / / 1.3 ± 0.3 (MSD) 231 ± 115 (MSD) Banaoui et al. (2004)
B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489

(MSD) (MSD)
WMWb 2.0 (M) 1.6 (M) 7.9 (M) / / / 2.2 (M) 5.0 (M) 130 (M) Cantillo (2003)
Legislation (wet
weight)
Spain 1 / 20 / 1 / / 5 /
Luxembourg 1 / / 0.5 / / 2 /
Brazil 1 / 30 / 0.5 / / 2 50
a
RJ State: Rio de Janeiro State; SC State: Santa Catarina State SP State: Sao Paulo State; RGS: Rio Grande do Sul State;
b
World Mussel Watch.
487
488 B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489
programmes in Rio de Janeiro and Santa Catarina States (Carvalho
et al., 1998; Gomes Ferreira et al., 2004; Saenz et al., 2010). They
are, however, in the same range of the impacted areas of Sepetiba
Bay, Angra dos Reis and Arraial do Cabo Beaches situated along the
Rio de Janeiro State coast (south-eastern Brazil) (Carvalho et al.,
1991). These observations suggest that Annaba Gulf is contami-
nated by this element. Though Carvalho and Lacerda (1992) and
Banaoui et al. (2004) found relatively similar results from P. perna
collected respectively in Guanabara Bay waters (Brazil) and along
the Moroccan coast, lower concentrations were reported by Szefer
et al. (1997) in the Aden Gulf (Yemen), by Avelar et al. (2000) in
Ubatuba Bay (Sao Paulo, Brazil) and by Otchere (2003) in the Benya
and Sakumo Lagoons of Ghana (Table 10). Data comparison with
worldwide studies using P. perna mussels as bioindicator suggests
serious heavy metal contamination of the Annaba Gulf. However,
except for Zn, the maximum concentrations of heavy metals found
in P. perna soft body tissues collected at the most polluted station
of the Annaba Gulf are lower than the maximum concentration
permitted for human consumption as determined by Brazilian,
Spanish and Luxembourg legislation (Table 10).
In conclusion, the use of mussels is sometimes controversial
due to the variability in their heavy metal accumulation. Their go-
nad maturation stages, age, temperature and feeding stages make
it difficult to compare investigation areas in different parts of the
world. However, according to data obtained in the present study,
P. perna seems to be an easy and useful bioindicator of man’s influ-
ence on the distribution of heavy metals in the Annaba Gulf coastal
ecosystem. This survey demonstrates that the concentrations of
heavy metal in specimens collected at the Annaba Gulf stations
are similar to values reported for impacted areas in studies in
Brazil or elsewhere in the world. Except for Zn at the most polluted
station, Sidi Salem Beach, this investigation shows that heavy me-
tal levels in mussels were lower than the maximum permissible
concentrations for human consumption as defined by the Brazilian,
Spanish and Luxembourg legislation for Cd, Cu, Hg, and Pb (wet
weight basis). Due to the lack of permissible standards for Cr, Fe,
Mn and Ni, the suitability of the Annaba mussels for human con-
sumption cannot be deduced. Differences in heavy metal levels be-
tween urban and harbour areas have been demonstrated through
statistical analyses. Therefore the high increase trend of metal con-
centration at Sidi Salem Beach is a warning, pleading in favour of
necessary sewage treatment facilities. From this study, heavy me-
tal continental (anthropogenic) pollution is obvious through near-
shore–offshore gradient evidence both in P. perna tissues and in the
sediment. Hydrodynamism, currentology and sedimentology seem
to have an important impact on heavy metal dissemination and
their transfer to the biota. The results of this study confirm the use-
fulness of the mussel P. perna as bioindicator for heavy metal pol-
lution. Referring to international guideline values, P. perna mussels
from the Annaba shore can be considered as uncontaminated but
the concentration levels detected in our samples indicate signifi-
cant contamination by heavy metals in comparison to those re-
corded elsewhere in the Mediterranean Sea. A pluriannual
sampling project enlarged to include other compartments of the
ecosystem may lead to a better understanding of the impact of this
local anthropogenic pollution. The population dynamics of P. perna
in the different populations will need to undergo further study to
evaluate the sub-lethal impacts of this kind of pollution on benthic
organisms.
Acknowledgements
This study results from a collaborative project between the Uni-
versity Badji-Mokhtar (Annaba, Algeria), the Institut National des
Sciences et Technologies de la Mer (La Goulette, Tunisia) and the
Chrono-Environment Laboratory UMR CNRS 6249, France. The
authors would like to devote a special thank to the members of
UMR CNRS 6249 for courtesy, kindness and help. We also thank
the fishermen of the Annaba Gulf for their logistical help.
References
Abdennour, C., Smith, B.D., Boulakoud, M.S., Samraoui, B., Rainbow, P.S., 2000. Trace
metals in shrimps and sediments from Algerian water. J. Catal. Mater. Environ.
3, 9–12.
Abdennour, C., Khelili, K., Boulakoud, M.S., Rainbow, P.S., 2004. Trace metals in
marine, brackish and freshwater prawns (Crustacea, Decapoda) from northeast
Algeria. Hydrobiologia 432, 217–227.
Arnone, R.A., Wiesenburg, D.E., Saunders, K.D., 1990. The origin and characteristics
of the Algerian Current. J. Geophys. Res. 95, 1587–1598.
Avelar, W.E.P., Mantelatto, F.L.M., Tomazelli, A.C., Silva, D.M.L., Shuhama, T., Lopes,
J.L.C., 2000. The marine mussel Perna perna (Mollusca, Bivalvia, Mytilidae) as an
indicator of contamination by heavy metals in the Ubatuba Bay, Sao Paulo,
Brazil. Water Air Soil Pollut. 118, 65–72.
Bakun, A., Agostini, V.N., 2001. Seasonal patterns of wind-induced upwelling/
downwelling in the Mediterranean Sea. Sci. Mar. 65, 243–257.
Banaoui, A., Chiffoleau, J.F., Moukrim, A., Azdi, M., Kaaya, A., Auger, D., Rozuel, E.,
2004. Trace metal distribution in mussel Perna perna along the Moroccan coast.
Mar. Pollut. Bull. 48, 378–402.
Baraj, B., Niencheski, L.F., Corradi, C., 2003. Trace metal content trends of mussel
Perna perna (Linnaeus, 1758) from the atlantic coast of southern Brazil. Water
Air Soil Pollut. 145, 205–214.
Belabed, B.E., Frossard, V., Dhib, A., Turki, S., Aleya, L., 2013. What factors determine
trace metal contamination in Lake Tonga (Algeria)? Environ. Monit. Assess.,
http://dx.doi.org/10.1007/s10661-013-3300-6.
Blackmore, G., Wang, W.X., 2003. Inter-population differences in Cd, Cr, Se, and Zn
accumulation by the green mussel Perna viridis acclimated at different
salinities. Aquat. Toxicol. 62, 205–218.
Blust, R., Kockelbergh, E., Baillieul, M., 1992. Effect of salinity on the uptake of
cadmium by the brine shrimp Artemia franciscana. Mar. Ecol-Prog. Ser. 84, 245–
254.
Boening, D.W., 1999. An evaluation of bivalves as biomonitors of heavy metals
pollution in marine waters. Environ. Monit. Assess. 55, 459–470.
Boyden, C.R., Phillips, D.J.H., 1981. Seasonal variation and inherent variability of
trace elements in oysters and their implications for indicator studies. Mar. Ecol-
Prog. Ser. 5, 29–40.
Byrne, R.H., Kump, L.R., Cantrell, K.J., 1988. The influence of temperature and pH on
trace metal speciation in seawater. Mar. Chem. 25, 168–181.
Campbell, P.G.C., 1995. Interactions between trace metals and aquatic organisms: a
critique of the free-ion activity model. In: Tessier, A., Turner, D.R. (Eds.), Metal
Speciation and Bioavailability in Aquatic Systems. John Wiley & Sons,
Chichester, UK, pp. 45–97.
Cantillo, A.Y., 2003. Comparison of results of Mussel Watch Programs of the United
States and France with worldwide Mussel Watch studies. Mar. Pollut. Bull. 39,
712–717.
Carvalho, C.E., Lacerda, L.D., 1992. Heavy metals in the Guanabara Bay biota: why
such low concentrations? Ciên. Cult. 44, 184–186.
Carvalho, C.E.V., Lacerda, L.D., Gomes, M.P., 1991. Heavy metal contamination of the
marine biota along the Rio de Janeiro coast, SE-Brazil. Water Air Soil Pollut. 57,
645–653.
Carvalho, C.E.V., Ovalle, A.R.C., Rezende, C.E., Molisani, M.M., Salomao, M.S.M.B.,
Lacerda, L.D., 1998. Seazonal variation of particulate heavy metals in the lower
Praiba do Sul River, RJ, Brazil. Environ. Geol. 621, 1–8.
Casas, S., Gonzalez, J.L., Andral, B., Cossa, D., 2008. Relation between metal
concentration in water and metal content of marine mussels (Mytilus
galloprovincialis): impact of physiology. Environ. Toxicol. Chem. 27, 1543–1552.
Chase, M.E., Jones, S.H., Hennigar, P., Sowless, J., Harding, G.C.H., Freeman, K., Wells,
P.G., Krahforst, C., Coombs, K., Crawford, R., Pederson, J., Taylor, D., 2001. Gulf
watch: monitoring spatial and temporal patterns of trace metal and organic
contaminants in the Gulf of Maine (1991–1997) with the Blue Mussel Mytilus
edulis L.. Mar. Pollut. Bull. 42, 491–505.
Chu, K.H., Cheung, W.M., Lau, S.K., 1990. Trace metals in bivalves and sediments
from the To1o harbor, Hong Kong. Environ. Int. 16, 31–36.
Chiffoleau, J.F., Auger, D., Boutier, B., Rouzel, E., Truquet, I., 2003. Dosage de certains
métaux dans les sédiments et la matière en suspension par absorption
atomique. Ifremer (Eds.), Brest, France.
Chipperfield, P.N.J., 1953. Observations on the breeding and settlement of Mytilus
edulis (L.) in British waters. J. Mar. Biol. Assoc. UK 32, 449–476.
Chiu, S.T., Lam, F.S., Tze, W.L., Chau, C.W., Ye, D.Y., 2000. Trace metals in mussels
from mariculture zones, Hong Kong. Chemosphere 41, 101–108.
Chong, K., Wang, W.X., 2001. Comparative studies on the biokinetics of Cd, Cr, and
Zn in the green mussel Perna viridis and the Manila clam Ruditapes
philippinarum. Environ. Pollut. 115, 107–121.
Debièche, T.H., 2002. Evolution de la qualité des eaux (salinité, azote et métaux
lourds) sous l’effet de la pollution saline, agricole et industrielle: Application à
la basse plaine de la Seybouse N.E algérien. PhD Thesis, Franche-Comté
University, Earth Science, p. 199.
Förstner, U., Wittmann, G.T.W., 1983. Metal pollution in the aquatic environment.
In: Förstner, U., Wittmann, G.T.W. (Eds.), Metal Pollution in the Aquatic
Environment, second ed. Springer-Verlag, Berlin, Germany, pp. 271–321.
 B.-E. Belabed et al. / Marine Pollution Bulletin 74 (2013) 477–489
Fowler, S.W., Oregioni, B., 1976. Trace metals in mussels from the N.W.
Mediterranean. Mar. Pollut. Bull. 7, 26–29.
Goldberg, E., 1954. Marine geochemistry I – chemical scavengers of the sea. J. Geol.
62, 249–265.
Goldberg, E.D., 1975. The mussel watch: a first step in global marine monitoring.
Mar. Pollut. Bull. 6, 111–132.
Goldberg, E.D., 1986. The Mussel Watch concept. Environ. Monit. Assess. 7, 91–
103.
Gomes Ferreira, A., Dos Santos Machado, A.L., Zalmon, I.R., 2004. Temporal and
spatial variation on heavy metal concentrations in the bivalve Perna perna
(Linnaeus, 1758) on the Northern Coast of Rio de Janeiro State, Brazil. Braz.
Arch. Biol. Technol. 47, 319–327.
Gonçalves, E.P.R., Boaventura, R., 1991. Sediments as indicators of the River Ave
contamination by heavy metals. In: Bau, J., Ferreira, J.P.L., Henriques, J.D.,
Raposo, J.O. (Eds.), Integrated approaches to water pollution problems. Elsivier,
London.
Green, G., Williamson, B., Bull, D., Oldman, J., MacDonald, I., Mills, G., 2001.
Prediction of Contaminant Accumulation in Auckland Estuaries. NIWA Client
Report: ARC01252, NIWA, Hamilton, New Zealand, p. 78.
Grosell, M., Brix, K.V., 2005. Introduction to the special issue on mechanisms in
metal toxicology. Aquat. Toxicol. 72, 3–4.
Hawkins, A.J.S., Bayne, B.L., 1992. Physiological interrelations, and the regulation of
production. In: Gosling, E. (Ed.), The Mussel Mytilus: Ecology, Physiology,
Genetics and Culture. Elsevier, Amsterdam, The Netherlands, pp. 171–212.
Hirst, D., 1962. The geochemistry of modern sediments from the Gulf of Paria.
Geochim. Cosmochim. Acta 26, 1147–1187.
Islam, M.D., Tanaka, M., 2004. Impact of pollution on coastal and marine ecosystems
including coastal and marine fisheries and approach for management: a review
and synthesis. Mar. Pollut. Bull. 48, 624–649.
Jackim, E., Morrison, G., Steele, R., 1977. Effects of environmental factors on
radiocadmium uptake by four species of marine bivalves. Mar. Biol. 40, 303–
308.
Joiris, C.R., Azokwu, M.I., Otchere, F.A., Ali, I.B., 1998. Mercury in the bivalve
Anadara (Senilia) senilis from Ghana and Nigeria. Sci. Total Environ. 224,
181–188.
Keck, T., Maurer, D., Lind, H., 1975. A comparative study of the hard clam gonad
development cycle. Biol. Bull. 148, 243–258.
Khelifi-Touhami, M., Ounissi, M., Saker, I., Haridi, A., Djorfi, S., Abdennour, C., 2006.
The hydrology of the Mafrag estuary (Algeria): transport of inorganic nitrogen
and phosphorus to the adjacent coast. J. Food Agric. Environ. 4, 340–346.
Kramer, K.J.M., Brockman, U.H., Warwick, R.M., 1994. Tidal Estuaries. Manual of
Sampling and Analytical Procedures. A.A. Balkema Publishers, Rotterdam, The
Netherlands, p. 304.
Krauskopf, K., 1956. Factors controlling the concentration of thirteen rare metals in
sea water. Geochim. Cosmochim. Acta 9, 1–32.
Latouche, Y.D., Mix, M.C., 1982. The effects of depuration, size and sex on trace
metal levels in Bay Mussels. Mar. Pollut. Bull. 13, 27–29.
Loring, D.H., Rantala, R.T.T., 1992. Manual for the geochemical analyses of marine
sediments and suspended particulate matter. Earth Sci. Rev. 32, 235–283.
Lunetta, J.E., 1969. Reproductive physiology of the mussels (Mytilus perna –
Mollusca Lamellibranchia). Boletim Facul. Filosofia, Ciencias e Letras, Univ. de
Sao Paulo 26, 33–111.
Luten, J.B., Bouquet, W., Burggraaf, M.M., Rauchbaar, A.B., Rus, J., 1986. Trace metals
in mussels (Mytilus edulis) from the Waddenzee, coastal North Sea and the
estuaries of Ems, Westerschelden and Eastern Scheldt. B. Environ. Contam.
Toxicol. 36, 770–777.
Marigomez, I., Ireland, M.P., 1990. A laboratory study of cadmium exposure in
Littorina littorea in relation to environmental cadmium and exposure time. Sci.
Total Environ. 90, 75–87.
Melwani, A.R., Gregorio, D., Jin, Y., Stephenson, M., Ichikawa, G., Siegel, E., Crane, D.,
Lauenstein, G., Davis, J.A., 2013. Mussel watch update: long-term trends in
selected contaminants from coastal California 1977–2010. Mar. Pollut. Bull..
http://dx.doi.org/10.1016/j.marpolbul.2013.04.025.
Mo, C., Neilson, B., 1994. Standardization of oyster soft tissue dry weight
measurements. Water Res. 28, 243–246.
Mubiana, V.K., Blust, R., 2007. Effect of temperature on scope for growth and
accumulation of Cd, Co., Cu and Pb by the marine bivalve Mytilus edulis. Mar.
Environ. Res. 63, 219–235.
Neuberger-Cywiak, L., Achituv, Y., Garcia, E.M., 2003. Effects of zinc and cadmium
on the burrowing behavior LC50 and LT50 on Donax trunculus Linnaeus
(Bivalvia – Donacidae). B. Environ. Contam. Toxicol. 70, 713–722.
O’Connor, T.P., Cantillo, A.Y., Lauenstein, G.G., 1994. Monitoring of temporal trends
in chemical contamination by the NOAA National Status and Trends Mussel
Watch Project. In: Kramer, K.J.M. (Ed.), Biomonitoring of Coastal waters and
Estuaries. CRC Press, Lewis Publishers, Boca Raton, Florida, USA, pp. 51–84.
Otchere, F.A., 2003. Heavy metals concentration and burden in the bivalves
(Anadara (Senilia) senilis, Crasostrea tulipa and Perna perna) from lagoons in
Ghana: model to describe mechanism of accumulation/excretion. Afr. J.
Biotechnol. 2, 280–287.
Páez-Osuna, P., Frias-Espericueta, M.G., Osuna-López, J.I., 1995. Trace metal
concentrations in relation to season and gonadal maturation in the oyster
Crassostrea iridescens. Mar. Environ. Res. 40, 19–31.
489
Pan, J.F., Wang, W.X., 2004. Influences of dissolved and colloidal organic carbon on
the uptake of Ag, Cd, and Cr by the marine mussel, Perna viridis. Environ. Pollut.
129, 467–477.
Parker, J.G., 1983. A comparison of methods used for the measurement of organic
matter in sediments. J. Chem. Ecol. 1, 201–210.
Phillips, D.J.H., 1976a. The common mussel Mytilus edulis as an indicator of
pollution by zinc, cadmium, lead and copper. 1. Effects of environmental
variables on uptake of metals. Mar. Biol. 38, 59–69.
Phillips, D.J.H., 1976b. The common mussel Mytilus edulis as an indicator of
pollution by zinc, cadmium, lead and copper. II. Relationship of metals in the
mussel to those discharged by industry. Mar. Biol. 38, 71–80.
Phillips, D.J.H., 1977. Effects of salinity on the net uptake of zinc by the common
mussel Mytilus edulis. Mar. Biol. 41, 79–88.
Phillips, D.J.H., Rainbow, P.S., 1994. Biomonitoring of Trace Aquatic Contaminants,
second ed. Chapman and Hall, London, UK, p. 371.
R Development Core Team, 2011. R: A Language and Environment for Statistical
Computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN: 3-
900051-07-0. <http://www.R-project.org>.
Rainbow, P.S., 1995. Biomonitoring of heavy metal availability in the marine
environment. Mar. Pollut. Bull. 31, 183–192.
Rajagopal, S., Venugopalan, V.P., Van der Velde, G., Jenne, H.A., 2006. Greening of the
coasts: a review of the Perna viridis success story. Aquat. Ecol. 40, 273–297.
Regoli, F., Orlando, E., 1993. Mytilus galloprovincialis as a bioindicator of lead
pollution: biological variables and cellular responses. Sci. Total Environ. 2,
1283–1292.
Regoli, F., 1998. Trace metals and antioxidant enzymes in gills and digestive gland
of the Mediterranean mussel Mytilus galloprovincialis. Arch. Environ. Contam.
Toxicol. 34, 48–63.
Ridgway, I.M., Price, N.B., 1987. Geochemical associations and post-depositional
mobility of heavy metals in coastal sediments: Loch Etive, Scotland. Mar. Chem.
21, 229–248.
Robisch, P.A., Clark, R.C., 1993. Sample preparation and analyses of trace metals by
atomic absorption spectroscopy. In: Lauenstein, G.G., Cantillo, A.Y. (Eds.),
Sampling and Analytical Methods of the National Status and Trends Program
National Benthic Surveillance Program and Mussel Watch Project 1984–1992:
Comprehensive Description of Elemental Analytical Methods, vol. III. NOAA
Tech. Memo. NOS ORCA 71, pp. 111–150.
Romankevich, E.A., 1984. Geochemistry of Organic Matter in the Ocean. Springer-
Verlag, Berlin, Heidelberg, New York, p. 344.
Roméo, M., Sidoumou, Z., Gnassia-Barelli, M., 2000. Heavy metal in various molluscs
from Mauritanian Coast. B. Environ. Contam. Toxicol. 65, 269–276.
Roméo, M., Gnassia-Barelli, M., 1988. Donax trunculus and Venus verrucosa as
bioindicators of trace metal concentrations in Mauritanian coastal waters. Mar.
Biol. 99, 223–227.
Saavedra, Y., Gonzalez, A., Fernandez, P., Blanco, J., 2004. The effect of the size on
trace metal levels in raft cultivated mussels (Mytilus galloprovincialis). Sci. Total
Environ. 318, 115–124.
Saenz, L.A., Seibert, E.L., Zanette, J., Fiedler, H.D., Curtius, A.J., Ferreira, J.F., De
Almeida, E.A., Marques, M.R.F., Bainy, A.C.D., 2010. Biochemical biomarkers and
metals in Perna perna mussels from mariculture zones of Santa Catarina, Brazil.
Ecotoxicol. Environ. Saf. 73, 796–804.
Sokołowski, A., Bawazir, A.S., Wołowicz, M., 2004. The effect of seasonal cycle,
weight and sex of individuals on trace metal concentrations in the brown
mussel Perna perna from the coastal waters of Yemen (Gulf of Aden). Arch.
Environ. Contam. Toxicol. 46, 67–80.
Stoffers, P., Summerhayes, C., Forstner, U., Patchineelam, S., 1977. Copper and other
heavy metal contamination in sediments from New Bedford Harbor,
Massachusetts: a preliminary note. Environ. Sci. Technol. 11, 819–821.
Stronkhorst, J., 1992. Trends in pollutants in blue mussel Mytilus edulis and Flounder
Platichthys flesus from two Dutch esturies, 1985–1990. Mar. Pollut. Bull. 24,
250–258.
Szefer, P., Ikuta, K., Kushiyama, S., Szefer, K., Frelek, K., Geldon, J., 1997. Distribution
and association of trace metals in soft tissue and byssus of mollusc Perna perna
from the Gulf of Aden, Yemen. Environ. Int. 23, 53–56.
Szefer, P., Glasby, G.P., StÜben, D., Kusak, A., Geldon, J., Berner, Z., Neumann, T.,
Warzocha, J., 1999. Distribution of selected heavy metals and rare earth
elements in surficial sediments from the Polish sector of the Vistula Lagoon.
Chemosphere 39, 2785–2798.
UNEP/FAO/IAEA/IOC, 1984a. Determination of Total Mercury in Selected Marine
Organisms by Cold Vapour Atomic Absorption Spectrophotometry. Ref. Meth.
Mar. Poll. Stud. No. 8 Rev. 1, p. 17.
UNEP/FAO/IAEA/IOC, 1984b. Determination of Total Cadmium, Zinc, Lead and
Copper in Selected Marine Organisms by Flameless Atomic Absorption
Spectrophotometry. Ref. Meth. Mar. Poll. Stud. No. 11 Rev. 1, p. 21.
Usero, J., Morillo, J., Gracia, I., 2005. Heavy metal concentrations in molluscs from
the Atlantic coast of southern Spain. Chemosphere 59, 1175–1181.
Wang, W.X., Fisher, N.S., 1997. Modeling metal bioavailability for marine mussels.
Rev. Environ. Contam. Toxicol. 151, 39–65.
Wangersky, P.J., 1986. Biological control of trace metal residence time and
speciation: a review and synthesis. Mar. Chem. 18, 269–297.
Zhong, A.P., Guo, S.H., Li, F.M., Li, G., Jiang, K.X., 2006. Impact of anions on the heavy
metals release from marine sediments. J. Environ. Sci. 18, 1216–1220.