Continental Shelf Research 152 (2018) 61–70

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Continental Shelf Research

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Controlling factors of harmful microalgae distribution in water column, T

biofilm and sediment in shellfish production area (South of Sfax, Gulf of
Gabes) from southern Tunisia
⁎
Amira Loukil-Bakloutia,b, , Wafa Feki-Sahnounb, Asma Hamzab, Moufida Abdennadherb,
Mabrouka Mahfoudhib, Abderrahmen Bouaina, Othman Jarbouib
a
Université de Sfax, Faculté des Sciences de Sfax, Route Soukra Km 3.5, BP 802, CP 3000 Sfax, Tunisia
b
National Institute of Science and Technology of the Sea, Centre of Sfax, BP 1035, CP 3018 Sfax, Tunisia

A R T I C L E I N F O A B S T R A C T

Keywords: The aim of this study was to investigate the spatio-temporal distribution of harmful microalgae coupled with
Southern coasts of Sfax environmental factors in the most important area for natural stocks of the grooved carpet shell Ruditapes de-
Harmful microalgae cussatus in southern Tunisia. Sampling was performed monthly from May 2010 to April 2011 in five stations
Water column through the Tunisian National Monitoring Stations Network of Phytoplankton and Phycotoxins along the
Sediment
southern coasts of Sfax (Gulf of Gabes). The presence of harmful microalgae species was explored in three
Biofilm
Physicochemical parameters
compartments: water column, biofilm and sediment. Our results revealed fourteen species were identified be-
longing to dinoflagellates and diatoms with higher densities during the summer period. The co-inertia plot
analysis exhibited that the seasonal fluctuations of these species were controlled by the temperature as well as
the nutrients (particularly nitrogenous). Ternary diagrams showed that biofilm was the most colonized com-
partment by toxic benthic dinoflagellates species, namely Amphidinium carterae, Prorocentrum rathymum,
Prorocentrum concavum, Prorocentrum lima, Ostreopsis cf. ovata and Coolia monotis. In addition, these species were
recorded simultaneously in the water column and the sediment, a fact that could be explained by the re-
suspension of these benthic dinoflagellates from the biofilm by hydrodynamics. The data suggest that harmful
microalgae could be the source of toxins in the studied stations, which provide support to the implication of
these results on the future sampling strategy of harmful microalgae in shellfish collecting areas in Tunisia.

1. Introduction are an important component of a significant number of photo-

Harmful microalgae species may have an increasing global dis-
tribution and become a real threat around the world (Wells et al.,
2015). Harmful algae proliferation is a complex phenomenon linked to
many interacting factors such as transport of resting cysts in ballast
waters or on floating debris (Smayda, 2007), coastal water eu-
trophication (Rekik et al., 2012), building harbors which increase water
residence-time (Masó and Vila, 2005) and global climate change
(Hallegraeff, 2010).
Many studies have focused on phytoplankton and cysts diversity in
the water column and sediment (Bravo et al., 2012), but little attention
has been paid to the phytoplankton community present in the water
adjacent to the sediment, the biofilm. These assemblages are known as
benthic microalgae or microphytobenthos (Round et al., 1990). They
synthesizers in intertidal and shallow marine waters (Queiroz et al.,
2004), able to grow at low light levels and taking advantage of high
nutrient concentrations in the sediment (Blanchard and Montagna,
1992). Tidal flushing is one of the main factors controlling phyto-
plankton abundance in estuarine systems. It constitutes a relevant
phytoplankton driving force since it induces substantial horizontal and
vertical mixing of the water column, as well as upstream and down-
stream displacement of water masses along the main longitudinal es-
tuarine axis (Domingues et al., 2010). While tidally-induced horizontal
mixing and advection have more mechanical than physiological effects
on phytoplankton (Legendre and Demers, 1984), vertical mixing can
seriously affect the phytoplankton physiology and growth due to its
strong impact on the availability of phytoplankton resources as nu-
trients and light (Demers et al., 1979). Tidally-induced vertical mixing

⁎
Corresponding author at: Université de Sfax, Faculté des Sciences de Sfax, Route Soukra Km 3.5, BP 802, CP 3000 Sfax, Tunisia.
E-mail addresses: amiraloukilbaklouti@yahoo.fr (A. Loukil-Baklouti), wafafeki@yahoo.fr (W. Feki-Sahnoun), asma.hamza@instm.rnrt.tn (A. Hamza),
moufidaabdennadher@yahoo.fr (M. Abdennadher), mabroukamahfoudi@yahoo.fr (M. Mahfoudhi), Abderrah.Bouain@fss.rnu.tn (A. Bouain),
othman.jarboui@instm.rnrt.tn (O. Jarboui).

https://doi.org/10.1016/j.csr.2017.11.003
Received 14 June 2016; Received in revised form 9 November 2017; Accepted 10 November 2017
Available online 11 November 2017
0278-4343/ © 2017 Elsevier Ltd. All rights reserved.
A. Loukil-Baklouti et al.
may also modulate phytoplankton loss rates either because it affects the
resuspension of benthic microalgae into the water column (MacIntyre
and Cullen, 1996) or because it effectively controls the grazing impact
of benthic filter feeders (Lucas and Cloern, 2002).
Harmful microalgae have been increasingly reported over the last
decade along the Sfax coasts (Ben Salem et al., 2015). The inshore
southern coasts of Sfax are an important exploitation area of the
grooved carpet shell (Ruditapes decussatus) in the Gulf of Gabes with
79% of the total Tunisian production (Ben Naila et al., 2012). These
bivalve populations can be found in sandy and muddy-sand sediments
in bays, estuaries, coastal lagoons and other sheltered environments,
living on tidal flats and below tidal marks (Derbali et al., 2015). Five
shellfish production stations are present along the Sfax coasts. Un-
fortunately, the exploitation of this stock has been frequently bunged
subsequent to successive alerts due to the presence of potentially toxic
microalgae. The analysis of national monitoring data indicated that
numerous alerts for the presence of biotoxines in shellfish weren’t as-
sociated with the presence of toxic species in the water column. Thus,
the presence of potentially toxic species within the shellfish organisms
could originate from the sediment or the biofilm. Understanding the
distribution of harmful microalgae species within these compartments
would be particularly helpful to avoid collection of contaminated
shellfish.
This study deals with the assessment of harmful microalgae species
in different habitats (water column, biofilm and sediment), and during
an annual cycle of sampling within shellfish harvest zones on the
southern coasts of Sfax. The relationships between physico-chemical
parameters and abundance of harmful microalgae were also examined
to identify the parameters affecting the abundance of these organisms.
2. Material and methods
The southern coasts of Sfax, in southern Tunisia, stretch for 15 km
from the fish harbor to Gargour (Fig. 1). The climate is arid and semi-
arid Mediterranean largely influenced by its mild topography and its
maritime exposure (Chamtouri et al., 2008). The substrate in the in-
shore area is mainly muddy sand (Derbali et al., 2015). The tide is
semidiurnal, with a high tide of +1.60 m and a low tide of +0.30 m in
spring tide (Zaghden et al., 2014).
Sampling stations were selected among the Tunisian National
Monitoring Stations Network of Phytoplankton and Phycotoxins: Tabia
“S2”, Mahres “S3”, Ras Younga “S4”, Jaboussa “S5” and Skhira “S6”
(Fig. 1). Stations S2, S3 and S4 were exposed to industrial effluents as
heavy metal and organic compounds (Zaghden et al., 2014) whereas S5
and S6 were affected by the petroleum pollution from the transport
harbor (Kobbi-Rebai et al., 2013).
The Southern Sfax area is characterized by a benthic community,
with an exceptional bionomy, made up of extensive magnoliophytes
62
Continental Shelf Research 152 (2018) 61–70
Posidonia oceanica and Cymodocea nodosa meadows (Ben Mustapha and
Hattour, 2013). Their leaves provide suitable substrate for the estab-
lishment and growth of a number of epiphytic microalgae.
Sampling was performed monthly, during spring tides, from May
2010 to April 2011 through the Tunisian National Monitoring Stations
Network of Phytoplankton and Phycotoxins. The sediment and the ad-
jacent water (biofilm) were sampled by means of a plastic core sampler
of 5 cm in diameter in mudflats before high tide on the day when the tide
coefficient was highest. Before cutting the sediment, the top cap from the
core was removed and the biofilm was recuperated in a bottle. Then the
sediment core was carefully pushed and the upper 3-cm cut
(Schwinghamer et al., 1991) and kept at 4 °C in the dark until analysis;
3 g of sediment were resuspended in 300 ml of filtered seawater and then
filtered using 0.45-μm filter membranes for microalgae identification and
enumeration. Water samples were collected at the same station, during
highest high tide with a Van Dorn bottle at a depth of 1 m.
Water samples collected from sediment, biofilm and water column
for microalgae analyses were preserved with acidic Lugol’s iodine so-
lution (4%) and stored in the dark at 4 °C until analysis. Cell counts
were conducted one day after sampling. A 10 ml aliquot was allowed to
settle in a sedimentation chamber according to Uthermöhl (1958). Cell
counts were performed under an inverted microscope (Olympus CK40)
at a magnification of x200. The entire surface of the sedimentation
chamber was scanned (Sournia, 1978), so the detection level was 100
cells l−1.
Environmental variables at the biofilm and the water column were
measured. Temperature, salinity and pH were measured at both surface
and near the bottom using a multiparameter kit (Multi 340 i/SET;
sensitivity ( ± 1 digit) especially important for pH ( ± 0.01 pH)). For
nutrient concentrations, samples of 125 ml were kept immediately
upon collection at −20 °C, in the dark. The inorganic nutrients (nitrite:
NO2-, nitrate: NO3-, ammonium: NH4+, orthophosphate: PO43- and si-
licate: Si (OH)4) were analyzed with a BRAN-LUEBBE type 3 auto-
analyzer (APHA, 1992) and their concentrations determined calorime-
trically according to Grasshoff (1983).
3. Statistical analyses
A simple log (x + 1) transformation was applied to data to stabilize
variance. Data were analyzed using a co-inertia analysis which is a
direct extension of multiple regressions to the modeling of multivariate
response matrix (Dray et al., 2003). This analysis examines the corre-
lation between an array of response variables (stations and sampling
periods) and independent explanatory variables (harmful microalgae
abundance) conditioned by a third matrix (environmental parameters),
keeping the environmental effect constant. Computing and graphical
displays were performed with the R-2.4.0 software (R Development
Core Team, 2006) using the packages ade4 1.4.2 (Chessel et al., 2004).
Fig. 1. Location of sampling stations in the southern
coasts of Sfax. S2: Tabia, S3: Mahres, S4: Ras Younga,
S5: Jaboussa, S6: Skhira.
A. Loukil-Baklouti et al.
Table 1
List of harmful microalgae species in Sfax area included in the IOC (Intergovernmental
Oceanographic Commission).
Taxa
Toxic dinoflagellates
Alexandrium catenella ((Whedon & Kofoid) Balech, 1985)
Alexandrium minutum (Halim,1960)
Alexandrium pseudognaulax ((Biecheler) Horiguchi ex Kita
& Fukuyo, 1992)
Amphidinium carterae (Hulburt, 1957)
Coolia monotis (Meunier, 1919)
Dinophysis acuminata (Claparède & Lachmann, 1859)
Gymnodinium catenatum (H.W.Graham, 1943)
Karlodinium veneficum (Ballantine, 1956)
Ostreopsis cf. ovata (Fukuyo, 1981)
Prorocentrum concavum (Fukuyo, 1981)
Prorocentrum lima ((Ehrenberg) F.Stein, 1878)
Prorocentrum rathymum (Loeblich, Sherley & Schmidt,
1979)
Toxic diatoms
Pseudo-nitzschia seriata ((Cleve) H.Peragallo, 1899)
Pseudo-nitzschia spp. (H.Peragallo, 1900)
A ternary diagram was used to assess the crucial compartment
dominated by harmful microalgae species using the Ms Excel software.
Indeed, ternary diagrams are frequently used in geosciences to visualize
compositional data characterized by three or more components, which
are amalgamated into three components. Problems with both visuali-
zation and graphical interpretation of these data particularly arise when
the compositions are close to the boundaries of the ternary diagram
(Eynatten et al., 2002). The coordinates of each point presenting the
species were determined by a simple projection parallel to each line of
the ternary diagram presenting the compartments. A species close to 1
indicates that it is further linked with the corresponding compartment
whereas a value close to 0 would mean that it is less present. Given that,
the toxic cells may exhibit a preferential distribution either in the water
column, the biofilm or the sediment.
4. Results
Water temperature was similar in both investigated compartments
and revealed a stable variation at different stations with mean values of
17.8 ± 1.18 °C and 18.9 ± 1.18 °C (Table 2). A slight difference was
noted on station S2, where the temperature variability was more pro-
nounced and exceeding 23 °C (18.9 ± 1.18 °C). Salinity was highest at
S4, with a mean value of 39.77 ± 0.17 psu and lowest at S6
(39.38 ± 0.13 psu) (Table 2). pH values showed a general increase in
S4 with mean values of 8.71 ± 0.20 (Table 2). Dissolved oxygen con-
centrations did not fluctuate widely at any station (Table 2), ranging
from 4.80 to 5.95 mg l−1, with the maximum at S2
(5.21 ± 0.10 mg l−1) and the minimum at S4 (4.93 ± 0.03 mg l−1)
(Table 2).
Nitrite concentrations were below 1.0 µmol l−1 during the sampling
period (Table 2). The minimal nitrite concentration in the water column
was about 0.18 ± 0.02 µmol l−1 at S5 and maximal at S3
(0.32 ± 0.08 µmol l−1) (Table 2). In the biofilm, nitrite concentration
ranged from 0.39 ± 0.05–0.47 ± 0.05 µmol l−1 with the highest con-
centration at S6 (0.47 ± 0.05 µmol l−1) (Table 2). Nitrate concentra-
tions ranged from 0.3 µmol l−1 to 8.48 µmol l−1. The highest values
were observed mainly in the water column where the maxima were
recorded at S4 (3.52 ± 0.79 µmol l−1) (Table 2). The highest value in
the biofilm was observed at S6 (4.76 ± 0.38 µmol l−1) (Table 2).
The dominant nitrogen form, ammonium, ranged from 0.09 to
13.25 µmol l−1 in the water column at S3 (2.24 ± 0.83 µmol l−1) and
S6 (4.35 ± 1.09 µmol l−1) (Table 2). Concentrations in the biofilm
were relatively constant with mean values of 1.08 ± 0.10 and
63
Continental Shelf Research 152 (2018) 61–70
2.10 ± 0.26 µmol l−1 at S4 and S5, respectively (Table 2). Orthopho-
sphate concentrations (0.05–1 µmol l−1) were generally low in the
biofilm (Table 2). The lowest values were recorded at S4
(0.30 ± 0.07 µmol l−1) and the highest at S3 (0.33 ± 0.09 µmol l−1).
In the water column, orthophosphate concentrations fluctuated be-
tween 0.09 and 12.55 µmol l−1 with a maximum at S3
(3.96 ± 1.27 µmol l−1) (Table 2).
Silicate concentrations were highly variable (0.39–19.80 µmol l−1)
particularly in S2, with mean values of 7.17 ± 1.45 µmol l−1 and
7.04 ± 1.81 µmol l−1 in the biofilm and water column, respectively
(Table 2).
The phytoplankton and microphytobenthos communities were
composed of four groups, namely Dinophyceae, Bacillariophyceae,
Euglenophyceae, and Cyanobacteria. The total microalgae abundance
was calculated for all stations at each compartment (Table 3). The
highest mean value was recorded in the biofilm at S3 (212.57 ± 209.89
× 104 cells l−1) and in the water column at S6 (3.68 ± 1.15 × 104 cells
l−1). The lowest mean values in all stations were recorded mostly in the
sediment compartment. The harmful microalgae fluctuated from 0.82
× 104 cells l−1 in the water column of S2 (0.82 ± 0.57 × 104 cells l−1)
to 0.09 ± 0.13 × 104 cells g−1 in the sediment of S4 (Table 3).
Fourteen harmful microalgae species were found during the entire
survey period (Table 1). Prorocentrum rathymum and Alexandrium min-
utum were the most abundant dinoflagellates, representing 47.1% and
32.6% of the total harmful microalgae abundance, respectively in all
stations (Table 4). Prorocentrum rathymum was more abundant in S3
(3356.94 ± 1894.95 cells l−1) than in S5 (1130.56 ± 603.30 cells l−1)
(Table 4). This epibenthic species was more frequent and abundant in
the sediment compartment from late spring to summer, particularly
during July (Fig. 2). Alexandrium minutum was recorded at S2 with an
average value of 3318.61 ± 1613.21 cells l−1, but was less abundant in
S5 with a mean value of 130.56 ± 36.16 cells l−1 (Table 4). The highest
density and abundance of this species was detected in the water-
column, especially in the spring and summer periods (Fig. 3). Two toxic
diatoms species Pseudo-nitzschia spp. and P. seriata were recorded but
were less abundant compared to other harmful microalgae species
(Table 4).
Ternary diagram allows visualizing frequencies of harmful micro-
algae species in different compartments (Fig. 4). Gymnodinium cate-
natum, Alexandrium pseudogonyaulax, Pseudo-nitzschia spp., P. seriata,
Ostreopsis cf. ovata, Coolia monotis, Amphidinium carterae and Pro-
rocentrum lima were mostly recorded in the biofilm with a frequency of
about 50% in almost all the stations. A. minutum, P. rathymum and K.
veneficum were linked to the water column with a frequency ranging
between 30% and 60%. In the sediment, the frequency of toxic species
was below 30%. Abundance of A. pseudogonyaulax in S4 and A. carterae
in S5 were exclusively linked to the sediment (Fig. 4c, d). For a typically
benthic species such as Amphidinium carterae, the ternary diagram of S2
showed that this species had a score between 0.4 and 0.6 (Fig. 4a). This
means that this species was linked to the sediment and the biofilm with
a frequency of 40% and 60%, respectively. On the other hand, this
species was typically recorded with the ternary value 1 in the sediment
of S5 (Fig. 4d) and in the biofilm of S6 (Fig. .4e). The species O. cf. ovata
was mainly found at the biofilm with ternary values of 0.65, 0.7 and 1
in S6, S2 and S4, respectively.
The co-inertia plot illustrates the relationships between biological
variables and abiotic features in the sampling stations (Fig. 5). The
overall model explained 59.2% of the total variation (permutation test,
p=0.02, 1000 replicates) attributed to harmful microalgae species
(19%) and to physical and chemical variability (16%) (Fig. 5b). Stations
S2, S3 and S4 showed close links between orthophosphate, dissolved
oxygen, pH, temperature, silicate and salinity and the harmful micro-
algae composition. This composition was illustrated around the X axis
by the position of the benthic (Prorcentrum lima, P. concavum and Am-
phidinium carterae) and pelagic dinoflagellates (Dinophysis acuminata,
and Gymnodinium catenatum). In contrast, along S5 and S6, this
 Table 2
Mean values and error standard mean (ESM) of physico-chemical parameters in Biofilm and Water column at the Southern coast of Sfax.

Stations S2 S3 S4 S5 S6

Compartments Biofilm Water column Biofilm Water column Biofilm Water column Biofilm Water column Biofilm Water column
Physical parameters Mean ± ESM Mean ± ESM Mean ± ESM Mean ± ESM Mean ± ESM Mean ± ESM Mean ± ESM Mean ± ESM Mean ± ESM Mean ± ESM
A. Loukil-Baklouti et al.

Temperature (°C) 18.90 ± 1.18 18.90 ± 1.18 18.43 ± 1.23 18.43 ± 1.23 18.12 ± 1.17 18.12 ± 1.17 17.82 ± 1.18 17.82 ± 1.18 18.01 ± 1.31 18.01 ± 1.31
Salinity (psu) 40.12 ± 0.11 40.12 ± 0.11 39.89 ± 0.16 39.89 ± 0.16 39.77 ± 0.17 39.77 ± 0.17 39.76 ± 0.99 39.76 ± 0.99 39.38 ± 0.13 39.38 ± 0.13
pH 8.37 ± 0.16 8.37 ± 0.16 8.31 ± 0.05 8.31 ± 0.05 8.71 ± 0.20 8.71 ± 0.20 8.19 ± 0.02 8.19 ± 0.02 8.18 ± 0.03 8.18 ± 0.03
Dissolved oxygen (mg l−1) 5.21 ± 0.10 5.21 ± 0.10 5.01 ± 0.09 5.01 ± 0.09 4.93 ± 0.03 4.93 ± 0.03 5.10 ± 0.09 5.10 ± 0.09 5.05 ± 0.06 5.05 ± 0.06
Chemical parameters
NO2- (µmol l−1) 0.37 ± 0.03 0.23 ± 0.04 0.39 ± 0.05 0.32 ± 0.08 0.32 ± 0.02 0.21 ± 0.03 0.38 ± 0.04 0.18 ± 0.02 0.47 ± 0.05 0.21 ± 0.04
NO3- (µmol l−1) 3.67 ± 0.38 3,08 ± 0.61 3.30 ± 0.28 3.15 ± 0.70 3.04 ± 0.33 3.52 ± 0.79 3.39 ± 0.29 2,35 ± 0.60 4.76 ± 0.38 2.70 ± 0.61
NH4+ (µmol l−1) 1.14 ± 0.12 1.97 ± 0.65 1.40 ± 0.07 2.24 ± 0.83 1.08 ± 0.10 3.28 ± 0.81 2.10 ± 0.26 4.11 ± 0.96 1.66 ± 0.19 4.35 ± 1.09
PO4- (µmol l−1) 0.37 ± 0.06 2.03 ± 0.82 0.33 ± 0.09 3.96 ± 1.27 0.30 ± 0.07 1.45 ± 0.94 0.28 ± 0.03 0.89 ± 0.25 0.31 ± 0.03 1.65 ± 0.63
Si(OH)4 (µmol l−1) 7.17 ± 1.45 7.04 ± 1.81 8.54 ± 0.64 3.61 ± 0.48 8.55 ± 1.00 3.60 ± 1.52 8.94 ± 1.17 5.73 ± 1.58 9.52 ± 0.51 3.07 ± 1.35

64
Table 3
Mean values and error standard mean (ESM) of biological parameters in the Southern coast of Sfax. (B: Biofilm; W: Water column and S: Sediment).

Stations S2 S3 S4

Compartments B (104 Cells l−1) W (104 Cells l−1) S (104 Cells g−1) B (104 Cells l−1) W (104 Cells l−1) S (104 Cells g−1) B (104 Cells l−1) W (104 Cells l−1) S (104 Cells g−1)

Total microalgae abundance 4.75 ± 0.7 1.66 ± 051 2.98 ± 0.35 212.57 ± 209.89 0.86 ± 0.10 1.11 ± 0.18 3.28 ± 0.20 1.19 ± 0.22 2.09 ± 0.24
Mean ± ESM
Harmful microalgae 0.36 ± 0.19 0.82 ± 0.57 0.45 ± 0.30 0.26 ± 0.21 0.49 ± 0.44 0.41 ± 0.37 0.13 ± 0.06 0.11 ± 0.05 0.09 ± 0.04
abundance Mean ± ESM

Stations S5 S6

Compartments B (104 Cells l−1) W (104 Cells l−1) S (104 Cells g−1) B (104 Cells l−1) W (104 Cells l−1) S (104 Cells g−1)

Total microalgae abundance 4.49 ± 0.43 2.19 ± 0.57 2.33 ± 0.40 3.93 ± 0.50 3.68 ± 1.15 5.67 ± 0.64
Mean ± ESM
Harmful microalgae 0.16 ± 0.07 0.23 ± 0.17 0.09 ± 0.06 0.16 ± 0.05 0.28 ± 0.14 0.14 ± 0.05
abundance Mean ± ESM
Continental Shelf Research 152 (2018) 61–70
A. Loukil-Baklouti et al. Continental Shelf Research 152 (2018) 61–70

Table 4
Composition of harmful microalgae species in the Southern coast of Sfax.

Stations S2 S3 S4 S5 S6 Relative abundance (%)

Mean ± ESM Mean ± ESM Mean ± ESM Mean ± ESM Mean ± ESM
Cells l−1 Cells l−1 Cells l−1 Cells l−1 Cells l−1

Alexandrium catenella 0±0 0±0 0±0 0±0 2.78 ± 2.78 0.02

Alexandrium minutum 3318.61 ± 1613.21 238.19 ± 58.40 203.47 ± 53.23 130.56 ± 36.16 637.50 ± 251.69 32.64
Alexandrium pseudogonyaulax 1.39 ± 1.39 1.39 ± 1.39 2.78 ± 2.78 0±0 5.56 ± 3.87 0.08
Amphidinium carterea 6.94 ± 4.06 0±0 0±0 2.78 ± 2.78 1.39 ± 1.39 0.08
Coolia monotis 18.75 ± 7.87 32.64 ± 14.915 103.47 ± 45.03 18.06 ± 7.22 40.28 ± 13.04 1.54
Dinophysis acuminata 1.39 ± 1.39 0±0 0±0 0±0 0±0 0.01
Gymnodinium catenatum 5.56 ± 5.56 5.56 ± 3.87 1.39 ± 1.39 0±0 0±0 0.09
Karlodinium veneficum 1220.56 ± 424.54 181.94 ± 48.13 177.78 ± 48.13 185.42 ± 54.36 350 ± 71.28 15.25
Ostreopsis cf. ovata 9.72 ± 6.24 11.11 ± 4.92 2.78 ± 1.94 29.17 ± 16.23 30.56 ± 14.12 0.60
Prorocentrum concavum 9.72 ± 4.37 5.56 ± 3.32 8.33 ± 4.23 5.56 ± 3.32 4.17 ± 3.07 0.24
Prorcentrum lima 90.97 ± 41.19 15.28 ± 8.87 4.17 ± 2.34 26.39 ± 8.09 18.06 ± 6.65 1.12
Prorocentrum rathymum 715.69 ± 414.32 3356.94 ± 1894.95 562.50 ± 228.07 1130.56 ± 603.30 768.06 ± 286.73 47.09
Pseudo-nitzchia seriata 0±0 0±0 0±0 1.39 ± 1.39 1.39 ± 1.39 0.02
Pseudo-nitzchia spp. 20.14 ± 11.04 22.22 ± 9.85 11.81 ± 4.93 68.06 ± 20.31 48.61 ± 33.66 1.23

Fig. 2. Boxplots depicting the spatial and temporal variability of Prorocentrum rathymum in different stations in Southern Sfax. Note the log-scale on the y-axes.

Fig. 3. Boxplots depicting the spatial and temporal variability of Alexandrium minutum in stations in the Southern Sfax. Note the log-scale on the y-axes.

component axis was surrounded by most of the harmful microalgae 5. Discussion

such as Pseudo-nitzschia spp., P. seriata, Alexandrium catenella, Karlodi-
nium veneficum, Coolia monotis, Ostreopsis cf. ovata, Prorocentrum ra-
thymum and dissolved nitrogen (NH4+, NO2- and NO3-) (Fig. 5a).
The monthly co-inertia analysis illustrated the relationship between
the harmful microalgae community composition and the abiotic factors
(Fig. 6). The overall model explained 83.7% of the total variation
(permutation test, p = 0.02, 1000 replicates). This variation was due to
harmful microalgae species (16%), physical and chemical variability
(16%) (Fig. 6b). From June through September close links were found
between dissolved oxygen, temperature, pH, silicate and nitrogen
concentrations as abiotic parameters and microalgae composition
which was illustrated by the position of harmful microalgae species
such as Coolia monotis, Prorocentrum concavum, P. lima, P. rathymum,
Karlodinium veneficum, Dinophysis acuminata, Ostreopsis cf. ovata, Gym-
nodinium catenatum, Alexandrium minutum and A. carterae around the Y
axis (Fig. 6a). In contrast, from January to May and from October
through December, this component axis was surrounded by Alexan-
drium pseudogonyaulax, A. catenella, Pseudo-nitzschia spp. and P. seriata
(Fig. 6a).
The present study is the first attempt to investigate the composition
of harmful microalgae species and their spatial distribution coupled
with environmental parameters at three compartments (biofilm, sedi-
ment and water column) during an annual cycle within a shellfish
harvesting area in the South of Sfax (Tunisia).
Our results showed that water temperature seems to be the main
factor affecting the proliferation of most harmful microalgae, especially
in summer (Fig. 6a). Most potentially toxic dinoflagellates, such as
Alexandrium minutum and Gymnodinium catenatum, recorded in Sfax
coasts were correlated with several abiotic parameters, in particular
with water temperature (Fig. 6a). The increase of the temperature could
potentially alter the phenology of certain species. These results are in
accordance with previous studies showing similar observations in
Skhira “S6” and Jaboussa “S5” for G. catenatum (Dammak-Zouari et al.,
2009). Besides, A. minutum outbreaks were correlated with sea surface
temperature at Jaboussa “S5” (Abdennadher et al., 2012). Furthermore,
several studies have reported that the temperature was the main en-
vironmental factor controlling harmful microalgae bloom occurrence

65
A. Loukil-Baklouti et al. Continental Shelf Research 152 (2018) 61–70

Fig. 4. Ternary plot illustrating the relative contribution of Biofilm, Sediment and Water-column) for all samples at five stations in Southern of Sfax (a) S2, (b) S3, (c) S4, (d) S5 and (e) S6.
The relative contribution, in percentage, of a component to total absorption can be read on the corresponding axis, Alexandrium catenella = A. catenella; Alexandrium minutum = A.
minutum; Alexandrium pseudogonyaulax =A. pseudogonyaulax; Amphidinium carterea = A. carterea; Coolia monotis = C. monotis; Dinophysis acuminate = D. acuminata; Gymnodinium
catenatum = G. catenatum; Karlodinium veneficum = K. veneficum; Ostreopsis ovata = O. cf. ovata; Prorocentrum concavum = P. concavum; Prorcentrum lima = P. lima; Prorocentrum
rathymum = P. rathymum; Pseudo-nitzchia seriata = P. seriata; Pseudo-nitzchia spp. = P. spp.

and physiological processes (Laabir et al., 2013). The studies under-
taken by Dammak-Zouari et al. (2009) and Feki et al. (2013) have also
highlighted the effect of temperature on harmful microalgae pro-
liferations, in summer, in the Gulf of Gabes.
Interestingly, the recorded harmful microalgae in our sampling
areas, such as A. minutum, A. pseudogonyaulax, Dinophysis acuminata and
Prorocentrum lima, were correlated with salinity (Fig. 5a). The main
inference we can draw from these results is that the temperature might
enhance evaporation leading to a salinity increase because of the
shallowness of the Gulf of Gabes. Thus, the effect of salinity on toxic
species might not be a direct cause-and-effect relationship but might
involve temperature as the main driving parameter. Similar results
were also found in Tunisian waters (Aissaoui et al., 2014; Armi et al.,
2012) and in other Mediterranean and northeast Atlantic ecosystems,
for instance, along the Greek coast (Aligizaki et al., 2009) and the
French Atlantic and English Channel coasts (Husson et al., 2016).
As regards to the analyses of nutrient concentrations in the water
column (Table 2), our results were quite similar to those reported in
previous studies in the Gulf of Gabes (Ben Salem et al., 2015; Kobbi-
Rebai et al., 2013). Nevertheless, the evaluation of orthophosphate
concentrations revealed high levels in most stations (Table 2). This
finding is in agreement with those of Ben Salem et al. (2015) contrary to
the results reported by Rekik et al. (2013) which showed lower con-
centrations than ours. The terrestrial inputs of phosphate originating
from chronic uncontrolled discharges generated by the chemical in-
dustry implemented in the Gulf of Gabes, seem to be involved in the
increased phosphate concentrations (Ben Salem et al., 2015). Moreover,
the studied area is urbanized and the coastline is industrialized and
characterized by eutrophic systems attributed to anthropogenic inputs
(Zaghden et al., 2014). The presence of anthropogenic nutrients can
also control the phytoplankton community structure and have an im-
portant role in promoting harmful algae blooms (Wells et al., 2015).
In the study area, we analyzed for the first time the abundance of
harmful microalgae species on the south coasts of Sfax simultaneously
in the three compartments (water column, biofilm and sediment)
(Fig. 4). Our findings showed the presence of potentially toxic

66
A. Loukil-Baklouti et al.
epibenthic dinoflagellate (Benthic HABs) in the water column, the
biofilm as well as the sediment, with the highest abundance noted in
the biofilm. Similar results confirmed the dominance of these species in
the biofilm in many intertidal and shallow marine environments,
especially since biofilm includes some nutrients necessary for micro-
algae growth (Underwood, 2005). These microphytobenthic organisms
can be transferred into the water column mainly through advection and
bioperturbation, particularly in shallow waters (Allison, 2000). The
effects of vertical migration on biofilm photophysiology have aroused a
growing interest and an assessment of the migration rate from one
compartment to another is still a hot topic (Perkins et al., 2010). Pre-
vious investigations in the Gulf of Gabes revealed that epiphytic mi-
croalgae communities were dominated by some potentially toxic di-
noflagellate species such as Amphidinium carterae, P. rathymum, P.
concavum, P. lima, Ostreopsis cf. ovata and Coolia monotis (Abdennadher
et al., 2017; Ben Brahim et al., 2013). These epiphytic microalgae were
present in the water column (Abdennadher et al., 2017; Ben Brahim
et al., 2013) and attached to phanerogamic plants represented by Po-
sidonia oceanica and Cymodocea nodosa (Ben Brahim et al., 2013;
Mabrouk et al., 2014). These species are significantly affected by re-
suspension and deposition events which determine the vertical
67
Continental Shelf Research 152 (2018) 61–70
Fig. 5. Co-inertia plot for the biotic and abiotic parameters
changes for the five stations of sampling (a) and partition of the
different harmful microalgae taxa and physico-chemical factors
(b), Alexandrium catenella = A. catenella; Alexandrium minutum =
A. minutum; Alexandrium pseudogonyaulax =A. pseudogonyaulax;
Amphidinium carterea = A. carterea; Coolia monotis = C. monotis;
Dinophysis acuminate = D.acuminata; Gymnodinium catenatum =
G. catenatum; Karlodinium veneficum = K. veneficum; Ostreopsis
ovata = O. cf. ovata; Prorocentrum concavum = P. concavum;
Prorcentrum lima = P. lima; Prorocentrum rathymum = P. ra-
thymum; Pseudo-nitzchia seriata = P. seriata; Pseudo-nitzchia spp.
= P. spp.
distribution of the organisms through the interface and the adjacent
water column (Leles et al., 2014; Queiroz et al., 2004). Therefore, the
succession of high and low tides gives rise to a series of oscillations
characterizing the short-term dynamics of intertidal benthic microalgae
biomass (Blanchard et al., 2001).
High densites of Prorocentrum species occurring in summer (from
June 2010 to September 2010) were correlated with temperature and
nitrogenous nutrients especially nitrate (Fig. 6a). Among Prorocentrum
species, P. rathymum exhibited the highest abundance with 47.1% of the
total harmful microalgae abundance (Table 4). Otherwise, this species
showed the highest density in the sediment and the water column
during the summer period particularly in July (Fig. 2). These results go
along with those of Aligizaki et al. (2009) revealing a high abundance
of P. rathymum within the same period (July) in the northern western
Mediterranan Sea (Greece). The epiphytic dinoflagellates are easily
resuspended in the water column due to the large tidal amplitude and
the hydrodynamic conditions in the Gulf of Gabes (Ben Ismail et al.,
2012). Furthermore, it is well known that a wedge-shaped deposit of
magnoliophytes leaf litter commonly referred to as “banquettes”
(Boudouresque and Meinesz, 1982) can cover vast areas of the coast
and deposit at the bottom. These phenomena represent a potential
A. Loukil-Baklouti et al.
source of nutrients and favor the development of species that have more
affinity to these nutrients as Prorocentrum spp. (Glibert et al., 2012).
Other benthic Prorocentrum spp. representing a serious hazard for
public health in Sfax southern coasts, such as P. lima, was recorded
during the summer period and showed a positive correlation with
temperature and a negative correlation with nitrogenous nutrient
concentrations (Fig. 6a). P. lima cells occurred in S2, S3 and S4 stations
correlated with orthophosphate concentration and salinity (Fig. 5a).
Similar findings on the correlation with the temperature were found in
Bizerte lagoon (Sahraoui et al., 2013), in the Gulf of Tunis (Aissaoui
et al., 2014) and in the western Adriatic (Ingarao et al., 2009). Fur-
thermore, P. lima was reported in several Mediterranean ecosystems
during August in the Gulf of Tunis (Turki, 2005) and in the Tunis
northern lagoon during spring and summer seasons (Armi et al., 2012).
In agreement with our results, the salinity and ammonium concentra-
tions were shown to strongly affect the proliferation of P. lima in the
Gulf of Tunis, South Mediterranean (Aissaoui et al., 2014), and as the
phosphate, affects its growth in Ría de Vigo, Galicia, northwestern
Spain (Varkitzi et al., 2010).
The second highest density among the total harmful microalgae
succeeding P. rathymum was noted for Alexandrium minutum (32.6%)
68
Continental Shelf Research 152 (2018) 61–70
Fig. 6. Co-inertia plot for the biotic and abiotic parameters
changes for the twelve months period of sampling (a) and parti-
tion of the different harmful microalgae taxa and physico-che-
mical factors (b), Alexandrium catenella = A. catenella;
Alexandrium minutum = A. minutum; Alexandrium pseudogo-
nyaulax =A. pseudogonyaulax; Amphidinium carterea = A. car-
terea; Coolia monotis = C. monotis; Dinophysis acuminate = D.
acuminata; Gymnodinium catenatum = G. catenatum; Karlodinium
veneficum = K. veneficum; Ostreopsis ovata = O. cf. ovata;
Prorocentrum concavum = P. concavum; Prorcentrum lima = P.
lima; Prorocentrum rathymum = P. rathymum; Pseudo-nitzchia ser-
iata = P. seriata; Pseudo-nitzchia spp. = P. spp.
(Table 4). This elevated density was recorded during spring (March-
April) and summer periods (Figs. 3 and 6a) and correlated with tem-
perature, salinity and phosphate especially at S2, S3 and S4 (Fig. 5a).
The occurrence of this species is in accordance with those reported by
Bravo et al. (2008) and Abdennadher et al. (2012) in the same periods.
This species appears to be euryhaline and eurythermal. These proper-
ties have been confirmed in culture experiments by Grzebyk et al.
(2003). The correlation found between A. minutum and phosphate can
be attributed to the increase of P released by industrial and anthro-
pogenic sewage. Previous works have shown that A. minutum blooms
occur primarily in coastal waters and nutrient discharge areas
(Abdennadher et al., 2012), while other authors found that phosphorus
deficiency in coastal areas can favor the development of species of the
genus Alexandrium (Imai et al., 2006). In fact, A. catenella was corre-
lated positively with nitrate, nitrite and negatively with ammonium in
Jaboussa and Skhira stations (Fig. 5a). Nonetheless, this species was
found to be affected by phosphate from January to May and from Oc-
tober to December (Fig. 6a). Taken together these results confirm the
studies showing that A. catenella was occurred during the winter season
in the North Lake of Tunis (Armi et al., 2011) and was able to use
several nitrogen (N) sources for growth with a preference for
A. Loukil-Baklouti et al.
ammonium in Thau lagoon, southern France (Collos et al., 2006).
Dammak-Zouari et al. (2006) showed an eventual transfer of A. cate-
nella near the shellfish production area in ballast water at Skhira (Gulf
of Gabes).
It is of note that the presence of some harmful microalgae, such as
Ostreopsis cf. ovata, Coolia monotis and Pseudo-nitzschia species pose a
risk of human intoxications by eating shellfish contaminated with their
toxins. So that, these species will be itemized hereafter. Data regarding
Ostreopsis species exhibited enhanced density in the biofilm and the
water column (Fig. 4) during the summer period that was correlated
with the temperature (Fig. 6a). These findings give support to several
studies highlighting an important role of the seawater temperature in
Ostreopsis proliferations in the Mediterranean Sea (Abdennadher et al.,
2017; Cohu et al., 2013). In contrast, in the North Adriatic Sea
(Accoroni et al., 2012) and the Sea of Japan (Selina et al., 2014), the
highest abundances of O. cf. ovata were always recorded in late summer
when temperature started to decrease. The abundance of Coolia monotis
was correlated with temperature, nitrite, nitrate and ammonium during
summer (Figs. 5a and 6a). This coincides with the observations of Armi
et al. (2012) in the northern Lake of Tunis, where a strong positive
correlation was highlighted between C. monotis density and high water
temperature. Similarly, Aligizaki and Nikolaidis (2006) detected a
summer peak during August of this species in the northern Aegean Sea,
Greece. Nevertheless, Ismael (2014) found a weak positive correlation
between C. monotis and temperature in Alexandria, Egypt. This corre-
lation goes along with the statement that C. monotis species can use a
range of organic and inorganic nitrogenous substrates (Armi et al.,
2010). Some potentially toxic diatoms, such as Pseudo-nitzschia spp. and
Pseudo-nitzschia seriata, were observed in winter, autumn and spring
seasons (Fig. 6a). These species were correlated with nitrogenous nu-
trients, especially ammonium in Skhira and Jaboussa (Fig. 5a). Ni-
trogen, especially nitrate and ammonium, were the main nutrients as-
sociated with proliferations of Pseudo-nitzschia spp. in northwestern US
(Trainer et al., 2012). Within the some context, various studies on
Pseudo-nitzschia species in the Bizerte Lagoon (northern Tunisia) re-
vealed a positive correlation between their abundance and nitrate
concentration during late spring (Sahraoui et al., 2012).
6. Conclusions
Our study provides the monthly spatial distribution of harmful mi-
croalgae in the water column, the biofilm and the sediment simulta-
neously revealing high density of harmful microalgae in the three
compartments on the Southern coasts of Sfax. Our results clearly con-
firmed that the water temperature influenced the distribution of most
potentially toxic dinoflagellate species. The highest density of benthic
dinoflagellates species, such as A. carterae, P. rathymum, P. concavum, P.
lima, O. cf. ovata and C. monotis, characterized the biofilm as it re-
presents the compartment accumulating the highest nutrient content.
Taking these findings into consideration, it is ascertained that these
species may be the source of toxins in wild clam’s beds along the coasts
of Sfax. A regular control of this compartment as well as the water
column is crucial in the framework of any phytoplankton monitoring
program.
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