Involvement of Cd Bioaccumulation in Spinal Deformities
Occurrence in Natural Populations of Mediterranean
Kaouthar Kessabi & Abdelhamid Kerkeni &
Khaled Saïd & Imed Messaoudi
Received: 11 August 2008 /Accepted: 30 September 2008 /
Published online: 25 October 2008
# Humana Press Inc. 2008
Abstract The aim of this study was to investigate the possible influence of environmental
exposure to cadmium (Cd) on the spinal deformities occurrence in the Mediterranean
killifish, Aphanius fasciatus (Pisces: Cyprinodontidae). For this purpose, some indicators of
skeletal bone mineralization, Cd, and calcium (Ca) concentrations in spinal column as well
as bioaccumulation of Cd from the water and the sediment have been compared in normal
and deformed fish collected from polluted (S1) and nonpolluted (S2) areas in the Gulf of
Gabès in Tunisia. When compared to the normal fish, the deformed fish showed signs of
spinal column demineralization such as significant decrease in the ash weight/dry weight
ratio, percentage of nonorganic components content, and Ca concentration. Cd concen-
trations in spinal column and liver were significantly higher in deformed fish than in normal
fish. A highly significant negative correlation (r=−0.915, p<0.01) between Cd and Ca
concentrations was noted in spinal column of deformed fish. Bioaccumulation factors of Cd
in the liver from the water and the sediment in deformed fish were also significantly higher
(p<0.0001) than in normal fish from S1 and S2. These findings suggest that the ability to
accumulate large amount of Cd may represent a potential risk to induce spinal deformities
in natural populations of Mediterranean killifish.
The Mediterranean killifish, Aphanius fasciatus (Nardo, 1827), is a Cyprinodontidae,
euryhaline fish that lives in geographically isolated waters and in environments of high
Biol Trace Elem Res (2009) 128:72–81
K. Kessabi:K. Saïd:I. Messaoudi (*)
UR 09/30: Génétique, Biodiversité et Valorisation des Bioressources, Institut de Biotechnologie,
Université de Monastir, 5000 Monastir, Tunisia
Eléments Trace, Radicaux Libres, Systèmes Antioxydants et Pathologies Humaines et Environnement,
Faculté de Médecine, Université de Monastir, 5000 Monastir, Tunisia
salinity, which are unsuitable for other species. It is an abundant species in lagoons, salt
marshes, brackish water habitats of relatively shallow depth, and also in freshwaters [1–3].
The Mediterranean killifish is included in Annex II and III of the European Council
Directive 92/43 “The Conservation of Natural Habitats, Wildlife and Flora” . This
species is of no commercial value but it holds as a significant position in the food chain of
coastal ecosystems, due to its distribution and abundance. In Tunisia, the Mediterranean
killifish is common along the whole coast. This species is found both in polluted and
nonpolluted areas. It is exposed to many physical and chemical variations, from
temperature and salinity changes to water pollution, in these most threatened ecosystems.
We have recently described, in natural populations of Mediterranean killifish collected
from the Gulf of Gabès in Tunisia, a complex spinal deformities consisting of a consecutive
repetition of scoliosis, lordosis, and kyphosis from the head to the caudal fin. Deformed fish
were eight times more frequent in the industrialized coast of Sfax than in the coast of Luza
which is unaffected by human activities, possibly indicating a relationship between
pollution levels and spinal deformities . However, a direct relationship between observed
spinal deformities and pollution was not determined in this species.
The Gulf of Gabès is located in the southeastern coast of Tunisia. It is characterized by
shallow waters, high temperature, and salinity. This region was reported to be contaminated
with heavy metals, especially cadmium (Cd) [5–9].
High frequencies of spinal deformities in wild specimens of teleost fish have been
observed only in polluted waters [10, 11] or in freshwater subjected to significant variations
of environmental parameters (such as temperature, i.e., ). A relation between Cd
pollution and occurrence of spinal deformities is well established essentially from
laboratory studies [13–15]. Hypocalcemia after waterborne and dietary Cd exposure has
been reported in both fresh and brackish water fish and seems to be a main mechanism of
Cd toxicity [16, 17]. During Cd-induced disruption of calcium (Ca) homeostasis, Ca
mobilization from bone to compensate for hypocalcemia has been suggested to be the main
cause of bone deformation [13, 18]. Therefore, the present study was conducted to compare
some indicators of skeletal bone mineralization as well as Cd bioaccumulation, in normal
and deformed Mediterranean killifish collected from polluted and nonpolluted areas in the
Gulf of Gabès in Tunisia, in order to investigate the possible influence of environmental
exposure to Cd on the spinal deformities occurrence in this species.
Materials and Methods
Study Area and Sampling
Sampling sites were selected in the Gulf of Gabès, in southeastern coast of Tunisia (Fig. 1).
Theindustrialized coast ofSfax(S1),surrounded byimportantindustrial activities,mainly crude
phosphate treatments and chemical industries, was chosen as a polluted site. This site is
contaminated with heavy metals, essentially Cd . The coast of Luza (S2), located ∼50 km
north of S1 apparently unaffected by human activities, was used as reference sample site.
Fish were collected in shallow water (0.5–1 m) from S1 and S2 using hand nets during
April 2007. Water samples were collected at a depth of 0.5 m in clean bottles from the same
area. The upper 5 cm of sediments were also collected. Sampling bottles were previously
cleaned by soaking with 10% nitric acid (HNO3) and rinsed with ultrapure water. Fish,
water, and sediment samples were transported to the laboratory in a thermos flask with ice
on the same day.
Involvement of Cd Bioaccumulation in Spinal Deformities73
Spinal deformities were visible on the fish body immediately upon catching (Fig. 2).
Analyses were carried out only in males to eliminate any possible interference of sex with
Cd bioaccumulation and bone mineralization. The sex was determined from external
characteristics, since the Mediterranean killifish exhibits sexual dimorphism [1, 3]. As far as
possible, fish of similar size and weight were selected for analyses (Table 1). The entire
spinal column from normal and deformed fish were cleansed of muscle tissue as much as
possible and weighed (W; wet weight) immediately after the dissection. Next, the spinal
column was dried at 100°C for 24 h and weighed to obtain dry weights (DW). Then, the
dried samples were ashed in the muffle furnace at 650°C for 8 h and weighed to obtain ash
weights (AW) .
On the basis of the obtained weights (W, DW, AW), the following calculation were made
in the spinal column:
Percentage of water content (Wwater=W−DW; % of water=Wwater×100/W).
Percentage of nonorganic components (Wnonorg. comp.=AW; % of nonorg. comp.=
Percentage of organic components content (Worg. comp.=DW−AW; % of org. comp.=
Ash weight to dry weight ratio (AW/DW).
The ash samples were dissolved in concentrated HNO3and made up to 5 ml by the
addition of ultra pure water. These solutions were then used for the determination of Cd and
The entire liver from each sample was removed, washed with redistilled water, and dried
in filter paper. Liver samples were dried to constant weight for 48 h at 60°C in Pyrex test
tubes. Dried tissues were weighed and digested with concentrated HNO3at 120°C. When
fumes were white and the solution was completely clear, the samples were cooled to room
Gulf of Gabès
Gulf of Gabès
Fig. 1 Localization of the sites sampling in the Gulf of Gabès (Tunisia)
74Kessabi et al.
temperatureand the tubes werefilled to5 mlwith ultrapurewater. Seawater samples were
stabilized at pH 2 with 1 M HNO3prior to direct determination of total Cd concentrations
. Sediment samples were oven-dried for 48 h at 100°C and 100 mg of each sample was
mineralized at 250°C with a set of acids composed of 1 ml of HNO3, 2 ml of fluorhydric
acid, and 0.5 ml of perchloric acid and then adjusted to 10 ml with ultrapure water .
Cd and Ca concentrations were determined by atomic absorption spectrometry method
(AAS) using an atomic absorption spectrophotometer model ZEEnit 700-Analytik-Jena
(Germany). Concentrations of these elements were measured by flame ASS with
atomization in air-acetylene burner (Ca, in spinal column) or flameless AAS (Cd, in all
samples) with electrothermal atomization in a graphite furnace. Samples were analyzed in
triplicate. The variation coefficient was usually less than 10%. Values are reported as μg/g
ash weight and mg/g ash weight, respectively, for Cd and total Ca in spinal column. Cd
concentrations are reported as μg/g dry weight for liver and sediment samples and as μg/
l for water samples.
Bioaccumulation factors of Cd in fish from water (BAFwater) or sediment (BAFsediment)
were calculated according to the following formula:
BAFwater=metal concentration in liver/metal concentration in water .
BAFsediment=metal concentration in liver/metal concentration in sediment .
All the data were expressed as mean±SE. Differences among normal and deformed fish
were assessed by one-way ANOVA followed by protected least significant difference
Fisher’s test. Values were considered statistically significant when p<0.05.
Fig. 2 Normal (a) and deformed
(b) A. fasciatus (males) caught in
the industrialized coast of Sfax in
the Gulf of Gabès (Tunisia).
Scale bar, 1 cm
Table 1 Details of Fish Samples Destined for Analyses
SitesPhenotypeTotal length (mm)Body weight (mg)
Means ± SE from six males fish in each case
S1 coast of Sfax, S2 coast of Luza
Involvement of Cd Bioaccumulation in Spinal Deformities 75
Cadmium Concentrations in Water, Sediment, and Liver of A. fasciatus
Cd concentrations in water, sediment, and liver of fish from S1 and S2 are summarized in
Table 2. The water and sediment collected from S1 contained 20.33 and 35.60 times more
Cd, respectively, than those from S2. The results showed that Cd concentrations in liver of
fish from S1 were significantly higher (p<0.0001) than those in liver of fish from S2.
Comparison between fish in S1 showed that liver concentration of Cd was significantly
higher in deformed fish than in normal fish (p<0.0001).
The results cited in Table 3 showed that the bioaccumulation factors of Cd in the liver
from the water (BAFwater) and from the sediment (BAFsediment) in deformed fish were
significantly higher than in the normal fish from S1 and S2 alike (p<0.0001). Comparison
between normal fish showed that BAFwater (p<0.001) and BAFsediment (p<0.05) were
significantly higher in fish from S1 than those from S2.
Spinal Column Wet, Dry, and Ash Weight
No significant difference was noted in wet (p=0.26) and dry (p=0.69) weights between all
fish samples (Table 4). However, the ash weight is significantly lower in deformed fish than
in normal fish from S1 (p<0.01) and S2 (p<0.0001). Normal fish from S2 showed a
Table 2 Cadmium (Cd) Concentrations in Water, Sediment and Liver of A. Fasciatus from the Coast of Sfax
(S1) and the Coast of Luza (S2)
S1 Water (μg/l)
Sediment (μg/g dry weight)
Liver (μg/g dry weight)
S2 Water (μg/l)
Sediment (μg/g dry weight)
Liver (μg/g dry weight)Normal
Means ± SE from six samples in each case
*p<0.0001 (significance from samples of S2); **p<0.0001 (significance from samples of S1)
Table 3 Bioaccumulation Factors of Cadmium in Liver Fish from the Water (BAFwater) and the Sediment
Means ± SE from six fish in each case
S1 coast of Sfax, S2 coast of Luza
*p<0.05 (significance from normal fish of S2); **p<0.001 (significance from normal fish of S2); ***p<
0.0001 (significance from normal fish of S2); ****p<0.001 (significance from normal fish of S1)
76Kessabi et al.
significant higher ash weight (p<0.05) when compared to normal fish from S1. A
significant decrease in ash weight/dry weight ratio is also noted in deformed fish compared
to normal fish from S1 (p<0.05) and S2 (p<0.01).
Spinal Column Water, Nonorganic and Organic Components Content
As shown in Table 4, water and organic components contents in the spinal column of
normal and deformed fish were not significantly different. However, a significant decrease
in nonorganic components content was found in deformed fish compared to normal fish
from S1 (p<0.001) and S2 (p<0.0001).
Cadmium and Calcium Concentrations in Spinal Column of A. fasciatus
Table 5 shows that Cd levels in spinal column of specimens from S1 were about eight- and
11-fold higher in normal and deformed fish, respectively, than those collected from S2.
Comparison between fish in S1 showed that spinal column concentration of Cd was
significantly higher in deformed fish than in normal fish (p<0.001). In contrast, Ca
Table 4 Spinal Column Wet, Dry, and Ash Weight as well as Percentage Content of Water, Nonorganic and
Organic Components in Normal and Deformed Fish from the Coast of Sfax (S1) and the Coast of Luza (S2)
Sites S1 S2
% Wnonorg. comp.
% Worg. comp.
Means ± SE from six fish in each case
W wet weight, DW dry weight, AW ash weight; % Wwaterpercentage content of water, % Wnonorg. comp.
percentage content of nonorganic components, % Worg. comp.percentage content of organic components
*p<0.05 (significance from normal fish of S2); **p<0.01 (significance from normal fish of S2); ***p<
0.0001 (significance from normal fish of S2); ****p<0.05 (significance from normal fish of S1); *****p<
0.01 (significance from normal fish of S1); ******p<0.001 (significance from normal fish of S1)
Table 5 Cadmium and Calcium Concentrations in Spinal Column of A. fasciatus from the Coast of Sfax
(S1) and the Coast of Luza (S2)
Sites FishCd (μg/g ash weight)Ca (mg/g ash weight)
Means ± SE from six fish in each case
*p<0.001 (significance from normal fish of S2); **p<0.0001 (significance from normal fish of S2); ***p<
0.01 (significance from normal fish of S1); ****p<0.001 (significance from normal fish of S1)
Involvement of Cd Bioaccumulation in Spinal Deformities77
concentrations in spinal column of deformed fish are significantly lower than those of
normal fish from S1 (p<0.01) and S2 (p<0.001). A highly significant negative correlation
(r=−0.915, p<0.01) between Cd and Ca concentrations was observed in spinal column of
deformed fish (Fig. 3).
Association of bone deformities with experimental exposure to Cd in both fish and mammals
has been extensively studied. The hypothesis that the environmental exposure to Cd is
involvedinspinaldeformities occurrencein natural populationsofthe Mediterraneankillifish
was examined in the current paper. In order to test this hypothesis, some indicators of skeletal
bone mineralization as well as Cd bioaccumulation were made on normal and deformed fish
collected from polluted and nonpolluted areas in the Gulf of Gabès in Tunisia.
The Mediterranean killifish is a widely distributed fish over the central and eastern
coastal zones of the Mediterranean. It is a small fish with various advantages for monitoring
environmental quality in coastal regions, particularly its high abundance and facility of
sampling all year round, its nonmigratory nature, and its easy identification [1–3].
The concentrations of metals in the liver represent the storage of metals from the water
and the sediment where the fish species live [25, 26]. Thus, the liver is more often
recommended as environmental indicator organ of water and sediment pollution than any
other fish organs. We have found that Cd concentrations in the water, in the sediment, and
in the liver of the Mediterranean killifish from the industrialized coast of Sfax were
significantly higher than those from the coast of Luza. Consistent with our findings, several
reports have also indicated high Cd concentrations in soft tissues of Reditapes decussatus
[5, 6] and in the fish Scorpaena porcus  from this coast. The pollution in this coast is
due essentially to the presence of continuous discharge of heavy metals from local
industrial activities  and from the phosphogypsum stock [29, 30].
The BAF indicates whether heavy metal bioaccumulation take place. A BAF greater
than 1 indicates bioaccumulation . In both sites, the results showed that the BAF of Cd
in the Mediterranean killifish from water and sediment were greater than 1, and this means
that the fish undergo bioaccumulation of this element from water and sediment.
Considering Cd concentrations in the liver and the BAF values, the deformed fish
exhibited higher accumulated tissues concentrations of Cd indicative of a differential ability
to handle this heavy metal. It is possible that the ability of deformed fish to accumulate
y = -158,8x + 139,1
R² = 0,839
0,275 0,325 0,3750,4250,475
Ca (mg/g ash weight)
Cd (µg/g ash weight)
Fig. 3 Cadmium (Cd) vs. calci-
um (Ca) relationship in spinal
column of deformed A. fasciatus
from the coast of Sfax (Tunisia)
78Kessabi et al.
large amounts of Cd, compared to normal fish from S1, is related to the absence or
deficiency of efficient detoxification mechanisms. Thus, Cd that has not been inactivated
may induce tissues damage such as spinal deformities.
Most of the evidence on Cd influence on induction of spinal deformities in fish is from
laboratory studies. An increase in spinal deformities rate induced by exposures of fish to Cd
were reported by many authors [13, 15, 31]. Myoxocephalus quadricornis exposed for
1 year to an artificial heavy metal-containing effluent displayed spinal curvature and
increased frequency of vertebral deformities . However, the actions of Cd in wild fish
leading to abnormal development of skeletal structures remain uncertain. A causal
relationship between high environmental levels of heavy metals, especially Cd, and
skeletal anomalies in fish has been suggested by Bengtsson et al. . Buhringer et al. 
found highest Cd levels in the bones in rainbow trout suffering from deformities compared
to healthy individuals. In a recent study, we have reported an association of spinal
deformities with heavy metals (Cd and zinc) bioaccumulation in natural populations of
Zosterisessor ophiocephalus collected from the Gulf of Gabès in Tunisia .
The decrease in ash weight/dry weight ratio, percentage of nonorganic components
content, and Ca concentration in spinal column clearly indicates skeletal bone demineral-
ization in the deformed fish. In fact, the ratio of ash weight/dry weight is a fraction of the
bone weight, which is composed of minerals and its decrease suggests the occurrence of
bone demineralization [13, 34]. The significant reduction in the percentage of nonorganic
components content in the spinal column of the deformed fish resulted mainly from the
marked decrease of Ca content which indicates an abnormal Ca metabolism. Because the
contents of the other minerals in the bone are low in comparison to Ca, the decrease in their
levels took a small part in the noted deficit of nonorganic components.
The most important finding of the present study is the highly significant negative
correlation between Cd and Ca concentrations observed in spinal column of deformed fish.
Consistent with our findings, studies on orally and waterborne Cd-exposed fish showed
bone deformities associated with Cd accumulation and bone decalcification [13, 18, 35].
Resorption of Ca from the bone to compensate for disturbed Ca homeostasis, under Cd
influence, is suggested as the main mechanism behind the observed deformities in these
studies. Bone demineralization in mammals under Cd influence was also reported by
several authors [34, 36, 37]. In contrast, in spite of the pronounced disturbance of the Ca
metabolism, the spinal columns of the Cd-exposed fish, such as Platichtys flesus  and
Salmo salar , did not show any signs of demineralization or deformities.
It is suggested that fish species with acellular bone tissue, such as Platichtys flesus, run a
minor risk of suffering from skeletal damage after Cd exposure than fish species with an
active cellular bone tissue . The first mentioned type is considered to be inactive bone
tissue with very limited or no exchange with the Ca pool in the blood. Thus, the Ca reserves
in the skeletal bone seem to be of little metabolic use to fish with acellular bone. The other
type of bone is an active tissue, which acts as an important reservoir for Ca and other
minerals . Like other teleosts belonging to the Cyprinidae family, the Mediterranean
killifish has cellular bone tissue [40, 41]. Therefore, a Cd-induced disturbance of the Ca
balance would probably lead to a situation, in which hypocalcemia is compensated by an
increased release of Ca from skeletal bone. As a result, the spinal column would become
more fragile and increasingly susceptible to deformities.
Alternative to disturbed Ca homeostasis and decalcification, Cd may also directly induce
bone deformities. Contamination of the bone with Cd can negatively affect the bone matrix
or the bone tissue itself, disturbing bone remodeling and mineralization. Dietary Cd
increases the solubility of collagen, an important substance in the spinal matrix in fish .
Involvement of Cd Bioaccumulation in Spinal Deformities79
In addition, Cd may interfere with the crystallization of the main bone mineral
hypoxyapatite  and osteoblast activity .
The causes of the occurrence of spinal deformities in natural populations of fish are
complicated and, to a large extent, unknown. They may be a response to a number of biotic
factors (e.g., hereditary defects, parasite infections) and abiotic factors (e.g., vitamin
deficiencies, electrical shock…) which are not related to pollution. Nevertheless, the
observed association of the skeletal bone demineralization and the important accumulation
of Cd, in terms of amounts and bioaccumulation factors, seems to be very interesting and
suggest that the ability to accumulate large amount of Cd may represent a potential risk to
induce spinal deformities in natural populations of Mediterranean killifish. However,
additional studies are necessary to throw further light upon this suggestion. The discussed
relationship between Cd bioaccumulation and spinal deformities in natural populations of
fish also deserves further investigations.
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