RESEARCH ARTICLE

Heavy metal accumulation and histomorphological alterationsin Aphanius fasciatus (Pisces, Cyprinodontidae) from the Gulfof Gabes (Tunisia)

Kaouthar Kessabi & Zohra Hwas & Asma Sassi &Khaled Said & Imed Messaoudi

Received: 28 February 2013 /Accepted: 24 June 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The present study illustrates an analysis of histo-logical changes; cadmium (Cd), copper (Cu) and zinc (Zn)accumulation; and metallothionein (MT) levels in normal anddeformed Mediterranean killifish, Aphanius fasciatus (Pisces,Cyprinodontidae), collected from unpolluted (S1) and pollut-ed areas (S2) in the Gulf of Gabes in Tunisia. Metal determi-nation in water and sediment showed that the concentrationswere significantly higher (p<0.0001) in S2 compared to S1.Deformed fish showed a significantly higher accumulation ofCd, Cu, and Zn and high levels of MTs in their tissuescompared to normal ones. Histopathological investigationsrevealed greater changes in gills, kidney, liver, and bonetissues of fish from the polluted area than those recorded infish from the reference area. In comparison to normal fish ofthe polluted area (S2), tissue alterations were more developedin deformed specimens of this site. A possible relationshipbetween metallic pollution, incidence of spinal deformities,and histological changes in A. fasciatus in the polluted sitewas discussed.

Keywords Aphanius fasciatus . Spinal deformities . Heavymetals . Histological changes . Gulf of Gabes . Tunisia

Introduction

Aquatic organisms such as fish are, in most cases, exposed tomultitudes of stressors that are either natural orantropogenically introduced into the environment. Heavymetal contamination in aquatic ecosystem is one of the mostcritical environmental issues. Heavy metals can disturb theaquatic environment and cause damage in aquatic organisms(Gupta and Neera 2006; Agtas et al. 2007; Shukla et al. 2007;Yoon et al. 2008).

It is difficult to confirm the etiology of deformities in fish,but it has been suggested that these deformities are good bio-indicators of pollution in fish (Bengtsson 1979; Klumpp et al.2002). High frequencies of spinal deformities have been re-ported in wild teleost collected from polluted waters (Slooff1982; Whittle et al. 1992). A relation between cadmium (Cd)pollution and occurrence of spinal deformities is wellestablished (Muramoto 1981; Bengtsson and Larsson 1986;Cheng et al. 2000; Sassi et al. 2010). These studies havedemonstrated that exposures to pollutants can affect eggs orlarvae and result in deformities and changes in physiologicalfunction.

In fish, heavy metals have been shown to make structuraland morpho-pathological alterations of varying severity invarious organs. As an indicator of exposure to contaminants,histology represents a tool to assess the degree of pollution.Results from histopathological studies are useful in establish-ing water quality criteria (FAO 1981) and has been widelyused as a biomarker in the evaluation of the health of fishexposed to Cd both in laboratory (Wester and Canton 1991;Randi et al. 1996; Thophon et al. 2003; Au 2004) and fieldstudies (Schwaiger et al. 1997; Teh et al. 1997).

The use of the fish liver as a monitor organ is wellestablished in ecotoxicology. Indeed, changes in hepatocytesare useful biomarkers to trace environmental pollution(Triebskorn et al. 1997; Gernhofer et al. 2001). Previous

Responsible editor: Philippe Garrigues

K. Kessabi (*) :A. Sassi :K. Said : I. MessaoudiLR11ES41: Génétique, Biodiversité et Valorisation desBioressources, Institut de Biotechnologie, Université de Monastir,Monastir 5000, Tunisiae-mail: kaoutharkessabi@yahoo.fr

Z. HwasLaboratoire d’Histologie, Cytologie et Génétique (02/UR/08-03),Faculté de Médecine de Monastir, Université de Monastir, Monastir,Tunisia

Environ Sci Pollut ResDOI 10.1007/s11356-014-3252-6

histopathological studies showed that fish gills could be effi-cient indicators of water quality. Gills are sensitive to waterpollutants due to their large surface area and external location.Moreover, renal tissues receive large volumes of blood flowand serve as a major route of excretion for metabolites ofvarious xenobiotics. Since renal tissues are potentially contin-uously exposed to toxic chemicals, the risk of effects is high(Au 2004).

We have previously described, in natural populations of theMediterranean killifish, Aphanius fasciatus (Valenciennes,1821), collected from the Gulf of Gabes in Tunisia, a complexspinal deformities consisting of a consecutive repetition ofscoliosis, lordosis, and kyphosis from the head to the caudalfin. Deformed fish were eight times more frequent in theindustrialized coast of Sfax than in the coast of Luza whichis unaffected by human activities, possibly indicating a rela-tionship between pollution levels and spinal deformities(Messaoudi et al. 2009a). The industrialized coast of Sfax inthe Gulf of Gabes (Tunisia) concentrates a great number ofindustrial activities, mainly related to the industry of phos-phates as well as other heavy metal transmitters such as saltworks, tanneries, lead foundry, textiles, ceramics industry,soap factories, and building materials. Several studies con-ducted in this region showed that the industrial activities areassociated to Cd pollution that touched terrestrial (Messaoudiand Ben Chaouacha-Chekir 2002) and aquatic (Hamza-Chafaiet al. 1995; Smaoui-Damak et al. 2003; Banni et al. 2007;Messaoudi et al. 2009b) fauna and flora. Therefore, the aim ofthis study was to investigate a possible relationship betweenmetallic pollution, incidence of spinal deformities, and histo-logical changes in A. fasciatus.

Material and methods

Study areas and samples collection

In this study, we compared the histological structure of liver,kidney, gills and bone, heavy metals, and MT levels in normaland deformed A. fasciatus collected from polluted andnonpolluted areas in the Gulf of Gabes in Tunisia.

Two sampling sites were selected in the Gulf of Gabes inthe southeastern coast of Tunisia (Fig. 1). The coast of Luza(S1) located ~50 km north of S2 and appeared to be unaffectedby human activities was used as reference sample site. Theindustrialized coast of Sfax (S2), which was surrounded byimportant industrial activities, mainly crude phosphate treat-ments and chemical industries, was chosen as a polluted site.This site is contaminated with heavy metals, essentially cad-mium (Cd) (Smaoui-Damak et al. 2003). Fish were collectedusing hand nets from S1 and S2.

Samples of water were collected in clean bottles from fivepoints within each site. The upper 5 cm of sediments were also

collected. Sampling bottles were previously cleaned bysoaking with 10 % nitric acid (HNO3) and rinsed with ultra-pure water. These samples were then conserved at 4 °C forheavy metal analyses. Fish were collected in shallow water(0.5–1 m) from S1 and S2 using hand nets. Water and sedi-ment samples were transported to the laboratory in a thermosflask with ice at the same day. Fish samples were immediatelycovered with dry ice until arriving at the laboratory where theywere weighed and dissected. Then, the liver was excised andstored at −80 °C until their analysis.

Analytical procedures

Chemical analysis

Fish of similar size and weight were selected for metalanalyses (Table 1). The whole body was washed withdistilled water and dried in filter paper. Then, the sam-ples were dried to constant weight for 48 h at 60 °C intest tubes. Dried tissues were weighed and digested withconcentrated nitric acid (Merck, 65 %) at 120 °C. Whenfumes were white and the solution was completely clear,the samples were cooled to room temperature and thetubes were filled with 5 ml ultrapure water. Sea watersamples were stabilized at pH 2 with 10 % nitric acidprior to direct determination of metal concentrations.Sediment samples were oven-dried for 48 h at 100 °C,and 100 mg of each sample was mineralized at 250 °Cwith a set of acids composed of 1 ml of HNO3, 2 ml offluorhydric acid, and 0.5 ml of perchloric acid and thenadjusted to 10 ml with ultrapure water (Rashed 2001).

Heavy metal concentrations were determined using agraphite furnace Atomic Absorption Spectrometry (AAS)method (ZEEnit 700- Analytik-Jena, Germany). Sampleswere analyzed in triplicate. The variation coefficient wasusually less than 10 %.

Metallothioneins measurement

MT protein levels in the liver were determined using aspectrophotometric assay for MTs using Ellman’s reagent(0.4 mM 5,50 dithio-nitro-benzoate (DTNB) in 100 mMKH2PO4) at pH 8.5 in a solution containing 2 M NaCland 1 mM EDTA (Viarengo et al. 1997). In brief, aliquotswere homogenized in three volumes of 0.5 M sucrose,20 mM Tris–HCl buffer, pH 8.6, with added 0.006 mMleupeptine, 0.5 mM phenylmethylsulphonylfluoride(PMSF) as antiproteolytic, and 0.01 % 2-mercaptoethanolas reducing agent. Total protein content in a sample of thehomogenate was measured by the Bradford method(Bradford 1976) at 595 nm using bovine serum albuminas standard. The homogenate was then centrifuged at15,000g for 30 min at 4 °C. The obtained supernatant

Environ Sci Pollut Res

was treated with ethanol/chloroform as described byViarengo et al. (1997) in order to obtain the MT-enriched pellet. The obtained MT pellet was resuspendedin HCl/EDTA in order to remove metal cations that stillbound to the MTs. Finally, 2 M NaCl was added to thesolution to facilitate thiol interactions with DTNB byreducing the interaction of divalent metals with theapothionein. MT content was estimated using a spectro-photometer at λ=412 nm. Data were expressed as micro-grams MT per milligram of protein.

Histological study

For histological examination, fish sampled from differ-ent sites were fixed in Bouin’s solution, dehydrated inan ethanol series, and embedded in paraffin. Five-micrometer-thick sections were made and stained withhematoxylin and eosin for light microscopic examina-tion, and each tissue was examined for the presence ofhistopathological lesions.

Histopathological alterations were assessed using ascore ranging from− to +++ depending on the degreeand extent of the alteration: (−) none, (+) mild occur-rence, (++) moderate occurrence, and (+++) severe oc-currence (Thophon et al. 2003). A total of 20 slideswere observed from each group.

Statistics analysis

For metal concentrations and MT content, the data wereexpressed as mean±SE. Differences among normal and de-formed fish were assessed by one-way ANOVA followed byprotected least significant difference Fisher’s test. Values wereconsidered statistically significant when p<0.05.

Results

Metals concentrations

Heavy metal concentrations in water, sediment, and fish tis-sues from S1 and S2 are presented in Fig. 2. The results showthat Cd, Cu, and Zn contents in both sediment and watercollected from S2 were significantly higher (p<0.001) thanthose from S1. The most important difference was noted forCd concentrations. Indeed, the water and sediment collectedfrom S2 contained 20.33 and 35.60 times more Cd, respec-tively, than those from S1.

Fish from S2 always had Cd, Zn, and Cu concentra-tions significantly higher (p<0.0001) compared to thosemeasured in fish from S1. Comparison between fish inS2 showed that tissue concentrations of Cd were signif-icantly higher in deformed fish than in normal ones(p<0.001).

Total metallothioneins accumulation

Total MT protein content was evaluated in the liver ofnormal and deformed A. fasciatus. A significant highlevels of MTs was observed in the liver of fish from S2(p<0.0001) than those in the liver of fish from S1(Fig. 3). Comparison between fish in S2 showed that

100 Km

N

Gulf of Gabès

Tunisia

Lybia

Gulf of Gabès

N

Alg

eria

Mediterranean Sea

Sfax

Luza(S1)

(S2)

Fig. 1 Location of the sitessampling in the Gulf of Gabes(Tunisia)

Table 1 Details of fish samples destined for analyses

Sites Phenotype Total length (cm) Body weight (g)

S1 Normal 3.14±0.67 0.525±0.28

S2 Normal 3.06±1.12 0.436±0.68

Deformed 2.97±0.7 0.389±1.36

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liver content of MTs was significantly higher in de-formed fish than in normal ones (p<0.0001).

Histopathological examination

Liver

The fish liver tissue is formed from the parenchyma calledhepatocytes. These were located among the sinusoids formingcordlike structures known as hepatic cell cords. The hepato-cyte has a polyhedral cell body with a central core containinggenerally one spherical nucleolus. In comparison to this typ-ical structure, the liver of A. fasciatus collected from thecontrol area (S1) appeared normal (Fig. 4a). However, theliver tissues of normal fish captured from S2 showed hyper-trophy and vacuolization of hepatocytes (Fig. 4b). In de-formed fish of polluted site, the structural alterations of theliver became more pronounced (Table 2) since desquamationof hepatic tissues, vacuolization, fusion, atrophy, and hyper-trophy of hepatocytes as well as hepatic central vein conges-tion, necrosis of hepatic tissue, and dilatation of blood sinu-soids were observed (Fig. 4c–f).

Fig. 2 Cadmium (Cd, a), copper (Cu, b), and zinc (Zn, c) concentrationsin water (μg/L), sediment (μg/g dry weight), and normal (N) and de-formed (D) fish in toto (μg/g dry weight) from the coasts of Luza (S1) and

Sfax (S2). *p<0.001 (significance from samples of S1); ap<0.01 (signif-icance between N and D from S2)

Fig. 3 Metallothioneins (MTs) accumulation (μg/mg proteins) in theliver of normal (N) and deformed (D) Aphanius fasciatus collected fromreference (S1) and polluted (S2) areas. Values are means±SE from eightsamples. *p<0.0001 (significance from samples of S1); ap<0.01 (signif-icance between N and D from S2)

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Gills

Gills of S1 specimens showed a typical histological structureof teleost fish. The secondary lamellae or gill lamellae lined upalong both sides of the primary lamellae or gill filament. Thesurface of the gill lamellae is covered with simple squamousepithelial cells and capillary separated by pillar cells runparallel along the surface. In between the secondary lamellae,the primary lamellae are lined by a thick stratified epithelium.The gill lamellae consisted of several cell types: chloride cells(at the base), pillar cells, mucous cells, and epithelial cells(Fig. 5a).

Histopathological results indicated that in comparison togills structure of specimens collected from the reference site(S1), those of the polluted site (S2) showed structure alter-ations consisting of partial detachment of epithelium (Fig. 5b).In comparison to normal fish of the polluted site (S2), gillstructure alterations were more pronounced in the deformedspecimens of this site (Table 2). Indeed, besides the hypertro-phy of chloride cells, we also noted total detachment ofepithelium, both a partial and total fusion of the secondarylamellae, as well as shortening of secondary lamellae anddisorganization of pillar cells (Fig. 5c–f).

Kidney

The kidney is made up of renal corpuscles and renal tubules.The glomerulus is a tuft of capillaries. Proximal tubules werecharacterized by cuboidal cells with brush border locatedalong the apices of the cells. Distal tubules were low cuboidalepithelium with basally round nucleus and eosinophilic cyto-plasm. The cells were parenchymatous, round to polygonal inshape, with nuclei in the center (Fig. 6a).

In normal fish collected from S2, kidney showed normalappearance similar to the control with the exception of sometubules dilatation (Fig. 6b). The most severe alterations wereobserved in deformed fish from the polluted site (Table 2).Indeed, histological examination of the kidney of these fishshowed damage consisting mainly to desquamation of thehematopoietic tissue and its necrosis. Congestion of bloodvessels, atrophy of the glomeruli, and vacuolization of epithe-lial cells were also recorded (Fig. 6c–f).

Bone

Histological examination of vertebral column of normalA. fasciatus showed a typical histological structure of teleost

H

BS

CHV

FHC

DHT

V

HH

AHCCCHV

DBS

NHT

a

b c

d e

Fig. 4 Liver structure of normalfish collected from S1 (a) and S2(b) and deformed fish collectedfrom S2 (c, d, e, and f) (×100). Hhepatocytes, CHV central hepaticvein, BS blood sinusoid, HHhypertrophy of hepatocytes, Vvacuolization, FHC fusion ofhepatic cells, DHT desquamationof hepatic tissue, AHC atrophy ofhepatic cells, NHT necrosis ofhepatic tissue, DBS dilatation ofblood sinusoid

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fish. Indeed, the vertebral column is formed by symmetricalvertebrae linked by intervertebral ligament and surmounted bythe neural tube, which extend the spines (Fig. 7a). Normalfish, both from reference and polluted areas, showed normaland thin vertebral walls. The notochord displayed the typicalvestigial appearance in both sides of trabecular (Fig. 7b).Conversely, deformed fish showed compressed vertebral discsand a general distortion of the vertebral column (Fig. 7c).

In addition, deformed fish showed various and frequentvertebral lesions (Table 2). Indeed, the histopathologicalchanges included marked hypertrophy of vertebral walls andzygapophyses, a prominent notochord, compression and frac-ture of vertebrae, fusion of some vertebral centra, bone necro-sis, and altered adjacent neural tube (Fig. 7d–i).

Discussion

The hypothesis that spinal deformities can correlate withheavy metal accumulation and histological damage in differ-ent tissues of A. fasciatus was examined in the current paper.In order to test this hypothesis, levels of Cd, Cu, and Zn weredetermined in water, sediment, and whole body fish. Hepaticcontent of MTs and histological structure of the liver, gills,kidney, and bonewere compared on normal and deformed fishcollected from the Gulf of Gabes in Tunisia.

Heavy metal pollution of aquatic ecosystems is becoming apotential global problem. These pollutants build up in the foodchain and are responsible for adverse effects and death in theaquatic organisms. Chronic exposure of fish to sub-lethal tracemetal levels causes among others disturbed ion regulationreduced swimming and reduced growth (Farkas et al. 2002).However, little information is available on the relationshipbetween internal tissue levels of metals and condition of fishunder natural exposure conditions.

Teleost liver is the primary organ for biotransformation oforganic xenobiotics and probably also for the excretion ofharmful trace metals, food digestion and storage, and metab-olism of sex hormones (Hinton et al. 2001). There have beennumerous reports of histo-cytopathological changes in liversof fish exposed to a wide range of organic compounds andheavy metals (Hinton and Lauren 1990; Hinton et al. 1992;Hinton 1994; Vandenberghe 1996; Braunbeck 1998). Metalscan either increase or decrease hepatic enzyme activities andcan lead to histopathological hepatic changes, depending onthe metal type and concentration, fish species, length ofexposure, and other factors (Paris-Palacios et al. 2000).Some studies showed that the accumulation of Cd leadsto histopathological changes in the liver, such asvacuolization of hepatocytes, fibrosis, polymorphism ofnuclei, necrosis of hepatocytes and parenchymal cells,and cell injury liver endothelial cells (Brown et al.1984; Van Dyk et al. 2007).

The gills of fish have vital roles because, besides being themain site of gas exchange (Hughes 1976), they are alsoinvolved in the process of osmoregulation (Verbost et al.1994), acid–base balance (Goss et al. 1992), and excretionof nitrogen compounds (Sayer and Davenport 1987). Mallat(1985) and Wood (2001) explained the structural changes ofgills in relation to exposure to toxic substances. The histo-pathological changes observed in the gills are not specific topollutants. Indeed, epithelial hyperplasia, fusion of lamellae,hypertrophy of the epithelium, telangiectasia, and epithelialdesquamation are typical histopathological changes of gillsexposed to contaminants including organochlorines, carba-mates, herbicides, and heavy metals (Au 2004). Since gillsare the first target of waterborne pollutants due to their con-stant contact with the external environment, it has been con-firmed that accumulation of metals may have an effect on

Table 2 Semi-quantita-tive scoring of the liver,gills, kidney, and bonelesions described in nor-mal and deformed fishcollected form coasts ofLuza (S1) and Sfax (S2)

(−) none, (+) mild, (++)moderate, and (+ + +)severe occurrence

N normal, D deformed

NS1 NS2 DS2

Liver Alterations

HH − + +++

CCHV − + +++

DHT − − ++

NHT − − ++

DBS − + +++

FHC − − +++

V − ++ +++

AHC − + ++

Gill alterations

HCC − + + +

HE − + + +

DPE − + + −DTE − − +++

PFSL − − ++

TFSL − − +++

T − − +++

SSL − − +++

Kidney alterations

TD − + +++

NHT − − ++

DHT − − ++

GA − − ++

CBV − − ++

VEC − − ++

Bone alterations

CV − − +++

FV − − ++

ANT − − ++

HVW − − +++

HZ − − ++

FVC − − ++

BN − − ++

PN − − ++

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these functions (Vernberg and O’Hara 1972; Thurberg et al.1973; Jones 1975).

In fish, the kidney is the organ preferential for the heavymetals accumulation (Brown et al. 1984; Allen 1995). It wasshown that in Dicentrarchus labrax living in contaminatedwater, heavy metals accumulate to a greater extent in thekidney and the liver and in third place in the gills(Cattani et al. 1996). In the liver and kidneys, the metalis mainly linked to MTs whose synthesis is strongly in-duced by Cd (Olsson et al. 1989). The accumulation ofMTs complex—Cd in renal tissue causes necrosis of renaltubular epithelial cells. Indeed, it was demonstrated byseveral authors (Komaba et al. 2008; Bover et al. 2009)that the alteration of kidney function in humans leads to adisease called "osteodystrophy "associated with alterationsin metabolism of Ca and phosphorus (P). In fact, thedeterioration of renal function can cause abnormalities inthe metabolism of Ca and phosphorus (P), a hyperparathy-roidism, vitamin D deficiency, and bone abnormalities. Allthese abnormalities may be causing serious health conse-quences by reducing the quality of life and causing cardio-vascular disease, bone fractures, and mortality.

Several reports have described histological changes inskeletal tissue of fish species exposed in vivo to differentcompounds, but few studies have associated these alterationsto spinal deformities in fish exposed environmentally to pol-lutants. Douglas et al. (1990) showed that following exposureof juvenile fish (Menidia beryllina) to a pesticide, terbufos,there was a very significant alteration of bone tissue comparedto normal fish. They noted a longitudinal compression of thevertebrae, hypertrophy of the walls and vertebral zygapophy-ses, a significant development of notochord as well as verte-bral fusion. Damage of the vertebral structure was also report-ed in Phoxinus phoxinus exposed to Zn (Bengtsson 1974).Indeed, following exposure to this metal, the alterations oc-curred mainly consists fracture, shortening of the vertebrae,and significant development of the notochord that becomesgelatinous.

Histological changes in the skeleton of Atlantic salmon(Salmo salar) were associated with increasing concentrationof carbon dioxide in water (Laura et al. 2006). They noted achange in one part of the bone surface indicating bone resorp-tion, and therefore, an important activity of osteoclasts. It wasreported that the observation of a significant stimulation of

PDE

ba

ePFSL

TFSL

SSLDPC

f

PL

SLEC

ErPC

a

HCC

c

TDE

d

Fig. 5 Gill structure of normalfish collected from S1 (a), S2 (b),and deformed fish (c, d, e, and f)(e×40) (a, b, c, d, and f×100). PLprimary lamellae, SL secondarylamellae, EC epithelial cells, PCpillar cells, Er erythrocytes, PDEpartial detachment of epithelium,TDE total detachment ofepithelium, PFSL partial fusion ofsecondary lamellae, TFSL totalfusion of secondary lamellae, SSLshortening of secondary lamellae,DPC disorganization of pillarcells

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bone resorption is due to reduced mineral content and reducedbone volume (Witten et al. 2001; Witten and Hall 2002). Inaddition, Helland et al. (2006) have associated histologicalchanges in the bone tissue in the Atlantic salmon with highconcentrations of phytic acid.

In the present work, vertebral lesions observed inA. fasciatus naturally exposed to heavy metals are similar tothe vertebral dysplasia in young sheepshead minnowsCyprinodon variegatus exposed to trifluralin (Couch et al.1979). In both cases, the histopathological changes includedmarked hypertrophy of vertebral walls, a prominent cellularnotochord, longitudinal compression of vertebrae, and fusionof some vertebral centra.

It is suggested that fish species with acellular bone tissue,such as Platichtys flesus, run a minor risk of suffering fromskeletal damage after Cd exposure than fish species with anactive cellular bone tissue (Larsson et al. 1981). The firstmentioned type is considered to be inactive bone tissue withvery limited or no exchange with the Ca pool in the blood.Thus, the Ca reserves in the skeletal bone seem to be of littlemetabolic use to fish with acellular bone. The other type ofbone is an active tissue, which acts as an important reservoirfor Ca and other minerals (Simmons 1971). Like other teleostsbelonging to the Cyprinidae family, the Mediterranean killi-fish has cellular bone tissue (Moss 1961, 1965). In this case,

histological alteration of bone tissue can make the spinalcolumn fragile and increasingly susceptible to deformities.

In this study, histological examination of gills, liver, kid-ney, and bone from normal A. fasciatus collected from areference area showed a typical structural organization ofteleostei fish. However, it could be observed that histologicallesions in A. fasciatus from the industrialized coast of Sfaxweremore severe than those derived from the reference site. Inaddition, the histological alterations were more severe in

a b

c d

e f

GA

VEC

CBV

NHT

DHT

GHPT

DTPT

TD

Fig. 6 Kidney structure of normal fish collected from S1 (a) and S2 (b)and deformed fish collected from S2; (a and b×40) (c, d, e, and f×100).DT distal tubule, G glomeruli, HPT hematopoietic tissue, PT proximaltubule, TD tubules dilatations, GA glomerular atrophy, NHT necrosis ofhematopoietic tissue, CBV congestion of blood vessels, DHT desquama-tion of hematopoietic tissue, VEC vacuolization of epithelial cells

a b

c d

e f

g h

i

VWZ

T

N

N

V

ILNT

CV

FV

ANTHVW

HZ

FVC

BN

PN

V

Fig. 7 Bone structure of normal fish (a, b) and S2 (b) and deformed fishcollected from S2 (c, d, e, f, g, h, and i) (a, b×10) (d, e, and g×40) (b, f,and h×100). V vertebrae, IL intervertebral ligament, NT neural tube, Zzygapophyse, VW vertebral walls, T trabecular, N notochorde, CV com-pressed vertebrae, FV fracture of vertebrae,ANTaltered neural tube,HVWhypertrophy of vertebral walls, HZ hypertrophy of zygapophyses, FVCfused vertebrae centra, BN bone necrosis, PN prominent notochord

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deformed fish than in normal ones derived from the pollutedarea.

Fish have the ability to accumulate heavy metal in theirtissues by the absorption along the gill surface and gut tractwall to higher levels than the toxic concentration in theirenvironment (Chevreuil et al. 1995). When fish are exposedto heavy metals, they synthesize proteins which the role isdetoxification such as MTs mainly in the liver (Kagi andSchaffer 1988). It was suggested that the synthesis of MTscan reduce the harmful effects of excess amounts of heavymetals (McCarter and Roch 1983). Zn and Cd are among themost potent inducers of MT transcription and proteinsynthesis.

Regarding our results, analyses for the heavy metals (Cu,Cd, and Zn) showed that the concentrations measured weresignificantly higher both in water and sediment of pollutedarea compared to the reference area. The concentrations ofthese metals, essentially Cd, measured in the whole body andthe MT content determined in the liver were significantlyhigher in deformed A. fasciatus compared to the normal ones.

To our knowledge, this study is one of the first attempts toinvestigate the possible relationship between observed spinaldeformities, accumulation of heavy metals, and histologicaldamages. We have previously (Kessabi et al. 2009) demon-strated that in deformed A. fasciatus, the levels of Cd in theliver and spinal column were significantly higher in the de-formed fish collected from the polluted area compared withthe normal one collected both from polluted and a referencearea, and a highly significant negative correlation was foundbetween Cd and Ca concentrations in spinal column of de-formed fish. Cd is considered to be one of most toxic heavymetals. It enters in the environment from natural and, essen-tially, anthropogenic sources (Burger 2008). Cd dissolved inwater or deposit in sediment constitutes a contaminationsource for the various aquatic food chain links (Romeo andGnassia-Barelli 1995). In this study, the observed damage indifferent tissues becomes more severe with increasing metallicpollution, essentially Cd.

Conclusion

The histopathological alterations observed in different tissuesand the high levels of MTs in deformed A. fasciatus derivedfrom the polluted area might be due to the heavy metalaccumulation, essentially Cd. These results seem very inter-esting and suggest a relationship between histological chang-es, vertebral column deformities, and heavy metal accumula-tion. However, complementary studies are necessary to con-firm this suggestion. Further molecular studies of vertebralabnormalities induced by exposure to heavy metals may pro-vide additional clues to the mechanisms responsible for thesevertebral deformities.

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