Int. J. Aquat. Biol. (2013) 1(2): 61-67 

E-ISSN: 2322-5270; P-ISSN: 2383-0956

Journal homepage: www.ij-aquaticbiology.com 

© 2013 Iranian Society of Ichthyology 

Histopathological effects of zinc (Zn) on mantle, digestive gland and foot in freshwater mussel, 

Anodonta cygnea (Linea, 1876) 
  

Fateh Moëzzi *1, Arash Javanshir1, Soheil Eagderi1, Hadi Poorbagher1 

 
1Department of Fisheries Science, Faculty of Natural Resources, University of Tehran, Iran. 

 

 
Article history: 
Received 12 April 2013 

Accepted 1 May 2013 

Available online 5 May 2013 

 
Keywords:  
Zinc 

Histopathology 

Swan mussel  

Mantle 

Digestive gland 

Foot 

Abstract: Heavy metals are the most important pollutants in aquatic ecosystems that may cause 
adverse effects on its biota. In this study, histopathological effects of zinc (Zn) and their incidence time 

on swan mussel, Anodonta cygnea, were studied. Exposure to Zn was done during 18 days and 

histopathological investigations were conducted in mantle, digestive gland and foot in days 0, 4, 9, and 

18. Histopathological changes observed were: damages of epithelium cells with increasing mucous 

cells (in mantle), atrophy of digestive tubules and haemocyte aggregation (in digestive gland), and 

hypoplasia, increasing mucous cells and myocyte swelling (in foot). Moreover, granuloma and tissue 

rupture were found in all organs. Primary histopathological changes were observed in fourth day of 

examination in all of studied organs. Results showed that sensitivity of digestive gland is lesser than 

mantle and foot in exposure to Zn. Also the results indicated the histopathological alterations in the 

organs of swan mussel can be considered as reliable biomarkers in biomonitoring of heavy metal 

pollution in aquatic ecosystems. 
 

Introduction 

Heavy metal is a general name for a group of metallic 

elements that have toxic effects in concentrations 

higher than tolerable physiological levels of animals 

(Forstner and Wittman, 1983; Rainbow, 2002; 

Banfalvi, 2011). They have two categories: essential 

(with structural and biological functions) and non-

essential (without any biological role) groups 

(Simkiss, 1981; Williams, 1981; Banfalvi, 2011). 

These pollutants may have adverse impacts on biota 

of aquatic habitats. Zinc (Zn) is one of the essential 

heavy metals contaminants of freshwater 

ecosystems. The major anthropogenic sources of this 

element are processing of ores, electroplating, 

domestic and industrial effluents, combustion of 

fossil fuels and soil erosion (Liobet et al., 1988; Buhl 

and Hamilton, 1990). The reported natural 

concentrations of Zn were in the range of 0.1-50  

                                                           
* Corresponding author: Fateh Moëzzi 

E-mail address: Fmoezi.fateh@gmail.com 

Tel: +989185177054 

µg l-1 in freshwaters and 0.002-0.1 µg l-1 in marine 

ecosystems (WHO, 2001).  

In last decades, aquatic organisms are used as 

bioindicators for monitoring chemical pollution of 

freshwater and marine environments. Bivalves are a 

group of molluscs that are appropriate for study of 

biological impacts of environmental pollutants in 

aquatic ecosystems (Livingstone et al., 2000) and are 

widely used as bioindicators in biomonitoring 

(Sanders et al., 1993). With high potential of 

accumulation of heavy metals in tissues and organs 

of bivalves, it is possible to detect and assess their 

occurrence using cellular and physiological 

responses (Van Duren et al., 2006). Long term 

exposure to heavy metals can increase susceptibility 

to disease and development of histopathological 

malformations (Zorita et al., 2006). Broad varieties 

of histopathological alterations in fish and bivalves 



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Moëzzi et al./ Int. J. Aquat. Biol. (2013) 1(2): 61-67 

have been developed and offered as biomarkers for 

monitoring of pollutants (Lowe, 1988; Cajaraville et 

al., 1992; Yevich and Yevich, 1994; Syasina et al., 

1997; Au, 2004).  

This study aimed to investigate the histopathological 

alterations in different organs of swan mussel, 

Anodonta cygnea, exposed to Zn. Swan mussel 
which belongs to family Unionidae, has a wide 

dispersal area including North America, Europe, 

northern Africa and Asia (Pourang et al., 2010). In 

Iran, this species is found in northern wetlands and 

rivers and in some coastal regions and due to its wide 

distribution area, can be considered as an appropriate 

bioindicator of heavy metal pollution in aquatic 

ecosystems. 

 

Material and methods 

Specimen collection and experimental condition: In 
October 2011, eighty specimens of A. cygnea were 
collected from the Tajan River estuary (36°48΄46΄΄N, 

53°6΄57΄΄E) (Mazandaran Province, Iran). These 

specimens were transferred to 60L fiberglass tanks 

for adaption to laboratory conditions. These tanks 

have been equipped to a circulatory system and 

Figure 1. Light micrographs of transverse sections (H&E) of control mantle and metal-exposed specimens. (A, C and E) control and (B, D and F) 

exposed to Zn. mc: mucous cell; ep: epithelium; ct: connective tissue; gr: granuloma; tr: tissue rupture; ha: haemocyte. 



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Moëzzi et al./ Int. J. Aquat. Biol. (2013) 1(2): 61-67 

aeration. During the experiment, dechlorinated tap 

water was used. Water temperature and pH were 

measured daily, which were 15-17°C and 6.8-7.4, 

respectively. Specimens were not fed through the 

experiment.  

Exposure plan: Twenty four specimens (length: 
12.7-13.3 cm) were introduced randomly into 3 

experimental tanks (8 specimens per tank). There 

were 3 replicates in every sampling time and a 

control tank with 4 specimens. Considering natural 

concentrations of Zn in aquatic environments and 

based on previous studies (Naimo, 1995), bivalves 

were exposed to 125 µg l-1 of Zn for 18 days. At days 

Figure 2. Light micrographs of transverse sections (H&E) of digestive gland of control and metal-exposed specimens. (A and C) control and (B 

and D) exposed to Zn. haa: haemocyte aggregation; gr: granuloma; ha: haemocyte. 

Figure 3. Light micrographs of transverse sections (H&E) of foot of control and metal-exposed specimens. (A and C) control and (B and D) 

exposed to Zn. mc: mucous cell; c: cilia; s: swelling; tr: tissue rupture; ls: longitudinal section of myocyte; ps: transverse section of myocyte. 

 
63 



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Moëzzi et al./ Int. J. Aquat. Biol. (2013) 1(2): 61-67 

4, 9 and 18, specimens of each tank were removed to 

collect obtaining tissue samples.  

Tissue preparation: The valves of specimens were 
opened by cutting anterior and posterior abductor 

mussels using a scalpel. Samples of ≈ 0.5 cm3 from 

each target organ (mantle, digestive gland and foot) 

were removed and fixed into Bouin’s solution 

(saturated picric acid: 75 parts, 40% formaldehyde: 

25 parts and glacial acetic acid: 5 parts) for 48h and 

then stored in 70% ethanol. The histological sections 

(5-7 µm thickness) were prepared based on Hewitson 

and Darby (2010) and stained using haematoxylin 

and eosin. Stained tissues were observed under a 

light microscope (Leica MS5).  

 

Results 

Histopathological observations:  
Mantle: Histological sections of the mantle of 
control group had normal status with contiguous 

external epithelium with no sign of alteration in 

connective tissue of sub-epithelial (Fig. 1A, C and 

E). Mucous cells were seen sporadically in 

epithelium and sub-epithelium with equal size (Fig. 

1A). The muscular cells were with conjunct structure 

(Fig. 1C) and the connective tissue was healthy (Fig. 

1E).  

Histological observations of treatments showed 

specific signs of tissue damage in mantle (Fig. 1B, D 

and F). The width of external epithelium was 

decreased and the number of the mucous cells in sub-

epithelial layer were increased (Fig. 1B). Granuloma 

(pigmented cells with yellow to brown color) was 

found in sub-epithelial layers (Fig. 1D). Tissue 

rupture and dissociation of regular cellular structure 

in the muscular tissue observed (Fig. 1B, D and E). 

In addition, the haemocyte infiltration and 

aggregation of the myocytes was occurred (Fig. 1D).  

Digestive gland: Figures 2A and C, display 
histological status of digestive gland and their 

junctions in control group with healthy basophilic 

columnar epithelial cells and digestive (dingy) cells 

in tubule structure. Also, connective tissue of 

interstitial space between tubules was normal with 

similar distribution of haemocytes (Fig. 2C). 

Digestive gland exposed specimens showed atrophy 

of digestive tubules and loss of digestive and 

basophile cells into the tubule (Fig. 2B). Intensive 

haemocyte aggregations in connective tissue and 

interstitial space (Fig. 2B and D), as well as, 

granuloma was found in connective tissue observed 

(Fig. 2D). 

Foot: In the control group, external epithelium with 
integrated ciliary structure was observed (Fig. 3.A). 

In subepithellial layer, the mucous cells had uniform 

distribution. In inner parts of foot, groups of 

myocytes were in various directions as their 

longitudinal and cross-sections were visible (Fig. 

3C).  

organ 
Time 

Day 4 Day 9 Day 18 

Mantle 
increase in mucous cells 

count 

Haemocyte infiltration and 

aggregation in sub-epithelial 

connective tissues; granuloma; 

sub-epithelial hyperplasia;  

Haemocyte infiltration and 

aggregation in connective and 

muscular tissues; tissue rupture  

Digestive gland 
Loss of digestive cells into 

tubules 

Haemocyte aggregation in 

connective tissue; Atrophy of 

tubules; granuloma in interstitial 

space between tubules. 

 

Foot 

Hypoplasia of external 

epithelium; increase in 

mucous cells count 

Haemocyte infiltration and 

aggregation in sub-epithelial 

tissues; epithelium rupture 

Myocyte inflammation 

(swelling); muscular tissue 

rupture.  

 

Table 1. Incidence time pattern of histopathological alterations in mantle, digestive gland and foot of Zn-exposed mussels during study 

period. Presented histopathological damages were observed in all 3 replicates of mussels in days 4, 9 and 18. 



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Moëzzi et al./ Int. J. Aquat. Biol. (2013) 1(2): 61-67 

The foot of Zn-exposed specimens represents an 

increase in the number of mucous cells and swelling 

of connective tissue of subepithellial layers (Fig.3B). 

Hypoplasia of external epithelium was occurred. 

There were tissue rupture in epithelium and layers 

below that (Fig. 3B). Also, inflammatory myocytes 

were observed (Fig. 3D).  

Incidence time pattern of changes: The 
chronological trends of histopathological effects of 

metal exposure are presented in Table 1. In the fourth 

day, histological changes were found in all organs. 

Intensity of damage in the mantle and foot was 

greater than the digestive gland. Haemocyte 

aggregation in the connective tissues was a common 

change in all studied organs in the ninth day. In day 

18, haemocyte aggregation in muscular tissue of 

mantle was found, while in other two organs, such 

damage was not occurred. In day 18, an intense 

histological damage was observed in mantle and 

foot. 

 

Discussion 

Exposure to Zn is led to histopathological alterations 

in mantle, digestive gland and foot tissues. 

Haemocytes and other lipopigmented cells 

(garanulocytomes) are related to sorption and storing 

of toxic chemicals (Johansson and Sӧderhäll, 1992). 

These cells were found in mantle and digestive gland 

of studied organism. Occurrence of this type of cells 

due to metal exposure has been reported in previous 

studies (Neff et al., 1987; Wolfe, 1992; Chakraborty 

et al., 2012). Haemocyte infiltration and aggregation 

in damaged area of different tissues is a common 

defensive response of organism against toxic agents 

(Oliver and Fisher, 1999) which was found in this 

study in connective and muscular tissues of mantle 

and digestive gland.  

Cilia of the external epithelial cells of mantle and 

foot are involved in dynamic activity of these organs. 

Exposure to Zn led to destruction of cilia. The loss 

of cilia (hypoplasia) of gill has been reported by Al-

Subiai et al., (2011) as a result of Cu exposure. 

Damaging of ciliary structures of epithelial cells 

weakens the moving performance of these organs. 

Increase in number of mucous cells in mantle and 

foot was occurred in subepithellial regions after loss 

of cilia. Likely, increase in number of these cells was 

occurred to compensate attenuation of dynamic 

operation. 

Our results showed apparent histopathological signs 

in different parts of digestive gland as result of 

exposure to Zn. Morphology of digestive tubules 

changed. Atrophy of digestive tubules in digestive 

gland in several studies reported in metal exposed 

organisms as a histopathological damage 

(Chakraborty et al., 2010; Sheir and Handy, 2010; 

Sheir et al., 2010; Al-Subiai et al., 2011). Digestive 

cells of tubules spilled into internal space of tubules. 

Usheva et al. (2006) reported such a histological 

damage in Crenomytilus grayanus in exposure to 
DDT.    

As the results of this study, the regions where 

damages were greater, tissue rupture and 

disintegration including connective and muscular 

tissues (in mantle and foot) were found. Tissue 

rupture has been reported by Al-Subiai et al. (2011) 

in Mytilus edulis, due to Cu exposure. Besides, it has 
been reported that heavy metals can be stored in 

haemocytes and interstitial storage tissues in bivalve 

organs (Regoli and Orlando, 1994; Soto et al., 1996). 

Thus, tissue rupture can be assigned to Zn storage in 

connective tissue of the mantle.  

Results showed swelling and inflammation of 

myocytes and mucous cells in foot in treatments. 

Previous studies reported swelling of myocytes in 

abductor muscles of Mytilus edulis (Al-Subiai et al., 
2010) and swelling of pore cells in Littorina litorea 
(Watermann et al., 2008) due to exposure of heavy 

metals. 

First signs of histopathological damages during 

experiment were appeared in mantle, digestive gland 

and foot in fourth day after exposure. Foot and 

mantle are directly contacted with pollutant, but 

exposure route of digestive gland is indirect. During 

experiment, by approaching to day 18, the extent of 

damages increased. In organs such as foot in which 

tissue mass intensity is greater, damages have been 

extended to inner parts at the end of experiment. 

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Based on finding of this study, exposure of 

A. cygnea to chronic concentrations of Zn is led to 
histopathological changes in their mantle, digestive 

gland and foot. Sensitivity of the mantle and foot are 

greater than digestive gland to Zn exposure. As a 

general conclusion, histopathological alterations in 

mantle, digestive gland and foot in A. cygnea are 
suggested as biomarkers for biomonitoring of heavy 

metal pollution in aquatic ecosystems.  

 

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