Iran. J. Ichthyol. (March 2015), 2(1): 35–42 © 2015 Iranian Society of Ichthyology P-ISSN: 2383-1561; E-ISSN: 2383-0964

Received: October 14, 2014 Accepted: February 01, 2015 doi: http://www.ichthyol.ir

Effects of Copper Sulfate on gill histopathology of grass carp (Ctenopharyngodon idella) Azadeh ATABATI1, Alireza KEYKHOSRAVI2, Majid ASKARI-HESNI*3, Jafar VATANDOOST2, Mina MOTAMEDI3 1

Department of Environment, Faculty of Geography and Environmental Sciences, Hakim Sabzevari University, Sabzevar, Iran. 2 Department of Biology, Faculty of Science, Hakim Sabzevari University, Sabzevar, Iran. 3 Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran. * Email: saeed. [email protected]

Abstract: The toxic impact of copper sulfate in lethal and sublethal concentrations was investigated on gill of grass carp, Ctenopharynogodon idella. In this investigation, 27 specimens were used as control group and three treatment groups were treated with 2.5 and 5mg/l of copper sulfate. Gill samples were collected from each treatment after 96hr and lesions were analyzed by light microscopy. In histopathological study of the gill tissues, hyperplasia was clearly obvious in treatment specimens. In all of the treated groups, heavy gill-mucus response was observed, which indicate a direct relation with high concentrations. Also, in histological study of the gill, epithelial cells faced to hyperplasia, which increased with high copper densities. Primary lamellar cells wrinkling and also changing in formation were observed in chloride cells. This lesion enhanced in higher densities and in concentrations of 2.5 and 5mg/l, primary and secondary lamellar epithelial cells were degenerated. Keywords: Heavy metals, Gills, Histopathological, Ctenopharynogodon idella, Teleostei.

Introduction Copper is an essential micronutrient and important for the normal healthy growth and reproduction of all higher plants and animals. This trace element serves an important role in cell physiology and consequently the nutrition and metabolism of vertebrates (Handy 2003). It is required and involved in the formation of collagen, bone, tissue pigmentation and the cardiac function. Although the essential role of the copper in several enzymatic processes has been cleared (Baker 1969; Li et al. 1998), this heavy metal can exert adverse toxicological effects, when present in high concentrations in water (Pelgrom et al. 1995). In fact, it is potentially toxic when the internal available concentration exceeds the capacity of physiological detoxification processes. Increasing agricultural production leads to

increasing the number of freshwater systems being impacted by the contaminants present in wastewater releases (Jafri & Shaikh 1998; Straus 2003). Copper sulfate is also used extensively in the aquaculture industry and in commercial and recreational fishponds as an algicide, molluscicide and as a therapeutant for protozoan ectoparasites because of its effectiveness and low cost. High concentration of this heavy metal was detected in some aquatic ecosystems found in vineyard runoff water and in ground water (Di Giulio & Hinton 2008). There are also anthropogenic sources of environmental contamination by copper including mining, smelting, foundries, municipal waste incinerators, burning of coal for power generation and a variety of copper-based products used in building and construction (Figueiredo35

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Fernandes et al. 2007). Heavy metal contamination has been reported for the aquatic organisms (Adham et al. 2002). These organisms are used extensively for biologically monitor deviations in the environmental levels of anthropogenic pollutants (Farkas et al. 2003). They can be used to identify potential environmental problems before the health of a system is critically altered or compromised (Figueiredo-Fernandes et al. 2007). Fishes and their physiological changes are widely used to evaluate the health of aquatic ecosystems. In addition, they served as biomarkers of environmental pollution (Kock et al. 1996). Grass carp, Ctenopharyngodon idella, is one of the most common freshwater fish used in toxicological studies. The organs of aquatic animals may accumulate copper, when they exposed to different toxic concentrations (Pelgrom et al. 1995; Mazon et al. 2002). The copper can lead to redox reactions generating free radicals and, therefore, may cause biochemical and morphological alterations. It is well-known that changes in fish gill are among the most commonly recognized responses to environmental pollutants (Figueiredo-Fernandes et al. 2007). Also, gills are the first target of waterborne pollutants because it is the main place for copper uptake, and because it has a constant and direct contact with the external environment. Therefore, this study was undertaken to examine the effect of different sublethal copper sulfate concentrations on histological aspects of the gill in grass carp.

photoperiod (12D:12L). Supplemental aeration was provided to keep dissolved oxygen near the saturation. The sexually mature specimens (35.3±5.4gr of the mean body weight) were randomly distributed through nine tanks. There were three specimens per tank and three tanks for each treatment (n=27). Three tanks containing nine specimens served as the control group. The remaining were exposed to copper with concentrations of 2.5 and 5mg/l, supplied as copper sulfate (CuSO4; MERCK), for 96hr. The experiments were carried out under constant temperature (25±1°C), controlled photoperiod (12D:12L) and constant filtration. The water had identical physical and chemical characteristics of the acclimation tanks and the experiments described comply with the Guidelines of the European Union Council (86/609/EU). The water quality parameters which are mentioned above were assessed in the experimental period. During the experimental period, fishes have not been fed. Four fish specimens per treatment (2 fishes per tank) were euthanized with 2- phenoxiethanol (Sigma) (1ml/l water), weighed and sampled. Gills were collected and weighed at 24, 48 and 96hr exposition. Histology: A gill arch of the right side of each fish was collected and fixed in Bouin’s fluid for 24hr, dehydrated in graded ethanol concentrations and embedded in paraffin wax. Sagittal sections (5μm of thickness) were cut and mounted on glass slides. Sections were deparaffinized in xylene, hydrated in ethanol and stained with hematoxylin-eosin (HE) and Toluidine Blue. Changes induced by treatment in the gill tissues were photographed and analyzed by light microscopy (Nikon® Labophot).

Materials and Methods

Fish and experimental system: The fish specimens

Results Representative light micrographs of the gills in control and the copper treated specimens are given in Fig. 1. The gill morphology of the control specimens is similar to that of other bony fishes. The gill is made up of double rows of filaments from which arise perpendicularly the lamellae. The lamellae are lined by a squamous epithelium composed by pavement

were obtained from the Aquaculture Station in city of Sabzevar. The experiment was performed in 100 liter recirculation tanks, filled with dechlorinated tap water (pH=6.5 to 7.5; alkalinity=60mg/l). Fishes were fed daily to satiation with a previously tested diet (Fontaínhas- Fernandeset al. 1999), kept at a constant temperature of 25±1°C and controlled 36

Atabati et al.-Effects of Copper Sulfate on gill histopathology of grass carp

Fig.1. Representative light micrographs of the gills in control and the copper treated grass carp specimens (B= 2.5mg -l CuSO4, B1=24hr, B2=48hr, B3=96hr) and (C=5 mg/l CuSO4, C1=24hr, C2=48hr, C3=96hr). (1A) Control fish, showing normal appearance of the gill filaments (F) and the lamellae (L), H&E; (1B1) gills from exposed fish showing an intense lamellar epithelium lifting (Lf) and exude of RBC, H&E; (1B2) section of the gill with lamellar axis vasodilation (cvs) and evident epithelium interstitial edema (**) in the filament near the lamellar axis and proliferation of filamentary epithelium with (Lfu), H&E; (1B3) fusion of adjacent lamellae, Toluidine Blue; (1C1) gill epithelium of the treated specimen showing necrosis, H&E; (1C2) gill epithelium of the treated specimen showing necrosis, Toluidine Blue; (1C3,4) gill epithelium of the treated specimen showing necrosis and lifting, H&E (Elaborations: cc= chloride cell, cvs= central venous sinus, fe= filament epithelium, pc= pillar cell, pv= pavement cell, RBC= Red Blood, **= edema and bars= 20μm) 37

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Fig.1. Continued.

and no differentiated cells. Below that epithelium are lamellar blood sinuses separated by pillar cells. Between the lamellae, the filament is lined by a thick stratified epithelium constituted by several cellular types, such as chloride, mucous and pavement cells (Fig. 1-A). Morphological changes Changes in the color of the gill: Gills of the control group have normal color (light red). The main changes in color observed on 5mg/l CuSO4. In the highest concentration, the gill color was gray. Secreted mucous: There was an increasing in the mucous secretion in gill of the fish specimens by

increasing of the copper concentration. Histological changes: 2.5mg/l CuSO4: The main changes observed after 24, 48 and 96hr of exposed to the 2.5mg/l CuSO4 were accentuated lifting of the lamellar epithelium (Fig. 1-B1, B2 and B3), edema in the filamentary epithelium, an intense lamellar vasodilatation and exuding of Red Blood Cell (RBC) from capillary of lamellar (Fig. B2). Exuding of RBC was due to lifting and necrosis of lamellar epithelium. The gills of grass carp in this treatment also exhibited lamellar fusion in numerous areas because of the filamentary epithelium proliferation (Fig. 1-B2 and 1B3). 38

Atabati et al.-Effects of Copper Sulfate on gill histopathology of grass carp

5mg/l CuSO4: The fish specimens showed the largest

Fingerlings exposed to copper sulfate solution for three weeks revealed Cu2+ induced lamellar edema followed by degeneration and lifting of epithelium. Thus, this signifies that these alterations are not specifically induced by copper or other heavy metals. The primary observed lesions (epithelial lifting, proliferation of pavement and chloride cells, necrosis) have also been described by several authors for those fishes exposed to the heavy metal (Mallat 1985; Karlsson-Norggren et al. 1986; Pane et al. 2004; Rajeshkumar et al. 2013; Hassaninezhad et al. 2014). Cell proliferation with thickening of gill filament epithelium is a histological change found in fishes exposed to the copper by several authors (e.g., Triebskorn et al. 1997; Arellano et al. 1999; Van Heerden et al. 2004), and may lead to the lamellar fusion observed in this study. Smart (1976) has also interpreted the epithelia lifting as an initial reaction of the gills to stress in response to variety of different pollutants (Alazemi et al. 1996). Epithelial lifting results in an enlargement of the water-blood diffusion barrier, which on one hand, might negatively influence respiration but, conversely might also prevent the penetration of the heavy metal (Morgan & Tovell 1973). Increasing of the mucus-containing vacuoles in copper-exposed specimens and the increased hyper secretion of mucus may also impede gas exchange (Ultsch et al. 1980; Pawert et al. 1998). A similar hyper secretion of the gill mucus has been described by Mallat (1985) for trout that exposed to the heavy metals or organic compounds and by Jagoe et al. (1993) for the trout after treatment with beryllium. Generally, it might be assumed that the gas exchange is negatively influenced in the specimens exposed to the copper, which probably could be related to a reduced swimming ability of these fishes (Triebskorn et al. 1997). The edema, epithelial lifting as well as lamellar fusion also are defense mechanisms that reduce the branchial superficial area in contact with the external milieu. These mechanisms also increase the diffusion barrier to the pollutant (Van Heerden et al. 2004).

signs of epithelial lesions when they exposed to the 5mg/l CuSO4 (Fig. 1-C1, C2, C3 and C4). In addition, few aneurisms were observed at gill lamellae (Fig. C4). Necrotic cells and macrophages often occurred in the filament and the lamellar epithelium (Fig. 1-C1, C2, C3 and C4). Mortality: The copper concentrations were selected based on preliminary results, shown to be sublethal after 96hr in 2.5mg/l and lethal after 96hr in 5mg/l CuSO4 period of exposure. Discussion Histopathological effects represent an intermediate level of biological organization between lower-level biochemical effects and higher-level population effects (Adams et al. 2000). These types of responses typically occur earlier than reproductive changes and are more sensitive than growth or reproductive parameters and, as integrative parameters, provide a better evaluation of organism health than a single biochemical response (Segner & Braunbeck 1988; Triebskorn et al. 1997). The histological investigations of the gill in this study showed a typical structural organization of the lamella in the untreated fish specimens. However, those fish specimens, which exposed to copper show several histological alterations, namely lamellar epithelium lifting, epithelium proliferation, lamellar axis vasodilatation, edema in the filament, fusion of lamellae and the lamellar aneurisms. The histology of the gill has been shown to reflect different environmental conditions for the fishes (Mallat 1985) and to be sensitive to copper exposure (Weinstein et al. 1997), to acids (Perry & Laurent, 1993), drugs (Schwaiger et al. 2004), diesel oil (Askari Hesni et al. 2011) and to heavy metals, such as aluminium (Karlsson-Norggren et al. 1986), cadmium (Reid & McDonald 1988), nickel (Pane et al. 2004) and zinc (Santos et al. 2012). Patel & Bahadur (2010) have investigated the copper ion toxicity in Catla catla with an emphasis on histopathological alterations in gill and kidney. 39

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approach. Environmental Research 126: 254-263. Adham, K.G.; Hamed, S.S.; Ibrahim, H.M. & Saleh, R.A. 2002. Impaired Functions in Nile Tilapia, Oreochromis niloticus (Linnaeus, 1757), from Polluted Waters. Acta Hydrochimica et Hydrobiologica 29(5): 278-288. Ahmed, K.M.; Habibullah-Al-Mamun, M.; Parvin, E.; Akter, M.S. & Khan, M.S. 2013. Arsenic induced toxicity and histopathological changes in gill and liver tissue of freshwater fish, tilapia (Oreochromis mossambicus). Experimental and Toxicologic Pathology 65 (6): 903-909. Alazemi, B.M.; Lewis, J.W. & Anddrews, E.B. 1996. Gill damage in the freshwater fish Gnathnonemus petersii (Family: Mormyridae) exposed to selected pollutants: An ultrastructure study. Environmental Technology 17: 225-238. Arellano, J.M.; Storch, V. & Sarasquete, C. 1999. Histological changes and copper accumulation in liver and gills of the Senegales sole, Solea senegalensis. Ecotoxicology and Environmental Safety 44: 62-72. Askari Hesni, M.; Dadolahi-Sohrab, A.; Savari, A. & Mortazavi, M.S. 2011. Gill Histopathological Changes in Milkfish (Chanos chanos) Exposed to Acute Toxicity of Diesel Oil. World Applied Sciences Journal 14 (10): 1487-1492. Baker, J.T.P. 1969. Histological and electron microscopical observations on copper poisoning in the winter flounder (Pseudopleuronectes americanus). Journal of the Fisheries Research Board of Canada 26: 2785-2793. Di Giulio R.T. & Hinton D.E. 2008. The toxicology of fishes. CRC Press, USA, Pp 28-35. Farhangi, M.; Gholipour Kanani, H.; Aliakbariyan, A. & Kashani, M. 2014. Effect of Copper sulfate on behavioral and histopathological changes in roach, Rutilus rutilus caspicus. Caspian Journal of Environmental Sciences 12 (1): 73-79. Farkas, A.; Salanki, J. & Specziar, A. 2003. Relation between growth and the heavy metal concentration in organs of bream, Abramis brama L. populating Lake Balaton. Arch. of Environ. Contaminant Toxicology 43(2): 236-243. Figueiredo-Fernandes, A.; Ferreira-Cardoso, V.; GarciaSantos, S.; Monteiro, M.; Monteiro, M.; Carrola, J.; Matos, O. & Fontainhas-Fernandes, A. 2007.

Lamellar axis vasodilatation was also found in grass carp exposed to copper. Garcia-Santos et al. (2006) suggested that this lesion can induce changes in pillar cell normal structure, with consequent loss of their support function and probably, was responsible for the emergence of lamellar aneurysms in the fish specimens exposed to cadmium. Similar results have also been observed in Rutilus rutilus caspicus and Clarias gariepinus which were exposed to copper sulfate (Farhangi et al. 2014; Wani et al. 2011). The same results have also been observed in Lates calcarifer (exposed to cadmium, Thophon et al. 2003), Chanos chanos (exposed to diesel oil, Askari Hesni et al. 2011), Oreochromis mossambicus (exposed to Arsenic, Ahmed et al. 2013), Epinephelus itajara (exposed to mercury, Sonne 2013), and in Acanthopagrus latus (exposed to mercury, Hassaninezhad et al. 2014). All these investigations showed that heavy metals have effect on fish gills. Based on the results achieved in this study, we concluded that the histological changes of the gills have related to the different copper concentrations. Moreover, gill alterations as a result of heavy metal exposition of the fishes may serve as a sensitive biomarker for the toxicity of sublethal concentrations of metals as well as the other pollutants. However, complementary studies are necessary for a better understanding of its deleterious effects. Acknowledgments The financial support was provided by Hakim Sabzevari University, Sabzevar, Iran. References Adams, S.M. & Ryon, M.G. 2000. Evaluating effects of contaminants on fish health at multiple levels of biological organization: Extrapolating from lower to higher levels. Human and Ecological Risk Assessment 6(1): 15-27. Adams, D.H. & Sonne, C. 2013. Mercury and histopathology of the vulnerable goliath grouper, Epinephelus itajara, in U.S. waters: A multi-tissue 40

Atabati et al.-Effects of Copper Sulfate on gill histopathology of grass carp

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