MORPHOLOGICAL INVESTIGATION ON GILLS AND LIVER OF FRESHWATER FISH FROM DAM LAKE STUDEN KLADENETS

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1 364 Bulgarian Journal of Agricultural Science, 16 (No 3) 2010, Agricultural Academy MORPHOLOGICAL INVESTIGATION ON GILLS AND LIVER OF FRESHWATER FISH FROM DAM LAKE STUDEN KLADENETS I. VELCHEVA 1, E. TOMOVA 1, D. ARNAUDOVA 2 and A. ARNAUDOV 1 * 1 University of Plovdiv Paisii Hilendarski, Faculty of Biology, BG 4000 Plovdiv, Bulgaria 2 University of Plovdiv P. Hilendarski, Branch L. Karavelov, BG Kardzhali, Bulgaria Abstract VELCHEVA, I., E. TOMOVA, D. ARNAUDOVA and A. ARNAUDOV, Morphological investigation on gills and liver of freshwater fish from dam lake Studen kladenets. Bulg. J. Agric. Sci., 16: The histostructure of gills and liver (hepatopancreas) of the bleak (Alburnus alburnus L.), rudd (Scardinus erythrophtalmus L.) and perch (Perca fluviatilis L.) inhabiting the dam Studen Kladenets was studied. Histopathological changes were found in the liver (hepatopancreas) and gills of all three investigated fish species. The morphological alterations in the liver were mainly degenerative, but necrotic and hyperemic changes in the parenchyma were also found. Morphological changes in gills are of hypertrophic type, most likely of a compensatory type. Key words: freshwater fish, gills, liver, histopathology Abbreviations: HE hematoxylin and eosin Introduction In recent years, the anthropogenic pollution of aquatic ecosystems increased the need for studies to identify the impact of heavy metals on the species living there. Fish are often exposed to highly polluted water, and that causes different disabilities, ranging from biochemical changes in single cells to changes in the whole organism (Bernet et al., 1999). Monitoring programs for bioaccumulation measuring serve as a biomarker for fish from contaminated places and provide information about the environmental conditions. They are designed for different aspects of risk assessment of the environment (Van der Oost et al., 2003). Histological changes are more sensitive and occur earlier. They provide a better assessment of fish arnaudov@uni-plovdiv.bg health, as well as the effects of pollution on each biochemical parameter. Histopathological changes have been integrated with the impact of various stressors (microbial pathogens, toxic compounds, nutritional and adverse environmental conditions) (Marchand et al., 2009). Until now, in the area of the dam Studen Kladenets, there were studies mainly related to tracking the content of heavy metals in fish organs (Batchvarov and Veltcheva, 1995; Velcheva, 1997, 1998). There are no data about the influence of anthropogenic pollution in the lake on morphology and functional state of organs of the fish inhabiting the waters. In our previous study (Arnaudova et al., 2008) we found anemic alterations in the blood of three of

2 Morphological Investigation on Gills and Liver of Freshwater Fish from Dam Lake Studen Kladenets 365 the most common species that inhabit the lake. Having in mind that erythrocytes are functionally closely related to fish gills and liver, and this motivated us to conduct this study. Its purpose is to study the influence of anthropogenic water pollution in the dam Studen Kladenets on histostructure of gills and liver (hepatopancreas) of the species bleak, rudd and perch. Materials and Methods As experimental animals were used three species of freshwater fish of similar age-size groups: bleak (Alburnus alburnus L.) cm; rudd (Scardinus erythrophtalmus L.) cm and perch (Perca fluviatilis L.) cm. 10 specimens of each species were tested. Samples were obtained throughout the year. Immediately after sectioning, the liver and gills were inspected. Attention was paid to the size, shape, consistency, color and presence of bleeding. The liver and gills of each specimen were taken out. Histological analyses by the means of classical methods described by Romeis (1989) were used. Preservation of samples was done in 10% neutral formalin. The material was put in a series of alcohols of increasing concentration. Clarifying was done in xylol. The treated samples were included in paraffin. From the paraffin blocks we prepared cuts of thickness 0.6 μ м with a microtome. Coloring of the cuts was made with hematoxylin and eosin. The sections were examined with a light microscope OLIMPUS CX21, using as reference Takashima and Hibiya (1995), and photographed using a digital camera. Results Macroscopic examination Many variations during the macroscopic examination of the different species were observed. In some bleak specimens, were noticed fading of hepatopancreas which often had mucilaginous texture. Darkcolored spotted areas were also observed. In the rudd, the hepatopancreas had greenish coloration, and in some specimens it was of hard texture. In the perch, almost no macroscopic changes were detected in the liver. The macroscopic view done by us can direct our attention toward some degenerative-necrotic processes in the hepatopancreas of bleaks and rudds. Macroscopically, any pathological changes in gills weren t found. Histological examination I. Histological finding in hepatopancreas The following changes occurred in the liver degeneration, necrosis and hyperemia. - Degeneration. It was observed it in all three species, and it mostly occurred as granular degeneration (Figure 1). There were a lot of small grains of Fig. 1. Hepatopancreas of bleaks. Granular degeneration. HE x 600 powder in hepatocytes and this gave a blurred appearance of the observed parenchymal tissue. In samples from the bleak and rudd, the parenchymal degeneration in some samples was combined with vacuolar (watery) degeneration (Figure 2). In the hepatocytes of affected tissue vacuoles and reticulate filaments (remnants of cytoplasm) were observed as well. In the samples, where both types of degeneration were found, the finding was of a mosaic type (a combination of sections with granular and vacuolar degeneration) (Figure 2).

3 366 I. Velcheva, E. Tomova, D. Arnaudova and A. Arnaudov Fig. 2. Hepatopancreas of rudds. Vacuolar and granular degeneration. HE x Necrosis. It occurred as karyolysis (Figure 3). The affected cells were ballooned, there was no nucleus in them, or in some cells it lost its specific shape, or it looked pale. This type of necrosis was found in individual samples from the three fish species, whose hepatocytes were affected by a strong vacuolar degeneration. Fig. 4. Hepatopancreas of bleaks. Hyperemia of sinusoidal capillaries. HE x 400 species were similar and they occurred in the following alterations: - Hyperplasia of filaments which was expressed in the expansion of their end sections (Figure 5). Fig. 3. Hepatopancreas of bleaks. Karyolysis. HE x Hyperemia. It was observed in different areas in the three species, and the sinusoidal capillaries were full of erythrocytes (Figure 4). II. Histological finding in gills The histological changes found in the three fish Fig. 5. Gills of bleaks. Hyperplasia of secondary lamellae and expansion of the cartilaginous base of gill arches. HE x Hyperplasia of epithelial cells. The spaces between two adjacent filaments were partially full of epithelial cells (Figure 6). - Fusion. Two or more lamellae (plates) were merged due to the hyperplastic processes in epithelial tissue (Figure 6). - Expansion of the cartilaginous base of gill arches (Figure 5). Blood vessels in some of the plates

4 Morphological Investigation on Gills and Liver of Freshwater Fish from Dam Lake Studen Kladenets 367 Fig. 6. Gills of bleaks. Hyperplasia of epithelial cells and fusion. Hyperaemia of secondary lamellae. HE x 400 were filled with blood. Hyperemic changes were also found in the secondary lamellae (Figure 6). We did not found atrophic and necrotic changes in filaments and lamellae. Discussion Uniform changes in the hepatopancreas of the three freshwater fish species were found such as degeneration, necrosis and hyperemia. Some authors also reported degenerative and necrotic changes in hepatocytes but in other species and under the influence of various pesticides and other toxic substances (Velisek et al., 2009, 2007; Jee et al., 2005). Similar are results for the fish inhabiting waters with different heavy metals pollution. Thus, in the liver of Tilapia zillii and Solea vulgaris Mohamed, 2009 (Lake Quarun, Egypt), found degenerative, necrotic, inflammatory, hyperemic and thrombosed alterations. El-Naggar et al., 2009, proved that bioaccumulation of some heavy metals (Fe, Cu, Zn, Mg, Pb and Cd) in the liver of Oreochronis niliticus in the Nile caused similar changes in the hepatopancreas like ours and the above mentioned. We think that the exposure of the bleak (Alburnus alburnus L.), rudd (Scardinus erythrophtalmus L.) and perch (Perca fluviatilis L.) in the polluted water of the dam Studen Kladenets causes histological changes in hepatopancreas which lead to stress in organisms. On the one hand, there is damage (degeneration and necrosis), on the other a compensatory activation of erythrocyte circulation occurring as hyperemia of sinusoidal capillaries. According to Lauren (1991) gills are sensitive subjects for identifying the effects of water pollutants on fish organisms. There are reports on various histopathological changes in gills, under the effects of metal pollution in the water - hyperplasia and hypertrophy of gills, lamellar fusion, epithelial lesions, and hyperactivity of mucous cells, lamellar aneurysm and rupture of epithelial cells (Alazemi et al., 1996; Camargo et al., 2007; Garcia-Santos et al., 2007; Mohamed, 2009). The histological changes that were found in gills of the three fish species in the dam Studen Kladenets are similar to the above mentioned data. As a whole, it can conclude that the histological finding in gills of the three tested fish species show typical alterations for metal pollution in the aquatic environment. The hyperplastic processes and blood filling in blood vessels are outstanding. Perhaps, these changes are of compensatory nature, so to neutralize the effects of anemia (Arnaudova et al., 2008). Unlike the results of Dulic et al. (2009), who found mild and reparable alterations in gills of carp farmed in Serbia using water from the Danube, in the present study the histological changes were severe and irreparable. Conclusion Therefore, it can be concluded that the histopathological changes found in gills and liver (hepatopancreas) of the examined freshwater fish are typical for the clinical finding in polluted with heavy metals water of habitat. In support of this, the results of Vinodhini & Nerayanan, 2009, done in laboratory conditions, proved that heavy metal mixtures cause similar histopathological changes.

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