MATERIALS AND METHODS

In the present study the healthy species of Cyprinus carpio were procured from the local fish farm from Vadalore, Cuddalore District, Tamil Nadu, India and it was acclimatized to the laboratory conditions for 20 days. Water was changed daily and fish were fed ad libitum with rice bran and groundnut oil cake twice a day. Fish ranging from 7-8 cm in length and weighing 8-10 g were selected for experimental purpose. The qualities of the water was determined according to the method of APHA (1976) and were as follows: dissolved oxygen 6.2±0.02 mg L^-1; pH 7.2±0.2; water temperature 25.0±2.0°C; salinity 0.2±0.07 ppm; total hardness 13±2.0 mg L^-1; calcium 5.0±0.1 mg L^-1; magnesium 8.0±2.0 mg L^-1 and total alkalinity 20.0±06 mg L^-1. Preliminary studies were carried out to find out the median lethal concentration (LC₅₀) for 96 h by probit analysis method of Finney (1978).

The concentration, at which 50% survival/mortality occurred, was taken as a median lethal concentration (LC₅₀) for 96 h which was 22 ppm L^-1 for ammonia, 27 ppm for ammonia with low pH (5.0) and 18 ppm for ammonia with high pH (8.0). The acute toxicity study was conducted for 96 h with four replicates of all the three treatments along with a common control (total of sixteen tanks). The first tank was filled with 20 L of water and 22 ppm L^-1 of ammonia was added and in the second tank the pH of the water was lowered to pH 5.0 by adding 0.1 N sulphuric acid drop by drop and 27 ppm L^-1 of ammonia and the third tank was filled with 20 L of water and the pH of the water was increased upto pH 7.5 by adding 0.1 N sodium nitrite drop by drop then 18 ppm L^-1 ammonia solution was added. Twenty fish were introduced into each tub. A common control (pH 7.2) was also maintained. Toxicant was renewed daily in all the experimental tanks. No mortality was observed throughout the experimental period. At the end of every 24 h, 10 fish from each experimental tank was collected for histopathological studies, gills were subsequently dissected using a sterile scalpel and were rinsed with distilled water in order to remove the adhering body fluid and were fixed in Bouin’s fixative and later processed following the methods of Pearse (1968); Roberts (1978) and Humason (1979). Histopathological changes were examined under a light microscope.

RESULTS AND DISCUSSION

The histopathological observations for control, ammonia and ammonia with low and high pH exposed fish gill images are displayed in Fig. 1, 2 and 3. In control fish, primary lamellae appeared normal and mucus free with well defined secondary lamellae branched from them (Fig. 1a). In ammonia treated fish, the gills showed necrosis, disintegration of epithelial cells and degenerative changes (Fig. 1b-e). In fish, exposed to ammonia with low pH concentration, lifting up of the epithelium, swelling, hyperplasia, hypertrophy and proliferation of chloride cells, were noticed (Fig. 2b-e). In ammonia with high pH treated fish, fused lamellar filaments, desquamated epithelium, hemorrhage and complete damage of epithelial cells of lamellae were noticed (Fig. 3b-e).

Histopathology provides information to detect effects of irritants in various organs (Altinok and Capkin, 2007). The exposure of fish to chemical contaminants is likely to induce a number of lesions in different organs. Gills are a well known target organs in fish, being the first to react to unfavourable environmental conditions. In the present study, hyperplasia, hypertrophy, deformation of the lamella and lamellar fusion were recorded in the gills of ammonia exposed fish. Bais and Lokhande (2012) noticed minor alterations like chloride cells of the secondary lamellae in zebra fish exposed to cadmium concentration of 3 μg L^-1. Osman et al. (2009) also noted congestion of gill arch and lamellar blood vessels, together with edema, hemorrhage, leukocytic in filtration, lamellar epithelial hyperplasia was observed at 2 weeks post-intoxication. Several authors have reported similar alterations on the gills of different fish species exposed to ammonia (Mallatt, 1985; El-sherif et al., 2008; Spencer et al., 2008). Larmoyeux and Piper (1973) determined aneurysms and fused lamella of rainbow trout (Salmo gairdneri) gill epithelium cells.

Fig. 1(a-e):	Transverse section of gills of fish Cyprinus carpio var. communis exposed to acute ammonia concentration for 96 h. (Magnification X 400), (a) control, (b) after 24 h as ammonia exposure, (c) after 48 h as ammonia exposure, (d) after 72 h as ammonia exposure and (e) after 96 h as ammonia exposure

Fig. 2(a-e):	Transverse section of gills of fish Cyprinus carpio var. communis exposed to acute ammonia with low pH concentration for 96 h. (Maganification X 400), (a) control, (b) affer 24 h as ammonia with low pH exposure, (c) affer 48 h as ammonia with low pH, (d) affer 48 h as ammon with low pH exposure and (e) affer 96 h as ammonia with low pH exposure

Fig. 3(a-e):	Transverse section of gills of fish Cyprinus carpio var. communis exposed to acute ammonia with high pH concentration for 96 h. ( Maganification X 400), (a) control, (b) affer 24 h as ammonia with high pH exposure, (c) affer 48 h as ammonia with high pH, (d) affer 48 h as ammonia with high pH exposure and (e) affer 96 h as ammonia with high pH exposure

Furthermore, Kirk and Lewis (1993) reported that the gills of the rainbow trout exposed to 0.1 mg^-1 ammonia for 2 h exhibited deformation of the lamellae. Salin and Williot (1991) observed that in Siberian sturgeon (Acipencer baeri) (270 g) exposed to more than 60 mg^-1 of ammonia revealed a modification of the epithelium of the secondary lamellae and the base of the filament was slightly turgescent.

Miron et al. (2008) observed the major changes in the gills of Silver catfish, Rhamdia quelen exposed to ammonia levels of 96 h- LC₅₀ (mainly in those exposed to pH 6.0) exhibited edema and fusion of the secondary lamellae. The above authors further stated that these lesions may reduce gill functional surface for gaseous exchange, impairing respiratory function. In the present study also, a similar mechanism might be operating, thus reducing the gill functional surface for gaseous exchange (Abbas and Ali, 2007). Wood et al. (1988), Venkataraman et al. (2007) and Moharram et al. (2011) reported that thickening of the lamellar epithelium increased diffusive distance of the gill. The thickening of the gill epithelium (via. cell hypertrophy) is sometimes considered to be an indicator of cell degeneration and eventually necrosis (Tietge et al., 1988; Peuranen et al., 1993; Jiraungkoorskul et al., 2006; Lamchumchang et al., 2007). The lifting and hypertrophy of cells greatly increases the diffusion distance (water-blood distance) (Ingersoll et al., 1990). A similar reason may be attributed for the fusion of primary and secondary lamella and hypertrophy in the present study too. In the present study the ammonia with low pH treated fish gills showed swelling of primary and secondary lamellae, fusion of adjacent secondary lamellae, increased mucus production, secondary lamellae appeared thickened and shortened with extremely rough surface and considerable mucus. A similar observation was also made by Tandjung (1982), Segner et al. (1988) and Lamchumchang et al. (2007) in brown trout, Salmo trutta and Oreochromis niloticus, respectively. Daye and Garside (1976) observed hypertrophy and separation of epithelial cells from the supporting pillar cells in brook trout, Salvelinus fontinalis in chronic acid pH (5.5) treatment and this condition greatly increased the diffusion distance (water-blood distance). The gill epithelium represents the primary site for oxygen exchange and ionoregulation (Lease et al., 2003; Jiraungkoorskul et al., 2006; Joseph and Raj, 2011) and disruption of these processes leads to decreased ability to function (Smart, 1976). Lamellar fusion which is the fusing of adjacent lamellae, affects gas exchange and can be a direct result of lifting pavement cells (Frances et al., 2000). In the present study, swelling of primary and secondary lamellae, fusion of adjacent secondary lamellae, increased mucus production, appearance of thickened and shortened secondary lamellae and necrosis of gill epithelium of fish may be due to direct deleterious effect of ammonia with low pH toxicity. Similar observation like gill edematosis, foamy vacuolation especially in the proximal part of the secondary lamellae, swollen, prominent in chloride cells, epithelial lifting and telangiectasis in the secondary lamellae were also found by Mahjoor and Loh (2008) and Mobarak and Sharaf (2011).

The gills of ammonia with higher pH exposed fish showed several histological changes like, separation of secondary lamellae, desquamated primary and secondary and lamellae and necrosis. Similar observations were made by several others (Tomasso et al., 1980; Frances et al., 2000; Lemarie et al., 2004). Meade (1985) in chinook salmon (Oncorhyncus tshawytscha) reported an extensive hyperplasia of gill epithelium after exposure to 0.005 mg L^-1 of NH₃-N for 6 weeks. Kirk and Lewis (1993) reported that the gills of the rainbow trout exposed to 0.1 mg L^-1 ammonia for 2 h exhibited deformation of the lamellae. The filamental and lamellar epithelium was covered with shallow, circular depressions in which the integrity of microridges was maintained. Salin and Williot (1991) observed that Siberian sturgeon (Acipenser baeri) (270 g) exposed to more than 60 mg L^-1 of ammonia revealed a modification of the epithelium of the secondary lamellae and the base of the filament was slightly turgescent. Smith and Piper (1975) and Smart (1976) found that the most characteristic feature for chronic exposure of rainbow trout to ammonia was the appearance of swollen, rounded secondary gill lamellae or telangiectatic capillaries in the secondary lamellae. Hemorrhage is a result of blood channel disruption and is indicative of severe physical damage. Smart (1976), Mallatt (1985) and Abdel-Moneim et al. (2008), who observed, desquamation, necrosis, the lifting of the lamellar epithelium, oedema, aneurism, hyperplasia of epithelial cells and fusion of the secondary lamellae were seen in the gills of fish Silver Sailfin Molly (Poecilia latipinna) after exposure to dyestuff and chemical wastewater. Edema with apparent separation of epithelium from the underlying basement membrane and an increase in the interstitial fluid spaces are common pathological findings in toxicity with heavy metals and many other chemical pollutants. In the present study, gill hyperplasia, degeneration of epithelium was noticed in ammonia with high pH, which might be due to direct deleterious effect of ammonia with high pH toxicity. Considering the above facts, the present study concludes that effect of ammonia toxicity was very high in ammonia with high pH condition.

CONCLUSION

Ammonia is an environmental toxicant that is especially problematic for aquatic organisms. Its concentrations in water systems can increase due to agricultural run-off and decomposition of biological waste. In addition, in intensive fish culture, high stocking densities and feeding rates also increase the probability of exposure of the animals to elevated concentrations of nitrogenous wastes, particularly ammonia. In the present study, exposures to ammonia concentrations at 22 ppm, ammonia with low pH (5.0) at 27 ppm and ammonia with high pH (8.0) at 18 ppm for 96 h resulted in definite histopathological changes in the gills. In the present study, swelling of primary and secondary lamellae, fusion of adjacent secondary lamellae, increased mucus production, appearance of thickened, shortened secondary lamellae and necrosis of gill epithelium of fish may be due to direct deleterious effect of ammonia toxicity. The histological results found in the present study indicate that the ammonia with high pH tends to be more harmful than ammonia and ammonia with low pH. Further intensive studies are needed to maintain the safe level of ammonia for aquaculture/aquarium practices.

ACKNOWLEDGMENT

The authors are grateful to the authorities of CAS in Marine Biology, Annamalai University, Tamil Nadu - India for providing all the necessary facilities to carry out the present work and deeply acknowledge to University grant commission for financial assistance.