Comparative Studies of Acute Toxicity and Histopathology of Orechromis niloticus and Tilapia zilli exposed to cadmium

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1 International Journal of Environmental Science and Toxicology Research (ISSN: ) Vol. 4(7) pp , August, 2016 Available online Copyright 2016 International Invention Journals Full Length Research Paper Comparative Studies of Acute Toxicity and Histopathology of Orechromis niloticus and Tilapia zilli exposed to cadmium Akaahan, T. J. 1*, Azua E.T 1, Ebah E.E. 2 1 Environmental Science and Zoology Unit, Department of Biological Sciences University of Agriculture, P.M.B Makurdi Benue state Nigeria. 2 Microbiology Unit Department of Biological Sciences University of Agriculture, P.M.B Makurdi Benue state Nigeria. Received 28 July, 2016; Accepted 23 August, 2016 This study was carried out to investigate the acute toxicity and histological damage of cadmium (Cd) on Tilapia zilli and Oreochromis niloticus using the 96-hour bioassay test to determine the LC 50 acute toxicity. Cadmium metal was used to prepare the stock solution. The concentrations of cadmium that the fish were exposed to were: 0.05, 0.10, 0.15, 0.20, and 0.25 mg/l which caused changes in the concentration dependent manner in the liver and gills of the fish. The LC 50 of 0.15mg/L of cadmium was determine for Tilapia zilli exposed to the metal. The probit transformation results of mortality plotted against the graded concentration of the metal with R2 value of and for Tilapia zilli and Oreochromis niloticus respectively indicating the ability of the concentration of the metal to lethality. The liver lost its characteristic structure of the liver with increased vacuolation, dilation, disarrangement and thickening of hepatocytes cells and tissue. The histology of the gills were altered by degenerative and necrotic changes in the epithelium of gill filaments and secondary lamellae. This results in curling of the secondary lamellae and shortening of the secondary lamellae of the gills of the two species of tilapia fish exposed to cadmium for 96h. Consequently, severe damage to the liver and gills of Tilapia zilli and Oreochromis niloticus were observed upon contamination of the fish environment with cadmium metal and efforts should be made to discourage the release of the metal into the aquatic environment. The metal has the ability of magnification and an implication for public health through human consumption of the fish that has been accumulated with cadmium metal in its tissue. Keywords: Tilapia zilli, Oreochromis niloticus, Cadmium metal. INTRODUCTION Cadmium is a naturally occurring non-essential trace element and its tendency to bioaccumulate in living organisms often in hazardous levels, raises environmental concern (Sfakianakis, 2015). Cadmium metal has toxic effect at high concentrations, and thus could be considered as risk factors for several diseases (Authman et al., 2015). Cadmium metal when brought into contact with the organs and tissues of the fish and *Corresponding Author terngu.adaga@gmail.com, akaahan.james@uam.edu.ng consequently accumulated at different degree in different organs and tissues of the fish (Kaoud and El-Dahshan, 2010). Cadmium metal released into the environment find their way into the aquatic environment as a result of direct input, atmospheric deposition and erosion due to surface run off (Kaoud and El-Dahshan, 2010). Therefore aquatic animals may be exposed to elevated levels of cadmium metal in surface water (Kalay and Canli, 2000). The accumulation patterns of heavy metals contaminants in fish depend upon their intake, storage and elimination from the body (Abdallah and Morsy, 2013). Significant changes in external features and behavioral activities of Oreochromisniloticus and Tilapia

2 Akaahan et al. 119 zilli can be observed as a result of cadmium metal pollution (Hinton and Lauren, 1990). The liver is one of the most susceptible organs to the harmful effects of cadmium metal, because it is a detoxification organ and is essential for the metabolism and the excretion of toxic substances (Hinton and Lauren, 1990). A number of studies have shown that cadmium may exert immunosuppressive effects in Tilapia zilli and Oreochromis niloticus. Fish have the ability to uptake and concentrate cadmium directly from the surrounding water or indirectly from other organisms such as small fish, invertebrates and aquatic vegetation (Polat et al., 2015). Fish accumulate pollutants preferentially in their fatty tissues like liver and the effects become apparent when concentrations in such tissues attain a threshold level (Omar et al., 2014). Cadmium can be taken up into fish either from ingestion of contaminated food via the alimentary tract or through the gills and skin (Drevnick et al., 2006; Sfakianakis, 2015). This study is imperative as it compared the susceptibility of the cadmium metal to two species of Tilapia and determines the one that is more prone to the graded concentration of the metal. MATERIALS AND METHODS Sourcing and acclimatization of fish juveniles Juveniles of Tilapia zilli and Oreochromis niloticus were obtained from the fish farm of Federal University of Agriculture Makurdi, Benue State. The fish were acclimatized to laboratory conditions for two weeks (14 days) in plastic bowls containing dechlorinated and aerated water at room temperature. The fish were fed daily with commercial fish feed (copens). The water was changed daily to prevent buildup of metabolic wastes and aerators were used to introduce oxygen in the bowls where the fish were kept. Preparation of stock and test solution of cadmium trioxonitrate tetrahydrate The test chemical used for the experiment was cadmium trioxonitrate tetrahydrate. The concentrations prepared for the experiment were 0.05, 0.10, 0.15, 0.20 and 0.25 mg/l. A stock solution of 1000 mg/l (1 g/l) of the cadmium trioxonitrate tetrahydrate was prepared by adding 1.0 g of the toxicant to 1 litre of distilled water. The amount of cadmium trioxonitrate tetrahydrate which contained 1.0 g of cadmium was determined from the molecular and atomic weight as: Molecular weight of cadmium trioxonitrate tetrahydrate Atomic weight Exposure of the fish to the cadmium metal One hundred and twenty each individuals of acclimatized Tilapia zill and Oreochromis niloticus were divided into five groups exposed to graded concentration of cadmium trioxonitrate tetrahydrate for four days (96 hours) with control experiment for each of the fish species where the toxicant was not introduced. Each group was replicated and an unexposed group without the toxicant (Cd) served as the control.the concentrations of cadium trioxonitrate tetrahydrate used were 0.05, 0.1, 0.15, 0.2 and 0.25 mg/l. The fish were not fed during the exposure period that lasted for 96 hours. Mortality of each group was observed and recorded within 96 hours. Fish were considered dead when gill movement ceased and no response upon gentle podding. Dead fish were removed from the test solution and incised for the gill and liver for histological analysis, the incision was also done on the fish in the control group. Histological Examination Preparation of slides The incised tissues from the fish samples were preserved using 10% formalin. The tissues were dehydrated using the ascending grades of alcohol, 70%, 80%, 90%, 95% and 100% between 2 to 4 hours each. Satisfactorily the tissues were cleared in xylene and were imbedded with paraffin wax of melting point between o C and moulds. The tissues were attached to the microtome holder. The blocked tissues were orientated on the holder and were cut after trimming with a different knife. The tissues were floated out by picking with a forceps (curved) seeker and floating it in hot water of 45 o C. The tissues were then attached to slide by dipping the slide into the hot water and picking the floating tissue on the slide. After then the tissues were dried in an incubator overnight at a temperature of 37 o C. Staining the slides with haematoxylin and eosin (H and E) stain The tissues were dewaxed in 2 changes of xylene for 2minutes. For each change the tissues were hydrated in absolute alcohol (ethanol) in the descending grades, 95%, 80%, and 70% for 2-3 minutes each. The section was washed in tap running water for 5mins. The section was stained with Harris Haematoxylin for 5-7mins. The stained section was washed in running tap water again. The section was briefly differentiated in 1% acid alcohol and in blue Scott tap water for 5minitutes. The section was briefly washed in water thereafter. It was then counter stained in 1% aqueous Eosin for 6mininutes and was washed briefly in water and dehydrated as follows; 70% alcohol for 2mininutes 80% alcohol for 3mininutes 90% alcohol for 3mininutes, absolute alcohol

3 120 Int. J. Environ. Sci. Toxic. Res. for 4minutes and absolute xylene for 3mininutes. It was cleared in 2 changes of xylene for 3mininutes each. The section was mounted in DPX and the slides were examined and snapped using photomicroscope. Determination of the physico-chemical characteristics of the water during the study period The physico-chemical characteristics of the water during the study period were examined and recorded using the multi-parameter water checker. The water parameters were; electrical conductivity (EC), total dissolved solute (TDS), ph, dissolved oxygen (DO) and water temperature Data Analysis The test concentrations were converted into logarithm and the corresponding mortality percentage into the probit value. The obtained probit values were plotted against the concentration of the cadmium metal. The physico-chemical results were subjected to student t test analysis and descriptive statistics. RESULTS The result presented in Table 1 is 96 hours acute toxicity test of Tilapia zilli juveniles exposed to different concentrations of cadmium. A perusal of the results showed that there was no mortality in the control group. However, highest mortality of 75% was observed at 0.25 mg/l concentration of the metal while the least mortality of 25% was recorded at the 0.05 mg/l concentration of the metal. The LC 50 concentration was 0.15mg/L of the metal. Similarly Table 2 is the 96 hours acute toxicity test of Oreochromis niloticus exposed to graded concentration of Cadmium metal. The highest mortality of 70% was recorded in the 0.25mg/L and the lowest mortality of 20% was in 0.05mg/L. However no mortality was recorded in the control where the toxicant was not introduced. The result presented in Figure 1 is the regression of probit values and graded concentration of cadmium trioxonitrate tetrahydrate exposed to the juveniles of Tilapia zilli. The obtained results have R 2 value of 0.65 which shows a moderate relationship between the concentrations of the metal and mortality of the fish. This indicates clearly that the metal is toxic to the fish and has the ability to kill at elevated concentrations. The regression probit mortality values and graded concentration of cadmium trioxonitrate tetrahydrate exposed to juveniles of Oreochromis niloticus is presented in Table 2. The R 2 value was 0.65 showing a moderate relationship between the metal and mortality of the fish at increased concentrations, Figure 2. The plate 1(A-F) shows the histopathological analysis of liver of Tilapia zilli. The liver of the control fish has no pathological lesion with hepatocytes (Plate 1A). The most alterations of the liver after cadmium exposure were dilation in hepatic cells and irregular shape of hepatocytes (Plate 1B, C). In addition increased vacuolation, increased blood vesseland disarrangement of hepatic cell, thickening of the hepatocyte, vascular dilation and cytoplasmic vacuolation (Plate 1D, E and F). While Plate 2(A-D) shows the histopathological analysis of gills of Tilapia zilli. The normal histological structure of the gills consisting of primary and secondary lamellae (Plate 2A). The lesions that were observed in the gills treated with cadmium metal were necrotic changes in the epithelium of gill filaments and secondary lamellae, curling of secondary lamellae and complete degeneration of secondary lamellae (Plate 2B, C and D). The results in Plate 3(A-F) shows the histopathological analysis of the liver of Oreochromis niloticus. The liver of control fish showing normal architecture with hepatocytes (Plate 3A). The observed changes of the liver of the fish that were exposed to cadmium are; increased vacuolation, disarrangement of hepatocytes, blood congestion (plate 3B and 3C) and degenerated nuclei (Plate 3D, E and F). While Plate 4 shows the histopathological analysis of gills of Oreochromis niloticus. The normal histological structure of the gills consists of primary and secondary lamellae (Plate 4A). The histological changes that were observed in the gills included shortening of secondary lamellae, curling of secondary lamellae, proliferation in the epithelium of gill filaments and secondary lamellae (Plate 4B, C and D). The result presented in Table 3 is the physico-chemical characteristics of the water samples during exposure of Tilapia zilli juveniles to cadmium metal. Generally all the parameters have their highest values in the highest concentration and lowest values in the control. A similar trend was observed in Table 4 which is the physico-chemical parameters of water samples exposed to juveniles of Oreochromis niloticus. The parameters examined have their highest values in the highest concentration of mg/l and the lowest in the control. All the water quality parameters were within the maximum permissible level. ANOVA was not significant between the graded concentration and the water quality parameters (P 0.05). DISCUSSION The result of this study indicates clearly that higher concentration of cadmium trioxonitrate tetrahydrate is lethal to the fish. This is evident in the relationship between concentrations of the cadmium andmortality with R 2 value of and for Tilapia zilli and Oreochromis niloticus respectively. Aquatic bioassay tests are indispensable in water pollution control to determine whether a potential toxicant is dangerous to

4 Akaahan et al. 121 Table hours acute toxicity test of Tilapia zilli juveniles exposed to cadmium trioxonitrate tetrhydrate concentration s/n Concentration Log of Number of fish Number of %Mortality Probit Value (mg/l) concentration exposed fish died Figure 1. Regression of Probit mortality values and graded concentration of cadmium trioxinitrate Tetrahydrate exposed to juveniles of Tilapia zilli. Table hours acute toxicity test of Oreochromis niloticus juveniles exposed to cadmium trioxonitrate tetrahydrate concentration s/n Concentration Log of Number of fish Number %Mortality Probit Value (mg/l) concentration exposed of fish died Figure 2. Regression of Probit mortality values and graded concentration of cadmium trioxinitrate Tetrahydrate exposed to juveniles of Oreochromis niloticus.

5 122 Int. J. Environ. Sci. Toxic. Res. Plate 1A. The liver of control fish showing normal architecture with hepatocytes. Plate 1B. Dilation in hepatic cells. Plate 1C. Irregular shape of hepatocytes Plate 1D. Vacuolation of hepatocytes. Plate 1E. Disarrangement of hepatic cells and thickening of hepatocytes Plate 1F. Vascular dilation and cytoplasmic vacuolation. Plate 2A. Normal histological structure of gills consists of primary and secondary lamellae. Plate 2B. Necrotic changes in the epithelium of the gill filaments and secondary lamellae.

6 Akaahan et al. 123 Plate 2C. Curling of the secondary lamellae. Plate 2D. Complete degeneration of the secondary lamellae Plate 3A. The liver of control fish showing normal architecture with hepatocytes Plate 3B. Disarrangement of hepatocytes Plate 3C. Vacuolation of hepatic cells and blood congestion Plate 3D. Increased vacuolation of hepatic cells Plate 3E. Increased vacuolation and disarrangement of hepatic cells. Plate 3F. Degenerated nuclei and blood congestion (BC).

7 124 Int. J. Environ. Sci. Toxic. Res. Plate 4A. Normal gill tissue with primary and secondary lamellae Plate 4B. Shortening of secondary lamellae. Plate 4C. Curling and shortening of secondary lamellae Plate 4D. Proliferation in the epithelium of gill filaments and secondary lamellae. Table 3. Physico-chemical characteristics of the water samples during exposure of Tilapia zilli juveniles to cadmium trioxonitrate tetrahydrate. Conc. mg/l Control Temp. ( O C) ph TDS (mg/l) DO (mg/l) EC (µs/cm) Table 4. Physico-chemical characteristics of the water samples during exposure of Oreochromis niloticus juveniles to cadmium trioxonitrate tetrahydrate. Conc. g/l Control Temp. ( O C) ph TDS (ppm) DO (mg/l) EC (µs/cm)

8 Akaahan et al. 125 aquatic life and if so, to find the relationship between the concentration of the metal and percentage mortality. During the course of this study it was observed the LC 50 of cadmium exposed to Tilapia zilli juveniles for 96 hours was 0.15mg/L. This result differs significantly from the result of an earlier study that reported LC 50 of cadmium exposed to Oreochromis sp to be 0.7mg/L for 96 hours (Aldoghachi et al., 2016). Similarly Raj et al. (2013) reported LC 50 concentration of cadmium of 6.08mg/L exposed to L. calrifer exposed to the metal for 96h period which is at variance to the result of this study. The effects of cadmium on the fish observed during this study showed that caution should be exercised in allowing cadmium into the aquatic environment. These effects included loss of balance, skin bleaching, and weakness of the fish thick layer of mucous on the skin covering the dead fish and air bubbles on the water. There was reduced activity evidenced by vertical positioning and less mobility of the fish in solution with concentration of cadmium (0.05, 0.10, 0.15, 0.20 and 0.25g/L) as was observed during the course of this study. The liver is the target organ altered by any foreign molecule through partial circulation and is subjected to damage. The liver is a vital organ of detoxification which break down toxic substances and metabolites of administered substances. This break down is done by endoplasmic reticulum of heptatocytes. In this study the effect of the cadmium concentration on the histology of the liver showed clearly the damages done to the heptatocytes with complete vaculation. These observations are similar to the findings of earlier studies of cadmium exposed to the fish (Babu, 2013; Salvanathan et al., 2013). Dyk et al. (2007) reported that Oreochromis mossambicus exposed to cadmium showed liver alternation in the form of hepatocyte, vacuolation, cellular swelling and congestion of blood vessels. Similar observations were made in the liver of Tilapia zilli and Oreochromis niloticus of this study exposed to the graded concentration of cadmium metal for 96 hours. Similarly Omeret al. (2012) reported histopathological alternation in the liver of tilapia fish Orechromis niloticus exposed to cadmium metal. This finding is consistent with the result of this present investigation. The results of the histopathological analysis of the liver of Tilapia zilli and Oreochromis niloticus shows, vacuolation of hepatocytes, dilation in hepatic cells, disarrangement and thickening of hepatic cells, blood congestion and degenerated nuclei. The liver showed degeneration of the hepatocytes and blood congestion in the hepatic cells. These findings were apparent, as the liver is considered as the organ of detoxification, excretion and binding proteins such as metallothionein (MTs). The proteins bind with the metal that is present in the nuclei of the hepatocytes cause increase in cell damage (De-Smet and Blust, 2001). The findings of this study conform to the result of previous study that have investigated the effects of different pollutants on fish liver (Abdel-Warith et al., 2011). Olojo et al. (2005) also observed the degeneration of the hepatocytes and focal necrosis in the liver of Clarias gariepinus exposed to lead which is also a toxic heavy metal. Babu (2013) reported that cadmium has induced marked pathological changes in the liver tissue and gills of the fish exposed to the cadmium metal as was the scenario in this study. The gills of the fish are highly specialized organ with several important functions. The gills of the fish are vital for the studies of the effect of toxicant because toxic substances enter the body of the fish through the gills. Studies have reported marked histopathological changes in the gills of the fish exposed to cadmium as was observed in the course of this investigation (Rani 1999., Babu 2013). There are reports on various histopathological changes in gills of fish found in polluted water such as, lamellar fusion, epithelial lesions, rupture of epithelia cells, curling and shortening of secondary lamellar (Camrgo et al., 2009). These findings are similar to the result of the present study. Cellular proliferation in the gill epithelium was observed in the two species of fish exposed to cadmium metal as described by Thophon et al. (2003). These findings are consistent of the result of this study. The gills of the both studied fish showed degenerative and proliferative changes in gill filaments and secondary lamellae. 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