Toxicity to Hematology and Morphology of Liver Brain and Gills during Acute Exposure of Mahseer (Tor putitora) to Cypermethrin.
The present study was aimed to evaluate the effects of acute dose of Cypermethrin (CYP) an extensively use synthetic pyrethroid on hematology and morphology of the liver gills and brain of Mahseer (Tor putitora). The effects were assessed on the basis of the previous results of acute toxicity test after exposing fish to acute concentration 63 g L-1 (LC50 of 96 h) of CYP. Light microscopic studies revealed severe histopathological changes in liver gills and brain tissues. The morphological alterations in liver involved glycogen vacuolation hemorrhage vacuolation congestion fatty infiltration and hepatic necrosis. In gills it resulted in cellular infiltration congestion swollen tip of the gill filament hetrophilic infiltration and damaged gill while in the brain it caused discoloration neuronal degeneration infiltration and severe spongiosis. Blood cell count also showed the toxic effect of CYP as RBCs count decreased while WBCs count increased with time in the treated group.
The results clearly classify CYP as a strong toxic agent for T. putitora.
Keywords: Cypermethrin; Acute toxicity; Tor putitora; Hematology; Histopathology
The rapid advancement of industrialization and green revolution has led to a number of environmental problems aquatic pollution being the most prominent. In Pakistan effluents from industries wastes from household activities and agricultural runoffs directly discharge into streams ponds and other aquatic bodies. These pollutants contain infectious pathogens oil hydrocarbon radioactive substances heavy metals pesticides herbicides and different corrosive substances such as acids and bases (Samantha et al. 2005). Yet these sources are used for supplying water to the local masses and culturing of economically important and luscious fish species (Stanitski et al. 2003).
Pesticides are among the major contributor of aquatic pollution. According to Latif et al. (2013) there are more than 200 types of organic pesticides being used in thousands of different products. These pesticides contain a number of heavy metals such as iron chromium cadmium nickel copper lead zinc and manganese etc. These elements ultimately reach the water bodies and adversely affect the growth reproduction physiology and even survival of the non target aquatic organisms including fish (Hayat et al. 2007).
Liver gills and brain are the most important organs of all vertebrates as these controls and maintain the most important life activities such as metabolism detoxification excretion and respiration etc. The chemical pollutants including heavy metals and pesticides adversely affected the morphology and functions of these organs and disturb the normal physiology of all animals (Atamanalp et al. 2008; Velmurugan et al. 2009a b; Ali et al. 2014).
In ecotoxicological studies various biomarkers are used for evaluating stress responses. Recently fish histopathology is gaining importance for rapid assessment of the toxic effects of pollutants in the laboratory. Histopathology can be utilized for the determination of the effect of stressor at biological organization level. According to Latif et al. (2013) studying histopathology is an important way to evaluate the effects of pollutants on fish; therefore it is used as a rapid tool for examining the effect of the pollutant in different organs and even tissue of the body.
The hematological parameters also reflect the animal response towards its environment and are useful indicators in assessing the toxic effects of chemicals in aquatic organisms such as fish (Gabriel et al. 2007; Ghaffar et al. 2014). Moreover surrounding environment where fish performing its activities put forth some impact on their hematological characteristics. Therefore hematological parameters are also helpful in examining the response of fish against stressors (Gabriel et al. 2007). These parameters have standardized reference values which can be increased or decreased due to stressor like pesticide and other pollutants and helpful in diagnosing the problem. Fish are directly linked to the aquatic environment therefore their hematological profile can provide information about their internal body conditions earlier than any noticeable disease indication (Fernandes and Mazon 2003).
Among many types of pesticides pyrethroid accounts 25% sales of pesticides in the world (Zhang et al. 2011). They are common in practice due to their low toxicity to avian and mammalian species but it appears that they are highly toxic to bees aquatic insects and fish (Aydin et al.2005). The difference in the rate of metabolic degradation and elimination from the body may be a major factor for the variations in the toxicity of pyrethroid between fish birds and mammals.
Cypermethrin is an important synthetic pyrethroid that has much higher biological activity and stability than its natural homolog pyrethrum (Khan et al. 2006). It is widely used throughout the world for controlling different types of insect pests of cotton fruits and vegetables (Khan et al. 2006) copepode parasite infestation (Athanassopoulou et al. 2001) aquatic and terrestrial ectoparasites (Treasurer and Wadsworth 2004) and for illegal fishing (Khan et al. 2006). It enters the water bodies mostly through agricultural run offs and affects the non target aquatic organisms such as fish (Werimo et al. 2009; Arjmandi et al. 2010).
Keeping in view the current scenario of polluted aquatic environments the present study was designed to investigate the adverse effects of cypermethrin on the hematology and histology of liver brains and gills of Tor putitora economically an important freshwater species of fish commonly used as a stapled food item in Pakistan.
Materials and Methods
About 250 healthy and uniform size seeds of Mahseer (Tor putitora) average body weight 3.23 0.34 g were purchased from Hattian Nursery Unit Attock Pakistan. These seeds were transported to Fisheries and Aquaculture laboratory Department of Animal Sciences Quaid-i-Azam University by closed system live hauling method. The fish were acclimatized for two weeks before the start of the experiment. During acclimatization water was changed on a daily basis and fish were offered 40% protein diet at 5% body weight.
The water quality parameters like temperature dissolved oxygen and pH were checked regularly on a daily basis and were ensured to remain in the optimum range. To avoid water foiling dead fish were removed as soon as possible. During acclimatization ammonia was less than 0.25 ppm while temperature pH and dissolved oxygen were 22C 8.44 5.02 mg L-1 respectively.
Preparation of CYP Solution
A stock solution of pesticide was prepared by dissolving 5 mg of technical grade CYP [Cyano (3-phenoxyphenyl) methyl 3-(2 2-dichloroethenyl)-2 2-dimethylcyclopropane- carboxylate] in 5 mL of 80% acetone. Then 1 mL solution was taken in 10 mL volumetric flask with the help of micropipette and 80% acetone was added up to mark to make concentration 1 mg CYP per 10 mL. The flask was shaken well to get homogenous solution. Then further dilutions were made from that stock solution. Based on previous studies on juvenile Mahseer (Atika 2012) of similar sized LC50 for 96 h 63 g L-1 was selected for acute study tests.
Experimental Detail and Treatment
The experiment was conducted in semi-static closed system. Healthy and uniform sized fish regardless of sex were selected and evenly distributed in six glass aquaria (60 A- 30A- 30 cm) at a stocking density of 1.5 kg m-3. The experiment was conducted in triplicate. The first three aquaria served as a control group while other three as treated group. All aquaria were fitted with air stones and heaters for constant temperature and dissolved oxygen. After 96 h of acclimatization fish in the treatment group were exposed to acute concentration 63 g L-1 (LC50 of 96 h) of CYP while the control group received 80% acetone equal to the volume used for exposing CYP in the treatment group. The water of each aquarium changed after every 24 h and concentrations of CYP was restored afresh.
The experiment was conducted for 4 days. After every 24 h up to 4 days one fish from every aquarium in the control and treated group was captured with a hand net and sacrificed. Their liver gills and brain tissues were removed by decapitation and placed in sera (absolute alcohol formaldehyde and glacial acetic acid in 6:3:1 ratio) for the further histology process.
Further 3 fish from each aquarium were captured and anesthetized with MS222 (70 mg L-1) and bled by caudal vein puncture and blood was collected for RBCs and WBCs counting.
Rosety et al. (2005) method was adopted for the preservation of tissues and preparation of slides for histopathological assessment. After staining with hematoxylin and eosin the slides were mounted with Canada balsam. Cover slips were placed on these and were kept in an incubator overnight. The extra Canada balsam was removed with xylene. The prepared slides of liver gills and brain tissues from both control and treated groups were studied under OPTIKA B-350 Microscope. Photography was done by using AIPTEK digital camera.
Blood cell counting: Blood was diluted by using commercially available diluting solutions (dilution for RBC 1:200; and for WBC 1:20) and blood cells were counted with the help of Neubauer hemocytometer.
RBCs and WBCs counting: Under light microscope counting chamber was adjusted and observed the smallest squares in the large center square where red cells lies. The chamber was examined at 10 X magnifications for the evenness of the red blood cell distribution. The 40X objective was turned in place; focused and the cells were counted in the designated
For WBCs counting 400 L of WBCs solution was taken in tube and a drop of blood was added to the WBCs solution. WBCs were counted with the help of hemocytometer. The cell counting was done under light microscope at 100 X magnification and only those cells touching the upper and left-hand boundary lines of the main squares were counted. For WBCs count cells in each square chambers were counted and then average per chamber was calculated. By using this average number WBCs per cubic mm was calculated.
Histological Changes in Body Parts
Hematological and Histopathological changes were observed in the gill liver and brain of juvenile T. putitora when exposed to an acute concentration of CYP for 96 h. The liver of fish in the control group revealed normal appearance having hepatocyte of polygonal shape central nuclei and granulated cytoplasm. Exposure of CYP for 96 h caused glycogen vacuolation congestion hemorrhage vacuolation fatty infiltration and hepatic necrosis (Fig. 1). No change was observed in the fish gills from the control group while the CYP exposure severaly damaged the gills by causing cellular infiltration congestion swollen tip of the gill filament hererophilic infiltration (Fig. 2). The histopathological examination of the fish brain from control group showed normal morphological structure while in CYP treated group discoloration neuronal degeneration mononuclear infiltration and severe spongiosis of the brain were obvious (Fig. 3).
In a control group of fish no significant changed in the number of RBCs during 24 48 72 and 96 h were observed. The RBCs value showed decreasing trend with time in CYP exposed group of fish (Table 1) therefore significantly (P less than 0.001) higher value was observed at 24 h and lower value was observed at 96 h. The WBCs count of juvenile fish significantly increased after exposure to an acute concentration of CYP (Table 1). The values in the control group of fish at 24 48 72 and 96 h were fluctuating between 10.54 and 11.86 A- 103 m-3. After exposure to CYP the WBCs count was significantly increased and shown an increasing trend with time and maximum value (36.11 1.78 A- 103 m-3) was observed at 96 h.
Table 1: Variations in RBCs and WBCs count of juvenile Tor putitora at different time period after exposure to an acute concentration of CYP
Time (h)###RBCs (106 m-3)###WBCs (103 m-3)
24###1.710.07a###1.260.10bc###11.29 4.8d###20.73 1.77c
48###1.650.16a###1.210.12cd###11.86 0.47d 27.33 1.84bc
72###1.760.16a###0.900.06cd###10.55 1.12d 33.893.27ab
96###1.590.18###0.830.12###10.63 0.76d 36.111.78a
In ecotoxicological studies histopathology is gaining importance for rapid evaluation of the toxic effect of pollutant and considered as an important tool for examining the effect in different organs and even tissue of the body (Latif et al. 2013). Many investigators reported lesions in different organs of fish in response to various chemical contaminants like heavy metals and pesticide (Omitoyin et al. 2006; Ayoola and Ajani 2008; Velmurugan et al. 2009a b). In the present study the histopathological examination of the brain gill and liver tissues of juvenile mahseer (Tor putitora) in response to CYP revealed that the liver and gills were the organs most affected compared to other organs.
In vertebrate including fish liver is the main organ that play important role in detoxification of pesticides. During metabolism liver has the ability to break down these harmful substances but beyond a certain limit these toxic compounds disturb the regulating mechanism of the liver and cause morphological alteration (Brusle et al. 1996). The histopathological changes observed in the present study were glycogen vacuolation hemorrhage fatty infiltration hepatic necrosis and congestion (Fig. 1). The glycogen vacuolization and fatty infiltration in the liver indicate the accumulation of fat and imbalance between rate of synthesis and release of substance in hepatocytes (Gingerich1982) whereas necrosis in some part of liver may appeared due to extra work load on hepatocyte during detoxification of CYP (Patel and Bahadur 2011). Many investigators reported the similar or different level of morphological changes in the liver in response to CYP in various fish species (Clarias gariepinus
Velmurugan et al 2009a; Labeo rohita Sarkar et al. 2005; Heteropneustes fossilis Joshi et al. 2007). The inconsistency in results may be due the fact that degree of lesion depends upon on the concentration and duration of exposure of pesticide (De Oliveira et al. 2002). Nevertheless many insecticides cause specific or non- specific histopathological damage. For example histopathological lesions in the liver tissue of freshwater fish (Cirrhinus mrigala) (Velmurugan et al. 2009b) and common carp (Cyprinus carpio) (Banaee et al. 2011) were observed after 10 and 30 days exposure to sublethal concentrations of dichlorvos and diazinon insecticides respectively while other researchers reported the same histopathological alterations in different tissues of fish treated with diazinon (Banaee et al. 2011) deltamethrin (Cengiz 2006) fenitrothion (Benli and Ozkul 2010).
It is well established that in fish gills are the main organs through which water enter into the body along with pesticides. According to Rankin et al. (1982) gills are a good indicator for studying the water quality and used as a model for the evaluation of environmental impact. Pesticides first affect the gills and once inside the body may damage various other organs of fish. When the gills are damaged the hypoxic condition occurs due to alteration in gas exchange mechanisms (Das and Mukherjee 2003). In the present study no significant changes were observed in the gill tissues of the control group of fish while fish exposed to CYP for 96 h showed cellular infiltration congestion swollen tip of the gill filament hererophilic infiltration and gill damaged. Cypermethrin cause necrosis hyperplasia of primary epithelial cells oedema epithelial hypertrophy epithelial lifting fusion of secondary lamellae and desquamation in the gills of African catfish Clarias gariepinus (Velmurugan et al. 2009a)
while Zeta CYP caused morphological changes like hyperplasia lifting of the epithelial layer from gill lamellae shortening of secondary lamellae exudation and necrosis in the gills of
Lebistes reticulates (Caliskan et al. 2003). Moreover in grass carp Ctenopharyngodon idella fenvalerate caused bulging of primary gill lamellae tips atrophy and complete fusion of secondary gill lamella club shaped secondary gill lamellae and severe necrotic changes in the epithelial cells (Tilak et al. 2001).
In the present study the light microscopy of the brain of control and pesticide exposed group of fish showed that beside liver and gills CYP also affected the brain structure and caused discoloration neuronal degeneration mononuclear infiltration and severe spongiosis. All these symptoms are the clear indication of brain damage. This agreed with the observations of other scientist who reported generalized spongiosis and severe congestion in brain of African catfish Clarias gariepinus in response to an acute concentration of CYP and Gramoxone (Omitoyin et al. 2006; Ayoola and Ajani 2008). All the histopathological observations in the liver gill and brain tissues of T. putitora after acute exposure of CYP indicated the destructive effect of this pesticide. These histopathological modifications could result in severe physiological problems those eventually end to the death of fish.
Fish as an aquatic vertebrate is in direct contact with the aquatic environment that could put forth some impact on the hematological characteristics (Gabriel et al. 2007). Therefore the hematological profile of blood can provide the information about the body internal condition of an animal earlier than any noticeable indication of disease. Many investigators studied the effects of toxicants on the hematology of different fish species and reported various degrees of hematological changes and suggested that reduction in hemoglobin RBC and PCV are related to oxygen carrying capacity of the blood (Adhikari et al. 2004; Gabriel et al. 2007).
In this study the RBCs count of T. putitora showed significant decreased after exposure to acute concentration of CYP while no significant difference at different periods was observed in control group of fish (Table 1). The RBCs count in the control group of fish at different time period ranged 1.59- 1.76 x 106 m-3. The values lies within the range observed in various other species (Maheswaran et al. 2008; Vasantharaja et al. 2012) but somewhat lower than 2.940.4 x 106 m-3 observed in Labeo rohita (Adhikari et al. 2004). After exposure to CYP RBCs count showed decreasing trend and lowest value (0.83 A- 106 m-3) was observed at 96 h. It was suggested that the reduction in the RBCs counts during treatment may be due to the development of hypoxia that lead to either increase in destruction of RBS or decrease in the genesis of RBCs due to non-availability of Hb content in cellular medium (Akinrotimi et al. 2012; Vasantharaja et al. 2012).
In vertebrates including fish WBCs are related to the defense mechanism and consist of lymphocytes thrombocytes monocytes and granulocytes. Monocytes and granulocytes play function in the removal of injured cell debris while lymphocytes related to the production of antibodies (Wedemeyer and Mcleay 1981). It was observed that the counted value of WBCs of juvenile mahseer significantly increased after exposure to acute concentration of CYP (Table 1). In treated group WBCs count showed positive relation with time and reached to maximum (36.11A-103 m-3) level at 96 h. Adhikari et al. (2004) also observed a significant increased in number of WBCs in L. rohita due to CYP and carbofuran treatment. Like our results other scientists also observed increased in WBCs counts in response to similar pesticide (C. mrigala Vasantharaja et al. 2012) and other pollutants like methyl mercury (Hoplias malabaricus Ribeiro et al. 2006).
The increase in number of WBCs in the present study or others reports in response to different type of toxicants may be related to stimulation of immune system due to tissue damage or may be related to compensatory response of lymphoid tissues to circulating lymphocytes (Shah and Altindag 2004).
The results of this study clearly indicated that CYP at acute concentration is toxic to juvenile mahseer Tor putitora. Hence restrictions on the indiscriminate use of pesticide can play a role in decreasing the wild population of fish in natural water bodies.
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|Publication:||International Journal of Agriculture and Biology|
|Date:||Feb 28, 2015|
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