INTRODUCTION

Endosulfan is one of the more toxic pesticides on the market today, responsible for many fatal pesticide poi- soning incidents around the world (Ahmad et al., 2000;

ARTICLE INFORMATION:

*Corresponding Author Received 10th March, 2015 Accepted after revision 30th June, 2015 BBRC Print ISSN: 0974-6455

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Akanji et al., 1993). Finally, this endosulfan makes their way to wetland and swamps of adjoining area. Both the beels and the swamps are ecologically and economically very important features (Arnold et al., 1996). These com- prise a major component of the area’s ecology. The beels

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are traditionally used as natural fisheries. Even today, the beels produce more fish per unit area than any many other man made fisheries. A large number of beels are connected with the rivers by one or more feeder chan- nels. These feeder channels are lifeline of such water bodies, which are getting polluted by pesticides, (Bisht and Agarwal, 2007 and Kaviraj & Gupta, 2014).

In addition to the direct impact of pesticides on aquatic life, bioaccumulation of contaminants through food chain in organisms is another important factor to be considered. Extensive investigations have been car- ried out all over the world including different parts of India for the effects of pesticides and metals on both ter- restrial and aquatic organisms, (Gill et al., 1991, Cripe, 1994; Dey & Gupta, 2002, Choudhary et al., 2003, Dae- sik et al., 2004, Cengiz, 2006; Boran et al., 2007; Choud- hary et al., 2008, Ali and Naaz, 2013 and Ali, 2014 ).

Lactic dehydrogenase (LDH) is an enzyme of interest in recent study of the glycolytic pathway. It catalyzes the interconversion of pyruvate and lactate with con- comitant interconversion of NADH and NAD+. It con- verts pyruvate, the final product of glycolysis to lactate when oxygen is absent or in short supply. LDH activity is present in all cells of the body and is invariably found only in the cytoplasm of the cell. Elevations of LDH may be measured either as a total LDH or as LDH isoenzymes. A total LDH level is an overall measurement of five dif- ferent LDH isoenzymes. isoenzymes are slightly differ- ent molecular versions of the LDH enzyme. A total LDH level will reflect the presence of tissue damage. LDH is released into the bloodstream when cells are damaged or destroyed. Because of this, the LDH test can be used as a general marker of injury to cells (Hiremath and Kaliwal, 2002 and Hernandez et al., 2006).

Endosulfan causes increase in Glucose 6 phosphate dehydrogenase (G6PDH) activity, blood glucose levels and phospholipid contents of the microsomal and sur- factant system and profoundly induce the activity of the alcohol dehydrogenase and cytosolic glutathione- S-transferases. It also decreases significantly Na-K and Mg-ATPases, plasma calcium level and alkaline phos- phatase in three intestinal epithelium (Holem et al., 2006, Johal et al., 2007).

A number of studies have documented that endosul- fan acts as an anti-androgen in animals (Joshi et al., 2007). It is also a xenoestrogen—a synthetic substance that imitates or enhances the effect of estrogens (Kala- vathy et al., 2001, Kumar et al., 2007) and it can act as an endocrine disruptor causing reproductive and developmental damage in both animals and humans. A 2009 assessment concluded that endocrine disruption occurs only at endosulfan doses that cause neurotoxicity (Malomo, 2000). Therefore, the recent studies have been carried out to explore the toxic effects of endosulfan

Akalesh K. Verma and Jashodeb Arjen

on locally available air breathing teleost fish, Channa gachna (Hamilton) in relation to lactic dehydrogenase, which is not well documented.

MATERIALS AND METHODS

Endosulfan was purchased from Meerut Agro Chemicals Industries Ltd., Meerut, India, Reduced nicotinamide adenine dinucleotide (βNADH), reduced nicotinamide- hypoxantine dinucleotide (β-dNADH), bovine serum albumin (BSA), nitrobluetetrazolium (NBT) and phena- zin emethosul fate (PMS), nicotinamide adenine dinucle- otide (β NAD) were obtained from the Sigma Chemical. All the other chemicals were analytical grade products of Merck (Darmstadt, Germany).

The bulk sample of the freshwater fish, Channa gachita (Hamilton) (ranging in weight 40gm to 45gm) was pro- cured from their natural habitat and transported to the laboratory in well aerated polythene bag and acclimated to the ambient laboratory temperature 26 ± 0.4 and pH

7.2in large glass aquarium. During the period of accli- mation, they were fed every day with mosquitos’ larva and artificial food diets. The period of acclimation lasted for 2 weeks. After acclimation healthy fish were selected from stock and transferred to another glass tank. Fur- ther the fishes were divided into different groups each containing 10 numbers of fishes. The control and treated fishes were collected from non endosulfan treated area (supply from Nagaon, India) whereas, experimental group fishes were collected from adjoining wetland and swamp area of tea garden( Karbi Anglong, India) where endosulfan is most extensively used in tea garden for insect pest control.

For determining LC50 values of the Endosulfan on Channa gachua, 10 fishes in each group were placed in a container of 15 liters capacity with single dose of 2.64μ1/lendosulfan. No food was given during the 4-day exposure. Mortality was recorded at 24, 48, 72 and 96 hrs. Dead organisms were removed and test solutions

was renewed every 24 hr. Finally LC50 was calculated by Percentage death Curve, experiment was tested in 5 replicates.

During bioassay the morality in control groups was adjusted according to the method of Ludke et al., (1993). Endosulfan was dissolved in acetone and a fresh medium of endosulfan was prepared by changing water on alter- nate day. The control group fishes were exposed to water containing 0.01% acetone only. Pilot experiment did not show any significant effect of 0.01% acetone exposure on biochemical changes in the fish. Whereas, treatment group was treated with sublethal concentration of endo-

sulfan (1.32μl/l, half of LC50 of 96hrs) for 21 days and experimental group was without any treatment, col- lected from endosulfan affected area (tea garden and

Akalesh K. Verma and Jashodeb Arjen

agriculture field). Tissues and blood were collected on 4th, 7th, 14th and 21st day for biochemical and histopatho- logical studies. The groups of exposure phase were as given below. Group I. Control exposed to 0.01% acetone only. Group II. Treated (1.32 μl/l), exposed to endosulfan for 21 days.Group III. Experimental, without any treat- ment and collected from endosulfan treated area.

The activity of LDH (L-lactate: NAD+ oxidoreduct- ase) was ascertained following the method of Schmidt et al., (1965) Lactate dehydrogenase catalyses the revers- ible reduction of pyruvate to lactate with the quantitative oxidation of NADH. The assay mixture without the sample was pre incubated for 5 minutes at 37°C in a 1ml quartz cuvette having lcm light path directly in an UV visible spectrophotometer (Beckman, Model DU 640), having a temperature regulator. In order to start the reaction, the sample was added to the preincubated reaction mixture.

The decrease in the absorbance was recorded at 340nm at an interval of 10 seconds, and the duration of linear decrease in the O.D. value was noted for calculating the activity of LDH. The molar extinction coefficient value of NADH at 340nm is 6.22 ×106. The enzyme activity of LDH (1 unit) is expressed as the amount of enzyme which catalyses the oxidation of 1 mole of NADH to NAD+ per minute at 37°C. The specific activity of the enzyme was calculated for hemolysate as U/gm Hb and for tissue homogenate as U/mg protein.

LDH isozymes pattern was studied in liver and serum only. For thisstudy 10% homogenate prepared in PBS in case of tissues, 6 fold dilution in case of serum and then centrifuged at 8,000g for 20mins (4°C). 50 μl of the super- natant was loaded on the polymerized gel. After loading the sample, the electrode buffer was filled gently in the upper and lower buffer chamber. Electrophoresis was run at a constant current of 50mA at 4° C (34 hrs.). The gel tubes were flushed out with the help of a syringe with dis- tilled water and stained for LDH in LDH staining solution for 15 mints at 37°C (Violet coloured LDH bands).

The gels were rinsed once in fixative (7% acetic acid). And then stored in the fixative and photographed. For histopathological study liver tissue was collected on 21st day of treatment and processed routinely. The liver tis-

sue was fixed in 10% formaldehyde. They were embed- ded in paraffin. 10 μm sections were obtained, stained with Harris hematoxylene-eosin and examined under light microscope. Results were Mean ± SE. Significant differences between control and treated groups were cal- culated using Student’s t-test. Number of replicates (N) = 5, p-values of ≤ 0.05 were considered significant.

RESULTS AND DISCUSSION

Mean LC50 values of endosulfan for C. gachua is depicted in Table 1, which reveals that the LC50 value at 96 hrs is

2.64μl/1. Accordingly, the sublethal concentration for endosulfan was selected as 1.32 μl/1, which is half of the LC50 of endosulfan.

LDH activity in the tissues and blood of control, treated and experimemal groups: Endosulfan treatment resulted in a progressive increase in the enzyme activity in the serum following the first day of treatment and showed a maximum increase of ~83% on day 14’1’ and- decrease (~8%) in the later treatment period (21st days). An initial increase in LDH activity (~24%) was noticed in the liver, after 4th days of treatment, maximum increase in activity (~81%) was observe on days 21st. In the kid- ney an increase in activity (~8%) was noted after 4th day of treatment and maximum activity (~89%) is found on 21st days.

Muscle shows continuous increase in activity with the increase of treatment duration the maximum activ- ity (~89%) is found on days 21st. The LDH activity in the gills was found to increase and on the 4th day, the activity was almost similar to the control whereas, the maximum LDF activity (~53%) was achieved on 21” days. In case of experimental group the LDH activity is found to increase significantly in all tissue including the serum as compared to control LDH isozymes pat- terns: The analysis of LDH isozymes patterns promi- nently revealed the presence of all the five isozymes forms (i.e. LDH- 1, LDH-2, LDH-3, LDH-4 and LDH- 5) in the liver and serum of all groups (Fig. 1). In the liver of control group (Fig. la) all the isozymes form in

different lanes are showing similar intensity hence show- ing almost similar activity on 4th, 7th, 14th and 21st days. Whereas, in the treatment group (Fig. lb) of liver tis- sue the intensity of all the isozymes increases reflecting increase in activity, the maximum intensity is observed in lane 4, the LDH 5 in lane land 3 were found to be least expressed. In case of experimental group (Fig. lc) band intensity of different isozymes form is slightly increases in comparison to control. In the blood serum of con- trol group (Fig. 1d) all the isozymes form in lane 1, 2, and 3 showingsimilar pattern but in lane 4 the intensity increases. The treated group (Fig. 1e) showed signifi- cant increased in band intensity in all lanes, very high intensity is observed in lane 2. In experimental group

(Fig. 1f) all LDH isozymes in lane I showing less inten- sity but are more prominently expressed in all other lane hence reflecting higher activity.

Histopathological examination: Histopathological examination of liver tissues of Control showed normal tissue architecture (Fig. 2A). Whereas, treated group (Fig. 2C) showed swelling of the hepatocytes in places with areas of diffuse necrosis and chronic toxic hepati- tis in liver. There was portal mononuclear inflammatory infiltration and some eosinophyl leucocytes and lobu- lary inflammation (liver cell necrosis). Histopathologi- cal examination of liver tissues of experimental group (Fig. 2B) showed the normal architecture of liver tissue was markedly disrupted. Sinusoids in most cases were

distended and central veins appeared severely dam- aged due to marked swelling and degeneration of the endothelial lining cells. It also showed some regenera- tive findings with mild hepatitis, nuclear hyperchromasy and minimal microvesiculary fatty degeneration

Fig A Photomicrograph of rat liver administered with 0.01% acetone (control), Showing normal hepatic plate and central vein. (100X)

Fig B Photomicrograph of rat liver treated with endo- sulfan at the dose of 1.32μl/l (treated), showing swelling of the hepatocytes in places with areas of diffuse necrosis and chronic toxic hepatitis in liver. (200X)

Fig C Photomicrograph of rat liver without any treat- ment, collected from adjoining area of endo- sulfan treated field (experimental) the normal architecture of liver tissue was markedly dis- rupted. Sinusoids in most cases were distended and central veins appeared severely damaged due to marked swelling and degeneration of the endothelial lining cells. (200X)

The measurement of the activities of ‘marker’ enzymes in tissues and body fluids can be used in assessing the degree of assault and the toxicity of a chemical com- pound on organ/tissues (Nelson & Cox, 2005; Pandey et al., 2009; Rahman & Siddiqui, 2005). In the recent study the result showed that the LDH activity is sig- nificantly higher in treated and experimental groups in comparison to control.

In the present study, the highest LDH activity has been found in kidney and lowest in blood serum in all groups. The LDH isozymes pattern of liver and blood

serum of treated and experimental group indicates the up regulation of LDH enzyme. This happened because under the condition of oxidative stress, there is a meta- bolic change in the tissue leading to increased activity of LDH, since under anaerobic conditions LDH catalyzes the reaction of transforming pyruvic acid into lactic acid and increased concentration of lactates. This indi- cates the loss of intracellular LDH and its release into the medium is an indicator of irreversible cell death due to cell membrane damage (Ramaneswari & Rao, 2008).

The significant loss of lactate dehydrogenase (LDH), an enzyme associated with the cytosolis quite under- standable since it is in close proximity to the plasmam- embrane (Schmidt et al., 1965). Slight damage to the plasma membrane will easily lead to leakage of LDH from the cell interior to the extracellular environment (Shanmugam et al., 2000). However, the increase in serum LDH activity (Table 2) in treated and experimental groups support the reasoning ofleakage of the cytosohc enzyme from the tissues into the serum due to labialized plasma membrane.

Histological examination of tissues could serve as complementary evidence (Shelby et al., 1996) to enzyme studies towards revealing any distortion/damage to the normal structure of the tissue cells (Singh & Srivastava., 1992, Singh & Singh, 2007; Tripathi & Verma, 2004). In this study, we have observed the toxic effects of endo- sulfan exposure on the liver tissues. As shown in Figure 2A, 2B and 2C, the livers of fig.2B and 2C suffered his- topathological damage.

All abnormal features displayed by the liver histol- ogy both in treated and experimental group following administration of Endosulfan may be due to damage to the hepatocyte by the endosulfan (Varayoud et al., 2008;

Velmurugan et al., 2007). This is an indication of cir- rhosis which usually disrupts the normal flow of blood through the liver; 7 . The liver histology of experimen- tal fishes also shows similar pattern with treated group this indicates that the experimental fishes collected from adjoining areas of endosulfan treated field is also affected by endosulfan (Venkatramana et al., 2006). Our results suggested that exposure of fishes to endosulfan caused liver tissue damage revealed by increased levels of liver LDH.

It is also established that Endosulfan is metabolized in the liver through cytochrome P450 system to hepatotoxic intermediates. The highly reactive free radicals generated during the course of the reaction cause damage to the organism (Wilson & LeBlanc, 1998). According to Gill et al. (2007) HSI of fresh water fish, Barbus conchonius was moderately increased after 2, 3 and 4 weeks of expo- sure to 6.72 ppb of organochlorine insecticide endosulfan. Chlorinated hydrocarbon insecticides (DDT, endosulfan, etc.) cause liver damage that ranges from increased liver weights and fat content to cell necrosis (Yakubu et al., 2003, and Kaviraj and Gupta, 2014).

However, some enzyme activity changes are related to hepatic alterations induced by pesticides, including induction of serum amino transferases, lactic dehydro- genase and alkaline phosphatase. Damage to liver cells also leads to changes in the organelles (Kurutsei et al., 2006). These morphological changes disturb various bio- chemical reactions which then can be measured in the serum or RBC.

CONCLUSION

Our results suggest that exposure of endosulfan to Channa gachna (Hamilton) caused liver tissue necrosis by increased levels of LDH in serum. The results of present study also suggest that the fishes collected from endo- sulfan treated areas are also notably affected by endo- sulfan. Moreover, we support that LDH enzyme may be an important biochemical marker for pesticide toxicity to mammalian system. Moreover, endosulfan is Banned in more than 62 countries, including the European Union and several Asian and West African nations, it is still used extensively in many other countries including India, Bra- zil, and Australia. Bearing in mind the adverse effect of endosulfan observedin recent study along with the sup- ported literature it is recommended that the application of endosulfan should be ban strictly in India as well.

ACKNOWLEDGEMENTS

We acknowledge the Head of department of Zoology, Lumding College for providing the research facility.

Akalesh K. Verma and Jashodeb Arjen

The authors are also thankful to the villagers of Karbi Anglong for their helping hands during fish collection.

REFERENCES

Ahmad S, Agarwal R, Agarwal D K and Rao G S (2000) Bio- reactivity of glutathionyl hydroquinone with implications to benzene toxicity Toxicology. 150, 31-43.

Ali AS (2014) Responses of the earthworm, Eisenia foetida coe- lomocytes to alumniuim chloride using the neutral red reten- tion assay Biosc. Biotech.Res.Comm Vol.7 (1) 42-45

Ali AS and I Naaz (2013) Earthworm biomarkers: The new tools of environmental impact assessment Biosc.Biotech.Res. Comm Vol.6 (2) 163-169

Akanji M A, Olagoke O A and Oloyede O B (1993) Effect of chronic consumption of metabisulphite on the integrity of rat cellular system. Toxicol. 81, 173-183.

Arnold S F, Klotz D M, Collins B M, Vonier P M, Guilette L J and McLachlan J A (1996) Synergistic activation of oestrogen receptor with combinations of environmental chemicals. Sci- ence 272, 1489-1497.

Bisht I and Agarwal S K (2007) Cytomorphological and histo- morphological changes in mucous cells of general body epi- dermis of Barilius vagra (Cyprinidae, Pisces) following expo- sure to herbicide-blue vitrol (CuSO4): A statistical analysis, J. Exp. Zoo. India.l0, 27-36.

Boran M, Altinok I, Capkin E, Karacam H and Bicer V (2007) Acute toxicity of carbaryl, methiocarb, and carbosulfan to the rainbow trout Oncorhynchus myskiss and guppy, Poecili retic- ulata Turk. .1. Vet Ani. Sci. 31, 39-48.

Cengiz E I (2006) Gill and kidney histopathology in the fresh- water fish Cyprinus carpio after acute exposure to deltameth- rin. EnvironToxicol Pharmacol.22, 200-212.

Choudhary N, Goyal R and Joshi S C (2008) Effect of malathion on reproductive system of male rats. J. Environ. Biol. 29, 259- 267.

Choudhary N, Sharma M, Verma P and Joshi S C (2003) Hepato and nephrotoxicity in rat exposed to endosulfan. J Environ. Biol. 24, 305-312.

Cripe G M (1994) Comparative acute toxicities of several pesti- cides and metals to Mysidopsis bahia and post larval Penaeus duorarum. Environ. Toxicol. Chem. 13, 1867-1873.

Daesik P, Minor M D and Proper C R (2004) Toxic response of endosulfan to breeding and non breeding female mosquito fish, J Environ. Bio.25, 119-125.

Dey M and Gupta A (2002) Acute toxicity of endosulfan on three anuran tadpoles. Ecotoxicol. Environ 12, 61-68.

Gill T S, Pande J and Tewart H (1991) Effects of endosulfan on the blood and organ chemistry of freshwater fish Ecotoxicol Enviromental Safety. 21, 80-92.

Hernandez A F, Gomez MA, Perez V, Garcia-Lario J V, Pena G, Gil F, Lopez 0, Rodrigo L, Pino G and Plan A (2006) Influence of exposure to pesticides on serum components and enzyme

Akalesh K. Verma and Jashodeb Arjen

activities of cytotoxicity among intensive agriculture farmers Environ. Res. 102, 70-75.

Hiremath M B and Kaliwal B B (2002) Effect of endosulfan on ovarian compensatory hypertrophy in hemi castrated albino mice Reproduct Toxicol. 16, 783-794.

Holem R R, Hopkins W A and Talent L G (2006) Effect of acute exposure to malathion and lead on sprint performance of the western fence lizard (Sceloporus occidentalis). Arch. Environ. Contam. Toxicol. 51, 43-52.

Johal M S, Sharma M L and Ravneet (2007) Impact of low dose of organop hosp hate, monocrotophos on the epithelial cells of gills of Cyprius carpio (Linn.)- SEM study. J. Environ. Biol. 28, 663-673.

Joshi N, Dharmalata and Sahu A P (2007) Histopathological changes in liver of Heteropneustes fossilis exposed to cyper- methrin. J. Environ. Biol. 28, 35-42.

Kalavathy K, Sivakumar A A and Chandran R (2001) Toxic effect of the pesticide Dimethoate on the fish Sarotherodon mossambicus. J Ecol. Res. Bioconserv. 2, 2735.

Kaviraj A., and A. Gupta (2014) Biomarkers of Type II Synthetic Pyrethroid Pesticides in Freshwater Fish, Biomed Research International PMC4017726, April 23rd 2014

Kumar M, Trivedi S P, Misra A and Sharma S (2007) His- topathological changes in testis of the freshwater fish, Het- eropneustes fossilis (Bloch) exposed to linear alkyl benzene sulphonate (LAS). J. of Environ. Biol. 28, 679-685.

Kuruts E B, Doran F and Qiralik H (2006) The effect of endo- sulfan on lactic dehydrogenase enzyme system in liver of Mus musculus a histochemical study. EIII.. J. Gen. Med.3, 148-156.

Ludke L, Finley M T and Lusk C (1971) Toxicity of mirex to crayfish, Procambarus blandigingi. Bull. Environ. Contain. Toxicol. 6, 89-98.

Malomo S O (2000) Toxicological implication of ceftriax- one administration in rats Nig. J. Biochem. A 461. Biol. 15, 33-45.

Maqvi S M and Vaishnai C (1993) Bioaccumulative potential and toxicity of endosulfan insecticide to non-target animals. Comp. Biochem. Physiol. 105, 347-354.

Moulin F, Copple B L, Ganey P E and Roth R A (2001) Hepatic and extrahepatic factors critical for liver injury during lipopol- ysacharide exposure. Am. J. Physiol 6, 1423-1432.

Nelson, D L and Cox M M (2005) Lehninger Principles of Bio- chemistry, fourth edn. W.H. Freeman and Company, NY.

Pandey R K, Singh R N, Singh S, Singh N N andDas V K (2009) Acute toxicity bioassay of dimethoate on freshwater air breathing catfish, Heteropneustes fossilis (Bloch).J. Environ Biol. 30, 2-11.

Rahman M F and Siddiqui M K F (2005) Sub-chronic effects of a phosphorothionate insecticide (R.P.R-II) on hemopoetic sys- tem and serum composition of male and female rats. Toxicol Envirom Chem. 87, 107-119,

Ramaneswari K and Rao L M (2008) Influence of endosulfan and monocrotophos exposure on the activity of NADPH cyto- chrome C reductase (NCCR) of Labeo rohita (Ham). J. Environ. Biol. 29, 183-192.

Schmidt C, Schmidt F W, Hoin D and Gelarch U C (1965) Lac- tate dehydrogenase enzyme assay: Methods of Enzymatic Analyses, Academic Press, NY.

Shanmugam M, Venkateshwarlu M and Naveed A (2000) Effect of pesticides on the freshwater crab Barytelphusa conicularis (West wood). J Ecotoxicol. Environ. Monit.10, 273-283.

Shelby M D, Newbold R R, Tully D B, Chae K and Davis V L (1996) Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays. Environ. Health Perspectives. 104, 1296-1304.

Singh N N and Srivastava A K (1992) Biochemical changes in the freshwater Indian catfish- Heteropneustes fossilis (Bloch.) following exposure to sublethal concentration of aldrin J. Environ. Bio1.22, 21-43.

Singh P B and Singh V (2007) Endosulfan induced changes in phospholipids in the fresh water female catfish Heteropneustes fossilis (Bloch.). J. Environmental Biol. 28, 605-616.

Tripathi G and Verma P (2004) Endosulfan-mediated biochem- ical changes in the freshwater fish Clarias batrachus. Biomed. Environ. Sci. 17, 47-50.

Varayoud J, Monje L, Bernhardt T, Munoz-de-Toro M, Luque E H and Ramos J G (2008)Endosulfan modulates estrogen- dependent genes like a non-uterotrophic dose of 17beta-estra- diol. Reproductive.Toxicol.26, 138-142.

Velmurugan B, Selvanayagam M, Cengiz E I and Unlu E (2007) The effects of Monocrotophos to different tissues of freshwater fish Cirrhinus mrigala. Bull. Environ. Contam. Toxicol. 78, 450-458.

Venkatramana G V Sandhya Rani P N and Moorthy P S (2006) Impact of Malathion on the biochemical parameters of gobiid fish, Glossogobius giuris(Ham). 27, 119-128.

Wilson V S and Le Blanc G A (1998) Endosulfan elevates testo- sterone biotransformation and clearance in CD-1 mice. Toxico. Appl. Pharmacol. 148, 158-168.

Yadav A, Neraliya S and Gopesh A (2007) Acute toxicity lev- els and ethological responses of Channa striatus to fertilizer industrial wastewater. J. Environ. Biol. 28, 159-164.

Yakubu M T, Bilbis L S, Lawal M and Akanji M A (2003) Effect of repeatedadministration of sildenafil citrate on selected enzyme activities of liver and kidney of male albino rats. Nig. J Pure and Appl. Sci. 18, 1395-1404.