ABSTRACT- The present investigation was carried out to the effect of Fenvalerate on haematological parameters in freshwater fish Channa marulius using standard methods. Fish was exposed to (1/4^th LC₅₀) sub lethal concentration of Fenvalerate (0.086ppm) for 96h.The blood sample were obtained from both, control and experimental fish and assayed haematological parameters (Total Erythrocytes count, TEC; Total Leucocytes count, TLC; Haemoglobin, Hb; Packed cell volume, PCV; Mean corpuscular volume, MCV; Mean Corpuscular Haemoglobin and Mean Corpuscular Haemoglobin Concentration; MCHC). The result revealed that TEC, Hb percentage, PCV and MCHC counts were significantly decreased, whereas TLC, MCV and MCH increased slightly in experimental fish. The study has thus indicated marked changes in blood of Channa marulius after exposure to Fenvalerate.
Key-words- Fenvalerate, Hematology, Channa marulius, Haemoglobin, Sub lethal

INTRODUCTION
The blood parameters in fishes are influenced by many factors viz. quality of water, temperature, the sex, size, season and age of fishes are directly reflected haematological parameters of the fishes (Bhagat et al., 1986). The study of fish blood parameters are important for determining factors related to its physiological capacity (Affonso, 2001; Wells et al., 2005).
Blood parameters are considered path physiological indicators of the whole body and therefore are important in diagnosis the structural and functional status of fish exposed to toxicants (Adhikari et al., 2004). A number of hematological parameters such as RBC, WBC, and haemoglobin content and so on, are used to assess the functional status of the oxygen carrying capacity of the blood. Pollution of aquatic ecosystem by chemical used in industry and agricultural is increasing day by day.
Due to injudicious and indiscriminate use of agrochemicals, fertilizers, pesticides, and insecticides to boost crop production with the sole aim of getting more yields. Water bodies like ponds, lakes and rivers are continuously getting polluted normally these pesticides reaches an aquatic environment through surface runoff and sediment transport from treated effluents (Bauman, 1981).
Direct application of pesticides to water bodies to control the inhabiting pests. Spray drift from normal agricultural operation and through heavy rainfall. In all cases these chemicals may directly toxic as well as deteriorate the water quality by changing its physicochemical nature and cause ecological imbalance leading to health hazards to different types of aquatic organisms in general and fishes in particular (Tripathi, 1992). The herbicide can cause death of fishes either directly or due to saturation by destruction of food chain many herbicides have shown to effect the growth and reproduction of fishes with evidence of tissue damage (Dhasarathan et al., 2000). The present study was under taken to analyze the impact of 1/4^th sub lethal concentration of Fenvalarate on haematological parameters of fresh water fish Channa marulius (Ham Buch).

MATERIALS AND METHODS
The fresh water fish Channa marulius weighing (15 ± 5 g) and length (10 ± 3 cm) were collected from Kan and Panzara river of Sakri Taluka (Dhule). Live fishes were brought to the laboratory and thoroughly washed under tap water and acclimatized in laboratory conditions for 15 days. They were fed with standard fish diet (Tokyu grow certified company). Water in the tank was changes after 2 days of interval. Technical grade Fenvalerate (ISAGRO ASIA), 20% emulsifiable concentration (EC) was purchased from Sushil Agricultural pesticide and fertilizer Agency, Sakri for present study.
The fishes were divided into a group, each group of ten healthy fishes were transferred to plastic tough having capacity of 10 litres and they exposed to1/4^th sub lethal concentration of Fenvalerate (0.086ppm). One group was kept as control. At the end of exposure period the blood was collected in glass tube by cutting the caudal peduncle, using EDTA as anticoagulant. Maximum 2ml blood was taken for haematological studies. The haematological parameters such as RBC, WBC, and Hb, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were estimated by using the methods described by Swarup et al., (2002).
The RBC and WBC were counted by haemocytometer, Hb was determined by Sahil’s haemoglobinometer and PCV was measured by using Wintrobe’s tube. The Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Haemoglobin Concentration were calculated by equation.

                                        PCV×10
MCV (Cubic micron) = ---------------------
                                                RBC (million /cu.mm)

                    Hb in g % ×10
MCH (Pg) = ---------------------------
                    RBC (million/cu.mm)

                    Hb in g %
MCHC (g/dl) = -------------X 100
                PCV

Result were expressed ± S.E. three replicates and differences between mean were considered to significance when p<0.05 (Bansal et al., 2004).

RESULTS AND DISCUSSION
In the present investigation, the effect of insecticide like Fenvalerate 1/4^th (0.086ppm) on haematological parameters such as RBC, WBC, Hb, PCV, MCV, MCH and MCHC in the fresh water fish C. marulius is shown in Table-1.

Effect on RBC: The amount of RBC present in the blood of fish after exposed to different hours in 0.086 ppm concentration of Fenvalerate .The blood of fishes exposed to 0.086 ppm Fenvalerate for 24 h, 48 h, 72 h and 96 h were found to contain 1.98, 1.7, 1.42, 1.32mm³ respectively and mean control was 2.51 mm3. The R BC values were significantly decreased than the control.
Lowering of RBC amount might be due to damaging action of insecticides on peripheral cell due to which viability of the cell was affected. Anemia could be due to effect of insecticide on haemopoisis or attraction of cell membrane and hydrolysis of acetyl chloride in body fluid by chlorine esterase of erythrocytes may be secondary result from a primary action of the insecticides on the erythropoetic tissue due to which there was a facture in the red cell production.

Effect on WBC: The amount of WBC present in the blood of fish after exposed to sub lethal concentration of 1/4^th (0.086ppm) 24h, 48h, 72h and 96h were found to contain 3.80, 4.69, 5.11, and 6.38 mm³ respectively and mean control was observed 4.25 mm³.
In the present investigation the WBC values showed significant higher in 48 h, 72h and 96 h than the mean control value while it was less in 24h. An increase in lymphocytes suggests that the immune mechanism of fish get stimulates and become adapted under insecticide stress to fight against the pollutants in the environment. Higher white blood cell count indicates damage due to infection of body tissue may be physical stress and leukemia. Increased in the in the number of leucocytes occurs due to immunological response. Similarly increased in total leucocytes counts has been observed by Binu Kumari, et al., (2010).This significant increase in total leucocytes count might be due to immunological reactions to produce more antibodies to cope with the stress induced by these toxicants. Increase in the number of WBC of C.marulius reflects impairment of the defense mechanism and manifested in to leucocytosis to cope with such a situation. Similar results were reported in teleost by Tyagi, et al (1989); Dutta, et al (1992); Kalair, et al (1993) and Ramesh and Saravan (2008) on exposure to different pesticide. Lymphocytes are numerically predominant white blood cells in fish (Kumar, et al., 1999). Lymphocytes of fish have been regarded as immune-competent. Thus they are responsible for the production of antibodies (Eills, et al., 1978).

Effect on haemoglobin: The amount of hemoglobin content in the blood of fish after exposed to sub lethal concentration of 1/4^th (0.86 ppm) of Fenvalerate for 24 h, 48 h, 72 h and 96 h were found to contain 9.2, 8.41, 7.02, 5.42 g/dl respectively and mean control was recorded as 10.22 g/dl. The hemoglobin level in all exposure period was significantly decreased over the control. Our work was supported by Thomas Nithyanandam, et al (2007). He was reported in C. carpio on exposed to pesticide, the reduction of hemoglobin might be due to blood coagulation. Similarly Gill,et al., (1992) reported reduction in RBC and Hb in Barbus conchohitus exposed to Endosulfan, Catla catla exposed to ekalux (Mustak and Natrajan,1992),Cyprinus carpio exposed to Dimethoate (Chauhan, et al.,1994). Ramesh and Manvalara Manujan, (1992) reported decrease in Hb, TEC in Malathion exposed fresh water carp cyprinus carpio and sumithion exposed to Clarias batrachus respectively.
Anaemia induced stress may also be due to blood cell injury and disrupted hemoglobin synthesis (Binna Kumari and Subhisha, 2010). The causes of depletion in the Hb content in Puntius conchonius are supposed to degeneration, depression and destruction of the blood forming material by the piscicidal compound (Patole and Bhoi, 2013).

Table-1: Haematological parameters of fresh water fish Channa marulius exposed to 1/4^th sub lethal concentration of Fenvalerate (0.086ppm) insecticide

Parameters	Exposure periods
	Control	24hrs	48hrs	72hrs	96hrs
RBC (×106/mm 3 )	2.51 ± 0.3	1.98 ± 0.22 (-26.76) **	1.71±0.29 (-46.78) **	1.42 ± 0.02 (-76.76) **	1.32 ± 0.14 (-90.15) ***
WBC (×103/mm 3)	4.25 ± 0.65	3.80 ± 0.18 (-11.84) *	4.69 ± 0.3 (9.38) *	5.11 ± 0.09 (16.15) *	6.38 ± 0.52 (33.38) **
Hb (g %)	10.22±0.98	9.2 ± 1.3 (-11.08) *	8.41±0.69 (-21.52) *	7.02 ± 0.48 (-45.58) **	5.42 ± 0.58 (-46.96) **
PCV (%)	30.9 ± 4.1	33.3 ± 2.7 (7.20) *	28.3 ±1.2 (-9.18)NS	26.25±0.75 (-17.71) *	23.93 ± 2.57 (-29.12) **
MCV(µm3)	122 ± 3.0	163 ± 8 (-25.15) *	165 ± 10 (26.06) *	172.4±16.6 (29.23) **	184.4± 24 (33.69) **
MCH(Pg)	42.1 ± 3.5	45.2 ± 3.3 (6.85) NS	41.3 ± 21 (-1.93)NS	50.2 ± 2.6 (16.13) *	43.36 ± 6.64 (2.90) NS
MCHC(g /dl)	34.1 ± 2.3	28.4 ± 1.6 (-20.07) *	29.6 ± 1.0 (-15.20) *	27.04±0.66 (-26.10) *	23.53 ± 1.77 (-44.92) **

Mean ± S.D. values differ significantly (p<0.05) within same columns.
*Significant value: p<0.05, ** p<0.01, *** p<0.001. NS = Non-Significant (p>0.05). Values in the parenthesis are percentage change over control treated as 100 per cent.

Effect on PCV: The amount of PCV of exposed C. marulius after the 24 h, 48 h, 72 h and 96 h exposure were 33.3, 28.3, 26.25, 23.93% and mean control was 30.9 %. It was increase in 24h.Whereas in 48h, 72 h and 96 h was significantly decreased from exposure period to control over.

Effect on MCV: The amount of MCV of the fish exposed to sub lethal concentration of Fenvalerate after the 24 h, 48 h, 72 h and 96 h exposed period revealed 163, 165, 172,184µm3 respectively and mean control was 122 µ³ respectively. The MCV contain in exposure period was significantly increased over control.

Effect on MCH: The amount of MCH in the blood of the fishes exposed to1/4^th sub lethal concentration (0.086ppm) of Fenvalerate for 24 h, 48 h, 72 h and 96 h was found to contain 45.2, 41.3, 50.2 and 43.36 pg respectively and mean control was 42.1 pg. The values were decreased in 48 h. While the values are increased in 24 h,72 h and 96 h than over control.

Effect on MCHC: The amount of MCHC in the blood of the fish recorded as 28.4, 29.6, 27.04, 23.53g/dl respectively and means control value was 34.1 g/dl was to be found. The MCHC was decreased from control to exposure period. Atamanalp et al, (2003), Atmanalp and Yanik, (2003) found a significant increase in the level of RBC and significant decrease in MCH, MCHC, thrombocytes count and erythrocyte sedimentation rate in rainbow trout (Oncorhynchus mykess) following Cypermethrin and Mancozeb acute exposure. A decrease in important blood parameters has been reported during exposure to various pesticides in fish (Saxena and Seth, 2002).
The decrease in the above parameters indicates stage of fish caused due to decreased erythropoetic activity or increased destruction of red blood cells further support in favors of this comes from the decrease noted PCV. Decrease in Hb indicates poor O2 transport by blood caused by damage or due to increased accumulation of CO₂ in blood. In the present investigation significant change was recorded in the RBC, WBC, Hb, MCV, PCV and MCHC. The reduction in this values might be due to spleen concentration after have been detected in fish. Cells released from spleen which is an erythropoetic organ would have lowered those values.

CONCLUSIONS
The present investigation show that Fenvalerate caused decrease in the haematological parameter in Channa marulius which suggest that the Fenvalerate (synthetic pyrethroids) may weak the immune system and result in severe physiological problem ultimately to the death of fish. Fenvalerate is a synthetic pyrethroid used to protect many fruits, vegetables and field crops against disease, hence farmer come direct contact it and may impair their health.

ACKNOWLEDGMENT
The authors are grateful to the principal, S.G. Patil Arts, Science and Commerce College, Sakri Dist. Dhule (M.S.) for providing necessary laboratory facilities. Also thankful to Zoological Survey of India, Western Regional Station, Akurdi, Pune (M.S.) for the identification of fish.

REFERENCES

1. Adhikari, S., Sarkar, B. (2004).Effect of Cypermethrin and Carbonfluron on certain haematological parameters and prediction of their recovery in a freshwater teleost, Laebeo rohita (Ham) Ecotoxicol. Environ. Safe. 58-220-226.
2. Affonso, E.G. (2001). Respiratory characteristics of Hoploternum littorale (Siluriformes, Callichthyidae); Acta Amazonica.31:249-262.
3. Atamanalp, M., and Yanik T. (2003). Alternation in hematological parameters of rainbow trout (Oncorynchus mykis) exposed to Mankozeb Tur. J. of veter. Ani. Sci. 27, 1213-1217.
4. Bansal, M. L., Singh, S., Bansal, M. L., Singh, T. and Kumar, R. (2004).Statistical methods for Research Workers.Kalyani publishers, New Delhi-2.
5. Bauman, F. (1981). Draft environmental report of India. Saota 1-77074 PN-AAH752 Arda 2:31-32.
6. Bhagwat, R.P. and Banerjee, V. (1986). Haematology of an Indian freshwater Cel Amphipnous cuchia (Hamilton) Erythrocyte counts related parameters with special reference to body length sex and season. Compt. physiol. Ecol. 2(1); 21-27.
7. Binu Kumari, S. and Subisha, M.C. (2010). Haematological responses in a fresh water fish Oreochromis mossambicus exposed to Chloripyrifos.The ekol.10 (1-2) 83-88.
8. Chauhan, R., Saxena, K. and Kumar, S. (1994).Rogor extract of the bark of Buchanian lanzanlin on behavior and chromatophores of freshwater fish Labeo rohita; J. Envion. Biol. 22(3):229-231.
9. Dhasarathan, P., Palanlappan, R. and Singh, A. (2000). Effect of Endosulfan and Butachlor on the digestive an enzyme proximate composition of the fish Cyprinus carpio. Indian J. Environ and Ecoplan 3(e): 339-446.
10. Dutta, H.M., Dogra, J.V., Sing, N.K., Roy, P.K., Nasar, S. T., Adhakari, S. Munshi, J.S. and Richmonds, C.K. (1992). Malathion induced changes in serum protein and haematological parameters of Indian catfish Heteropneustes fosslis (Bloch);Bull.Environ.Contam.Toxicol. 49:91-97.
11. Ellis, A.E., Roberts, R.J., and Tytler, P. (1978). The anatomy and physiology of teleost In: Roberts RJ (Ed) Fish pathology.Bailliere Tindall, London, P.23-54.
12. Gill, T.S., Pande, J. and Tiwari, H. (1992). Effect of Endosulfan on the blood and organ chemistry of freshwater fish, Barbus conchonitus (Ham). ,i>Ecotoxicol. Environ.Saf.21:80-89.
13. Kalair, J.S., Mishra, V.S. and Sing, R. K. (1993). Pesticide sumithion induced haematological changes in mud eel Amphipnous cuchia; Bio.Mem.19:41-48.
14. Kumar, S., Lata, S. and Gopal, K. (1999). Deltamethrin induced physiological changes in freshwater catfish Heteropneustes fossils; Bull.Envioron.Contam.Toxicol.62: 254-258.
15. Mustak, M. and Natarajan, K. (1992). Altered physiological parameters due to the pesticide Ekalux in the fish Catla catla. Environ and Ecol., 10 (2): 445-447.
16. Patole, S. S. and Bhoi, S.S. (2013). Comparative studies on haematological responses in the fresh water fish, Punctius conchonius exposed to two piscicidal plants extracts.
17. Ramesh, M., Manaralara Manujan, R. and Sivakumari, K. (1992). Effect of water hardness and toxicity of Malathion on haematological parameters of the fresh water fish, Cyprinus carpio, J. Ecotoxicol. Environ. Monit. 2(1): 31-34.
18. Ramesh, M. and Saravanan, M. (2008). Haematological and biochemical responses in a freshwater fish Cyprinus carpio exposed to Chloripyrifos; Int. J. Inter. Biol. 3 (1):80-83.
19. Saxena, K.K. and Seth, N. (2002). Toxic effect of Cypermethrin on certain haematological aspects of fresh water fish Channa punctatus Bull.Environ.Contam. Toxicol. 69; 364-369.
20. Swarup, N., Arora, S. and Pathak, S.C. (2002).Laboratory techniques in modern biology. Kalyani publishers, New Delhi-2.
21. Thomas Nithyanandam, G., Maruthanayagam, C. and Viswanathan, P. (2007). Effect of sub lethal level of pesticide, Monochrotophos on haematology of Cyprinus carpio during the exposure and recovery periods .Nat. Environ. Poll. Publications, 6: 615-621.
22. Tripathi, G. (1992). Relative toxicity of Aldrin, Fenvalerate, Captan and Diazon to freshwater for fish Clarias batranchus.Biomedical and environmental science 5(1): 33-38.
23. Tyagi, M., Agrawal, V.P. and Nandini, S. (1989). Haematological abnormalities induced in Ophiocephalus puntatus by Endosulfan; Environ. Ser.Recent. Trends. Toxicol. 1:81-84.
24. Well, R., Baldwin, J., Seymour, R., Chritian, K. and Brittain, T. (2005).Red blood cell function and haematology in two tropical freshwater fishes from Australia; Comp. Biochem, Physiol. A 141: 87-93.

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