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8(3): 989-992, 2013 (Supplement on Toxicology)www.thebioscan.in

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HISTOPATHOLOGICAL CHANGES IN THE OVARY OFFRESHWATER FISH PUNTIUS TICTO (HAM) UNDERDIMETHOATE TOXICITY

GANESHWADE RAJU MARUTIRAOZoology Department, P. D.V. P. College Tasgaon – 416 312, Sanglie-mail: rmganeshwade@gmail.com

INTRODUCTION

Pesticides are the major source of water pollution, as it eradicatethe economically important species either indirectly throughbreaking the biological chains or directly produces toxic stressand chemical changes. Predominantly, as a result of extensiveapplication of pesticide, large scale mortalities of fish occurredwidely (Srivastava and Srivastava, 1994). Fishes are verysensitive to a wide variety of toxicants in water, various speciesof fish show uptake and accumulation of many contaminantsor toxicants such as pesticides (Herger et al., 1995) Due toaccumulation of these pesticides in tissues produces manyphysiological, histological and biochemical changes in thefishes and freshwater fauna by influencing the activities ofseveral enzymes and metabolites (Nagarathnamma andRamamurthi, 1982). The pervious histopathological studiesof fish exposed to pollutants revealed that fish organs areefficient indicators of water quality (Cardoso et al., 1996 andCengiz et al., 2001). Therefore, it is necessary to study indetail on the histopathological alterations in different organsof fishes and thoroughly investigate them in order to assessthe extent of damage.

Dimethoate is an organophosphorous insecticide widely usedagainst vegetable and fruit sucking aphids, mites and saw flies.Exposure to chemical pollutants may cause many molecular,biochemical changes in the fish which precede cellular andsystemic dysfunctions. So that, if appropriate parameters aremonitored, early warning signs of distress may be detected(Palmes, 1976).

Similarly pesticides are also known to cause varioushistopathological effects on the ovary of fish (Singh and Sahai,1985; Rastogi and Kulshrestha, 1990, Deshmukh andKulkarni, 2005; Verma and Srivastava, 2008 andPugazhvendan et al., 2009). However there has been littleinformation on the histopathological impact of dimethoateon an ovary of Puntius ticto. Therefore, the presentinvestigation was undertaken with a view to study in detailabout histopathological changes in the ovary of Puntius ticto,under dimethoate toxicity.

MATERIALS AND METHODS

The freshwater fish P. ticto were selected from the freshwatersources around Aurangabad city. They were acclimatized inaged, dechlorinated and well aerated water for two weeks inthe laboratory. During acclimatization they were fed on alter-nate days with pieces of live earthworms. The LC50 values aredetermined by following the guidelines given by committeeof toxicity tests with aquatic organism (Annon, 1975) and ProbitAnalysis Method (Finney, 1971). The acclimated fish wereexposed to lethal concentration (5.012ppm) for 96h and sub-lethal concentrations (2.506ppm and 1.253ppm) for 60 days.Simultaneously a control group of healthy fishes were main-tained under identical conditions. The 20 healthy fishes show-ing normal activity were exposed for chronic study. After com-mencement of exposure period fishes were killed by decapi-tation and ovaries are removed and fixed in Bouins fluid for24h and processed according to standard procedure of rou-tine microtechnique. For staining double stain method was

ABSTRACTHistopathological changes induced by dimethoate on the ovary of Puntius ticto (Ham) were studied. Puntius tictoa freshwater fish exposed to lethal (5.012ppm) and two sublethal (2.506 and 1.253ppm) concentration ofdimethoate for acute (four days) and chronic (sixty days) exposure. After lethal exposure ovary showed significantchanges. There was partial disruption of ovarian follicles and vaculation in cytoplasm of germinal cells. Theinterfollicular connective tissue was damaged. The cytomorphological structure of ovarian follicles got deformedand elongated, losing their typical configuration. Necrosis and fibrosis in connective tissue and damage to yolkvesicles of maturing oocytes was observed. Degenerative oocytes became phagocytic and exhibited atresia.Chronic exposure results also shows significant changes in the ovary and are dose dependent

KEYWORDSHistologyAcute and chronictoxicityDimethoatePuntius ticto

Received on :07.06.2013

Accepted on :21.08.2013

*Correspondingauthor

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followed by using Haematoxylin and Eosin and mountingwas done in DPX.

RESULTS AND DISCUSSION

In the ovary of control fish Puntius ticto, shows large numberof mature and maturing oocytes. The immature oocytes aretransparent with nucleus and cytoplasm. Mature oocytes areround opaque and contain large and small yolk globules. Thevitelline membrane and follicular layer become moreprominent. (Fig. 1 and 2) Puntius ticto when subjected tolethal (Acute) and two sub-lethal (chronic) concentrations ofdimethoate, showed histological changes in the ovary. Afterlethal (Acute) exposure ovary showed significant changes.There was partial disruption of ovarian follicles andvacuolation in cytoplasm of germinal cells. The inter-follicularconnective tissue was damaged. The cytomorphologicalstructure of ovarian follicles got deformed and elongated,losing their typical configuration. Necrosis and fibrosis inconnective tissue and damage to yolk vesicles of maturingoocytes was observed. Degenerative oocytes becamephagocytic and exhibited atresia. In maturing oocytes thegranulosa layer gets separated and complete or partial rupturewas observed. In mature oocytes, clumping of cytoplasm andkaryolysis was observed. (Fig. 3)

Two sub-lethal (Chronic) exposure results also showssignificant changes in the ovary of Puntius ticto and are dosedependent. Severe damage was observed in 2.506 ppmexposure. After 2.506 ppm exposure, it was observed thatonly immature and maturing oocytes were present and matureoocytes are completely absent. Atresia was seen in the maturingfollicles, ovarian follicles separated due to loss of inter-follicularconnective tissue. Inter-follicular spaces were larger andvacuolation in developing oocytes were also observed. Thedegenerating oocytes became phagocytic and formed atreticoocytes. In certain oocytes inversion of granulosa layer wasobserved (Fig. 4).

After 1.253 exposures, ovary shows, it has few numbers ofoocytes and small inter-follicular spaces. Also showsvacuolation and atretic oocytes. Clumping of cytoplasm, wallrupture, displacement of nucleus, yolk vesicles & nucleolusand disappearance of nucleolus was observed in some matureoocytes.( Fig. 5 and 6).

The above result shows the retardation of oocytes proliferationand increase in the number of atretic follicles, fibrosis andnecrosis or degeneration in the ovary was more in higherconcentration exposure. The mature oocytes were totallyabsent after sub-lethal exposure (2.506 ppm), which may bedue to changes in oogenesis cycle i.e. vitellogenic growth ofoocytes must have been stopped. Dimethoate affects thesecondary growth of primary oocytes due to the impairmentof vitellogenesis.

Histological abnormalities in ovaries may be caused by severalfactors, viz., ionizing radiations, electric current, parasiticinfections, mechanical injuries, xenobiotic toxicants (Sarojiniand Victor, 1985) and by a variety of effluents and aquaticpollutants (Shukla et al., 1984; Davis and Cook, 1993; Farmeret al., 1995; Kumar et al., 2000). Almost similarhistopathological findings were reported by Hossain et al.

(2002) in the ovaries of Anabas testudineus and C. punctatusafter the exposure 0.5 and 5.0 ppm concentration of pesticide,dimecron 100SCW. Giri et al. (2000) reported the effects ofinsecticide basathrin induced histoanatomical insult of ovariantissue of catfish, H. fossilis. They reported marked damage ingerminal epithelium, atresia of oocyte, stromal hemorrhage,vacuolization of oocytes and general inflammation.

Kling (1981) reported that cells assimilation of yolk granulesduring the exposure and simultaneous arrest of vitellogenesiscaused the reduction in size of oocytes, resulting in a totalatresia of ovaries of Tilapia leucostica exposed to labycid for14 days. During present study numbers of mature oocytes arereduced. Similar result was observed by Pawar and Katdare(1983) in Garra mullya under sumithion toxicity. Muley andMane (1987) observed ruptured follicles, completely dissolutenucleus and nucleoli in the gonads of Lamellibranch mollusksunder Cythion and Malathion toxicity.

Saxena and Agrawal (1986) have shown that cadmium chlorideblocked all the oogonial activity at the vitellogenic phase in C.btrachus and suggested it that it might be due to retardation,oocytes proliferation and increase in number of atretic follicles.Similar results were found during present study. The rate ofoocytes degeneration in the ovary was more in higherconcentration of dimethoate i.e. dose dependent. Similarresults observed by Ramchandra Mohan (2000) in G. giuris.Rastogi and Kulshrestha(1990) reported necrosis and fibrosisin connective tissue along with dilation of blood vessels anddamage to yolk vesicles of maturing oocytes in carp minnowRasbora daniconius under endosulfan, carbofuran and methylparathion toxicity. During present study atretic oocytes andincrease in the interfollicular spaces are observed. Same resultwas supported by findings of Nath and Kumar (1990) in C.fasciatus under nickel toxicity. Benarji and Rajendranath(1991) studied cyto-architectural changes in the oocytes,including pronounced vacuolation, degeneration anddeformation, clumping of the cytoplasm and karyohypertrophyin Clarias batrachus exposed to dichlorvos. This supportsauthor’s findings in Puntius ticto.In Puntius ticto, control or normal ovary was fully maturedhaving large number of mature oocytes. After exposure periodoocytes exhibits degenerative changes, liquification ofperinuclear cytoplasm and condensation of nucleus,disappearance of nuclear membrane, cytoplasmic clumping,degenerated granulose layer, and degenerated ovarian walland wrinkled oocytes. Similar results were also found byKhillare (1992), Sukumar and Karpagaganpathy (1992).Prominent inter-follicular spaces were observed in the ovarywhich was probably formed due to shrinkage of the oocytes.A large numbers of atretic follicles were also observed in theovary of Puntius ticto. Same result was observed by Srivastavaand Srivastava (1994). Hazarika and Das (1998) studied effectof BHC on the ovary of the H. fossilis and observed vacuolationin cytoplasm of germinal cells and reduction in number ofmatured ovum and secondary oocytes. They observed ovarianfollicles were separated due to loss of interfollicular connectivetissue. Jyothi and Narayan (1999) observed vacuolation andnecrosis, arrested ovarian recrudescence and inter-follicularoedema, in the ovary of Clarias batrachus (L) under carbaryltoxicity.

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Sakthival and Gaikwad (2001) observed significant increasein the level of atresia in the ovary of dimecron treated fishGambusia affinis. Baruah and Das (2002) noted partial lysis,swelling, atresia and changes in nucleus and Cytoplasmicorganization after exposure of Heteropneustes fossilis to papermill effluent for 20 days. Dutta and Meijer (2003) studied theeffect of the insecticide diazinon (op) on the ovaries of bluegill(Lepomis micromeres). He noticed adhesion of primaryfollicles, cytoplasmic retraction in oocytes II, cytoplasmicdegeneration, increased atretic oocytes, damages to theoocytes IV, partial destruction of the ovigarous lamellae andvitellogenic membrane, destruction of follicles, increased

intrafollicular spaces, vacuolated cytoplasm, extrusion ofkaryoplasms and necrosis in the cytoplasm.Gupta and Guha (2006) reported less yolk in developingoocytes, follicle cells were deformed and necrosis in the ovaryof Heteropneustes fossilis under mycrocystin toxicity. Olfatand El-Greisy (2007) observed extensive necrosis of oolema,hypertrophy and hyperplasia of the follicular cells of oocytes,atresia in the large vacuolated mature follicles of the ovary inSiganus rivulatus after exposure to different waste sourcescontaining Zn. Pugazhvendan et al.. (2009) observed manydisturbed oogonial, break downed germinal vesicles and yolkgranules were disappeared in the ovary of Ophiocephalus

Figure 1: T. S. of ovary of Puntius ticto (Control). [HE X40]. N: NucleusOW:Ovarian wall RO:Resting oocyte, MO :Mature oocytes YV:Yolk vesicle; YG:Yolkgranules

Figure 2: T.S. of ovary of Puntius ticto (Control). [HE 400X]. T: ThecaYV: Yolk vesicle YG: Yolk granules; ZG: Zona granulose; ZR: Zona radiata

Figure 3: T. S. of ovary of Puntius ticto after 5.012 ppm exposure todimethoate.[HE 100X]. AC: Atretic oocyte CC : Cytoplasmic clumping DGL:Degenerated granulose layer DOW :Degenerated ovarian wall N : Nucleus WO: Wrinkledoocyte

Figure 4: T. S. of ovary of Puntius ticto after 2.506 ppm exposure todimethoate [HE 100X]. AF;Atretic follicle CC:Cytoplasmic clumpingCL;Cytoplasmic liquification DOW:Degenerated ovarian wall IFS:Inter-follicular space;IGL:Invasion of granulosa layer LGL:Lifting of granulose layer

Figure 5: T. S. of ovary of Puntius ticto after 1.253ppm exposure todimethoate [HE 100X]. AF: Atretic follicle CL: Cytoplasmic liquefaction DOW:Degeneration ovarian wall IFS: Inter-follicular space IGL: Invasion of granulosa layer

Figure 6: T.S. of ovary of Puntius ticto after 2.506 ppm exposure todimethoate [HE 100X]. AF: Atretic follicle CC: Cytoplasmic clumping CL:Cytoplasmic liquification; DOW: Degenerated ovarian wall; DGL: Degenerated granuloselayer IFS: Inter-follicular space; IGL: Invasion of granulosa layer

HISTOPATHOLOGICAL CHANGES IN OVARY

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punctatus under Malathion toxicity. Deka and Mahanta(2012) reported adhesion of primary follicle, cytoplasmicretraction & clumping, cytoplasmic degeneration, increasedatretic oocytes, partial destruction of ovigerous lamellae andvitellogenic membrane in the ovary of Heteropneustes fossilistreated with Malathion.

Significant Histopathological alterations were observed in theovaries of Puntius ticto under dimethoate toxicity. Theprominent changes are occurrence of atretic oocytes andincrease in interfollicular spaces. These changes also notedby large number of workers Murugesan and Haniffa (1992);Nath and Kumar (1990); Verma and Srivastava (2008). Largeinterfollicular spaces have been formed due to the shrinkageof the oocytes and several oocytes were observed in theprocess of absorption i.e. atretic oocytes.

ACKNOWLEDGEMENT

I am thankful to the Head, Department of Zoology, Dr.Babasaheb Ambedkar Marathwada University who allowedme all necessary facilities to do my work. I am also grateful toDr. R. R. Kumbhar, Principal, Padmabhushan Dr.Vasantraodada Patil Mahavidyalaya Tasgaon and Dist. Sanglifor their constant encouragement and appreciation.

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