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Int. J. Life. Sci. Scienti. Res., 2(6): 708-711 NOVEMBER- 2016
http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 708
Histopathological Impact of Dimethoate on the
Liver of Freshwater Fish, Garra mullya (Sykes)
Borane V R*
Department of Zoology Jijamata Arts, Science and Commerce College, Nandurbar, Maharashtra, India
*
Address for Correspondence: Dr. Borane V R, Head cum Associate Professor, Department of Zoology, Jijamata Arts,
Sci. and Comm. College, Nandurbar, India
Received: 15 Sept 2016/Revised: 30 Sept 2016/Accepted: 19 Oct 2016
ABSTRACT- The present investigation was carried out to the effect of Dimethoate on histopathological changes in liver
of freshwater fish, Garra mullya using standard methods. Fish was exposed to sub lethal concentration of Dimethoate
(0.0238ppm of 96hrs) for 7, 14, and 21days. Administration of pesticide to determine lesion of liver as indicators of tissue
damage. Histopathological changes in liver ranged from vacuolization, necrosis, fibrosis of perivascular region and
disposition of yellow brown grains at different time of exposure. Liver histology exhibited various abnormalities,
including hyperplasia, nuclear pyknosis, fatty necrosis and degeneration of hepatocytes leading to tumor and syncytium
formation, which are the indicative of carcinogenesis. In chronic treatment of dimethoate exposure may pose serious
threat to fish health and affect their population.
Key-words- Dimethoate, Histopathology, Liver, Garra mullya
-------------------------------------------------IJLSSR-----------------------------------------------
INTRODUCTION
Pesticides are occasionally used indiscriminately in large
amounts causing environmental pollution and potential
health hazards. Dimethoate is systemic insecticides
produced by reacting salts of Dimethyldithio-phosphoric
acid with N-methylchloroaecetamide, in aqueous medium
in the presence of some organic solvents is widely used
against a broad range of insects and mites and is also used
for indoor control of houseflies. The extensive use of DM
poses a health hazard to animals and humans because of its
persistence in soil and crops [1]. One of the major
agricultural chemical groups is pesticide which play
important role in increasing agricultural productivity
through controlling pest. But on the other hand, they cause
much damage to the non-target organisms both in terrestrial
and aquatic environment. Fish accumulate pollutants
directly from contaminated water and indirectly via food
chain [2]. The runoff from treated areas enters the river and
aquaculture ponds that are supplied by rivers and adversely
affect the quality of water surfaces and creates hazards for
aquatic life resulting in serious damage to non-target
species, including fishes [3].
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DOI: 10.21276/ijlssr.2016.2.6.10
Histopathology deals with the study of pathological changes
induced in the microscopically structure of body tissue. Any
alteration in normal structure of tissue indicates presence of
disease or the effect of toxic substances like heavy metal
and pesticides. [4] described histopathology as important
tool for evaluating the action of any toxicant at tissue level.
Histopathology provides data concerning tissue damage.
Histopathological alterations can be used as indicators for
the effect of various anthropogenic pollutants on organisms
and are a reflection of overall health of the entire population
in the ecosystem. The present study was under taken to
analyze the impact of chronic concentration of dimethoate
in liver of fish, Garra mullya.
MATERIALS AND METHODS
Healthy adult fish 4-5cm. in length Garra mullya were
collected from local river Shivan Dist. Nandurbar, India.
Fishes were washed with 0.1% of potassium permagnate
(Km No 4) solution to avoid dermal infection. They were
then rinsed in water and acclimatized to the laboratory
conditions in the department of zoology for two weeks in
1000 l. capacity glass aquaria. Dead fish were removed
immediately, Such as mortality may deplete dissolved
oxygen with resultant effect on other fishes. During
acclimatization fishes were fed with pieces of live
earthworm on alternate days. Water also changed once in
every day. The experiment was conducted natural and
photoperiod of temperature 25·1 ± 3·20
C. Water quality
was measure as per by [5], Conductivity 0.64 ± 0.3,
Dissolved O2
-
6.3±1.1 (ml/L), pH 8.60 ± 0.3,
Research Article (Open access)
Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 6
http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 709
Acidity 2.5± 0.1, Alkalinity 44.1 ± 0.5, Total
hardness 67.5 ± 0.3. LC50 of dimethoate for 96 hours was
determined by probit analysis method [6]. The animals
were dissected and liver tissue carefully removed. Tissues
were immediately washed in 1% saline solution to remove
the adherin mucus and blood and soaked between the
blotting papers. The tissue from the control and exposed
batches were taken out and preserved in aqueous Bouin’s
fluid for 24 to 48 hrs. This was followed by successive
dehydration in ascending grades of alcohol. Tissues were
cleared in xylene and embedded in paraffin wax
(at 58˚- 60˚C). The tissue was then processed routinely and
prepared into paraffin block cut at 6μm thickness using
microtome and stained with Haematoxyline and Eosin [7].
Standard histopathological procedures were followed for
histopathological investigations [8]. Observations were
taken under light microscope.
RESULTS AND DISCUSSION
Histopathological changes have been widely used as
biomarkers in the evaluation of the health of fish exposed
to contaminants, both in the laboratory [9] and field studies
[10]. In normal liver the hepatocytes form a cord-like
pattern. These cords are arranged around tributaries of the
hepatic vein. The liver cells are large in size, polygonal in
shape with homogenous eosinophilic cytoplasm and
centrally located nuclei. A large number of blood sinusoids
are observed and separates the hepatic cords one from
another. Exposure of dimethoate to Garra mullya induced
histopathological changes in the liver. The hepatocytes
have lost their normal architecture. The lumen of sinusoid
contains mainly erythrocytes and macrophages. Numerous
hepatocytes show marked cytoplasmic vacuolization. The
liver cells are degenerated with necrosis because of
lymphocytic infiltration. The histopathological changes of
liver were more pronounced after the exposure period of
fenvalerate [11], similar observation by [12]. Fish exposed
to sub lethal concentration of dimethoate during 7, 14 and
21 days shown considerable degree of alteration in the
liver.
Fig. 1 normal structure of liver showing blood sinosoids
and hepatic cells, centrally placed nucleus containing
nuclei (H&E, 450X)
Fig. 2 On Dimethoate treatment after 7 days structure
of liver shows hemorrhage at some places vacuolation in
the hepatic cells (H&E, 450X)
Fig. 3 On Dimethoate treatment after 14 days of liver
shows haemorrhage at some places sinusoids are
enlarged with vacuolation in the hepatic cells. Extensive
degeneration of hepatocytes (H&E, 450X)
Fig. 4 On Dimethoate treatment after 21 days of liver
shows extensive hemorrhage, necrotic hepatocytes,
cloudy swelling and cytoplasmic vacuolization of
hepatocytes (H&E, 450X)
Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 6
http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 710
Liver being the main organ of various key metabolic
pathways, the effects of a chemical usually appear
primarily on liver. This in turn, provides toxicologists, a
significant site for investigation. In the experimental
studies, liver histology exhibited various abnormalities,
including hyperplasia, nuclear pyknosis, fatty necrosis and
degeneration of hepatocytes leading to tumor and
syncytium formation, which are the indicative of
carcinogenesis.
The histopathological changes were more evident in
specimens exposed to dimetoate and were not observed in
the control fish. The liver cells in G.mullya are polygonal
containing spherical central nucleus (Fig.1). After 7 days of
exposure the hepatocytes became irregular and loose their
polygonal shape. Some cells exhibited cloudy swelling,
their contour becoming indistinguishable. There were many
regions in the liver where cells were highly vacuolated.
Many cells had exhibited pycnosis (Fig. 2 and 3). At the
end of 21 days treatment, pronounced structural changes in
hepatocytes such as focal necrosis, pycnosis, cloudy
swelling and darkly stained specks of necrotic nuclei were
observed (Fig. 4). The majority of insecticides are
bio-transformed in metabolites by liver through various
enzyme systems and as a consequence of this process, liver
undergoes different levels of damages. Alteration like
irregular shaped hepatocytes, cytoplasmic vacuolation and
laterally placed nuclei were also observed in the siluriform
Corydoras paleatus after exposure to organophosphate
pesticide for 96 hrs observed by [13]. [14] also observed
lipid vacuoles, hepatocytes swelling and pyknotic nuclei in
Oreochromic nilotitus exposure to alachlor for 96 hrs.
[15] also observed that dimethoate is strongly hepatotoxic
and severely affect histology, carbohydrate and protein
metabolism on liver of fish, Cyprinus carpio. [16] reported
histopathological lesions induced in the hepatopancreas of
Channa punctatus and Clarius batrachus exposed to
industrial pollutants.
CONCLUSION
The present investigation shows After 7, 14 and 21 days of
exposure of Dimethoate the hepatocytes became irregular,
cloudy swelling and loose their polygonal shape. Liver
cells were highly vacuolated, pycnosis and necrotic nuclei
were observed in the histological structure of liver in Garra
mullya. The changes in liver are biomarker in the
evaluation of health of fish. The metabolic activities of the
fish are affected which in turn become lethal to the fish.
Dimethoate used to protect many field crops against
disease, hence farmer come direct contact it and may affect
their health.
REFERENCES
[1] WHO/IPCS (1996) Principles and methods for assessing
direct immune toxicity associated with exposure to
chemicals. Environ. Health Criteria 180: 110-112.
[2] Sasaki, Y., Izumiyama, F., Nishidate, E., Ishibashi, S.,
Tsuda, S., Matsusaka, N., Asano, N., Saotome, K., Sofuni,
T. and Hayashi, M. (1997) Detection of genotoxicity of
polluted sea water using shellfish and the alkaline
single-cell gel electrophoresis (SCE) assay: A preliminary
study.Mutation. Res. 393; 133-139.
[3] Bondarenko, S., Gan, J., Haver D.L. and Kabashima, J.N.
(2004) Persistence of selected organophosphate and
carbamate insecticides in waters from a coastal watershed,
Environmen. Toxicol. and chemistry, 23(11); 2649-2654.
[4] Sprague J.B. (1973) The ABC’s of pollutant bioassay using
fish. In biological methods for the assessment of water
quality, Am. Soc. Test. Mater. Tech. Publ., 528, 6-36.
[5] APHA AWWA and WPCF (2005) Standard methods for the
examination of water and wastewater. 21st
Edn.Washington
DC USA.
[6] Finney, D.J., (1971) ‘Probit analysis’ Third edition,
Cambridge University, Press.
[7] Luna L.C (1968), manual of histologic staining methods.
Armed Forces Institute of Pathology, 3 rd Ed. McGraw Hill
Book Company, New York.
[8] Robert, R.J. (1989) Fish pathology (2’nd edition), London,
Billier, Tindall. 11, pp 453.
[9] Thophon, S., Kruatrachue, M., Upathan, E.S.,
Pokethitiyook, P., Sahaphong, S. and Jarikhuan S. (2003)
Histopathological alteraions of white seabass Lates calcarifer
in acute and subchronic. Environ. Poll., 121: 307-320.
[10]Teh, S. J., Adams, S.M. and Hinton, D.E. (1997)
Histopathological biomarkers in feral fresh water fish
populations exposed to different types of contaminant stress.
Aquatic Toxicol., 37:51-70.
[11]Sakr, S. A. and Jamal Al lail, S.M. 2005. Fenvalerate Induced
Histopathological and Histochemical changes in the Liver of
the Cat fish Clarius Gariepinus. Egypt. J. Aquatic Biol.
Fish.,6 (2):103-124.
[12]Sridhar, K. and Esther Joice, P. (2012) Carbendazim
induced histopathological and histochemical changes in liver
tissue of common carp, Cyprinus carpio. Inter. J. of Life Sci.
5(1); 65-70.
[13]Fanta, E, F. Ritos, S., Ramao, S. Vinna, A.C.C. and
Friberger, S. (2003) Histopathology of the fish, Corydoras
paleatus contaminated sub lethal of organophosprus in water
and food. Ecotoxicol. Environ. Saf., 54;119-130.
[14]Peebua, P.M., Kruatrachue, Pokethitiyook P. and
Singhakaew, S.(2008) Histopathological alteration in Nile
thilapia, Oreochromic nilotitus in acute and sub chronic
alachlor exposure. J. of Environ. Biology, 29, 325-331.
[15]Ram Narayan Sing. (2013) Effect of dimethoate (30% EC),
an organophosphate pesticide on liver of common carp,
Cyprinus carpio, J. of Environ. Biology. 34,657-661.
[16]Bhattacharya, T., Ray, A.K. and Bhattacharya, S. (1984)
Histopathological lesions in the haepatopancreas of Channa
punctatus (Bloch.) exposed to mixture of mercuric chloride,
phenol and a factory effluent. Matsya.11:1.
Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 6
http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 711
International Journal of Life-Sciences Scientific Research (IJLSSR)
Open Access Policy
Authors/Contributors are responsible for originality, contents, correct
references, and ethical issues.
IJLSSR publishes all articles under Creative Commons
Attribution- Non-Commercial 4.0 International License (CC BY-NC).
https://creativecommons.org/licenses/by-nc/4.0/legalcode
How to cite this article:
Borane V R: Value Histopathological Impact of Dimethoate on the Liver of Freshwater Fish, Garra mullya (Sykes). Int. J.
Life. Sci. Scienti. Res., 2016; 2(6): 708-711. DOI:10.21276/ijlssr.2016.2.6.10
Source of Financial Support: UGC (WRO) Pune, Conflict of interest: Nil

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Histopathological Impact of Dimethoate on the Liver of Freshwater Fish, Garra mullya (Sykes)

  • 1. Int. J. Life. Sci. Scienti. Res., 2(6): 708-711 NOVEMBER- 2016 http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 708 Histopathological Impact of Dimethoate on the Liver of Freshwater Fish, Garra mullya (Sykes) Borane V R* Department of Zoology Jijamata Arts, Science and Commerce College, Nandurbar, Maharashtra, India * Address for Correspondence: Dr. Borane V R, Head cum Associate Professor, Department of Zoology, Jijamata Arts, Sci. and Comm. College, Nandurbar, India Received: 15 Sept 2016/Revised: 30 Sept 2016/Accepted: 19 Oct 2016 ABSTRACT- The present investigation was carried out to the effect of Dimethoate on histopathological changes in liver of freshwater fish, Garra mullya using standard methods. Fish was exposed to sub lethal concentration of Dimethoate (0.0238ppm of 96hrs) for 7, 14, and 21days. Administration of pesticide to determine lesion of liver as indicators of tissue damage. Histopathological changes in liver ranged from vacuolization, necrosis, fibrosis of perivascular region and disposition of yellow brown grains at different time of exposure. Liver histology exhibited various abnormalities, including hyperplasia, nuclear pyknosis, fatty necrosis and degeneration of hepatocytes leading to tumor and syncytium formation, which are the indicative of carcinogenesis. In chronic treatment of dimethoate exposure may pose serious threat to fish health and affect their population. Key-words- Dimethoate, Histopathology, Liver, Garra mullya -------------------------------------------------IJLSSR----------------------------------------------- INTRODUCTION Pesticides are occasionally used indiscriminately in large amounts causing environmental pollution and potential health hazards. Dimethoate is systemic insecticides produced by reacting salts of Dimethyldithio-phosphoric acid with N-methylchloroaecetamide, in aqueous medium in the presence of some organic solvents is widely used against a broad range of insects and mites and is also used for indoor control of houseflies. The extensive use of DM poses a health hazard to animals and humans because of its persistence in soil and crops [1]. One of the major agricultural chemical groups is pesticide which play important role in increasing agricultural productivity through controlling pest. But on the other hand, they cause much damage to the non-target organisms both in terrestrial and aquatic environment. Fish accumulate pollutants directly from contaminated water and indirectly via food chain [2]. The runoff from treated areas enters the river and aquaculture ponds that are supplied by rivers and adversely affect the quality of water surfaces and creates hazards for aquatic life resulting in serious damage to non-target species, including fishes [3]. Access this article online Quick Response Code: Website: www.ijlssr.com DOI: 10.21276/ijlssr.2016.2.6.10 Histopathology deals with the study of pathological changes induced in the microscopically structure of body tissue. Any alteration in normal structure of tissue indicates presence of disease or the effect of toxic substances like heavy metal and pesticides. [4] described histopathology as important tool for evaluating the action of any toxicant at tissue level. Histopathology provides data concerning tissue damage. Histopathological alterations can be used as indicators for the effect of various anthropogenic pollutants on organisms and are a reflection of overall health of the entire population in the ecosystem. The present study was under taken to analyze the impact of chronic concentration of dimethoate in liver of fish, Garra mullya. MATERIALS AND METHODS Healthy adult fish 4-5cm. in length Garra mullya were collected from local river Shivan Dist. Nandurbar, India. Fishes were washed with 0.1% of potassium permagnate (Km No 4) solution to avoid dermal infection. They were then rinsed in water and acclimatized to the laboratory conditions in the department of zoology for two weeks in 1000 l. capacity glass aquaria. Dead fish were removed immediately, Such as mortality may deplete dissolved oxygen with resultant effect on other fishes. During acclimatization fishes were fed with pieces of live earthworm on alternate days. Water also changed once in every day. The experiment was conducted natural and photoperiod of temperature 25·1 ± 3·20 C. Water quality was measure as per by [5], Conductivity 0.64 ± 0.3, Dissolved O2 - 6.3±1.1 (ml/L), pH 8.60 ± 0.3, Research Article (Open access)
  • 2. Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 6 http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 709 Acidity 2.5± 0.1, Alkalinity 44.1 ± 0.5, Total hardness 67.5 ± 0.3. LC50 of dimethoate for 96 hours was determined by probit analysis method [6]. The animals were dissected and liver tissue carefully removed. Tissues were immediately washed in 1% saline solution to remove the adherin mucus and blood and soaked between the blotting papers. The tissue from the control and exposed batches were taken out and preserved in aqueous Bouin’s fluid for 24 to 48 hrs. This was followed by successive dehydration in ascending grades of alcohol. Tissues were cleared in xylene and embedded in paraffin wax (at 58˚- 60˚C). The tissue was then processed routinely and prepared into paraffin block cut at 6μm thickness using microtome and stained with Haematoxyline and Eosin [7]. Standard histopathological procedures were followed for histopathological investigations [8]. Observations were taken under light microscope. RESULTS AND DISCUSSION Histopathological changes have been widely used as biomarkers in the evaluation of the health of fish exposed to contaminants, both in the laboratory [9] and field studies [10]. In normal liver the hepatocytes form a cord-like pattern. These cords are arranged around tributaries of the hepatic vein. The liver cells are large in size, polygonal in shape with homogenous eosinophilic cytoplasm and centrally located nuclei. A large number of blood sinusoids are observed and separates the hepatic cords one from another. Exposure of dimethoate to Garra mullya induced histopathological changes in the liver. The hepatocytes have lost their normal architecture. The lumen of sinusoid contains mainly erythrocytes and macrophages. Numerous hepatocytes show marked cytoplasmic vacuolization. The liver cells are degenerated with necrosis because of lymphocytic infiltration. The histopathological changes of liver were more pronounced after the exposure period of fenvalerate [11], similar observation by [12]. Fish exposed to sub lethal concentration of dimethoate during 7, 14 and 21 days shown considerable degree of alteration in the liver. Fig. 1 normal structure of liver showing blood sinosoids and hepatic cells, centrally placed nucleus containing nuclei (H&E, 450X) Fig. 2 On Dimethoate treatment after 7 days structure of liver shows hemorrhage at some places vacuolation in the hepatic cells (H&E, 450X) Fig. 3 On Dimethoate treatment after 14 days of liver shows haemorrhage at some places sinusoids are enlarged with vacuolation in the hepatic cells. Extensive degeneration of hepatocytes (H&E, 450X) Fig. 4 On Dimethoate treatment after 21 days of liver shows extensive hemorrhage, necrotic hepatocytes, cloudy swelling and cytoplasmic vacuolization of hepatocytes (H&E, 450X)
  • 3. Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 6 http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 710 Liver being the main organ of various key metabolic pathways, the effects of a chemical usually appear primarily on liver. This in turn, provides toxicologists, a significant site for investigation. In the experimental studies, liver histology exhibited various abnormalities, including hyperplasia, nuclear pyknosis, fatty necrosis and degeneration of hepatocytes leading to tumor and syncytium formation, which are the indicative of carcinogenesis. The histopathological changes were more evident in specimens exposed to dimetoate and were not observed in the control fish. The liver cells in G.mullya are polygonal containing spherical central nucleus (Fig.1). After 7 days of exposure the hepatocytes became irregular and loose their polygonal shape. Some cells exhibited cloudy swelling, their contour becoming indistinguishable. There were many regions in the liver where cells were highly vacuolated. Many cells had exhibited pycnosis (Fig. 2 and 3). At the end of 21 days treatment, pronounced structural changes in hepatocytes such as focal necrosis, pycnosis, cloudy swelling and darkly stained specks of necrotic nuclei were observed (Fig. 4). The majority of insecticides are bio-transformed in metabolites by liver through various enzyme systems and as a consequence of this process, liver undergoes different levels of damages. Alteration like irregular shaped hepatocytes, cytoplasmic vacuolation and laterally placed nuclei were also observed in the siluriform Corydoras paleatus after exposure to organophosphate pesticide for 96 hrs observed by [13]. [14] also observed lipid vacuoles, hepatocytes swelling and pyknotic nuclei in Oreochromic nilotitus exposure to alachlor for 96 hrs. [15] also observed that dimethoate is strongly hepatotoxic and severely affect histology, carbohydrate and protein metabolism on liver of fish, Cyprinus carpio. [16] reported histopathological lesions induced in the hepatopancreas of Channa punctatus and Clarius batrachus exposed to industrial pollutants. CONCLUSION The present investigation shows After 7, 14 and 21 days of exposure of Dimethoate the hepatocytes became irregular, cloudy swelling and loose their polygonal shape. Liver cells were highly vacuolated, pycnosis and necrotic nuclei were observed in the histological structure of liver in Garra mullya. The changes in liver are biomarker in the evaluation of health of fish. The metabolic activities of the fish are affected which in turn become lethal to the fish. Dimethoate used to protect many field crops against disease, hence farmer come direct contact it and may affect their health. REFERENCES [1] WHO/IPCS (1996) Principles and methods for assessing direct immune toxicity associated with exposure to chemicals. Environ. Health Criteria 180: 110-112. [2] Sasaki, Y., Izumiyama, F., Nishidate, E., Ishibashi, S., Tsuda, S., Matsusaka, N., Asano, N., Saotome, K., Sofuni, T. and Hayashi, M. (1997) Detection of genotoxicity of polluted sea water using shellfish and the alkaline single-cell gel electrophoresis (SCE) assay: A preliminary study.Mutation. Res. 393; 133-139. [3] Bondarenko, S., Gan, J., Haver D.L. and Kabashima, J.N. (2004) Persistence of selected organophosphate and carbamate insecticides in waters from a coastal watershed, Environmen. Toxicol. and chemistry, 23(11); 2649-2654. [4] Sprague J.B. (1973) The ABC’s of pollutant bioassay using fish. In biological methods for the assessment of water quality, Am. Soc. Test. Mater. Tech. Publ., 528, 6-36. 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  • 4. Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 6 http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 711 International Journal of Life-Sciences Scientific Research (IJLSSR) Open Access Policy Authors/Contributors are responsible for originality, contents, correct references, and ethical issues. IJLSSR publishes all articles under Creative Commons Attribution- Non-Commercial 4.0 International License (CC BY-NC). https://creativecommons.org/licenses/by-nc/4.0/legalcode How to cite this article: Borane V R: Value Histopathological Impact of Dimethoate on the Liver of Freshwater Fish, Garra mullya (Sykes). Int. J. Life. Sci. Scienti. Res., 2016; 2(6): 708-711. DOI:10.21276/ijlssr.2016.2.6.10 Source of Financial Support: UGC (WRO) Pune, Conflict of interest: Nil