Int. J. Int. J. Cur. Tr. Res (2014) 3 (2):163-174 ISSN:2278-8042 www.injctr.com Impact anthropogenic activities on the histological parameters Labeo calbasu caught from Betwa River in Raisen District (M.P) Altaf Hussain Mir* Mohd Younus Department Zoology, Barkatullah University, Bhopal (M.P) - 462 026, India. Received: 5 December, 2014 Accepted: 29 December 2014/ Published online 30 December 2014. INJCTR 2014 Abstract The present investigation was undertaken to assess the effect human hazards on the histology investigated organs like gills, liver and kidney Labeo calbasu. The results showed that the histological changes induced in gills, were mainly represented by dilation blood capillaries in gills, swelling epithelium, hyperplasia epithelial lining, ruptured basement membrane and necrosis in respiratory epithelium. Liver tissues revealed compactly arranged hepatocytes with eccentric or pyknotic nuclei, degeneration endothelial lining cells, clumped hepatic portal vein, necrosis in hepatic cells, vacuolation in hepatic cells was observed. Normal kidney showed large number nephrons, each consisting renal corpuscles, proximal and distal convoluted tubules. However affected kidney showed marked shrinkage and breakage haemopietic tissues, degeneration renal epithelium. Glomeruli showed shrinkage and breakage in Bowman s capsule, necrosis in renal tubules, hypertrophy epithelial cells renal tubules. Keywords: Betwa River, Labeo calbasu, Histological parameters. Introduction As all us are aware about the fact that water is a primary driving force for major physical, chemical and biological changes all over the world. It is pertinent that oceans and seas contain approximately 97% where as fresh water resources contain 3% the entire water reserve the earth. It is impossible to sustain life more than few days without water (WHO, 2005). Water also played a pivotal role in the evolution human civilization e.g. Rivers like Nile, Tigris, Indus and Ganges have been recognized as the life lines for ancient civilizations. Until recent population explosion from few decades, that has caused an immense pressure on water resources, fresh water as a source has never been an issue concern. However, due to intensive agricultural practices, extensive urbanization, rapid industrialization and burning fossil fuels are amongst anthropogenic activities, which have increased rapidly and have been considered important for changing the natural conditions an aquatic ecosystem. Thus degradation natural Corresponding author* E.mail: altafraja07@gmail.com ecosystems ultimately alters the structure and functions aquatic biota due to the adverse effect human activities. Not only in one part or in one area, but also throughout the world, fresh water resources are facing number environmental problems largely associated with anthropogenic activities in their catchment areas. Especially in poor and developing countries, the pollution fresh water resources is a matter great concern because without considering environmental protective measures, pollutants are discharged directly or indirectly into rivers and streams. There are various types agents which are responsible for degrading the water quality e.g additives like fertilizers, pollutants and other soil improving agents which are used to increase the crop production, after heavy rain fall get dissolved and leachdown to ground water or moves to rivers/ streams with surface run f after soil saturation. Similarly, metals are also one the important contaminant groups responsible for deterioration surface water quality, which
164 either originate naturally from parent rock material as a result weathering or contributed from anthropogenic processes. Heavy metals like iron, copper, nickel, chromium and zinc are essential in living organisms because their structural and functional roles in various physiological processes (Wepener et al., 2001), where as non-metals have no known role in metabolic functions the organisms and are toxic even in trace amounts. The absorbed heavy metals in organisms can bind with cellular components (i,e nucleic acids and proteins) and interfere with metabolic processes that lead to genotoxic, neurotoxic, mutagenic effects. So accumulation heavy metals disrupts the physiology and histology aquatic organisms that may lead to death. For analysis water pollution in a river, aquatic organisms are used because they are sensitive to any physical and chemical change in river water. Aquatic organisms such as algae, invertebrates, fishes and amphibians are important organisms for understanding the impacts human activities in river ecosystem. Among aquatic organisms, fishes are good indicators pollution stress and have wide range tolerance. Fishes are sensitive to any type human disturbance such as industrial effluents, municipal waste, river discharge and strongly influence the distribution, migration, colonization fishes (Plafkin et al., 1989). So from few years importance protection, restoration and management aquatic resources has been realized all over the globe. Int. J. Cur. Tr. Res (2014) 2 (1):163-174 Hence the present study was taken to study the effect anthropogenic activities in Betwa river in Raisen district(m.p) on gills, liver and kidney Labeo calbasu which will provide first hand information regarding their histological damages caused by anthropogenic activities. Area study The Betwa or Vetravati is a river great antiquity and immense mythological and religious values for the people the Malva region M.P for hundreds years. This second largest river the Malva region is not only important from the geo-ecological point view but also has a significantly potent socioeconomic impact on the area through which it flows. Betwa is an important tributary the Yamuna, which in turn is a tributary the river Ganga. This makes the Betwa as an important river sacred gangetic river system. The river Betwa rises from the main Vindyan range in the extreme southwest the Raisen district at jhirribarod village (Longitude 77024 E & latitude 23.20N). It flows for estimated total length 573 km which 216km in M.P and 98 km in U.P and finally joins the river Yamuna at Hamirpur in U.P (Longitude 80.130 and Latitude 25.550N). The river has a huge catchments area around 46580 sq.kms. During the course its flow, Betwa receives 14 tributaries which as many as 11 are located in the Madhya Pradesh. A site named as Ramghat on the Betwa River is a sacred ghat situated in the Vidisha district M.P. Fig: Map Betwa River having catchment area 216 Km² in M.P.
Int. J. Cur. Tr. Res (2014) 2 (1): 163-174 Nayapura, Mandideep (site 1) Industrial nalla, Mandideep (site 2) Kaliyasot confluence, Mandideep (site 3) 165 It is a religious ghat and many temples are situated on both the banks the river. About 1km length this stretch is restricted for fishing and fishermen avoid to fish in this stretch. The river Betwa plays a significant role in the human life the villages located in Mandideep, Bhojpur, and Raisen areas. It has become polluted at some places Mandideep due to industrial activities and the confluence sewage, domestic wastes and industrial effluents many big and small enterprises with various types organic compounds and heavy metals detrimental to human health and aquatic organisms. Urban areas, farms, factories and individual households- all contribute to the contamination this river. Moreover, water Betwa River is widely used for drinking, agricultural and power generation purposes. The water quality in the stretch the river Betwa extending from its origin near Mandideep industrial area up to Bhojpur remains poor because the regular inflow domestic wastes the Bhopal city through the Kaliyasot river and industrial/domestic water from Mandideep. Owing to the above facts and public complaints from local agencies the overall quality the river water in this area has been marked as poor. The quality the Betwa river water improves after Bhojpur due to the confluence some smaller rivers like Ricahan, Dawar etc. flowing from the forest area located in the central part the district. The average quality in the north-western part the district i.e. towards Vidisha District falls under a medium category with some patches low quality attributed to the industrial/domestic contamination from isolated large industries and scattered settlements (Lesser, 1978). The present study was undertaken to assess the impact anthropogenic activities in the catchment area Betwa River in Raisen district on Labeo calbasu (Ham.) Materials and Methods Road Bridge Pugneshwar, Raisen (site 4) For evaluation biological parameters four fish samples at different sites were collected from the highly polluted belt and less polluted belt the Betwa River. One fish sample was collected from the area Nayapura, Mandideep (site 1).The second fish sample was taken at Industrial Nalla, Mandideep(site 2) while as other two fish samples were collected from Kaliyasot confluence, Mandideep (site 3) and Road Bridge Pugneshwar, Raisen (site 4).All the above samples collected from sites1,2,3 and 4 Betwa river were considered fish samples from polluted water (test fish samples) and were compared with the fish sample collected from Patra fish farm, Bhopal( Madhya Pradesh) which was considered as the control fish sample.
166 The live specimens Labeo calbasu measuring about 10-20 cms and weighing approx. 80-125 gms were collected and brought to laboratory in polythene bags for studying the combined effect agrochemicals and heavy metals due to anthropogenic activities. However, control fishes were transferred into the glass aquaria 50 liters capacity containing well-aerated, unchlorinated ground water for 15 days acclimatization. The fishes were screened for any physical damage, disease and mortality. The immobilized, injured, abnormal and dead fishes were discarded immediately. Control fishes were fed every day twice with wheat flour pellets, boiled egg protein and grounded dried shrimps purchased from local fish market. Before stocking, the aquaria were washed with 0.1% KMnO 4 to free the walls from any possible fungal infections if any and acclimatization was judged satisfactory when the incidence fish monolith was less than 10% total fish during one week prior to the commencement the experiment. The fishes were also treated with 0.1% KMnO 4 solutions to check any possible bacterial infections. The media in aquaria for control fish was renewed on alternate days to prevent accumulations metabolites. In addition, the individual fish in the control aquaria were considered dead when they failed to respond to touch stimulus. After the death each fish, its body was removed immediately from the container along with the aquaria water allotted. After 15-day acclimatization control fish, survived individuals in the container were dissected in the laboratory. Both the test and control fish samples were compared for various histological parameters to assess the effect water pollution on the fish health with extrapolation to hazards to human health. The fishes were sacrificed by decaefication the head and their gills, liver and kidney were dissected out quickly and were fixed in Bouin s fixative, then paraffin sections were cut at 5-7 µm thick. Paraffin sections were stained with haematoxylin using eosin as the counter stain for normal histology. Results and Discussion In the present investigation, the following histological alterations were observed in Labeo calbasu caught from Betwa River at different sites. Gills The histology gills from control group showed two rows elongated gill filaments attached to the gill arch. Each it bears the primary gill lamellae. Primary gill lamellae bear secondary lamellae on both sides; each Int. J. Cur. Tr. Res (2014) 2 (1):163-174 secondary lamellae consists a central vascular layer surrounded by thin layer connective tissues and respiratory epithelium. The vascular layer consists a network capillaries. A number pilaster cells are arranged in rows between the capillaries. The mucous and mast cells are present in the gill lamellae. In gills Labeo calbasu caught from site1 (Nayapura, Mandideep), primary gill lamellae had mild congestion blood vessels, fusion lamellae and marked hyperplasia the branchial arches. Dilation the blood capillaries, hyperplasia the epithelial lining, abnormal raising or swelling epithelium. Necrosis and shortening as well as fusion the secondary lamellae were also observed. Histology the gills Labeo calbasu caught from site 2 (Industrial nallah, Mandideep), showed that the basement membrane was found ruptured and respiratory epithelium was separated from the vascular layer. The necrosis and cytoplasmic vacuolation was observed. The tips gill lamellae were found bulged. Histology the gills Labeo calbasu caught from site 3(Kaliyasot Confluence, Mandideep), showed necrosis in the respiratory epithelium and secondary gill lamellae were found bulged. Similarly histology gills Labeo calbasu caught from site 4 (Road Bridge, Pugneshwar, Raisen), the gill lamellae showed erosion the respiratory epithelium. The pilaster cells were found degenerated. Hyperplasia was observed in the vascular layer and necrosis was also observed in respiratory epithelium.the above study is close to the results found by Konar, (1970) and Srivastava et al., (1988) who have observed the effect DDT on the gills Heteropneustes fossilis. Hypertrophy mucus cells, blood corpuscles and cartilaginous axis cells along with severe necrosis and epithelial separation from the basement membrane were observed. Tilak, Veeraiah and Yacob, (2001) studied the histopathological changes in the tissues gill,liver and kidney the fish Ctenopharyngodon idellus when exposed to sublethal concentration 20% EC fenvalerate, a synthetic pyrethriod and observed the necrosis, vacuolar degeneration and atrophy. Velmurugan et al., (2007) studied the effect lamda-cyhalothrin on gills, kidney, liver and intestine Crihinnus mrigala and observed that the epithelial hyperplasia, epithelial necrosis, epithelial odema, shortening secondary lamellae and lamellar fusion in gills. Saravanan et al., (2010) studied the effect chronic exposure endosulphan on Labeo rohita and observed that in gills treated fishes, the tip primary lamellae were shapeless and seemed to be eroded and RBCs were ten observed between secondary lamellae and primary lamellae. Riji John and Jayabalan, (2007) have observed hyperplasia, lamellar fusion, curling and bulging tips primary lamellae, exudation erythrocytes, when the fish Cyprinus Carpio was exposed to sublethal concentration endosulphan.
Int. J. Cur. Tr. Res (2014) 2 (1): 163-174 167 HEL= Hyperplasia epithelial lining, SEC=Swelling epithelial cells, N= Necrosis, CBV=Congestion blood vessels, FL=Fusion lamellae MC PC SGL PGL HEL= Hyperplasia epithelial lining, SEC=Swelling epithelial cells, N= Necrosis, CBV=Congestion blood vessels, FL=Fusion lamellae. SEC N FL HEL CBV
168 Int. J. Cur. Tr. Res (2014) 2 (1):163-174 N= Necrosis, BGL=Bulged gill lamellae, RBM= Ruptured basement membrane N BGL RBM N= Necrosis, BSGL= Bulged secondary gill lamellae N N BSGL
Int. J. Cur. Tr. Res (2014) 2 (1): 163-174 169 DGP= Degeneration pilaster cells, EGL=Erosion gill lamellae, NRE= Necrosis in renal epithelium, H= Hyperplasia EGL H NRE DGP NH= Normal hepatocytes, CT= Connective tissue, P= Phagocytes in connective tissue P CT NH CV= Cytoplasmic vacuolation, HCN= Hepatocellular necrosis, DEC= Degeneration endothelial lining cells DEC CV HCN HCN
170 Int. J. Cur. Tr. Res (2014) 2 (1):163-174 HCN= Hepatocellular necrosis, VHC= Vacuolation hepatic cells V HCN VHC VHC HCN= Hepatocellular necrosis, HPVC= Hepatic portal vein clumped HCN HCN VHC HPVC DCT=Damaged connective tissue, DH= Damaged hepatocytes, HTR= Hepatic tissue ruptured, HCN= Hepatocellular necrosis HCN DCT HTR DH
E Int. J. Cur. Tr. Res (2014) 2 (1): 163-174 171 GC=Glomeruli capsule, PCT= Proximal convoluted tubule, N= Nephron GC N GC PCT GC SGC= Shrinkage glomeruli capsule, DRE= Degeneration renal epithelium SGC DRE SBC= Shrinkage Bowman s capsule, NRT= Necrosis renal tubules, DDCT= Degenerated distal convoluted tubule, DPCT= Degenerated proximal convoluted tubule NRT DPCT DCT SBC SG VBC DDCT
172 Int. J. Cur. Tr. Res (2014) 2 (1):163-174 DCT= Degenerated convoluted tubules, SG= Shrinkage glomeruli, VBC= Vacuolation Bowman s capsule SG DCT VBC NTE= Necrosis tubular epithelium, DG= Degeneration glomeruli, HT=Hypertrophy, CBC=Contraction Bowman s capsule NTE DG NTE HT CBC Liver Histology the liver from control group showed normal homogenous mass hepatocytes with centrally located nuclei and granular cytoplasm. Hepatocytes are polyhedral in shape and are arranged in groups. The connective tissue is in definite pattern. Phagocytes are present between connective tissue. The histology the liver Labeo calbasu caught from site 1(Nayapura, Mandideep) revealed compactly arranged hepatocytes but with a strong cytoplasmic vacuolization with eccentric or pyknotic nuclei. Hepatocellular necrosis with parenchymal vacuolization, breakdown cell boundaries, NTE CBC HT
173 swelling and degeneration the endothelial lining cells. Histology the liver Labeo calbasu caught from site 2 (Industrial Nallah, Mandideep), exhibited destruction and heavy necrosis in hepatic cells. Empty spaces appeared in the cytoplasm, degeneration and vacuolation in hepatic cells was observed. Likewise, histology the liver Labeo calbasu caught from site 3 (Kaliyasoot Confluence, Mandideep): necrosis was noticed in the exocrine part the liver. Hepatic portal vein was found clumped. The histology the liver Labeo calbasu caught from site 4 (Road Bridge, Pugneshwar, Mandideep), the hepatocytes and connective tissue were found damaged and blood vessels were separated. The hepatic tissue was found ruptured. The hepatocytes showed necrosis and degeneration with small spaces in between the hepatic cords. Liver is the major metabolic center, and any change to this organ would subsequently do so many physiological disturbances leading to subsequent mortality fish. The histological alterations observed in the present investigation are in accordance to the investigations made by different workers during exposure to different pollutants. Dalela et.al. (1984) reported necrosis, hypertrophy and atrophy in the liver tissues, loss polygonal shape liver cells, splitting the cells and formation spaces in the tissues after exposure Cyprinus carpio to lethal and sublethal concentration copper and cadmium. Bakre, (1985) reported cellular damage, nuclear hypertrophy hepatocytes, vacuolation and necrosis leading to lysis, increase in blood sinuses, bile canaliculi and bile pigments become prominent in Gambusia affinis exposure to mercury chloride. Anita kumara and Ram Kumar, (1995) observed the histopathological lesions caused by industrial effluents in kidney, liver and gills fish Heteropneustes fossilis in Hussain sagar and reported that the degeneration hepatic cells, necrosis, vacuolation hepatocytes and rupture nuclear and cell membrane in liver. The histological alternations noticed in the present study are in accordance to the chronic exposure to the different pollutants. The lesions developed in the liver might be due to the cumulative action toxicant on blood and ultimately to other cellular structures. Kidney The kidney is a vital organ body and proper kidney function is to maintain the homeostasis. It is not only involved in removal wastes from blood but it is also responsible for selective reabsorption, which helps in maintaining volume and ph blood and body fluids and erythropoiesis. The kidney is one Int. J. Cur. Tr. Res (2014) 2 (1):163-174 the first organs to be affected by contaminants in the water. Thus, the present investigation will help in elucidating the normal histology kidney Labeo calbasu (Ham.) brought from Patra fish farm and effect pollutants caused by anthropogenic activities on the histology fishes collected from selected sites Betwa River. The histology the kidney from control group Labeo calbasu is made up a large number nephrons, each consisting renal corpuscles, proximal and distal convulated tubules. The intertubular space is full lymphoid tissue which is uneventually distributed and showing well vasculorised glomerulus capsule and renal tubules. The kidney histology Labeo calbasu caught from site 1 (Nayapura, Mandideep), showed marked glomeruli shrinkage and breakage the outer wall haemopoietic tissues and also exhibited degeneration and flattening renal epithelium. Histology the kidney Labeo calbasu caught from site 2 (Industrial Nalla, Mandideep), also showed glomeruli shrinkage and breakage leaving a large space in Bowman s capsule. Haemopoietic tissues also showed degeneration. Necrosis was observed in renal tubules. The epithelial cells the proximal convoluted tubule were degenerated and their nuclei were found enlarged. Histology the kidney sections Labeo calbasu caught from site 3(Kaliyasot, Mandideep), showed disintegration convoluted tubules with large intra- cytoplasmic vacuoles in the epithelial cells. Shrinkage and degeneration the glomeruli, increase space within the Bowman s capsule was also seen. Histology the Kidney Labeo calbasu caught from site 4 ( Road Brigde, Pugneshwar, Raisen), showed necrosis tubular epithelium, degeneration the epithelial cells the renal tubules, degeneration the glomeruli, hypertrophy the epithelial cells the renal tubules, narrowing the tubular lumen and glomerular contraction in the Bowman s capsule. Similar histopathological lesions were recorded previously in kidney tissues treated fishes with different pollutants. They include necrosis and degeneration hemopoietic tissue and vacuolation renal tubular cells in winter pounder (Pseudopleuronectes americanus) poisoned by copper (Baker, 1969). Sastray and Sharma,(1979) observed a number striking changes in the histological structure the kidney Channa punctatus exposed to sublethal concentration 0.01ppm endrin for a span 30 days and found that the shrinkage glomerulus was the visible sign intoxicatication. Most common alterations
Int. J. Cur. Tr. Res (2014) 2 (1): 163-174 found in the kidney fishes exposed to water contamination are tubule degeneration (cloudy swelling and hyaline droplets) and changes in the corpuscle, such as dilation capillaries in the glomerulus and reduction Bowman s space (Takashima and Hibiya, 1995). The present study is in proximity with the results observed by Tilak et al., (2001) who noticed severe necrosis, cloudy swelling in the renal tubules, cellular hypertrophy, granular cytoplasm and vacuolization in kidney tissues Ctenopharyngodon idella after exposure to fenvalerate. Cengiz, (2006) observed degeneration in the epithelial cells renal tubules, pycnotic nuclei in the hematopietic tissue, dilation glomerular capillaries, degeneration glomerulus, intercytoplasmic vacuoles in the epithelial cells with hypertrophied cells and narrowing the tubular lumen was observed in the kidney tissues fish exposed to detamethrin. Our results are in agreement with the changes in the kidney Zebra fish, Danio rerio exposed to sublethal concentrations chlropyriphos (Scheil et al., 2009). References Anitha Kumari and Shree Ram Kumar (1995). Effect polluted water on histochemical localization carbohydrates in a fresh water teleost Heteropneustes fossilis (Bloch.) from Husssain Sagar Lake, Hyderabad, Andhra Pradesh. Poll. Res. 16(3): 197-200. Baker, J.T.P. (1969). Histological and electron microscopical observations on copper poisoning in the winter flounder (Pseudopleuronectes americanus). J. Fish. Res. Bd. Can. 26:2785-2793. Bakre, V.P. (1985). Role environmental variables on the biology aquatic vertebrates in relation to mercury pollution.ph.d Thesis, University Rajasthan, Jaipur, India. Cengiz, E. I. (2006). Gill and kidney histopathology in the freshwater fish Cyprinus carpio after acute exposure to deltamethrin. Environ. Toxicol. Pharm. 22, 200-204. Dalela, K.A. Kumar and R.B. Sharma (1984). Toxicity copper and cadmium to fish Cyprinus carpio. Histopathological approach paper presented in National Symposium on assessment Environmental pollution due to industrialization and urbanization at Aurangabad; India. December 20-2, in abstract: pp37. Konar, S.K. (1970). Pesticides and aquatic ecosystems. Indian. J. Fish. 22(1&2):80-85. Lesser, H. (1978) Medium scale geomorphological mapping and landscape ecology // Guide to medium scale geomorphological mapping IG.U commission Geomorphological survey and mapping/ Department Geography. Basel, P. 149-151. Plafkin, J.L., M.t. Barbour, K.D. Porter, S.K. Gross and R.M. Hughes (1989). Rapid bioassessment protocols for use in streams and rivers: Benthic 174 macro vertebrates and fish. Environmental protection Agency EPA/ 440 /4-89 /001. Washington DC, USA. Riji John, K. and N. Jayabalan (2007). Sublethal effects endosulfan on the histology and protein pattern Cyprinus carpio gill. J. Appl. Ichth. 9 (1): 49 56. Sastry, K.V. and S.K. Sharma (1979). Toxic effects endrin on liver and kidney a fresh water teleost fish. Proc. Symp. Environ. Biol. 337-342. Srivastava, S.J., N.D. Singh, R. Sinha, A.K. Srivastava (1988). Malachite green induced histopathological lesions in the liver a fresh water Catfish, Heteropneustes fossilis (Bloch). J. Adv.Zool. 19(1),46-49. Scheil, V., A. Zurn, H. Kohler and R. Triebskorn (2009). Embryo development, stress protein (Hsp70)responses, and histopathology in Zebra fish, Danio rerio following exposure to nickel chloride, chlorpyrifos, and binary mixtures them. Environ. Toxicol. 25(1): 83-93. Saravanan, T.S., P. Rajesh and M. Sundaramoorthy (2010). Studies on effects chronic exposure endosulfan to Labeo rohita. J. Environ. Biol. 31: 755-758. Takashima, F. and T. Hibiya (1995). An atlas fish histology: normal and pathological features. 2. ed. Kodansha, Tokyo. Tilak, K.S., K. Veeraiah and S. Vijaya Lakshmi, (2001) Biochemical changes induced in fresh water fish Labeo rohita (Hamilton) exposed to pesticide mixture. Asian J. Microbiol. Biotech. Environ. Sci. 3, 315-319. Velmurugan, B., Selvanayagam, M., Cengiz, E.I. and Unlu, E., (2009) Histopathological changes in the gill and liver tissues freshwater fish, Cirrhinus mrigala exposed to dichlorvos. Braz. Arch. Biol. Technol. 52: 1291-1296. Wepener, V.J., Van Vuren, H.J. and du Preez, H.H. (2001) Uptake and distribution a copper, iron and zinc mixture in gill, liver and plasma a freshwater teleost, Tilapia sparrmanii. Water SA 27: 99-108. WHO, (2005) Ecosystems and human well-being: health synthesis: a report the Millennium Ecosystem Assessment. WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland.