Effect of Microcystin on Hepatotoxicity of Molly Fish (Poecilia sphenops) | Open Access Journals

ISSN ONLINE(2319-8753)PRINT(2347-6710)

Effect of Microcystin on Hepatotoxicity of Molly Fish (Poecilia sphenops)

R.Sonia1 and R.Ramanibai2*
  1. Aquatic Biodiversity Unit, Department of Zoology, University of Madras, Guindy campus, Chennai, India
  2. Professor, Aquatic Biodiversity Unit, Department of Zoology, University of Madras, Guindy Campus, Chennai, India
Related article at Pubmed, Scholar Google

Visit for more related articles at International Journal of Innovative Research in Science, Engineering and Technology

Abstract

Cyanobacterial blooms are the most common problem in eutrophic or hyper eutrophic water bodies. The blue green algae, Microcystis aeruginosa occurred and dominated during the summer season in Velacherry Lake. Along with M. aeruginosa, M. flos-aquae and M. wesenbergii co inhabit the freshwater habitats. Molly fish were used as animal models to detect the toxicity of microcystins consumed along with the diet intake. Fish were exposed to a known quantity of Microcystis (6 X 104 cells/mL) mixed in an aquarium tank. Survival rate and growth rates were monitored daily. Soon after death, various vital organs of the experimental fish were removed for histopathological analyses. Results revealed that the mortality of the Molly fish may have occurred due to the toxic effect of microcystins. Signs of toxicity exhibited by Molly fish indicated that the bloom samples were predominantly hepatotoxic in nature.

Keywords

Poecilia, Microcystis, microcystins, toxicity, cyanobacteria.

INTRODUCTION

Cyanobacteria are prokaryotes that possess cell walls composed of peptidoglycon and lipopolysaccharide layers inside the cellulose of green algae. Morphological diversity of cyanobacteria ranged from unicellular levels, to small colonies of cells, to simple and branched filamentous forms [9].Toxic blooms of cyanobacteria has lead to death of livestock and has caused illness and sometimes even death in human beings. [3, 7].
Cyanobacteria have a number of special properties besides their ability to fix N2 using the enzyme nitrogenase. In general, the nutrient overload favours the cyanobacterial bloom development. The freshwater cyanobacteria, such as Microcystis, Anabaena, Oscillatoria and Nostoc have been reported to produce toxins known as hepatotoxic microcystins[6].
Microcystin have also accumulated in aquatic vertebrates [14,19]. Mohammed Hussain [18] studied the accumulation of microcystins present in different vital organs particularly in the liver of fish.
A brief experimental study was carried out to evaluate the toxic effects of microcystins produced by cyanobacteria on Molly fish (Poecilia sphenops) through histopathological examinations.

MATERIALS AND METHODS

Study Area

Velacherry Lake, located in the southern part of Chennai city, is an example of an aquatic ecosystem suffering from various kinds of environmental impacts. This lake is situated at 12º 59’ 15” latitude and 80 º 30’ 45” longitude with a capacity of 63 mc ft covering an area of about 962 acres and its full tank level is 6.550 m. It is utilised by the public for various purposes other than drinking.

Microcystis Collection

The sampling was conducted during the high algal bloom from the Velacherry lake using 50 μm plankton net. From the algal samples the toxic cyanobacteria were identified and isolated (Table1).
Image

Poecilia sphenops Capture and Maintenance

Healthy fish with a 5±1 cm length devoid of cyanobacteria as well as other parasitic infections were purchased from a nearby aquarium shop. The fish tanks were set up with continuous aeration under room temperature. The fish were fed with commercial fish food and were acclimatized for one week prior to the start of the experiments.

Experimental Design

One control and two experimental fish in each tank were maintained for the experiment. The cyanobacteria Microcystis aeruginosa, M. flos-aquae and M. wesenbergii were given as the diet at a concentration of 6 X 104 cells/mL to each fish individually. Survival rate was monitored and body weight of the fish measured daily. Soon after mortality, the fish were sacrificed and the tissues (liver, gills and ovary) were fixed in aqueous Bovines fluid before histochemical analyses. Finally, 5μ sections were stained with Haematoxyllin and observed under the microscope for histopathological changes. Serum bilirubin was estimated in the liver by the method of Malloy and Evelyn [17].

RESULTS

A decrease in the body weight of the experimental fish was observed. Body weight of the fish measured 2.3±2 gm initially and decreased to a final weight of 1.6±5 gm (Figure1). There was no mortality of fish noticed among the control fish. The experimental fish survived up to seven days. On the eighth day fish mortality occurred (Figure 2).
Image
Image

Bilirubin Estimation

The experimental fish were fed with Microcystis sp. The concentration of bilirubin was 0.1 mg/dl for control fish whereas the experimental fish showed 0.45 mg/dl (Figure 3). The breakdown of heme in red blood cells results in the formation of bilirubin - the main bile pigment. Hepatocyte necrosis occurred when Microcystis was given as a diet.
Image

Histopathological studies

Histopathological examination carried out on the liver of the experimental fish (Poecilia sphenops) showed mainly architectural changes in the liver (Figures 4A and 4B). Parenchyma architecture of the liver was disturbed and hepatocytes appeared to be swollen with granular cytoplasm. The hepatocyte cells showed condensed chromatin material with sinusoidal epithelium. Blood spilling into the liver tissues and hepatocyte necrosis was observed. Normal nucleus and hepatocytes were seen in the control fish tissues.
Control fish exhibited straight gill lamellae with compact cellular structure and large numbers of mucous cells at the tip of primary lamellae. Experimental fish showed hypertrophy in epithelial cells, lamellar aneurism and vascular congestion. The mucous cells were large and prominent (Figures 4C and 4D). Necrosis, epithelial lifting and lamellar fusion, lamellar aneurism, hypertrophy, apoptosis in the cells, and lamellar blood dilation were observed in the gills.
Control fish showed mature oocytes with nucleus and nucleolus and oocytes filled with yolk granules. Experimental animals showed ruptured follicle walls in the oocyte and the compactness of the tissue was also disturbed. The developing oocyte had less yolk, follicle cells were deformed and necrosis was also observed at some places in the ovary (Figures 4E and 4F).
Image
E. Ovary Section of Experimental Fish
F. Ovary Section of Control Fish

DISCUSSION

Mohammed and Hussein [18] reported an accumulation of microcystins in different organs, particularly in the liver of tilapia fish (Oreochromis niloticus) from Egyptian fish farms with toxic blooms of Microcystis aeruginosa. Microcystins also serve as tumour promoters. Microcystis blooms occur in quiet warm water with nutrient rich conditions. Serious illnesses such as hepatoenteritis, symptomatic pneumonia and dermatitis may result if humans consume or have contact with water contaminated by the toxin producing cyanobacteria [10, 27]. The broad specificity anion transport bile acid carrier plays a main role in the entry of toxins into hepatocytes of the liver and other targeted organs [24]. Microcystins are very stable in water and show resistance to pH extremes and temperatures up to 300ºC.
The impact of cyanobacteria on fish populations is not only due to the toxins that they release but they also create anoxic conditions and release of compounds during and after bloom collapse [2,1]. Nevertheless, Shebulsky [25] proved that fish death occurred in the presence of a toxic strain of M. aeruginosa with and without an oxygen supply indicating that toxins were also involved in the fish kill.
Tancella et al., [27] proved that microcystin administered orally to fish caused massive hepatic necrosis followed by death. The same results were also observed in this study. Obrehm et al., [20] reported that microcystin may affect the development of fish embryos. Cyanotoxins are usually found inside the cells being degraded very slowly [21,26,23]. Animal intoxication may occur either by direct ingestion of liver cells or by drinking contaminated water after the bloom collapses and toxins are released from the cells.
The liver was the most affected organ in all such cases because microystins were found to be potent hepatotoxins for mammals and fish [8,11]. Microcystins accumulate mainly in the liver but also in other organs, for example, the kidneys, gills and intestine[22,30,13,16]. In the present study, the accumulation of microcystins was observed in the liver, gills and gonads.
Cabris et al., [5] reported an increase in the level of bilirubin concentration in Cyprinus carpio due to microcystin intoxication. The same results were obtained in the Molly fish (Poecilia sphenops). In the present study, apoptotic cells were formed in the liver, gills and ovary of the experimental fish.

CONCLUSION

Oral doses of cyanobacteria can cause enhanced physiological stress as well as continued pathological changes in the liver, gills and ovaries possibly leading to decreased functionality of these organs. The combination of stress and organ damage might explain the reduced weight and mortality of the fish.

Acknowledgement

One of the authors (R.R) thank UGC New Delhi for funding (MRP) support and R. Bharathi for the laboratory assistance.

References

1. Ayles G.B., J.G.I. Lark, J. Barica and H.Kling, “Seasonal mortality in rainbow trout (Salmo gairdneri) planted in small eutrophic lakes of central Canada”: J.Fish. Res. Board Can., 37: 647 – 655. 1986.

2. Barica.J., “Collapses of Aphanizomenon flos aquae blooms resulting in massive fish kills in eutrophic lakes: Verein.” Limnol., 20: 208 -213. 1978.

3. Billings W.H., “In the water environment: Algal toxins and Health;’’ (eds W.W. Caramichael) pp. 243-250 Plenum Press: New York. 1981.

4. Briand T.F., S.Jacket, C.Bernard and Humbert. J.F., “Health Hazards for terrestrial vertebrates from toxic cyanobacteria in surface water ecosystem:” Vet. Res., 34: 361-377. 2003.

5. Cabris C.R., G.T.Rawlin, P.Grant, G.F.Mitchell, J. W.Anderson, And Mc Cauley J, “A study of feral carp, Cyprinus carpio L. Exposed to Microcystis aeruginosa at take Mokvan Australia and possible implications for fish health:” J.Fish Dis., m, 20: 81. 1997.

6. Caramichael W.W, “Health effects of toxin producing cyanobacteria “The cyanoHAB’s”: Human and Ecological risk manag. 7(5): 1359 – 1401. 2001.

7. Falconer I.R., “Toxicity Assessment” 4, 175-184. 1989.

8. Fischer W.J. and D.R.Dietrich., “Pathological and biochemical characterization of microcystin induced hepatopancreas and kidney damage in carp (Cyprinus carpio)” : Toxicol. Appl. Pharmacol. 164(1): 73-81. 2000.

9. Gupta U.S and Shubra Guha, “ Microcystin toxicity in a fresh water fish, Heteropneustes fossilis (Bloch)”: Curr.Sci. 91(9): 1261 – 1271. 2006.

10. Hawkins P.R., M.T.C. Runnegar, A .R.B. Jackson and I.R. Falconer, “Severe hepatotoxicity by the tropical cyanobacterium (Blue green algae) Cylindrospermopsis racihorskii (Woloszynska) Seenaya and Subha Raju isolated from a domestic water supply reservoir:” Appl. Environ. Microbiol., 50 : 1292 – 1295. 1985.

11. Jos A , “Toxic cyanobacterial cells containing microcystins induce oxidative stress in exposed Tilapia fish (Oreochromis sps.) under laboratory conditions”: Aquat. Toxicol, 72(3) : 261-271. 2005.

12. Landsberg J.H., “The effects of harmful algal blooms on aquatic organisms:” Reviews in Fisheries Science. 10(2): 113-390. 2002.

13. Li, L., P. Xie and J.Chen, “In vivo studies on toxin accumulation in liver and ultrastructural changes of hepatocytes of the phytoplanktivorous big head carp i.p. injected with extracted microcystins:” Toxicon 46: 533-545. 2005.

14. Magalhaes V.F., M.M. Mainho, P.Damingos, A.C. Oliviero, S.M. Costa and Azevedo, “ Microcystins (Cyanobacteria hepatotoxins ) bioaccumulation in fish and crustaceans from Sepetiba Bay (Brasil, RJ)” :Toxicon 42: 284-295. 2003 .

15. Magalhaes, V.F., R.M. Soares and S.M.F.O. Azevedo, 2001. “Microcystin contamination in fish from the Jacerpegua lagoon (Rio de Janerio, Brazil) ecological implication and human health risk:” Toxicon 39: 1077-1085.

16. Malbrouck , C and P. Kestemont, “Effects of microcystins on fish:”Environ. Toxicol. Chem., 25(1): 72-86. 2006.

17. Malloy H.T and K.A. Evelyn, “The determination of bilirubin with the photometric taximeter:” J. Biochem., 119: 481 – 490. 1937.

18. Mohammed Z. A., and A.A. Hussein , “Depuration of microcystins in Tilapia fish exposed to natural populations of toxic cyanobacteria: a laboratory study:” Ecotoxicol. Environ. Sat. 63(3): 424-429. 2006.

19. Mohammed Z.A, Z.A. Carmichael and A.A. Hussein, “Estimation of microcystins in the fresh water fish Oreochromis niloticus in an Egyptian fish farm containing Microcystis bloom:” Environ. Toxicol, 18: 137-141. 2003.

20. Obrehmm A., J. Fastner and C.Steinberg, “Effects of MC – LR and crude cyanobacterial extracts on embryo larval development of Zebra fish:”Water Res 31: 2918 – 2921. 1997.

21. Orr P.T and G.J. Jones , “Relationship between microcystin production and cell division rates in nitrogen limited Microcystis aeruginosa cultures:” Limnol. Oceanogr. 43: 1604 – 1614. 1988.

22. Radbergh C.M.I., G. Bylund and J.E. Erikkson , “Histopathological effects of microcystin LR a cyclic peptide toxin from the cyanobacterium (Blue green algae) Microcystis aeruginosa in common carp (Cyprinus carpio):”Aquatic. Toxicol. 20 131-146. 1991.

23. Rapala A., K.Sivonen, C . Lyra and S.I. Niemala, “ Variation of microsystins, cyanobacterial hepatotoxins in Anabaena sp, as a function of stimuli:” Appl. Environ. Microbiol. 63: 2206-2212. 1997.

24. Runnegar M.T., R.G. Gerdes and I.R. Falconer,. “The uptake of the cyanobacterial hepatotoxin microcystin by isolated rat hepatocytes:” Toxicon, 29: 43-51. 1991

25. Shebulsky, M., “Observations on the properties of a toxin produced by Microcystis :” Verh. Internat.Verein.Limnol., 11: 363-366. 1951.

26. Sivonen .K., “Effects of light, temperature, nitrate, orthophosphate and bacteria on growth and hepatotoxin production by Oscillatoria agardhii strains:” Appl. Environ. Microbiol., 56: 2658- 2666. 1990.

27. Tancella F.G and D.R.Dietrich, “Biochemical characterization of Microcystis aeruginosa peptide toxin to yearling rainbow Trout (Oncorhynchus mykiss):” Aquatic Toxicology 30 (3): 215-224. 1994.

28. Turner P.C., A.J. Gammio, K. Hollinrake and G.A. Codd., “Pneumonia associated with cyanobacteria”: Mr. Med. J., 300: 1440-1441. 1990.

29. Wannemacher R.W., “Chemical stability and laboratory safety of naturally occurring toxins” Fort Detrick, Fredrick MD, US Army Medical Institute of Infectious Disease. 1989.

30. Xie L, P. Xie and K. Ozawa ,” Dynamics of microcystins LR and RR in the phytoplanktivorous silver carp in a sub chronic toxicity experiment:” Environ. Pollut. 127: 431-439. 2004.