HISTOLOGICAL RESPONSE OF AQUEOUS EXTRACT OF …



HISTOLOGICAL CHANGES IN GILL, LIVER AND KIDNEY OF NILE TILAPIA, Oreochromis niloticus, FINGERLINGS AND ADULTS EXPOSED TO AQUEOUS EXTRACTS OF DRUMSTICK, Moringa oleifera, SEEDS

E.O. AYOTUNDE1, O.A. FAGBENRO1, O.T. ADEBAYO1 and B.I. OSHO2

1Department of Fisheries and Wildlife

Federal University of Technology, Akure, Nigeria.

2Department of Animal Production and Health

Federal University of Technology, Akure, Nigeria.

ABSTRACT

Histological changes in gill, liver and kidney tissues of Oreochromis niloticus fingerlings and adults exposed to aqueous extract of Moringa oleifera seeds at different sub-lethal concentrations under static bioassay procedure are described. There were pathologic lesions observed which included different levels of degeneration of cells. In the gills, lamellar hyperemia, and hypertrophy of gill arch occurred, while hyperplasia, disarrangement of hepatic cell, necrosis and vacuolation occurred in liver and kidney tissues of O. niloticus fingerlings and adults. These damages became severe with increasing concentration of aqueous extract of M. oleifera seeds and time of exposure.

Key words: Histology, aqueous extract, Moringa oleifera seeds, Oreochromis niloticus.

INTRODUCTION

Moringa oleifera Lam. (Moringaceae) (drumstick, horse-radish) tree is a multipurpose tree that thrives in tropics and sub-tropics (Foidl et al.,2001). M. oleifera seed is a good water purifier; it leaves water clear with 90-99% of the bacteria removed (Acquaye et al., 2000). It contains polyelectrolytes, which constitute active ingredients in water treatment (Jahn,1986). The seed powder is also be used to harvest algae from wastewaters. Although Moringa seeds have been successfully used as water purifier, little has been done on their toxicity to cultivated fishes (Ayotunde et al.,2004, 2005).

Water pollution/contamination induces pathological changes in fish. Bernet et al.(1999) noted that as an indicator of exposure to contaminants, histology represents a useful tool to assess the degree of pollution. The description and assessment of histological changes in different organs represent a very sensitive and crucial parameter in determining cellular changes that occur in target organs, such as the gills, liver and gonads (Dutta,1996). Histological investigations may therefore be a cost-effective tool to determine the health of fish populations, hence reflecting the health of an entire aquatic ecosystem.

This study examined the histology of gill, liver and kidney of Nile tilapia, Oreochromis niloticus, fingerlings and adults exposed to various sub-lethal concentrations of aqueous extract M. oleifera seeds.

MATERIALS AND METHODS

The study was conducted under standard static bioassay procedure (Reish & Oshida, 1987; Ayotunde et al.,2004). Live and apparently healthy O. niloticus fingerlings (7.5-8.3 cm total length; 11.6-17.6 g) and adults (15.8-17.9 cm total length; 89.6-105.6 g) were collected from a government fish farm in Akure, Nigeria, and were transported to the Fisheries Laboratory of Federal University of Technology Akure; where they were acclimated for seven days inside transparent rectangular glass tanks (75 x 45 x 45 cm). The fish were fed to apparent satiation twice daily (0900 h, 1600 h) with a commercial pelleted fish diet during the acclimation period. Feeding was discontinued 48 hours prior to the commencement of the toxicity tests in order to minimize the production of wastes.

Freshly mature M. oleifera seeds were sun-dried at ambient temperature, their seed coats and wings were manually removed. The white kernel was ground to a fine powder, using the coffee mill attachment of a Moulinex domestic food blender and the powder was kept in desiccators for later use in stock solutions. Ten O. niloticus fingerlings were batch-weighed with a top-loading Mettler balance and stocked into triplicate tanks per treatment. The tanks were covered; there was no aeration, no water change nor feeding throughout the toxicity test period. For the fingerlings, the toxicant was introduced at 200, 210, 220, 230, 240 and 250 mg/l with a control of 0 mg/l in triplicate tanks; while in the adults, the toxicant was introduced at 300, 310, 320, 330, 340 and 350 mg/l with a control of 0 mg/l in triplicate tanks. Both toxicity tests lasted for 96 hours.

One fish per tank was removed after 96 hours of exposure for histological examination. The fish was dissected to remove the gill, liver and kidney. The organ tissues were fixed in 10% formalin for three days after which the tissue was dehydrated in periodic acid Schiff’s reagent following the method of Hughes & Perry (1976), in graded levels of alcohol for three days, to allow paraffin wax to penetrate the tissue during embedding. The tissues were then embedded in malted wax. The tissue was sectioned into 5-7(m sections using a rotatory microtome and were dehydrated and stained with Harris haematoxyllin-eosin (H&E) stain (Bancroft & Cook,1994), using a microtone and each section was cleared by placing in warm water (38oC), and oven-dried at 58oC for 30 minutes to melt the wax. The slide containing sectioned tissue was cleared using xylene and graded levels of alcohol for two minutes each. The section was stained in H&E stain for ten minutes. The stained slide was observed under a light microscope at varying magnifications; sections were examined and photographed using an Olympus BH2 microscope fitted with a camera and automatic light exposure unit.

RESULTS AND DISCUSSION

a. Histological changes in O. niloticus fingerlings

Histological changes in the gill, liver and kidney tissues of O. niloticus fingerlings exposed to aqueous extract of M. oleifera seeds are described and presented in Tables 1 (plates 1-3), Table 2 (Plates 4-9) and Table3 (Plates 10-15), respectively.

Table 1: Histological changes in the gill of O. niloticus fingerlings exposed to aqueous extract of M. oleifera seeds.

|Concentration (mg/l) |Plate |Histological changes |

|Control |1 |No pathological lesion was observed. |

|200 |2 |Degenerated filaments and necrosis on the gill arches. |

|210 |3 |Vacuolation of the gill arches and total removal of lamellae. |

|220, 230, 240, 250 |- |Complete degeneration of filaments of the gill arches. |

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Table 2: Histological changes in the liver of O. niloticus fingerlings exposed to aqueous extract of M. oleifera seeds.

|Concentration (mg/l) |Plate |Histological changes |

|Control |4 |No pathological lesion was observed. |

|200 |5 |Disorientation of the liver parenchyma structure. |

|210 |6 |Disorientation of the liver parenchyma structure. |

|220 |7 |Vacuole formation and the cell hepatocyte enlarged. |

|230 |8 |Hyperplasia and disorientation of hepatic cells. |

|240 |9 |Vacuole formation, shrinkage of cells. |

|250 |- |Shrinkage of cells. |

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Table 3: Histological changes in the kidney of O. niloticus fingerlings exposed to aqueous extract of M. oleifera seeds.

|Concentration (mg/l) |Plate |Histological changes |

|Control |10 |No pathological lesion was observed. |

|200 |11 |Vacuolation and shrinkage of cells. |

|210 |12 |Shrinkage of cells. |

|220 |13 |Shrinkage of cells. |

|230 |14 |Karyolysis of the hepatocytes. |

|240, 250 |15 |Complete degeneration of cells. |

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Babatunde et al.(2001) reported that the gills of O. niloticus fingerlings exposed to 12.0 and 14.2 mg/l of Paraquat® showed a dose-dependent destruction of the lamellae architecture, filament hyperplasia and atrophy, leading to impairment in oxygen uptake. Visoottiviseth et al.(1999) observed that the liver, kidney and gill of O. niloticus exposed to 3 mg/l of triphenyltin hydroxide showed a variety of changes such as congestion and dilatation of sinusoidal space, pallor of cytoplasm, vacuolation/accumulation of hyaline droplets, sub-capsular and scattered focal necrosis in the hepatocytes. In the kidney, hydropic degeneration/accumulation of hyaline droplets in the tubular epithelial cells, congestion of pertubular capillaries and detachment of tubular epithelial cells were observed. Alterations in the gill filaments and lamellae included hyperplasia of the covering gill epithelium, congestion of gill capillaries, and aneurismal formation of gill lamellar capillaries. Histological changes observed in the kidney of tilapia fingerlings in this study were similar to those previously reported for formalin-treated rainbow trout (Salmo gairdneri) (Smith & Piper,1972).

b. Histological changes in O. niloticus adults

Changes in the gill, liver and kidney histology of adult O. niloticus exposed to aqueous extract of M. oleifera seeds are described and presented in Tables 4 (Plates 16-21), Table 5 (Plates 22-27) and Table 6 (Plates 28-30), respectively.

Table 4. Histological changes in the gill of adult O. niloticus exposed to aqueous extract of M. oleifera seeds.

|Concentration (mg/l) |Plate |Histological changes |

|Control |16 |No pathological lesion observed. |

|300 |17 |Half of the gill arches have filaments and lamellae degenerated. |

|310 |18 |Hypertrophy of gill arches and degenerated gill filaments and lamellae. |

|320 |19 |Degeneration of lamellae and filaments in central parts of gill arches. |

|330 |20 |High degenerated lesion observed in gill arch, filaments and lamellae. |

|340 |21 |Hypertrophy of gill arches and degenerated gill filaments. |

|350 |- |Complete degeneration of gill arch and filaments. |

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Table 5. Histological changes in the liver of adult O. niloticus exposed to aqueous extract of M. oleifera seeds.

|Concentration (mg/l) |Plate |Histological changes |

|Control |22 |No pathological lesion observed. |

|300 |23 |Disarrangement of hepatic cells. |

|310 |24 |Vacuole formation. |

|320, 330 |25 |Necrosis and thickening of the hepatocyte cells. |

|340 |26 |At x40 the karyolysis of the hepatocytes. |

|350 |27 |Necrosis and faded hepatic cells. |

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Table 6. Histological changes in the kidney of adult O. niloticus exposed to aqueous extract of M. oleifera seeds.

|Concentration (mg/l) |Plate |Histological changes |

|Control |- |} No pathological lesion observed. |

|300 |- | |

|310 |- | |

|320 |28 |Hyperplasia cell and vacuole formation. |

|330 |29 |Shrinkages of hepatocyte cells. |

|340, 350 |30 |Total degeneration of the cells. |

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Histological changes in the gill, liver, and kidney tissues were observed in all treatments and pathologic lesions were similar for all treatments, although the intensity of cell damage increased with increasing concentration and time of exposure. Abd El-Gawad (1999) studied the histology on the liver and gill of O. niloticus (15.0 g, 10.0 cm total length) exposed for 14 days to different concentrations of lead acetate (0.25, 0.50, 1.00, 2.00 ppm) or zinc sulphate (8, 10, 12, 14 ppm). The gills were pale and congested. The liver showed vacuolated hepatocytes with frequent necrosis, evidenced by pyknosis. The bile canaliculi were dilated. The epithelial covering of the gill filaments was hyperplastic and edematous with vacuolated epithelial covering of the gill rakers. The lamellar blood spaces showed telangiectasis. Wade et al.(2002) studied the toxicity of cassava (Manihot esculenta) effluent on O. niloticus. He histological examination of the gill, liver and kidney tissues indicated damage ranging from oedema and talengiectasis of the gill lamella and gill hyperplasia to vacuolation of the liver cells and necrosis.

Cruz et al.(1988) studied the effects of two molluscides, Aquatin® and Brestan®, on adult O. mossambicus. Gills of adult O. mossambicus exposed to Aquatin® exhibited varying degrees of epithelia and hyperplasia in filaments. Hyperplasia resulted in the fusion of many lamellae markedly reducing the respiratory surface area of some filaments (Cruz et al., 1988). Heavy mortalities occurred in fish, showing severe gill epithelia layer from supportive tissue, necrosis of intestinal mucosa, liver hepatocytes, and renal tubules. Tilapia exposed to Brestan® had similar histological alterations. Simpson et al.(2000) reported that in flounder (Platichthys flesus) impacted by endocrine-disrupting chemicals; the liver, kidney and gonad showed hepatic tubular vacuolation, cellular alterations, and hepatocellular tumors, while renal pathology showed enlarged and abnormal glomeruli.

REFERENCES

Abd-El-Gawad, A.M.(1999) Histopathological studies on the liver and gills of Tilapia nilotica (Oreochromis niloticus) exposed to different concentrations of lead acetate and zinc sulphate. Journal of Egyptian Zoological Society 30 (C): 13-22

Acquaye, D., Smith, M., Letchamo, W. & Simon, J.(2000) The miracle tree: Center for new use agriculture and natural plant products. Rutgers University, College, New Brunswick.

Ayotunde, E.O., Fagbenro, O.A., Adebayo, O.T. & Amoo, A.I.(2004) Toxicity of aqueous extract of drumstick, Moringa oleifera, seeds to Nile tilapia, Oreochromis niloticus, fingerlings and adults. Proceedings of the Sixth International Symposium on Tilapia in Aquaculture (ISTA 6), pp.200-208. (R.B. Bolivar, G.C. Mair & K. Fitzsimmons, editors). Manila, Philippines. September 12-16, 2004.

Ayotunde, E.O., Fagbenro, O.A., Adebayo, O.T. and Amoo, A.I.(2005) Toxicity of aqueous extracts of drumstick, Moringa oleifera, seeds to African catfish, Clarias gariepinus, fingerlings and adults. Biological and Environmental Sciences in the Tropics 2: 39-42.

Babatunde, M.M., Oladimeji, A.A. & Balogun, J.K.(2001) Acute toxicity of gramoxone to Oreochromis niloticus (Trewavas) in Nigeria. Water, Air and Soil Pollution 131: 1-10.

Bancroft, J.D. & Cook, H.C.(1994) Manual of histopathological techniques and their diagnostic application. Churchill Livingstone, London. 305pp.

Bernet, D., Schmidt, H., Meier, W., Burkhardt-Holm, P. & Wahli, T.(1999) Histopathology in fish: proposal for a protocol to assess aquatic pollution. Journal of Fish Diseases 22: 25-34.

Cruz, E.R., de la Cruz, M.C. & Sunaz, N.A.(1988) Haematological and histopathological changes in Oreochromis mossambicus after exposure to the molluscicides Aquatin® and Brestan®. Proceedings of the The Second International Symposium on Tilapia in Aquaculture (ISTA 2), pp.99-110. (R.S.V. Pullin, T. Bhukaswan, T. Tonguthai & J.L. Maclean, editors). Bangkok, Thailand. March 16-20, 1987.

Dutta, H.M.(1996) A composite approach for evaluation of the effects of pesticides on fish. In: Fish morphology (J.S.D. Munshi & H.M. Dutta, eds). Science Publishers Inc., India.

Foidl, N., Makkar, H.P.S. & Becker, K.,(2001) The potential of Moringa oleifera for agricultural and industrial uses, pp 45-76, In: The miracle tree: the multiple uses of Moringa. L.J. Fuglie (ed.), CTA, Wageningen, The Netherlands.

Hughes, G.M. & Perry, S.F.(1976) Morphometric study of trout gills: a light microscopic method for the evaluation of pollutant action. Journal of Experimental Biology 63: 447-460.

Jahn, S.A.A.(1986) Monitored water coagulation with Moringa oleifera seeds in village household. Journal of Analytical Science 1: 40-41.

Reish, D.L. & Oshida, O.S.(1987) Manual of methods in aquatic environment research. Part 10. Short-term static bioassay. FAO Fisheries Technical Paper No 427. Rome. 62pp.

Simpson, M.G., Parry, M., Kleinkauf, A., Swarbreck, D., Walker, P. & Leah, R.T.(2000) Pathology of the liver, kidney, and gonad of flounder (Platicthys flesus) from UK estuary impacted by endocrine disrupting chemicals. Marine Environment Research 50 (1-5): 283-287.

Smith, C.E & Piper, E.G.(1972) The pathological effects in formalin-treated rainbow trout (Salmo gairdneri). Journal of the Fisheries Research Board of Canada 29: 328-329.

Visoottiviseth, P., Thamamaruitkum, T., Sahaphong, S., Reiengroipitak, M. & Kruatrachua, C.(1999) Histopathological effect of triphenyltin hydroxide on liver, kidney and gill of Nile tilapia (Oreochromis niloticus). Applied Organometallic Chemistry 13 (10): 749-763.

Wade, J.W., Omoregie, E. & Ezenwaka, C.(2002) Toxicity of cassava (Manihot esculenta Crants) effluent on the Nile tilapia Oreochromis niloticus (L.) under laboratory conditions. Journal of Aquatic Sciences 17 (2): 14-18.

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