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Studies on Cassava Mill Effluent and its Toxicological

Impact using Histopathological Technique

ADEWOYE S.O; SAWYEER H.O & 1*OPASOLA O.A

1*Department of Environmental Management and Toxicology,

Kwara State University, Ilorin, Nigeria

2Department of Environmental Health Science,

Kwara State University, Ilorin, Nigeria

*Corresponding Author: Olaniyi.opasola@kwasu.edu.ng

ABSTRACT This study was carried out to determine the pathological effects of cassava mill effluent on Clarias

gariepinus. The histological changes in the gills and liver of C. gariepinus exposed to cassava mill

effluent at different sub-lethal concentration under static bioassay procedure were experimentally

determined. In this study, the most generally encountered type of degenerative changes was congestion,

vacuolization of hepatocyte, cellular infiltration and necrosis. The liver of the exposed organisms revealed

slight vacuolated cells which is an indication of fatty degeneration of hepatocytes. This study was able to

establish that exposure of C. gariepinus to even low concentration of cassava effluent can induce various

toxicological effects and histological degradation which depends on the period of exposure and

concentration of the pollutant. In the view of the toxicity effect of this effluent, it can be inferred that,

indiscriminate discharge of cassava effluent can induce damage to the tissue and organs which might

make all the living entities in the polluted environments vulnerable to diseases and eventually resulted to

death.

Keywords: Histopathology, sub-lethal, effluent, necrosis

INTRODUCTION

Histopathology is the microscopic study of tissues affected by disease. The procedures adopted for the

preparation of material for such studies are known as histological or histopathological techniques.

Considerable interest has been shown in recent years in histopathological study while conducting sub-

lethal tests in fish (Akinsanya, 2007).Tissue changes in test organisms exposed to a sub-lethal

concentration of toxicantare a functional response of organisms which provides information on the nature

of the toxicant. Histological changes associated with toxicants in fish have been studied by many authors

(Mercy et al., 1996).

Histopathological investigations have proved to be a sensitive tool to detect direct effects of chemical

compounds within target organs of fish in laboratory experiments (Schwaiger et al., 1996). Fish are

widely used to evaluate the health of aquatic ecosystem and physiological changes serves as biomarkers

of environmental pollution. Cassava is a major starchy food for more than 300 million people in many

tropical countries of the world and many cultivars are toxic. Cassava food products are the most important

staples of rural and urban households in southern Nigeria. In Nigeria, traditional foods processed at home

or in small-scale cottage operations constitute the principal mode of utilization of cassava (Adeyemo,

2005). Therefore, this study was primarily carried out to determine the histopathological effects of

cassava mill effluent to C. gariepinus gills and liver.

International Journal of Innovative Environmental Studies Research 4(3):24-33, July-Sept., 2016

© SEAHI PUBLICATIONS, 2016 www.seahipaj.org ISSN: 2354-2918

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METHODOLOGY

To observe the impact of cassava mill effluent on the histopathology of the fish, One hundred (100)

apparently healthy adult Clarias gariepinus of average body weight of 350g and length ranging from

25.0-27.0cm were purchased from Ministry of Agriculture, Fisheries Division, Ogbomoso. Transportation

of the fishes was done in properly aerated container to the Biology laboratory of Ladoke Akintola

University of Technology, Ogbomoso, and Oyo State. The fishes were acclimatized for 14 days in the

laboratory inside transparent container filled with 50 litres of well dechlorinated water. This experiment

was conducted under standard static bioassay procedure (AOAC, 1987). This involves carefully

controlled environmental conditions as to define the response of the test organism to the effect of cassava

mill effluent. The cassava effluent used for this study was collected from a local ‘garri’ producing

industry at Arada Cassava Processing industry in Ogbomoso South Local Government area, Ogbomoso,

Nigeria. Collection of the cassava effluent was done every morning to ensure that the cassava effluent

collected is fresh and potent i.e active ingredient in the effluent is at its maximum potency.

Sublethal Test

All the tested organisms (C.gariepinus) into six groups, each group contained ten fishes. The five varying

concentrations used ranged from 0.020ml/L, 0.016ml/L, 0.012ml/L, 0.008ml/L, and 0.004ml/L. The

volumes of effluent measured into 10Litres of water are 200mls, 160mls, 120mls, 80mls, and 40mls.The

experiment was carried out for 14days. The behaviours and morphological features of the fishes were

observed every 24hrs. The mortalities were collected and stored in freezer. The sublethal test was done to

determine the mean results.

Histopathology Liver, kidney and gill were collected in 10% neutral buffered formalin and processed for paraffin blocks

(56-58 °C) and sectioning at 3-5 μm. Stained sections were examined under a Zeiss compound binocular

microscope fitted with a photomicrographic attachment.

RESULTS

Behaviour Responses of the test organisms

Exposed fish became darker in colour and showed signs of respiratory distress, increased opercular

movement were also observed.

Results of histological examination of selected organs

The results of histological examination of gill and liver of exposed fish as revealed by Zeiss compound

binocular microscope fitted with a photomicrographic attachment are presented in the following plates 1-

10. The results obtained showed that the gill and liver sections in the control group shows no lesion in the

primary and secondary lamellae. Meanwhile, gill and liver sections of the exposed fish showed significant

alterations and abnormalities.

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Plate 1: Tissue of gill of C. gariepinus(×400) in control.

Plate 2: Gill section of C. gariepinus (x400) at 0.004mg/L showing mild cellular infiltration of hepatocytes

(CIH).

PLATE 3: Gill structure of C. gariepinus (x400) at 0.008mg/L showing more pronounced cellular infiltration

of hepatocytes exposed to cassava effluent.

SCIH

CIH

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Plate 4: Tissue of gill structure of C. gariepinus (x40) at 0.012mg/L showing irregular lamellae epithelium and

severe hepatocytes degeneration afterbeing exposed to cassava effluent.

Plate 5: Gill section of C. gariepinus (x400) exposed to 0.016mg/L of cassava effluent showing a complete

degeneration of hepatocytes (CDH) hence a necrotic condition.

SHD

ILE

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Plate 6: Tissue of liver section of C. gariepinus (x400) in the control group showing no significant lesion

exposed to cassava effluent.

Plate 7: Liver section of C. gariepinus (x400) at 0.004mg/L showing mild glycogen vacuolation and fairly

congested central vein after been exposed to cassava effluent.

FCCV

MGV

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Plate 8: Liver section of C. gariepinus (x400) at 0.004mg/L showing mild glycogen vacuolation (MGV) and

fairly congested central vein (FCCV).

Plate 9: Tissue of liver section of C. gariepinus (x400) at 0.008mg/L showing diffuse hepatic necrosis (DHN)

with severe cellular infiltration (SCI).

DHN

SCI

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Plate 10: Liver section of C. gariepinus (x400) at 0.012m/L showing severe infiltration of leukocytes (arrow)

and lipid vacuolation (L).

Plate 11: Tissue of liver section of C. gariepinus (x400) at 0.016mg/L showing more pronounced hepatic

necrosis.

DISCUSSION

The exposure of fish to chemical contaminants is likely to induce a number of lesions in different organs

(Bucke et al., 1996). Gills (Poleksic et al., 1994), kidney (Bucher and Hofer, 1993), and liver (ICES,

1997) are suitable organs for histological examination in order to determine the effect of pollution. The

exposure of aquatic organisms to very low levels or sublethal concentration of pollutants in their

environment may result in various biochemical, physiological, and histological alterations in vital tissues.

In this study histological investigation of the liver tissues of C. gariepinus not exposed to sublethal

concentrations of cassava effluent showed a typical structural organization of the parenchymatous cell

appearance of the hepatocytes in the untreated fishes. However, the fishes exposed to the cassava effluent

L

L

L

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showed major histological abnormalities. The abnormalities observed include cellular infiltration,

congestion of central vein and cellular necrosis, which showed a progressive architectural distortion at

varied concentrations, this observation is in agreement with the submission of Strivastava, (1994).

In this study, the most generally encountered type of degenerative changes was congestion, vacuolization

of hepatocyte, cellular infiltration and necrosis. The liver of the exposed organisms revealed slight

vacuolated cells which is an indication of fatty degeneration of hepatocytes. Cellular necrosis as observed

in this study might have resulted from excessive work required by the fish liver to get rid of the toxicants

from its body during the process of detoxification. High accumulation of several components of the

cassava effluent in the liver is a pointer to the fact that, liver plays a major role in the accumulation and

detoxification.

Necrosis became evident as the concentration increases and this could be attributed to the inability of

fishes to regenerate new liver cells. It was also observed that the histopathological changes in the liver

caused metabolic problems; this lesion is characterized by the remains of the bile in the form of droplets

in the cytoplasm of the hepatocyte and this convincingly supported the submission of Pacheco and

Santos, (2002) that stated that bile is not being released from the liver which is also an indication of

possible damage to the hepatic metabolic functions of the liver. An increased in the degree of damages

done to the liver tissue of the fishes (Clarias gariepinus) held in 0.004 mg/L, 0.008 mg/L, 0.012mg/L,

0.016mg/L and 0.02mg/L cassava effluent, is generally related to important hepatic lesions such as

degenerative and necrotic processes, this observation was in line with the submission of Chang et al.,

(1998).

Additionally, the presence of bile stagnation or accumulation and melanomacarophages in great quantity

in the liver of exposed C. gariepinus is strong evident in that this organ suffered structural and metabolic

damage due to the exposure to the cassava effluent. This is in a way signalling the fact that this

environment where the effluent is discharged is grossly polluted and impaired. The histological alterations

identified within the hepatocytes in this study may have been the results of various biochemical lesions.

Anomalies such as irregular shaped central vein, cellular vacuolation and infiltration may be attributed to

the accumulation of lipids and glycogen due to liver dysfunction as a result of exposure to the effects of

cassava effluent, this is in conformity with the submission of Fanta et al., (2003).

Pacheco and Santo (2003) also described increased level of vacuolation of the hepatocytes as a signal to

the degenerating process that suggest metabolic damage, possibly related to exposure to contaminated

water. The liver parenchyma cells of all exposed fish showed signs of degeneration and this has been

reported by many authors to be associated with the exposure of fishes to certain toxicants (Chang et al.,

1998).

Evidently the damage done to the tissues of the liver and the gills seemed to increase with increase in

concentration as shown by the plates 1-11. This result is similar to the work of Wade et al. (2002) who

worked on the toxicity of cassava (Manihot esculenta) effluent on the Nile tilapia Oreochromis niloticus,

histopathological examination of gill, kidney, and liver indicated damage ranging from oedema and

talengiectasis of the gill lamella and gill hyperplasia to vacuolation of the liver cells and necrosis. The

Present findings are strongly supported by the works of Cengiz (2006) who observed and reported the

histopathological effects of δ-methrin on the gill of the common carp; Cyprinus Carpio (Linn.), the

changes at all doses of the test compound were desquamation and necrosis besides aneurism in secondary

lamellae, lifting of lamellar epithelium, edema, hyperplasia and fusion of the secondary lamellae were

reported.

The present findings are in conformation with the work of Dhanapakiam et al. (1998) who studied

histopathological changes in the gills of Channa punctatus (Bloch.) exposed to industrial effluents for 45

days, revealing deformities such as; secondary lamellae of primary filaments showing hyperplasia as

compared to the gills of controlled fish indicating that the industrial effluents of river Cauvery induced

considerable chemical stress on fish populations. Adhikari (1996) also studied the histopathological

changes in the gills of a cat fish; Heteropneustes fossilis on exposure to malathion at different

concentrations and different exposure times finding degenerative changes like complete secondary

lamellae fusion, edema, hypertrophy and hyperplasia of lamellar cells.

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Similarly, the present finding are in agreement with Tilak et al. (2001), Velmurugan et al. (2007) who

also observed the same histopathological changes in the gills of various fishes on exposure to different

toxins resulting in necrosis, vacuolar degeneration, dystrophy, desquamation, epithelial lifting, edema,

shorting of secondary lamellae and lamellar fusion. All these histopathological research studies on the

gills of fishes proved to be supporting the results of the present investigation. The observations in the

present investigation are also supported by the work of Nagarajan and Kumar (2006) who reported the

same histopathological changes in the gills of Labeo rohita (Ham.), exposed to sago effluent, which

included worn out gill filaments of primary and secondary gill lamellae.

The works of Pandey et al. (1997) provided confirmation to the present investigation who worked on

histopathological changes due to the effect of heavy metals on the gills of fishes. Fernandes et al. (2006)

observed the histopathological changes in gills epithelium of Oreochromis niloticus exposed to copper;

they reported edema, lifting of lamellae epithelia, lamellar fusion, thus also providing support for the

present investigation.

CONCLUSION This study has been able to establish the fact that; exposure of C. gariepinus to even low concentration of

cassava effluent can induce various toxicological effects and histological degradation which depends on

the period of exposure and concentration of the pollutant. In the view of the toxicity effect of this effluent,

it can be inferred that, indiscriminate discharge of cassava effluent can induce damage to the tissue and

organs which might make all the living entities in the polluted environments vulnerable to diseases and

eventually resulted to death.

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