Histological study of the intestine and liver tissues in...

JOURNAL OF MODERN BIOTECHNOLOGY, VOL. 3, NO. 4, pp 48–54, July 2014 Copyright © 2014, by Madras Institute of Biotechnology. All Right Reserved. www.peer-reviewedjournals.com

48 JOURNAL OF MODERN BIOTECHNOLOGY VOLUME 3· NUMBER 4· JULY 2014

Research Article

Histological study of the intestine and liver tissues in the fish Oreochromis mossambicus exposed to cypermethrin

Thayappan Karthigayani1, Maghil Denis1, Annadurai Rexlin Andrew Remy1* and Narayanasamy Shettu2

1Department of Zoology, University of Madras, Chennai – 600 025, Tamil Nadu, India.

2Department of Zoology, Pachaiyappa's College, Chennai – 600 030, Tamil Nadu, India.

*Corresponding Author e-mail: [email protected]

Received 10 May 2014; Revised 28 May 2014; Accepted 11 June 2014

Abstract

Cypermethrin, the synthetic pyrethroid commonly used as a pesticide, contaminates the aquatic ecosystem as a toxic pollutant from agricultural and domestic washouts. The aim of the study was to investigate the toxic effect of cypermethrin on intestine and liver tissues in the tilapia fish Oreochromis mossambicus exposed to sublethal concentration of 0.008ppm at different exposure time from 24 to 192 hrs. The histological observations of the intestine showed deleterious effect of cypermethrin at 96 hrs exposure time, the epithelial cells and the cells of the outer wall disintegrate which would eventually result in the breakdown of the intestinal functions. Similarly the histological

preparation of the liver tissue showed damaging effect of cypermethrin on the hepatocytes that were altered with pycnotic nucleus. The break down in the function of the hepatocytes influences the distribution of nutrients to the other vital tissues and the survival of the fish. The deleterious effect of pesticide cypermethrin on increase in concentration or time of exposure may affect the survival of the fish. Hence the LC50 of the fish tilapia which was calculated at 96hrs exposure as 0.04ppm clearly indicated the lethal effects due to alterations in the metabolic pathways of the tissues by direct and indirect exposure of cypermethrin to the fish

Keywords: Oreochromis mossambicus, Cypermethrin, LC50, Toxicity

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INTRODUCTION

Cypermethrin a pyrethroid insecticide extensively used to control insects pests of agricultural crops and vegetables and at domestic level used for eradication of cockroaches (Smith and Stratton 1986, Moore and Waring 2001) though the control of pesticides are achieved by use of pesticides yet it may contribute to the pollution of the environment by accumulation of toxic substances (Hill, 1989). Thereby the potent insecticide

cypermethrin through wash out waters find their way into water reservoirs streams and rivers, lakes and inland fishery ponds that may have an adverse effect on the aquatic organisms including fishes. (John and Prakash, 2003), The assessment of the ecotoxicological risk caused by pesticides is based on its toxic effect on the survival and on its toxic effect on its vital tissues of the organisms (Demoute, 1989; Srivastav et al 1997; Bernet et al 1999; Milam et al 2000).

Karthigayani et al Effect of Cypermethrin Toxicity in the intestine and liver of the fish

49 VOLUME 3· NUMBER 4· JULY 2014 JOURNAL OF MODERN BIOTECHNOLOGY

The present study was aimed to study the effect of pyrethroid insecticide cypermethrin on the intestine and liver of the fish Mozambique tilapia, Oreochromis mossambicus. Histological changes in the organs that are directly exposed to the pesticide such as the intestine was taken as a parameter to assess the impact or toxic effect of cypermethrin. Also histological studies of the liver were undertaken to understand the effect of absorbed cypermethrin on the internal soft tissue.

MATERIALS AND METHODS

Chemical

Cypermethrin is a synthetic pyrethroid molecule used to control agricultural pests, moth pests of cotton, fruits and vegetable crops. Technically Cypermethrin is a mixture of eight different forms of isomers. Cypermethrin is photo stable and at temperature below 220ºC does not disintegrate. It is commonly used as a pesticide as it is a neurotoxin in insects. Analytical grade chemicals was purchased from Sigma Chemicals Co. (USA)

Molecular Structure

The molecular structure of cypermethrin is given below (Dr. Raymond J. Heitzman Compton, Newbury Berkshire, United Kingdom)

(RS).-alpha-cyano-3-phenoxybenzyl(1RS).-cis-,trans-3-(2,2-dichlorovinyl).-2,2-dimethylcyclopropane carboxylate (IUPAC chemical name);

Molecular formula: C22H19Cl2NO3

Molecular weight: 416.3g/mol

Solubility -0.009mg/l at 20ºC (WHO 1989)

Structure of cypermethrin

Bioassay tests

Determination of lethal concentration (LC50)

Bioassay or toxicity tests were carried out for the determination of LC50 values by following FAO procedure for short term bioassays (Reish and Oshida, 1987). The duration of the test was 96 hours. Stock solution of Cypermethrin 10% Effective Concentration (EC) was prepared by diluting 1ml insecticide in 100ml of distilled water, and was diluted to different concentrations of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 and 0.08 ppm, which were used as experimental waters for toxicity study of tilapia. The experiment was set in triplicate and healthy fishes (n=10) with an average weight of 450 mg and an average length of 3.5 cm were maintained in 10 litre of experimental water having different concentrations of Cypermethrin. Similarly a control was set up with water devoid of Cypermethrin. Feeding was stopped one day prior to the experiment and also during the experimental period, as recommended by Ward and Parrish (1982) and Reish and Oshida (1987), The LC50 values were calculated as average from the three replicates for each experimental concentration of water by arithmetic graph method (Reish and Oshida, 1987).

Tilapia fish

The Mozambique tilapia, Oreochromis mossambicus, is an African tilapia cichlid fish, used as food and thereby introduced in aquaculture for commercial production and may be (erroneously) called "Java tilapia" in trade was chosen for the present study on toxicity of cypermethrin.

Collection and maintenance of tilapia

About 300 fishes with the average body length and the body weight of the fishes varying between 3 to 5 cm and 400 to 800 mg respectively, was obtained from a local aquarium in Chennai, India and maintained in the laboratory in aerated aquarium tanks with ambient temperature of 26 ± 2ºC. The fish were fed with a mixture of artificial pellets and acclimated for a period of 15 days.

Preparation of tissue sample

Histological Techniques

The method for histological study followed was that of

McManus and Mowry, (1964). The intestine and liver

Effect of Cypermethrin Toxicity in the intestine and liver of the Fish Karthigayani et al


tissues of the control and the experimental fishes were

dissected and fixed in Bouin’s fluid for 24 hrs The fixed

tissues were dehydrated in graded ethanol series, cleaned

in xylene and embedded in paraffin wax (congealing

point 58-60ºC). The longitudinal and transverse sections

each of 6µm thickness were stained in haematoxylin and

eosin (HandE). The sections were deparaffinised in

xylene each and rehydrated using a graded ethanol series

and the dehydrated sections were blotted once again and

cleared in two changes of xylene with the first change of

ten minutes duration and second change of fifteen

minutes duration and further blotted and mounted in

DPX (Diestrene Plasticizer Xylene) to be examined

under light microscope

RESULTS

Median Lethal Concentration (LC50 -96 hours)

The tilapia fish, O. mossambicus, exposed to

cypermethrin was subjected to a test of survivability and

at 96 hours the LC50 value was found to be 0.04 ppm for

Replicate I, II and III.

Histology of Liver tissue in control fish

The microscopic observations of histological sections of

liver from fishes taken as control in the experiment

showed the typical structure of hepatic tissue. The

hepatic cells appeared as a continuous mass and

distinctly shaped as round or polygonal, containing clear

spherical nucleus. They were located among sinusoids

forming cord like structure known as hepatic cell cords.

Bile canaliculatus was centrally located in each cord. A

large number of blood sinusoids were found in the

hepatic mass of these cords. Thin bile canaliculi are

observed between the hepatic cells (Figure 1A).

Histology of Liver tissue in fish exposed to

cypermethrin

The histological observation of the liver tissue in

experimental fish exposed to 0.008 ppm cypermethrin at

24 hrs revealed the hepatic cells with the characteristic

round or polygonal shape with insignificant alterations in

the other components of the tissues as shown in (Figure

1B).

On increasing the exposure time to 48 hrs, the

histological sections of the liver of the fish was observed

to undergo minor alterations such as the change in shape

of hepatocytes due to complete vacuolation (Figure 1C).

However the changes became more significant with the

time of exposure. The histopathological changes noticed

in the liver of exposed fish at the same concentration

with the 96 hrs exposure witnessed complete

disintegration of hepatocytes and decrease in size of

nucleus (pycnotic) (Figure 1D) and after 192 hrs of

exposure there was extensive vacuolation of all the

hepatocytes and finally lost the polygonal shape (Figures

1E and 1F).

Histology of intestinal issue in control fish

The histological sections of the intestine of the tilapia

fish O. mossambicus exhibited simple columnar

epithelium, lamina propria, submucosa, tunica

muscularis and serosa as well defined layers in the order

from internal to external surface of the intestine. The

epithelial cells appeared as long and thin columnar cells

and the mucosa showing surface microvilli. A few goblet

cells containing lymphocytes were located between the

epithelial cells. The mucosa was highly branched and

folded (Figure 2A).



Figure 1A: Transverse section of the intestine of the control fish Oreochromis mossambicus showing the normal organization of intestine. Columnar Epithelium (CE), sub mucosa (SM) and serosa (S). Bouin, 6 µm, HE. X 110.

Figure 1B: Transverse section of the intestine of the fish Oreochromis mossambicus exposed to 24 hrs, sublethal exposure of cypermethrin at 0.008ppm concentrations showing less damage cell wall of the intestine columnar epithelium (EC) sub mucosa (SM) and Serosa (S) in higher magnification Bouin, 6 µm, HE. X 950.

Figure 1C: Transverse section of the intestine of the fish Oreochromis mossambicus, exposed to 48 hrs, sublethal exposure of cypermethrin at 0.008 ppm concentration showing slight damage in the periphery of the cell wall (indicated in arrows). Bouin, 6µm, HE.X 950.

Figure 1D: Transverse section of the intestine of the fish Oreochromis mossambicus exposed to 72 hrs, sublethal exposure of cypermethrin at 0.008 ppm concentration showing disintegration of the cell wall. (indicated in arrows) Bouin, 6 µm, HE. X 950.

Figure 1E: Transverse section of the intestine of the fish Oreochromis mossambicus, exposed to 96 hrs, sublethal exposure of cypermethrin at 0.008ppm concentration showing shrinkage of columnar epithelial cells. Bouin, 6 µm, HE. X 950.

Figure 1F: Transverse section of the intestine of the fish Oreochromis mossambicus exposed to 192 hrs, sublethal exposure of cypermethrin at 0.008 ppm concentration showing severe damage of columnar epithelial cells. Bouin, 6 µm, HE. X 950.



Figure 2A: Transverse section of the liver of the control fish Oreochromis mossambicus showing the normal organization of the hepatic cell (HC) and blood capillaries (BC) (indicated in arrows). Bouin, 6 µm HE. X 110.

Figure 2B: Transverse section of the liver of the fish Oreochromis mossambicus exposed to 24 hrs, sublethal exposures cypermethrin at 0.008 ppm concentration showing less damage in hepatocytes. (indicated in arrows) Bouin, 6 µm, HE. X 110.

Figure 2C: Transverse section of the liver of the fish Oreochromis mossambicus exposed to 48 hrs, sublethal exposure of cypermethrin at 0.008 ppm concentration showing few hepatocytes, with completely vacuolated nuclei (indicated in arrows). Bouin, 6 µm, HE. X 950.

Figure 2D: Transverse section of the liver of the fish Oreochromis mossambicus, exposed to 72 hrs, sublethal exposure of cypermethrin at 0.008 ppm concentration showing vaculation in few hepatocytes (indicated in arrows). Bouin, 6 µm, HE. X 950

Figure 2E: Transverse section of the liver of the fish Oreochromis mossambicus, exposed to 96 hrs, sublethal exposure of cypermethrin at 0.008 ppm concentration showing complete disintegration of hepatocytes and decrease in size of nucleus (Pycnotic) (indicated in arrows). Bouin, 6 µm HE. X 950.

Figure 2F: Transverse section of the liver of the fish Oreochromis mossambicus, exposed to 192 hrs, sublethal exposure of cypermethrin at 0.008ppm concentration showing complete vacuolation of hepatocytes, polygonal shape of hepatocytes completely disturbed. Bouin, 6 µm, HE. X 950.



Histology of intestinal tissues in fish exposed to cypermethrin

The observation of the histological preparations of intestine of fish O. mossambicus exposed to 0.008 ppm of cypermethrin for 24 hrs showed no definite alterations except for an increase in number of goblet cells (Figure 2B) and on increasing the exposure to 48 hrs the intestinal wall, the serosa was eroded (Figure 2C). However, at 72 hrs exposure period the goblet cells increased in number and the disintegration of intestinal serosa continued up to 96 hrs of exposure (Figure 2D). The epithelial columnar cells were observed to change in structural form from well-defined columnar cell to shrunken cells from 96 hrs of exposure to finally disintegrate at 192 hrs of exposure (Figures 2E and 2F).

DISCUSSION

The LC50 value of 0.04 ppm at 96 hrs exposure of cypermethrin a synthetic pyrethroid pesticide has been reported in the present study for tilapia fish O. mossambicus. The reported LC50 value of 14, 50µg/l of deltamethrin (synthetic pyrethroid) in Oreochromis niloticus fingerlings suggested acute pyrethroid sensitivity and toxicity to fish (Glow and Godzi 1994; Karthigayani et al (2014). In Poecilia reticulata the pyrethroid dieldrin was considered more two times more toxic to the fish species than other pesticides (Mittal et al 1994). Many reports have demonstrated the acute toxicity of pyrethroid insecticides on fish species and its developmental stages (Mestres and Mestres 1992 ; Koprucii and Aydin 2004). Evidently the pyrethroid pesticide exposure to fish had deleterious effects on tissues that eventually had lethal effects (Yildririm et al 2006, Singh and Singh 2007).

The present study confirms and demonstrates the toxic effect of cypermethrin on intestine of tilapia fish O. mossambicus that was directly exposed to cypermethrin by ingested water and liver which was exposed to the absorbed cypermethrin. Since, cypermethrin is a chemical of different iso forms and is stable in extreme conditions can withstand disintegration which enhances

its capacity to react with the living tissues without undergoing degradation. The present study was designed to study the toxic effect of cypermethrin with an increase of exposure time to analyse the histological alterations of intestine and liver of tilapia fish O. mossambicus. Recently Cengiz and Unlu, (2006) reported histopathological effects of deltamethrin on the gills, liver, and gut tissues in mosquito fish (Gambusia affinis) after exposure to sublethal concentrations of 0.25 and 0.50 g/L. These results are in agreement with our findings on histological alterations observed in intestine and liver tissues.

The exposure of sublethal concentration of 0.008 ppm of cypermethrin on histological preparations of intestine demonstrated alterations in the intestinal wall. On increasing the exposure of cypermethrin, had a deleterious effect on the internal epithelial lining cells and such changes in intestine, probably induce changes in biological functions of the fish that may contribute to lethargic behavioural responses.

The histological alterations of the liver tissues increase with the exposure time of cypermethrin also indicate that absorption of cypermethrin was normal up to 48hrs and at 96hrs of exposure and the cells were unable to differentially diffuse cypermethrin and thereby higher concentration of cypermethrin possibly accumulated in the liver. The hepatocytes were able to withstand the toxic effect of cypermethrin at sub lethal concentration (0.008ppm) up to 48hrs as the concentration of cypermethrin increased in the liver cells, the hepatocytes apparently showed alterations in shape with more cells changing from polygonal to round shape and the cells becoming more vacuolated due to probable change in an osmatic stress induced by cypermethrin.

Also the pycnosis of nucleus proved the disintegration of regulative functioning of the nucleus and resulting changes in the metabolism of the nutrients reaching the liver cells. This change in metabolic pathway of the essential nutrients such as glucose and amino acid may have direct or indirect influence on the other vital tissue of the fish such as brain and kidney.



The results obtained by the study on toxic effect of cypermethrin clearly demonstrate that, at sublethal concentrations itself the pesticide has deleterious effect on the tissues exposed to it directly or even indirectly. Hence, cypermethrin can be considered as a potent toxic pollutant capable of destroying the balance of aquatic ecosystem.

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