TOXICITY STUDY OF DIETHYL PHTHALATE ON … Project...Plate 6: Gill section of group C exposed to...

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TITLE PAGE

TOXICITY STUDY OF DIETHYL PHTHALATE ON CLARIAS

GARIEPINUS FINGERLINGS (BURCHELL, 1882)

BY

IKELE CHIKA BRIGHT

PG/M.Sc/07/42622

A PROJECT SUBMITTED IN PARTIAL FULFILMENT FOR THE

AWARD OF MASTER DEGREE IN FISHERIES BIOLOGY IN THE

DEPARTMENT OF ZOOLOGY, FACULTY OF BIOLOGICAL

SCIENCES, UNIVERSITY OF NIGERIA NSUKKA

SUPERVISOR: PROF. B. O. MGBENKA

AUGUST 2010

ii

APPROVAL PAGE

I approve that this project was carried out under my supervision by Ikele Chika Bright, in

the Department of Zoology, University of Nigeria Nsukka.

In partial fulfillment of the requirement leading to the award of Masters degree in

Fisheries Biology.

------------------ ---------

Prof. B. O. Mgbenka Date

Supervisor

------------------------ ------------------

Prof. J. E. Eyo Date

Head of Department

--------------------------- ---------------------

External examiner: Date.

iii

DEDICATION

This work is dedicated to my beloved parents Ven. Dr. and Mrs. I. I. Ikele.

iv

ACKNOWLEDGMENTS

I thank God for His loving kindness, protection and knowledge He has given to me so as

to see this work is a success. I am grateful to my supervisor, Prof. B. O. Mgbenka for his

wonderful effort and supervision made to see this work is done well. Words only are

grossly insufficient in expressing my immense and profound gratitude to him. He was

indeed the latent force behind the prompt success of this research work, through his ever

ready assistance, prodigious encouragement and constructive advice that sustained me

throughout the duration of this project.

My appreciation goes to my parents Ven. Dr. and Mrs. I. I. Ikele for their prayerful,

encouragement and financial support. I pray that God will bless them and continue to

protect them. Also, to my wonderful siblings Andrew, Obinna, Ikenna, Chinenye, and

Onyeka for their love and prayers through the research. To all my good and most

cherished friends I say thank you for your prayers and spiritual support.

v

TABLE OF CONTENTS

Title Page … … … … … … … … … i

Approval Page … … … … … … … … ii

Dedication … … … … … …. … … … iii

Acknowledgements … … … … … … … … iv

Table of Contents … … … … … … … … v

List of Tables … … … … … … … … … vii

List of Figures … … … … … … … … … ix

List of Plates … … … … … … … … … xi

Abstract ... … … … … … … … … xiii

CHAPTER ONE

1.1 Introduction … … … … … … … … 1

1.2 Aim of study … … … … … … … … 7

1.3 Literature review … … … … … … … 7

1.4 Brief description and taxonomy of Clarias gariepinus … … 11

CHAPTER TWO: MATERIALS AND METHODS

2.1 Collection of the fish … … … … … … … 13

2.2 Collection of the test compound … … … … … 13

2.3 Acute toxicity test (short term exposure) … … … … 13

2.4 The sub lethal or chronic test (long term exposure) … … … 14

2.4.1 Assay for enzyme … … … … … … … 14

2.4.2 Determination of Acetylcholinesterase (AChE) activity in the brain and

muscle of fishes in various groups. … … … … … 14

2.4.3 Determination of liver and muscle acid phosphatase enzyme activity of

fishes in various groups. … … … … … … 18

2.4.4 Determination of liver aspartate enzyme activity. … … … 20

2.4.5 Determination of liver alanine transaminase enzyme activity … … 20

2.5 Histopathology. … … … … … … … 21

2.6 Haematological assay. … … … … … … 22

2.7 Statistical analysis … … … … …. … … 22

vi

CHAPTER THREE: RESULTS

3.1. Acute toxicity test … … … … … … … 23

3.2. Biochemical assay … … … … … … … 35

3.2.1. Acetylcholinesterase enzyme activity in the brain of various groups … 35

3.2.2. Acetylcholinesterase enzyme activity in the muscle of various groups 36

3.2.3. Acid phosphatase enzyme activity in the liver of various groups … 37

3.2.4. Acid phosphatase enzyme activity in the muscle of various groups … 39

3.2.5. Aspartate transaminase enzyme activity in the liver of various groups 40

3.2.6. Alanine transaminase enzyme activity in the liver of various groups … 41

3.3. Haematology assay … … … … … … … 42

3.4. Packed cell volume level in various groups … … … … 43

3.5. Red blood cell count in various groups … … … … … 45

3.6. White blood cell count in various groups. … … … …. 46

3.7. Mean cell volume in various groups. … … … …. …. 48

3.8. Mean cell haemoglobin in various groups … … … … 49

3.9. Mean cell haemoglobin concentration in various groups … … 51

3.10. Histopathology. … … … … … … … … 55

3.10.1. Histopathology of gill tissues in various groups. … … … 55

3.10.2. Histopathology of kidney tissues in various groups … … … 60

3.10.3. Histopathology of liver tissues in various groups. … … … 65

CHAPTER FOUR: DISCUSSION

Discussion … … … … … … … … … 71

Conclusion … … … … … … … … … 79

References … … … … … … … … … 81

Appendix … … … … … … … … … 91

vii

LIST OF TABLES

Table 1: Physiological properties of diethyl phthalate. … … … … 2

Table 2: Collected experimental data for acute toxicity (mg/l) of some phthalate esters

of fish … … … … … … … … … … 10

Table 3: Toxicity of diethyl phthalate to fish. … … … … … 11

Table 4: Procedure for determining of acetylcholinesterase enzyme activity … 18

Table 5: Acute toxicity test result for 10 Clarias gariepinus per replicate for a total of

96 h. … … … … … … … … … … 24

Table 6: Mean descriptive for acute toxicant concentration. … … … … 25

Table 7: Percentage mortality table at 24h exposure of toxicant concentration to

Clarias gariepinus fingerlings. … … … … … … 26







Table 11: Cumulative percentage mortality table. … … … … … 34

Table 12: Percentage survival at the end of the exposure time. … … … 34

Table 13: Mean value of Ache activity in brain of fingerlings exposed to DEP. … 35

Table 14: Mean value of Ache activity in the muscle of fingerlings exposed to DEP 36

Table 15: Mean value of ACP activity in liver of fingerlings exposed to DEP … 38

Table 16: Mean value of ACP activity in the muscle of fingerlings exposed to DEP 39

Table 17: Mean value of AST activity of fingerlings exposed to diethyl phthalate … 40

viii

Table 18: Mean of liver ALT activity of fingerlings exposed to diethyl phthalate … 41

Table 19: Mean of haemoglobin (g/dl) of various groups exposed to diethyl phthalate 43

Table 20: Mean of the Packed cell volume (%) of various groups exposed to diethyl

phthalate. … … … … … … … … … 44

Table 21: Mean of the red blood cell count (106

mm3) of various groups exposed

to diethyl phthalate. … … … … … … … … 46

Table 22: Mean of the total white blood cell count (x104/mm

3) of various groups

exposed to diethyl phthalate. … … … … … … … 47

Table 23: The mean value of mean cell volume (fl) of various groups exposed

to diethyl phthalate. … … … … … … … … 49

Table 24: Changes in the mean cell heamoglobin (Pg) of various groups

exposed to diethyl phthalate. … … … … … … … 50

Table 25: Changes in the mean cell heamoglobin concentration (g/dl) of various

groups exposed to diethyl phthalate … … … … … … 52

Table 26: The changes in the Lymphocyte count of Clarias gariepinus exposed to diethyl

Phthalate ….. … … … … … … … … 53

Table 27: The changes in the monocyte count of clarias gariepinus fingerlings exposed

to diethyl phthalate … … … … ... … … … 54

Table 28: The changes in the neutrophils of Clarias gariepinus fingerlings exposed to

diethyl phthalate … … … … … … … … 55

ix

LIST OF FIGURES

Fig.1: Probit transformed responses for total death. … … … … … 25

Fig.2: Probit transformed responses for 24 h exposure. … … … … … 27

Fig.3: Probit transformed responses for 48 h exposure. … … … … … 29

Fig.4: Probit transformed responses for 72 h exposure … … … … 31

Fig.5: Probit transformed responses for 96 h exposure … … … … 33

Fig.6: Effect of different concentration of diethyl phthalate on brain AchE … … 36

Fig.7: Effect of different concentration of diethyl phthalate on muscle AchE … 37

Fig. 8: Effect of different concentration of diethyl phthalate on liver Acid

Phosphatase activity … … … … … … … … 38

Fig.9: Effect of different concentration of diethyl phthalate on muscle Acid

Phosphatase activity … … … … … … … … 39

Fig.10: Effect of different concentration of diethyl phthalate on the liver Aspartate

Amino transferase … … … … … … … … 41

Fig.11: Effect of different concentration of diethyl phthalate on the liver Alanine

Amino transferase … … … … … … … … 42

Fig.12: Effect of different concentration of diethyl phthalate on the haemoglobin level 43

Fig. 13: Effect of different concentration of diethyl phthalate on the Packed cell

Volume … … … … … … … … … 45

Fig.14: Effect of different concentration of diethyl phthalate on the red blood cell … 46

Fig. 15: Effect of different concentration of diethyl phthalate on the white blood cell 48

Fig.16 Effect of different concentration of diethyl phthalate on the mean cell volume 49

x

Fig.17: Effect of different concentration of diethyl phthalate on the mean cell

Haemoglobin … … … … … … … … … 51

Fig.18: Effect of different concentration of diethyl phthalate on the lymphocyte count 52

Fig.19: Effect of different concentration of diethyl phthalate on the monocyte count 53

Fig.20: Effect of different concentration of diethyl phthalate on the Neutrophil count 54

xi

LIST OF PLATES

Plate 1: Gill section of control fish showing no changes magnification (H&E) x40. 56

Plate 2: Gill section of group A exposed to 30µg/l for 15 days mag. (H&E) x40 a.

Prominent lamellar showing the acidophil cells. … … … … 56

Plate 3: Gill section of group A exposed to 30µg/l for 30 days mag (H&E) x 40 a.

Haemorrhaging of the gill filament. … … … … … … 57

Plate 4: Gill section of group B exposed to 40µg/l for 15 days mag (H&E) x40 a.

Fatty cells prominent. … … … … … … … … 57

Plate 5: Gill section of group B exposed to 40µg/l for 30 days mag (H&E) x 40 a.

Enlarged filament … … … … … … … … … 58

Plate 6: Gill section of group C exposed to 60µg/l for 15 days mag (H&E) x 40 a.

Filaments are not enlarged, they are narrow with disjointed lamella and the

width is not uniform. … … … … … … … … 58

Plate 7: Gill section of group C exposed to 60µg/l for 30 days mag (H&E) x 40 a.

There is enlargement of the filament and the lamella are disjointed … … 59

Plate 8: Gill section of group D exposed to 80µg/l for 15 days mag (H&E) x40.a.

Severe destruction of the lamella. … … … … … … … 59

Plate 9: Gill section of group D exposed to 80µg/l for 30 days mag (H&E) x40 a.

Extensive lamellar fusion. … … … … … … … … 60

Plate 10: No recognizable changes were observed in the kidney of control fish … 61

Plate 11: Kidney section of group A exposed to 30µg/l for 15 days mag (H&E) x40 a.

Tubules are in tact. … … … … … … … … 61

Plate 12: Kidney section of group A exposed to 30µg/l for 30 days mag (H&E) x40 a.

Tubules are in tact (CS) and Sequensiation of kidney architecture. … … 62

Plate 13: Kidney section of group B exposed to 40µg/l for 15 days mag (H&E) x 40 a.

Destruction or fusion of the tubules. … … … … … … 62

xii

Plate 14: Kidney section of group B exposed to 40µg/l for 30 days mag (H&E) x 40 a.

Severe destruction of the tubules. … … … … … … … 63

Plate 15: Kidney section of group C exposed to 60µg/l for 15 days mag(H&E) x40 a.

Pyknosis (pyvnotic nuclei present, degenerated kidney tubule pyknosis. … 63

Plate 16: Kidney section of group C exposed to 60µg/l for 30 days mag(H&E) x40 a.

Destruction of tubules. b. Tubules are not continuous. … … … … 64

Plate 17: Kidney section of group D exposed to 80µg/l for 15 days mag(H&E) x40 a.

Condensation of the glomeruli content. … … … … … … 64

Plate 18: Kidney section of group D exposed to 80 µg/l for 30 days mag (H&E) x40 a.

Condensation of the glomeruli content. … … … … … … 65

Plate 19: No recognizable changes were observed in the kidney of control fish … 66

Plate 20: Liver section of group A exposed to 30µg/l for 15 days mag x40 a. cellular

proliferation. … … … … … … … … … 66

Plate 21: Liver section of group A exposed to 30µg/l for 30days mag x40 a. liver

exhibits normal morphology. … … … … … … … 67

Plate 22: Liver section of group B exposed to 40µg/l for 15 days mag x40 a. Severe

cellular proliferation. … … … … … … … … 67

Plate 23: Liver section of group B exposed to 40µg/l for 30 days mag x40 a.

Congestion … … … … … … … … … 68

Plate 24: Liver section of group C exposed to 60µg/l for 15 days mag x40 a. severe

necrosis. … … … … … … … … … … 68

Plate 25: Liver section of group C exposed to 60µg/l for 30 days mag x40 a.

Sinusoid enlargement. … … … … … … … … 69

Plate 26: Liver section of group D exposed to 80µg/l for 15 days mag x 40 a.

parenchymateous degeneration and extravasation. … … … … 69

Plate 27: Liver section of group D exposed to 80µg/l for 30 days mag x40 a. Fatty or

glycogen degeneration. … … … … … … … … 70

xiii

ABSTRACT

Diethyl phthalate (DEP) is used as a plasticizer, a detergent base, in aerosol sprays, as a

perfume binder and after shave lotion. It is known to be a contaminant of fresh water and

marine ecosystem. Therefore, a study was designed to determine the acute toxicity effects

of DEP on a fresh water fish, Clarias gariepinus fingerlings. The fish was treated with 50

µg/l, 75 µg/l, 100 µg/l, and 150 µg/l. DEP was dissolved in distilled water to determine

the LC50. There was 100 % mortality observed in 150 µg/l, 56.6 % mortality in 100 µg/l

treated fish, 46.7 % mortality at 75 µg/l, and 29.9 % mortality in 50 µg/l within 24h to

96h of exposure. The LC50 of DEP was estimated at log toxicant concentration as 2.217,

2.734, 3.435 and 3.931 µg/l at 24, 48, 72, 96h and 1.871µg/l for the total death. This

shows that the impacts are dose and time dependent with respect to marked reduction in

mortality rate. At sub-lethal concentrations of the test substance at 30 µg/l, 40 µg/l, 60

µg/l, and 80 µg/l in a renewal bioassay system, the water and the test compound were

changed intermittently. One group was maintained as a control in dechlorinated water.

Fish were killed and dissected to obtain liver, muscle, and brain samples. Brain and

muscle acetylcholinesterase (AchE) activity was measured. Liver and muscle acid

phosphatase (ACP) were measured. Liver aspartate (AST) and alanine transaminase

(ALT) were measured. There was significant difference (P ≤ 0.05) in brain and muscle

AchE activity compared to the control. The liver ACP activity was statistically significant

(P ≤ 0.05) at day 15 respectively compared to the control. The muscle ACP in other

treatment groups showed no significant difference (P > 0.05). Liver AST showed no

significance in all treated groups (P > 0.05) and liver ALT activity was statistically

significance (P ≤ 0.05) at day 30 only. The haematological parameters (HB, PCV, RBC

and WBC) carried out showed that haemoglobin level estimated in all treatment groups to

the duration of exposure showed no significance different (P > 0.05) compared to the

control. The park cell volume showed a significance different (P ≤ 0.05) at day 30 only.

The erythrocyte count mean values were not statistically significant (P > 0.05) compared

to the control throughout the duration of exposure. The leucocyte count throughout the

exposure period showed that the mean values are statistically significant (P ≤ 0.05) at day

15 only compared to the control. The mean cell volume (MCV), showed a significant

different at day 15 (P ≤ 0.05) whereas mean cell haemoglobin (MCH) and mean cell

haemoglobin concentration (MCHC) showed no significance difference (P > 0.05)

throughout the exposure period. The mean values in day 0 for differential counts

xiv

(lymphocytes, monocytes and neutrophils) showed that monocytes are significantly

different P ≤ 0.05 compared to the control. No significant was seen between the

lymphocytes and the neutrophils. In day 15 only, the monocytes and the lymphocytes

showed a significant difference (P ≤ 0.05) whereas no significance was seen in day 30

between the lymphocytes, monocytes and neutrophils. The histopathological changes of

DEP on the liver, gill, and kidney were determined by light microscopy. The most

common gill changes at all concentrations of DEP were destruction of lamella, disjointed

and enlargement of lamella, haemorrhaging of the gill filament and fatty cells. Pyknotic

nuclei, destruction or fusion of tubules, condensation of the glomeruli and severe

destruction of the tubule were observed in kidney tissue of fish. Cellular proliferation,

congestion, necrosis, sinusoid enlargement, parenchymatous degeneration and fatty or

glycogen degeneration were observed in the liver tissue of fish.

1

CHAPTER ONE

INTRODUCTION

Diethyl phthalate (C12H1404) is a man made colourless liquid with a slight

aromatic odour, bitter and disagreeable taste. Its relative molecular mass is 222.3. The

trade name of diethyl phthalate includes neantine, peilantol A, and solvanol. Its structural

formula is given in Fig.1.

Fig.1. Structure of diethyl phthalate

O

C – O – CH2 CH3 (ATSDR, 1995)

C – O – CH2 CH3

0

O

2

Table 1: Physiological properties of diethyl phthalate

(Agency for toxic substances and Disease registry (ATSDR) (1989))

a From Hazardous Substance Data Bank (1994)

b Temperature not specified.

c Assuming a temperature for the dimensioned value at around 20˚ C.

Property Value

Water solubility 1000 mg/litre

Solubility in

organic solvents

Soluble in alcohol, acetone,

ether, benzene, ketones,

esters, aromatic,

hydrocarbons, aliphatic

solvents and vegetable oil.

Partition

Coefficients

Log Kocb 2.65

Vapour pressure

at 20°C 4.59x10-

2pa

at 28o C 2.19x10-1pa

Henry’s law

constantb

7.9x10-5 kpa

Dimensionless

Henry’s law

constant (Air/water

pollution

coefficient)c

4.310-8

3

Diethyl phthalate is produced industrially by the reaction of phthalic anhydride

with ethanol in the presence of concentrated sulfuric acid catalyst (HSDB, 1994). The

purity of manufactured phthalate esters is reportedly between 99.70% and 99.97%, with

the main impurities being isophthalic acid, tetephthalic acid, and maleic anhydride

(Peakall, 1975). Diethyl phthalate is manufactured for many uses such as insecticide,

mosquito repellants, as a camphor substitute, plasticizer for cellulose ester plastic films

and sheets, bathing soaps, cosmetics, aftershave lotion, hair sprays, nail polish and

enamel removers, eye shadow, perfumes, detergent, skincare preparation, etc. (Kamrin

and Mayor, 1991).

Diethyl phthalate may enter the environment in industrial wastewaters by

evaporation into the air from disposal sites, directly from consumer products, burning of

plastic products and by leaking from landfills into soil or water including ground water

(ATSDR, 1995). It may also be deposited on the ground or in the water by rain. In more

slowly moving waters, microorganism in the water or sediment may break down some of

the deadly phthalate into nontoxic products.

However, if there is a little organic matter in the soil, diethyl phthalate may move

down through the soil and enter the ground water. Small amount of diethyl phthalate can

build up in animals that live in such waters like fish, oyster, etc. Based on 1994 toxics

release inventory data, USEPA, (1995) estimated that 72 tonnes and 341 kg of diethyl

phthalate would be released annually.

Diethyl phthalate is likely to undergo biodegradation in the environment. The

abiotic degradation processes such as hydrolysis, oxidation, and photolysis are unlikely to

play significant role in the environmental fate of diethyl phthalate (US EPA, 1979).

4

Volatilization of diethyl phthalate is expected to be slow based on its low vapour

pressure of 4.59 x 102 at 20° C (Grayson and Fosbraey, 1982). Diethyl phthalate reacts

photochemically with hydroxyl radicals in the air, with an estimated half-life of 2 – 22 h

(HSDB, 1994). The distribution of diethyl phthalate between the gaseous and particulate

phases in air has been estimated by the Jungle Pankow model, which determines the

fraction of diethyl phthalate in the particulate creosol phase to be 0.00039 (Staples et al.,

1997).

However, it has been estimated that approximately 1% of the phthalate ester

content of plastics materials in direct contact with water or other liquids may be released

into the aquatic environment (Peakall, 1975). Diethyl phthalate can absorb into

suspended particles in marine waters with the maximum occurring into particles ranging

353 - 698 m in size (Al-Omran and Preston, 1987). Diethyl phthalate have been

detected in aquatic organisms and have been found to bioconcentrate modestly in these

organisms (Camanzo et al., 1983; Devault, 1985; Mcfall et al., 1985). Biodegradation of

diethyl phthalate in soil has been shown to occur as a series of sequential steps. 0.1-100

mg/g of diethyl phthalate with a half life of 0.75 days at 20°C was biodegraded rapidly in

soil and was not expected to persist in the environment (Cartwright et al., 2000b).

Diethyl phthalate has been detected in ambient in-door air waste waters from

industrial facilities, subspace waters and sediments, and marine waters. Diethyl phthalate

has been measured in the indoor air of a telephone switching office and in out door air in

Newark, USA, at a concentration ranging from 1.60 to 2.30 g/m3 and from 0.40 to 0.52

g/m3 respectively, during a 43-day sample period (Shields and Weschler, 1987).

Diethyl phthalate has been detected in treated waste waters from various

manufacturing plants. For example 3.2 g/liter was detected at textile manufacturing

5

plants (Walsh et al., 1980), 60 g/litre was detected at a tyre manufacturing plant

(Jungclaus et al., 1976), and 50 g/litre was detected at a pulp and paper manufacturing

plant (Brownlee and Strachan; 1977, Voss, 1984). Diethyl phthalate has been found at a

median concentration of < 10 g/L in 10% of the industrial effluent samples and in 3.0%

of the ambient water samples in the storage and retrieval (STORET) database maintained

by the US Environmental Protection Agency (EPA) (Staples et al, 1985). A river water

sample from the lower Tennessee River, USA, was found to contain diethyl phthalate at a

concentration of 11.2 g/l (Goodley and Gordon, 1976) and 21 g/litre in tap water from

the Kitakyushu area of Japan. The source of exposure was considered to be domestic

sewage and industrial waste (Akiyama et al, 1980). River water samples and sewage

effluent collected in 1984 from the Rivers Irwell and Etherow near Manchester, England,

contained 0.4 – 0.6 g/l diethyl phthalate (Futoki and Vernon, 1990).

Diethyl phthalate was detected in 10% of aquatic sediment samples at a median

concentration of < 2.5 mg/kg wet weight (Staples et al., 1985). Diethyl phthalate was

detected in 4.26% of the soil samples taken from the U.S. National Priorities List of

Hazardous Waste Sites, at a mean concentration of 39 mg/kg in the positive samples

(Contract Laboratories Programme Statistical Data Base, 1989). Human exposure to

diethyl phthalate can result from eating foods into which diethyl phthalate has leached

from packaging materials, eating contaminated sea food, drinking contaminated water,

breathing contaminated air or as a result of medical treatment involving the use of PVC

tubing for example dialysis of patient (Blount et al., 2000b). The use of diethyl phthalate

as an ingredient in a variety of cosmetic formulations at concentrations raging from <

0.1% to 28.6% however, are likely to be the primary sources of human exposure (Api,

2001). A 2001 survey of fragrance manufacturers in the USA provided maximum

concentrations of 1 – 11% diethyl phthalate in perfume, 1.0% in deodorants, and other

6

personal cleanliness products, (Anonymous, 1985). The products when applied to skin,

eyes, hair, and nails will come in contact with mucous membranes and with the

respiratory tract frequently and for prolonged duration (Kamrin and Mayor. 1991).

Diethyl phthalate was detected in 42% of the human adipose tissue samples from children

and adults (cadavers and surgical patients) in various regions of the USA during 1982

(USEPA, 1986).

Diethyl phthalate was detected in 1 of 1 drinking water sample, 1 of 8 ambient air

sample and 2 of 12 exhaled breath samples. Diethyl phthalate concentrations ranging

from 0.01 g/l (in 6 of 10 US cities) to 1.0 g/litre (in Miami, Florida) were found in

drinking water samples from water treatment plants in the USA (Keith et al.,

1976).Diethyl phthalate is not known to cause cancer in humans or animal. It can

however be mildly irritating when applied to the skin of animals.

Freshwater fish collected from the Great Lake tributaries in Wisconsin and Ohio,

USA, in 1981, contained diethyl phthalate in composite whole body tissue samples at

concentration ranging from < 0.02 mg/kg to < 0.30 mg/kg (Devault, 1985). Lake trout

(Salvelinus namaycush) and white fish (Coregonus clupeaformis) taken from Lake

Superior near Isle Royale, Michigan, USA, had elevated levels of diethyl phthalate (0.5

and 2.2 g/g, respectively compared with lake trout and white fish taken from other part

of Lake Superior (US EPA, 1980). Fish taken from Siskiwit Lake on Isle Royale,

Michigan, a pristine area supposedly unaffected by human activity, also had relatively

high concentrations of diethyl phthalate in their tissue, 0.4 mg/kg for lake trout and 1.7

mg/kg for white fish.

However, diethyl phthalate has been seen to be toxic to the environment and most

especially the aquatic organism found in aquatic environment. The effect of different

7

doses of diethyl phthalate on histopathology and haematological parameters has not been

studied earlier. This study therefore aims at investigating the acute and chronic effect of

this compound, diethyl phthalate on the enzymatic activities and haematology. To be

studied are haematological parameters such as haemoglobin, packed cell volume, red

blood cell count, white blood cell count, differential count, and histopathology of the fish

liver, kidney, and gills.

It is justified to embark on these further studies so as to check the extent of

damage done in a dose of dependent manner at given point in time following the

administration of the compound on the fishes.

1.2 Aims of Study

The objectives of this study were to:

1. Determine whether the compound diethyl phthalate is lethal to test organism

Clarias gariepinus (Burchell, 1882) and at what concentration the number of half the

treated fish would die;

2. Determine the effect of this chemical on their biochemical parameters; and

3. Check the effects of diethyl phthalate on haematological parameters and

histopathology of the test organism when exposed to sublethal concentrations.

1.3 Literature Review

Diethyl phthalate has been found to be used as a component in insecticide sprays

and mosquito repellents and has been shown to have deleterious effects in animals and

aquatic organism such as fish etc. (Devault, 1985). A study on toxicity of diethyl

phthalate in rats was shown when diethyl phthalate (10 or100mg) was administered to

8

each three Wister rats by stomach intubations. Their daily urine collections were

analyzed for 10 days by Gas chromatography (Kawano, 1980). Seventy seven to seventy

eight percent of the administered dose was excreted in urine within 24 hours as

monoester derivatives (67 - 70% of the dose), phthalic acid (8 - 9% of the dose) and

about 35 - 93% was excreted within 11 weeks after administration.

Male rats exposed to a single dermal application of 14

C diethyl phthalate (5 – 8

mg/cm2) excreted 24% of the administered dose in the urine and 1% dose in faeces

within 24h (Elsisi et al., 1989). Studies have reported increase in absolute and relative

liver weights in rats after 1-16 weeks of exposure of diethyl phthalate (Brown et al.,

1978; Moody and Reddy, 1978; Oishi and Hiraga, 1980). According to NTP (1995) in a

4-week study, diethyl phthalate was dermally applied to rats. A Group of 10 male and 10

female rats were administered with 0 ml, 35 ml, 75 ml, 150ml and 300ml of diethyl

phthalate (corresponding to 0, 200, 400, 800, or 1600 mg/kg for males and 0, 300, 600,

1200 or 2500 mg/kg body weight per day for females. Increased relative weights were

observed in 300 ml (male 9% and Female rats 7%). However, no adverse effects on the

histopathology of heart, lung, liver, kidney, esophagus, gall bladder, large intestine, small

intestine and stomach in rats were observed (NTP, 1995).

A comparison of the results of in vitro mutagenic assays of diethyl phthalate in

strains of Salmonella typhimurium shows contradictory findings. Diethyl phthalate has

been shown to be mutagenic for S. typhimurium strains TA100 and TA 1535 only without

metabolic activation (Kozumbo et al., 1982; Agarwal et al., 1985). The maximum ratio of

induced revertants to control was about 2 - 3 (Kozumbo et al., 1982; Agarwal et al.,

1985) and about 2 (Agarwal et al., 1985) for TA100 and TA1535, respectively. No

induced revertants were observed for TA98 and TA1537 with or without metabolic

activation (Rubin et al., 1979). Contrary to positive findings, diethyl phthalate has been

9

found to be non-mutagenic in S. typhimurium strains TA98, TA 100, TA1537 with or

without metabolic activation (Zeiger et al., 1982).

Chronic toxicity studies were performed with commercial phthalate esters on

Daphnia magma (14 phthalates) and rainbow trout (Oncorhynchus mykiss ) using the

lower molecular-weight phthalate esters - dimethylphthalate (DMP), diethyl phthalate

(DEP), di-n- butyl phthalate (DBP), and butylbenzylphthalate (BBP). The results of the

studies indicated a general trend to which toxicity for both species increased as water

solubility decreased. Early life-stage toxicity studies with rainbow trout indicated that

survival (DMP) and growth (DBP) were affected at 2.1 and 0.19 mg/l, respectively (Jon

et al., 1994).

Preliminary results of quantitative structure-activity relationship (QSAR) showed

acute toxicity of fathead minnow (Pimephales promelas) for phthalate esters. The result

showed that 10 mg/L < LC50 < 100 mg/L was harmful, 1 mg/L < LC50 <10 mg/L was

toxic; LC50 < 1 mg/L was very toxic. This prediction effort resulted in classification of the

vast majority of the phthalates in the very toxic group (Parkerton and Konkel, 2000).

Nivedita et al., (2002) report on the toxic effect of diethyl phthalate on Cirrhina

mrigala indicated that DEP brings about significant changes in the activity of certain

liver and muscle enzyme and at 75 ppm DEP (w/v) 100% mortality occurred within 24h.

However, the alteration in enzyme activity may have long term effect when DEP is

introduced in low doses. Fatoki and Vernon (1990) opined that when the enzyme

activities in vital organs of fishes are altered due to long exposure, it may be dangerous to

the fishes and their survival is at risk.

10

Table 2: Data on acute toxicity (mg/L) of some phthalate esters on fish*

Chemical Sheephead

minnowflow

through 96 h

Rainbow

troutflow

through

96 h

Bluegill

static

96h

Fathead

minnow

static

96h

Fathead

minnow,

flow

through

96 h

Di-methyl

phthalate**

29 56 67 120 39

Diethyl

phthalate**

99 12 22 17 17

Butyl

benzyl

phthalate

NATBLS

NATBLS

15

Butly-2-

ethylhexyl

phthalate

NATBLS NATBLS NATBLS NATBLS NATBLS

(ATSDR, 1989)

11

Table 3. Toxicity of diethyl phthalate to fish

(ATSDR, 1995)

*NOEC- no observed effective concentration

1.4 Brief Description and Taxonomy of Clarias gariepinus

Clarias gariepinus is an omnivorous bottom feeder, feeding on detritus, plankton,

insect larvae, worms, gastropods, crustacean and small fishes such as Tilapia and Alestes.

Abundance: It is an indigenous species in Africa. It is most abundant and widely

distributed in African lakes and rivers e.g. River Nile. It naturally inhabits in tropical

swamps, lakes, rivers and flood plains. It is naturally called Africa magur. In recent years

Name of fish End point Concentration

(mg/L)

Blue Gill

(Lepomis macrochirus)

96-h LC50 98

96-h NOEC* 1.7

Rainbow trout

(Oncorhynchus mykiss)

96-h LC50 12

96-h NOEC 3.8

Fat head minnow

(Pimephales promelas)

96-h LC50 17

96-h NOEC 4

Golden orfe (Leuciscus

idus melanotus)

48-h LC50 53-61

Sheep head minnow

(Cyprinodont variegates)

96--h LC50 30

96--h LC50 29

96-h NOEC 20

12

it has been introduced in Europe, Asia and South American. There are about 13 genera

and 100 species of clariids (Idodo-Umeh, 2003).

Utilization:

(1) The flesh is of high quality, greatly prized and consumed by many people.

(2) It is cultured artificially in fish ponds or enclosures.

(3) Big-sized Clarias gariepinus is usually used to control the population of

tilapia when cultured together (Idodo-Umeh, 2003).

13

CHAPTER TWO

MATERIALS AND METHOD

2.1 Collection of the Experimental Fish

The fingerlings of Clarias gariepinus use in this study was obtained from the

Awka outlet of Aquafish limited, Anambra State, Nigeria. The fish were transported to

the University of Nigeria, Nsukka and acclimated for three weeks in the laboratory before

the commencement of the study. The water was aerated continuously using aerators

throughout the study period. The fish were fed with Dizengoff fish feed containing 55%

crude protein.

2.2 Collection of the Test Compound

The test compound, diethyl phthalate that was used in this study was of analytical

grade 99.97% purity and was obtained from Sigma Chemicals, Ohio, USA.

2.3 Acute Toxicity Test (Short Term Exposure)

Experimental Setup

One hundred and fifty fingerlings of 13.13 ± 2.27 g were used for this

experiment. The fingerlings were distributed randomly to fifteen containers each

containing ten fingerlings. Each container was covered on top with nylon mesh tied

firmly with rubber strap to prevent the fish from jumping out. Each treatment group was

replicated into three containing 10 fish per replicate with five different treatments (0

µg/L, 50 µg/L, 75 µg/L, 100 µg/l, and 150 µg/l). Mortality was observed at interval for

some days and the LD50 was detected. Water quality characteristics of temperature, pH,

dissolved oxygen (DO) and total hardness as equivalent of calcium carbonate were

determined. The temperature was 27.1˚ C, pH 7.9, the DO 6.4 mg/L and total hardness

14

100 mg/L equivalent of CaCO3. The water contained (mg/L) Ca2+

, 4.01; Mg2+

, 9.73; Na+,

4.9; Cl-, 7.5; SO4

2-, 15.6; NO3

-, 0.96, and total phosphorus, 0.04 (Ozoko, 1988).

2.4. The Sublethal or Chronic Test (Long Term Exposure)

Experimental Setup

One hundred and eighty fishes of 13.13 ± 2.27 g were used in this experiment.

Diethyl phthalate was administered at concentrations of 0 µg/L, 30 µg/L, 40 µg/L, 60

µg/l, and 80 µg/L. In a renewal bioassay system, the water and the test compound were

changed daily to maintain the toxicant concentration. The temperature, pH and the

dissolved oxygen of the tap water used in the study were 27.5˚ C, 7.2 mg/L and 6.4 mg/L

respectively while the total hardness was 100 mg/L CaCO3. The liver, muscle and brain

tissues were assayed for enzymatic activities, the blood samples were collected for

haematological studies, the liver, kidney and gills of the fingerlings were excised and

subjected for histopathological studies.

2.4.1 Assay for Enzyme

Several parameters were assayed using the tissues of the fish obtained from

various groups. The parameters are

1. Acetylcholinesterase enzyme (AchE).

2. Acid phosphatase (ACP).

3. Aspartate Aminotransferase (AST).

4. Alanine Aminotransferase (ALT).

15

2.4.2. Determination of Acetyl Cholinesterase (AChE) Activity in the Brain and

Muscle of Fishes in Various Groups

(a) Collection of Tissue Sample

The brain and muscle tissues of the fish were extracted, homogenized, with equal volume

of normal saline, centrifuged and the supernatant collected for the assay.

b) Assay system

Substrate: Benzoycholine

Incubation time: 60 minutes

Reagents for acetylcholinesterase enzyme –The reagents were of analytical grade.

(1) Sodium veronal (sodium barbital)

(2) Sodium acetate, crystalline

(3) Acetylcholine chloride

(4) Benzoylcholine chloride

(5) Sodium hydroxide

(6) Hydroxyl ammonium chloride, hydroxylamine

(7) Ammonium Ferric sulphate, Fe+ (NH4 SO4)2 .12 H2 0)

(8) Potassium nitrate

(9) Citric acid, monohydrate

(10) Hydrochloric acid, 1N.

16

D. Preparation of Solution

(i) Veronal buffer (0.1 m, pH 8.6)

49.2 g sodium veronal and 32.4 g, sodium acetate were dissolved in 3000 ml of

distilled water, added to 30 ml 1N HCl and diluted to 5000 ml with distilled water.

(ii) Benzoycholine Stock Solution (200 mM)

2.4374 g of Benzoylcholine chloride was poured into a 50 ml volumetric plastic,

dissolved in distilled water, make up to 50 ml and stored at 4°C.

(iii) Acetylcholine stock solution (200 mM)

1.8167 g acetylcholine chloride was added to a 50 ml volumetric flask, dissolved in

distilled water and made up to volume.

(iv) Substrate benzoylcholine (1.33 mM)

150 ml veronal buffer and 1 ml benzoylcholine stock solution were mixed in a mixer.

(v) Substrate acetylcholine (1.33 mM)

150 ml veronal buffer and 1ml acetylcholine stock solution were thoroughly mixed in a

mixer.

(vi) Sodium hydroxide (2.5 N)

100 g NaOH was dissolved in distilled water and made up to 1000 ml.

(vii) Hydroxylamine

70 g of hydroxyl ammonium chloride was dissolved in distilled water and made up to

1000 ml. The solution was stored in well-stoppered polyethylene flasks in a refrigerator.

17

(viii) Alkaline hydroxylamine solution

Equal volumes of solutions (vi) and (vii) above were mixed.

(ix) Iron solution (0.7 m)

337.5 g Fe(NH4))2(SO4)2 .12H2 0 were dissolved in 700 ml distilled water with

gentle warming. 25 g potassium nitrate was dissolved in a little distilled water

transferred to a 1000 ml volumetric flask and diluted to the mark.

(x) Citrate buffer (1 m; pH 1.4)

10.5 g citric acid was dissolved in 4.0 g NaOH in the distilled water. The mixture

is shaken and diluted in 10 ml distilled water in a volumetric flask to 100 ml.

Procedure: In the reference tube, the initial concentration of the substrate was measured

and in the test the final concentration was also taken.

Incubation temperature of 37°C, incubation volume of 27 ml

Colorimetric measurement of 490 nm and light path of 1 cm. The procedure followed is

tabulated as shown in Table 4.

18

Table 4. Procedure of determining acetylcholinesterase enzyme activity

Pipette into 50 ml

volumetric flask

Reference Test Blank Concentration assay

mixture

Sample

Substrate solution (iv or

v)

-

25 ml

2 ml

25 ml

-

-

1.23 mM

Mix and incubate

Alkaline hydroxylamine 0.125 N Na0H

Solution (viii) 5 ml 5 ml 5 ml 5cmM NH20H

Sample 2 ml - -

Citrate buffer (x) 5 ml 5 ml 5 ml 0.10 m

Ferric solution (ix) 10 ml 10 ml 10 ml 0.14 m

The ferric solution was allowed to run slowly down the walls of the flask. It was

Diluted to mark with distilled water, shaked thoroughly and allowed to stand for 20 min

at room temperature. The solution was filtered through a double folded filter paper and

the first portion of filtrate discarded. The extinction of the filtrates was measured. The

extinction difference between ER (Reference) and ET (test) was measured and used for

the calculations.

2.4.3. Determination of Liver and Muscle Acid Phosphatase Enzyme Activity

(Babson and Read, 1959)

Reagents

A. 1 x 50 tablets of substrate.

B. 2 x 100 ml citrate buffer.

C. 1 x 6 ml tartaric acid.

19

Working Reagents

The substrate was dissolved with the buffer solution, one tablet per 4 ml buffer, stood for

5 - 10 min and mixed until completely dissolved.

The concentrations in the reagent solutions were:

Citrate buffer pH 4.9, 40 mM, p-Nitrophenyl phosphate 5.5 mM. The tartaric acid

solution is ready for use and the concentration in the medium was 15 mM.

Assay for Acid Phosphatase Enzyme Activity

The liver and muscle tissues of the fingerlings at various groups were collected,

weighed, homogenized with equal volume of normal saline centrifuged, the supernatant

removed and stored at 0o

C to maintain the high level of the enzyme, before the assay.

Into the labeled test tube B - blank and T - test, 0.5 ml of the working reagents were

pipetted in each labeled tube and incubated at 37° C in the water bath and the timer set at

3 min. After the incubation, 0.1 ml of the sample was added to the T labeled test tube

samples, mixed and incubated again at 37° C for exactly 30 min. 5.0 ml of 0.02 N NaOH

was pipetted in each labeled tube. The absorbance of the content of T labeled test tube

were read against blank at 450 nm and the calculation made.

2.4.4. Determination of Liver Aspartate Aminotransferase (Reitman and Frankel,

1957)

Assay System

Materials: Buffer/aspartate substrate solution, water bath, 2-4-dinitrophenylhydrazine

(chromogen solution) sodium hydroxide and colorimeter.

Assay of Aspartate Aminotransferase

The liver was collected from the fish using forceps and scissors and was weighed,

homogenized with equal volume of normal saline and centrifuged. The supernatant was

20

transferred into another test tube and immediately refrigerated so as to maintain the high

level of enzyme activity until further analysis. 0.1 ml of the sample was pipetted into a

test tube, mixed with 0.5 ml of buffer and incubated for exactly 30 min at 37° C. 0.5 ml

of the chromogen solution was mixed with the solution and allowed to stand for 20 min at

20° C to 25° C. After the time elapsed, 5.0 ml of 0.4N NaOH was added. The solution

was allowed to stand for 5 minutes at room temperature, the absorbance was read against

a blank using a colorimeter at a wavelength of 546 nm.

2.4.5. Determination of Liver Alanine Aminotransferase (Reitman and Frankel,

1957)

Assay System

Materials: Buffer/alanine substrate solution, Water bath, 2, 4-dinitrophenylhydrazine,

sodium hydroxide (0.04N) and colorimeter

Procedure

The liver was collected from the fish using forceps and scissors and was weighed,

homogenized with equal volume of normal saline and centrifuged. The supernatant was

transferred into another test tube and immediately refrigerated so as to maintain the high

level of enzyme activity until further analysis.

0.1 ml of the sample was pipette into a test tube, mixed with 0.5 ml of buffer and

incubated for exactly 30 min at 37° C. 0.5ml of the chromogen solution was mixed with

the solution and allowed to stand for 20 min at 20° C to 25° C. After the time elapsed,

5.0 ml of 0.4N NaOH was added. The solution was allowed to stand for 5 minutes at

room temperature. The absorbances were read against a blank using colorimeter at

wavelength of 546 nm.

21

2.5 Histopathology

The tissue samples (kidney, liver and gill) were quickly excised from the

fingerlings and fixed at 10% formal-saline. Slices of the organs were quickly prepared for

histological examination to show if there were morphological change in the organs during

the treatment (intoxication). Processing started with parking of the tissues in the tissue

capsule. The tissues were dehydrated in graded levels of ethanol (70 - 100%) in

ascending order. The alcohol was changed after soaking the tissues in them for 1-2 h.

The tissues were cleared in chloroform and impregnated with paraffin wax and

sectioned at 4 - 5 micron thickness using rotary microtone. The sections were floated on a

water bath maintained at 2 - 3° C below melting point of paraffin wax. They were on a

hot plate thermostatically maintained at a temperature of 2 - 3° C above the midpoint of

paraffin wax. When properly dried (15 – 30 minutes), they were stained with

haematoxylin and eosin (H and E), dehydrated, cleared and mounted (D.C.M.) in a

mountant, avoiding air bubbles. E staining was used for the demonstration of general

tissue structures in various colours. The nuclei as well as some calcium salts and ureates

were to take blue colour. Other tissue structures were to appear red, pink or orange in

color (eosinophilic).

Microscopic Observation of Slides

The permanent slides prepared were mounted one after the other and were

viewed at different magnifications of the microscope. Photographs of each slides was

taken and the results are shown.

22

2.6 Haematological Evaluation

The blood sample was collected by the method of caudal ablation. The blood

samples were dispensed into tubes containing EDTA anticoagulant. The haemoglobin

was estimated by cyanoheamoglobin method. Red blood cells and white blood cells were

counted by neubauers improved heamocytometer using Hayems and Turks solution as

diluting fluids, respectively. Packed cell volume (PCV), mean corpuscular haemoglobin

concentration (MCHC), mean corpuscular haemoglobin (MCH), mean cell volume

(MCV), were calculated respectively using standard formula described by Blaxhall and

Daisley (1993).

2.7 Statistical Analysis

Mean values were analyzed for significant differences (P ≤ 0.05) using the

ANOVA. Differences between means were partitioned using the Duncan New Multiple

Range test. The Statistical Package for Social Sciences (SPSS) version 16 was used. The

probit value was determined from the probit model developed by Finney (1971).

23

CHAPTER THREE

RESULTS

3.1. Acute Toxicity Test

The fingerlings showed behavioural responses to the diethyl phthalate. They

showed slight adaptive colouration turning from dull grey to light grey in response to

diethyl phthalate in their external environment. However, although they showed signs of

body weakness before death, swiftness of movement was detected and assessed after a

tap at the plastic aquaria.

The LD50 of diethyl phthalate on the fingerlings determined at 95% confidence

limit was 2.217, 2.734, 3.435, and 3.931 at log toxicant concentrations for 24 h, 48 h, 72

h, and 96 h, respectively and 1.871µg/l at log toxicant concentration for total death. The

positive correlates between the cumulative percentage mortality (Probit scale of the test

organism) and the various concentration of diethyl phthalate is as shown below (Fig 2,

Fig 3, Fig 4 and Fig 5). The LD50 (2.217) of acute toxicity test at 24 h exposure to test

substance was followed by sharp increase in mortality rate after 24 h and (2.734, 3.435,

3.931) at 48 h, 72 h, and 96 h respectively of dose dependent result. A chi-square

goodness of fit did not indicate significant difference between the observed and the

expected responses, the model is appropriate and the LC50 was calculated from the data.

Acute toxicity test result for total death and percentage survival of the fish to DEP is

shown in Table 5 and Table 12 respectively.

24

Table 5. Acute toxicity test result for 10 Clarias gariepinus per replicate for a total of

96 h

Mean acute toxicity of toxicant concentrations (µg/l) of diethyl phthalate to Clarias

gariepinus after different periods (24 h, 48 h, 72 h, and 96 h) is as shown in Table 6. The

LD50 for total death has been estimated at 95 % confidence limits for toxicant

concentration at 74.37 µg/l and 95 % confidence limit for log toxicant concentration

Toxicant

concentration

(µg/l)

Log10

concentration

Exposure

time

24h

48h

72h

96h

Total

dead

Total

Number

survived

50 1.69 0

1

0

2

1

1

1

1

1

1

0

0

4

3

2

6

7

8

75 1.87 1

0

1

2

2

1

1

2

1

1

0

2

5

4

5

5

6

5

100 2 2

2

3

3

2

2

1

1

0

0

0

1

6

5

6

4

5

4

150 2.17 6

3

4

2

3

3

1

3

2

1

1

1

10

10

10

0

0

0

Control 0 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

10

10

10

25

(µg/l) as 1.871 as shown in Fig 1. The model explains the data at 97.6% i.e. the model is

highly efficient.

Table 6. Mean Descriptive Statistics for Acute Toxicity Test Result for Total Death

Diethyl phthalate (µg/l) Mean mortality

50 5.75 ± 3.59a

75 8.50 ± 5.20b

100 13.50 ± 4.51c

150 22.75 ± 7.50d

*The means were statistically significant at p < 0.05 level

Fig.1: Probit transformed responses for total death of Clarias gariepinus fingerlings

exposed to graded concentrations of diethyl phthalate (DEP)

26

Percentage Mortality at 24 h

Percentage mortality at 24 h increased with increase in toxicant concentration.

Fingerlings exposed to 50 µg/l and 75 µg/l had 3.3 % and 6.7 % mortalities, respectively,

while 23.3 %, 43.3% mortalities were observed in aquaria with concentration of 100 µg/l

and 150 µg/l, respectively as shown in Table 7. The 24 h LD50 at 95 % confidence limit

at log toxicant concentration which is estimated at 2.217 µg/l as shown in Table 7 and

Fig.2. The model explain the data at 97.6% i.e. the model is highly efficient.

Table 7. Percentage mortality table at 24h exposure of toxicant concentration to

Clarias gariepinus fingerlings

Toxicant

concentration

µg/l

Log10

concentration

Total

number of

fishes in

each

replicate

Exposure time

24 h mortality in

each replicate

Total

dead

Percentage

mortality

50 1.69 10

10

10

0

1

0

1 3.3

75 1.87 10

10

10

1

0

1

2 6.7

100 2 10

10

10

2

2

3

7 23.3

150 2.17 10

10

10

6

3

4

13 43.3

Control 0 10

10

10

0

0

0

0 0

27

Fig.2: Probit transformed responses for 24h exposure of Clarias gariepinus fingerlings

exposed to graded concentrations of diethyl phthalate (DEP).


The percentage mortality at 48 h increased in the toxicant concentration.

Fingerlings exposed to 50 µg/l and 75 µg/l had 13.3% and 16.7 % mortality whereas 23.3

% and 26.7 % mortality, respectively occurred at 100 µg/l and 150 µg/l as shown in

Table 8. The LD50 at 95 % confidence limit for toxicant concentration was estimated at

542.34 µg/l and the 95% confidence limit for log toxicant concentration was estimated at

2.73 µg/l is as shown in Table 8 and Fig.3. The model explains the data at 97.6% i.e. the

model is highly efficient.

28



Toxicant

concentration

µg/l

Log10

concentration

Total

number of

fishes in

each

replicates

Exposure

time 48hr

mortality in

each

replicates

Total death Percentage

mortality

50 1.69 10

10

10

2

1

1

4 13.3

75 1.87 10

10

10

2

2

1

5 16.7

100 2 10

10

10

3

2

2

7 23.3

150 2.17 10

10

10

2

3

3

8 26.7

Control 0 10

10

10

0

0

0

0 0

.

29




The percentage mortality at 72 h reduced at 100 µg/l at 6.7% and increased at 75

µg/l at 13.3%. The LD50 at 72 h at 95 % confidence limit for toxicant concentration

(µg/l) was estimated at 2721.6 µg/l and 95 % confidence limit for log toxicant

concentration was estimated at 3.44 µg/l as shown in Table 9 and Fig 4.The model

explains the data at 97.6% i.e. the model is highly efficient.

30



Toxicant

concentration

µg/l

Log10

concentration

Total

number of

fishes in

each

replicates

Exposure time

72hr mortality in

each replicate

Total

dead

Percentage

mortality

50 1.69 10

10

10

1

1

1

3 10

75 1.87 10

10

10

1

2

1

4 13.3

100 2 10

10

10

1

1

0

2 6.7

150 2.17 10

10

10

1

3

2

6 20

Control 0 10

10

10

0

0

0

0 0

31

y = 0.2081x - 0.2147

R2 = 0.976

0

0.5

1

0 1 2 3 4 5 6 7

Log toxicant concentration (µg/l)

Pro

bit

re

spo

nse




The percentage mortality was minimum (3.3 %) at the concentration of 50 µg/l

and 100 µg/l and maximum at (10 %) at the concentrations of 75 µg/l and 150 µg/l. The

LD50 at 95 % confidence limit was estimated at 8538.78 µg/l and 95 % confidence limit

for log toxicant concentration estimated at 3.93 µg/l as shown in Table 10 and Fig 5. The

model explains the data at 97.6% i.e. the model is highly efficient.

32



Toxicant

concentration

µg/l

Log10

concentration

Total

number of

fishes in

each

replicates

Exposure

time 96 h

mortality in

each

replicates

Total dead Percentage

mortality

50 1.69 10

10

10

1

0

0

1 3.3

75 1.87 10

10

10

1

0

2

3 10

100 2 10

10

10

0

0

1

1 3.3

150 2.17 10

10

10

1

1

1

3 10

Control 0 10

10

10

0

0

0

0 0

33


exposed to graded concentrations of diethyl phthalate (DEP)

Total Percentage Mortality Table

Total percentage mortality of the Clarias gariepinus fingerlings exposed to the

acute toxicant of diethyl phthalate at various concentrations and duration of exposure is

shown in Table 11 below. Total percentage mortality increased with increase in toxicant

concentration. This shows that mortality is concentration dependent to the time of

exposure. There was a high mortality at 100µg/l and 150µg/l at 24h and 48h this is as a

result of stress caused by the toxicant which led to the death of the fingerlings.

34

Table 11. Cumulative percentage mortality

Toxicant

conce

ntratio

n µg/l

Percentage

mortality

24 h

Percentage

mortality

48 h

Percentage

mortality

72 h

Percentage

mortality

96 h

Mean

mortality

50 3.3 13.3 10 3.3 29.9

75 6.7 16.7 13.3 10 46.7

100 23.3 23.3 6.7 3.3 56.6

150 43.3 26.7 20 10 100

Control 0 0 0 0 0

Table 12. Percentage survival at the end of the exposure time

Toxicant

concentration

Log10

concentration

Total number

of fish in

each

replicates

Total number

survived

Sum total

survived

Percentage

survival

50 1.69 10

10

10

6

7

8

21 70

75 1.87 10

10

10

5

6

5

16 53.3

100 2 10

10

10

4

5

4

13 43.3

150 2.17 10

10

10

0

0

0

0 0

Control 0 10

10

10

10

10

10

30 100

35

3.2 Biochemical Results

Acetylcholinesterase Enzyme (I/U)

3.2.1 Acetylcholinesterase Enzyme Activity in Various Groups (Brain)

Treatment of Clarias gariepinus fingerlings with diethyl phthalate caused changes

in the acetylcholinesterase enzyme in the brain and muscle tissue as shown in Table 13

and Fig.6 below. The groups treated with 30 µg/l, 40 µg/l, 60 µg/l, and 80 µg/l at day 0,

showed a significant increase as shown in Fig.6. At day 0 and day 30 in Table shown

below it can be deduced that the Ache enzyme activity in all treatment groups maintained

the same mean value whereas at day 15 there is tend to be inhibition of the

acetylcholinesterase enzyme in the brain in group B, C and D. The mean values were

statistically significant (P ≤ 0.05).

Table 13. Mean value of Ache activity in brain of fingerlings exposed to DEP

Exposure Duration (DAYS)

GROUPS 0 days 15 days 30 days

Group A 30 µg/l 2049.20±325.72a 5777.70±54.43

a 2049.20±325.72

a

Group B 40 µg/l 6048.50±371.96bc

13433±119.71b 6048.50±371.96

bcd

Group C 60 µg/l 9226.10±214.53c 6086.70±90.21

a 9226.10±88.53

b

Group D 80 µg/l 8327±136.12cd

22014±28.53c 8327±136.12

bdc

Control 0 µg/l 5615.10±196.35b 2903±96.30

d 5615.10±196.35

b

*Mean values having the same alphabets as superscripts along the column do not show

significant difference (P≥0.05). Values having different alphabets as superscripts along

the column show significant difference (P≤0.05).

36

Fig.6: Effect of different concentration of diethyl phthalate on brain AchE

3.2.2. Acetylcholinesterase Enzyme Activity in Various Groups (Muscle)

Acetylcholinesterase activity in the control groups is shown in Table 14 below

for day 0, day 15 and day 30. The Ache activity in the muscle is shown in Fig.7, showed

that 60 µg/l- and 80 µg/l-treated groups increased at day 15 compared to the control

whereas 30 µg/l and 40 µg/l which showed decrease in Ache activity in the muscle. The

mean values were statistically significant (P ≤ 0.05).

Table 14. Mean value of Ache activity in the muscle of fingerlings exposed to DEP

Exposure Duration (DAYS)

GROUPS 0 15 30

GROUP A 30

µg/l

3322.10±522.54a 2446.40±758.63

a 3322.1±522.54

d

GROUP B 40

µg/l

1697.20±266.81ab

126380±263.52c 1697.2±266.80

ab

GROUP C 60

µg/l

2527.70±145.01cd

9505.90±120.78b 2527.7±145.01

cd

GROUP D 80

µg/l

2879.80±141.47cd

5777.6±334.61a 2879.8±141.47

cd

Control 1110.40±55.74a 1184.40±178.87

bc 1110.4±206.74

a




37

Fig.7: Effect of different concentration of diethyl phthalate on muscle AchE

Acid Phosphatase (I/U)

3.2.3. Acid Phosphatase Enzyme Activity in Various Groups (Liver)

Results from Table 15 and Fig.8, below show that the acid phosphatase levels in

the liver tissues were statistically significant (P ≤ 0.05) at day 15 compared to the control

and all diethyl phthalate treated groups. However, at day 0 and day 30, the acid

phosphatase level of the treated groups was not statistically significant (P > 0.05)

compared to the control.

38

Table 15. Mean value of ACP activity in liver of fingerlings exposed to DEP

Exposure duration (DAYS)

GROUPS 0 15 30

GROUP A 30 µg/l 1.21±.14a 2.22±.939

b 143.70±5.27

ab

GROUP B 40 µg/l 1.62±.28a 3.11±.097

c 127.85±14.2

a

GROUP C 60 µg/l 1.16±.21a 1.09±.097

a 136.63±17.7

ab

GROUP D 80 µg/l 1.67±.35a 1.84±.36

ab 140.40±16.8

ab

Control 1.11±.14a 1.54±.34

ab 1.53.±0.31

c

Fig.8: Effect of different concentration of diethyl phthalate on liver ACP




39

3.2.4. Acid Phosphatase Enzyme Activity in Various Groups (Muscle)

Acid phosphatase level in the muscle tissues were not statistically significant (P >

0.05) between the control and all diethyl phthalate treated groups as shown in Table 16,

Fig 9, at day 0, day 15, and day 30.

Table 16. Mean value of ACP activity in the muscle of fingerlings exposed to DEP


GROUPS 0 15 30

GROUP A 30 µg/l 3.59±.78a 2.121±.27

a 175.30±7.29

b

GROUP B 40 µg/l 3.84±.43a 2.55±.78

a 142.2±9.87

a

GROUP C 60 µg/l 2.58±.78a 3.93±.21

a 140.65±13.64

a

GROUP D 80 µg/l 2.53±.14a 2.02±.14

a 153.70±20.87

ab

Control 1.06±.07a 2.39±.62

a 171.93±0.08

b

*Means within the same column followed by different letters are significantly

different (P≤0.05). *Means within the same column followed by the same letters are not

statistically significant (P≥0.05).

Fig.9: Effect of different concentration of diethyl phthalate on muscle ACP.

40

Aspartate Transferase (AST) Activity (I/U)

3.2.5. Liver Aspartate Enzyme Activity in Various Groups

Aspartate transferase activity (AST) level of Clarias gariepinus fingerlings

exposed to diethyl phthalate had a sequential increase in day 0 and day 15 and was at

highest level at day 30 in group B, C, D as shown in Table 19, Fig.12. The mean values

are not statistically significant P > 0.05.

Table 17. Mean value of AST activity of fingerlings exposed to diethyl phthalate


GROUPS µg/l 0 15 30

Control 15.55±2.89a 35.80±2.83

ab 33.75±9.55

a

GROU P A 30 µg/l 13.08±3.82a 32.40±3.82

ab 27.80±9.42

a

GROUP B 40 µg/l 16.20±3.64a 27.00±.00

c 64.85±5.20

a

GROUP C 60 µg/l 19.05±5.06a 39.15±9.55

c 62.15±2.98

a

GROUP D 80 µg/l 15.30±1.69a 47.30±1.253

a 81.50±1.91

a

*Means within the same column followed by different letters are significantly different

(P≤0.05).Means within the same column followed by the same letters are not

significantly different (P≥0.05).

41

Fig.10: Effect of different concentration of diethyl phthalate on liver AST.

Alanine Transferase (ALT) Activity (I/U)

3.2.6. Liver Alanine Transferase Enzyme Activity in Various Groups

The ALT enzyme activity in the liver of Clarias gariepinus fingerlings exposed to diethyl

phthalate is shown in Table 18, Fig 11 .The liver ALT activity in all treated groups

increased at day 0 and day 30 and decreased at day 15. The ALT activity increased

progressively with increased in toxicant concentration compared to the control. The mean

values are statistically significant P ≤ 0.05 at day 30.

Table 18. Mean of liver ALT activity of fingerlings exposed to diethyl phthalate


GROUPS 0 15 30

Control 11.70±3.67a 28.15±3.98

a 11.80±1.27

c

GROUP A 30µg/l 18.15±3.71a 14.55±5.16

a 2.70±1.27

a

GROUP B 40µg/l 18.15±3.71a 14.55±5.16

a 6.00±.56

a

GROUP C 60µg/l 22.10±2.58a 17.30±1.273

a 7.30±2.55

ac

GROUP D 80µg/l 21.80±2.54a 14.55±2.62

a 7.30±2.55

ac


(P≤0.05).Means within the same column followed by the same letters are not significantly

different (P≥0.05).

42

Fig.11: Effect of different concentration of diethyl phthalate on liver ALT

HAEMATOLOGY

3.3. Haemoglobin (g/dl) Levels of Various Groups Exposed to Diethyl Phthalate

The haemoglobin concentration in the treatment groups was significantly lower

than the control (P < 0.05) and differed also between the treatment groups (P < 0.05) as

shown in Table 19 and Fig 12.The control fish showed mean values of 4.92 g/dl at day 0

and day 15, 4.42g/dl at day 30 for haemoglobin. The fish were exposed to chronic

concentration of diethyl phthalate and showed the haemoglobin mean values of 4.64,

4.76, 4.7 and 4.96 g/dl haemoglobin at 30, 40, 60 and 80 µg/l at day 0, 3.64, 2.76, 3.96

and 2.86 g/dl haemoglobin at 30 µg/l, 40 µg/l, 60 µg/l, and 80 µg/l at day 15 and 3.36,

3.5, 2.96 and 2.66 g/dl haemoglobin at 30 µg/l, 40 µg/l, 60 µg/l, and 80 µg/l respectively

at day 30. Generally, the haemoglobin concentration decreased with increased DEP

concentration and with the duration of exposure.

43

Table 19. Mean of haemoglobin (g/dl) of various groups exposed to diethyl phthalate


GROUPS 0 15 30

GROUP A 30

µg/l

4.64±.98a 3.64±1.25

ab 3.36±1.07

ab

GROUP B 40

µg/l

4.76±1.40a 2.76±.95

a 3.5±1.17

ab

GROUP C 60

µg/l

4.7±.62a 3.96±1.33

ab 2.96±1.10

ab

GROUP D 80

µg/l

Control

4.96±1.24a

4.92±1.47

2.86±1.98ab

4.92±1.61

2.66±1.68a

4.42±.45



significantly different (P≥0.05)

Fig.12: Effect of different concentration of diethyl phthalate on the heamoglobin level.

3.4. Packed Cell Volume (PCV) levels in Various Groups

The packed cell volume (PCV) of DEP-treated fish was lower than the control (P <

0.05). The decrease in PCV was concentration dependent and with increasing duration of

44

treatment. The packed cell volume (PCV) of healthy controls showed mean values of

18.2 %, 18.4 %, and 18.2 %. The fish exposed to chronic concentration of diethyl

phthalate showed mean values of PCVs as 19.6, 16.4, 17.8, and 19.6 % for 30 µg/l, 40

µg/l, 60 µg/l, and 80 µg/l treatment respectively was observed at day 15 and 13.4 %, 13.8

%, 11.6 % and 8.88 % for 30 µg/l, 40 µg/l, 60 µg/l and 80 µg/l treatment respectively at

day 30 as shown in Table 20, Fig.13. The mean values mentioned above showed a

significant difference P ≤ 0.05 at day 30 compared to the control.

Table 20. Mean of the packed cell volume (%) of various groups exposed to diethyl

phthalate

Duration of exposure

z 0 15 30

GROUP A 30 µg/l 19.60±2.40a 17.20±2.38

ab 13.40±2.79

b

GROUP B 40 µg/l 16.40±3.91a 15.20±4.43

ab 13.80±3.35

b

GROUP C 60 µg/l 17.80±3.11a 14.80±3.76

ab 11.60±2.96

ab

GROUP D 80 µg/l 19.60±1.94a 12.40±4.39

a 8.88±3.44

a

Control 18.20±1.30a 18.40±4.15

b 18.20±1.64

c




45

Fig.13: Effect of different concentration of diethyl phthalate on the packed cell volume.

3.5 Total Red Blood Cell Count in Various Groups

The erythrocyte count of the controls showed mean values of 2.71, 2.69, and 2.26,

106 mm

-3 at day 0, day 15 and day 30. The fish exposed to the chronic concentration of

the toxicant diethyl phthalate showed mean values of RBCs as 2.20, 2.12, 2.36, and 2.54,

106 mm

-3 for 30 µg/l, 40 µg/l, 60 µg/l, and 80 µg/l treatments at day 0, 2.34, 1.61, 2.15,

2.01, and 2.69, 106 mm

-3 for 30 µg/l, 40 µg/l, 60 µg/l, and 80 µg/l treatment at day 15;

1.93, 1.83, 1.94, and 1.62, 106mm-

3 for 30 µg/l, 40 µg/l, 60 µg/l and 80 µg/l treatment

respectively at day 30 as shown in Table 21, Fig.14. The mean values are not statistically

significant P > 0.05 compared to the control.

46

Table 21. Mean of the red blood cell count (x106/ mm

3) of various groups exposed to

diethyl phthalate

Duration of exposure

Fig.14: Effect of different concentration of diethyl phthalate on the Red blood cell

3.6 Total White Blood Cell Count (TWBC) in Various Groups

The result of the total count of white blood cell revealed that the blood of the

control fish showed a mean value of 1.33, 2.18, and 2.72, 103/mm

3 for day 0, day 15 and

GROUPS 0 15 30

GROUP A 30 µg/l 2.20±.578ab

2.34±.695ab

1.93±.487a

GROUP B 40 µg/l 2.12±.374ab

1.61±.468a 1.84±.45

a

GROUP C 60 µg/l 2.36±.2018a 2.15±.6103

ab 1.94±.50

a

GROUP D 80 µg/l 2.54±.695ab

2.01±.6718ab

1.62±.64a

Control 2.71±.598b 2.69±.4556

b 2.27±.33

a

*Means within the same column followed by different letters are significantly

different (P≤0.05).Means within the same column followed by the same letters are

not significantly different (P≥0.05).

47

day 30. The fish exposed to chronic concentration showed the mean value of WBC as

2.26, 2.05, 1.92 and 1.92, 103/mm

3 for 30 µg/l, 40 µg/l, 60 µg/l, and 80 µg/l at day 0,

2.04, 1.82, 2.35 and 2.38, 103/mm

3 for 30 µg/l, 40 µg/l, 60 µg/l and 80 µg/l treatment at

day 15 and 2.58, 2.64, 2.45 and 2.86, 103/mm

3 for 30 µg/l, 40 µg/l, 60 µg/l and 80 µg/l

treatment respectively at day 30 as shown in Table 22, Fig.15. The mean values

mentioned above are statistically significant P ≤ 0.05 at day 15 when compared to the

control.

Table 22. Mean of the total white blood cell (x104/mm

3) of various groups exposed

to diethyl phthalate

Duration of exposure (Days)

GROUPS 0 15 30

GROUP A 30µg/l 2.26±0.5b 2.04±0.24

ab 2.58±0.30

a

GROUP B 40µg/l 2.05±0.19ab

1.83±0.26a 2.64±0.36

a

GROUP C 60µg/l 1.92±0.18ab

2.35±0.13b 2.46±0.48

a

GROUP D 80µg/l 1.92±0.98ab

2. 38±0.40b 2.87±0.32

a

Control 1.332±0.10a 2.19±0.35

ab 2.73±0.19

a


(P ≤ 0.05).Means within the same column followed by the same letters are not

significantly different (P ≥ 0.05).

48

Fig.15: Effect of different concentration of diethyl phthalate on the White blood cell.

3.7. Mean Cell Volume in Various Groups

The mean cell volume of the control fish showed a mean value of 9.68, 6.79, and

8.12, fl . The fish exposed to chronic concentration of diethyl phthalate showed a mean

value of MCV’s as 7.7, 9.6, 6.3 and 6.5, fl for 30 µg/l, 40 µg/l, 60 µg/l and 80 µg/l at day

15 and 7.1, 7.9, 5.4, and 6.24, fl for 30 µg/l, 40 µg/l, 60 µg/l and 80 µg/l treatments

respectively at day 30 as shown in Table 23, Fig.16. The means were statistically

significant P ≤ 0.05 at day 15 compared to the control

49

Table 23. The mean value of mean cell volume (fl ) of various groups exposed to

diethyl phthalate


GROUPS 0 15 30

GROUP A 30 µg/l 9.19±1.71a 7.7±1.85

ab 7.1±1.19

a

GROUP B 40 µg/l 8.56±2.13a 9.6±1.60

b 7.88±2.96

a

GROUP C 60 µg/l 7.68±1.14a 6.28±.16

a 5.42±1.02

a

GROUP D 80 µg/l 8.09±1.89a 6.5±1.02

a 6.24±2.69

a

Control 9.69±1.25a 6.79±.63

a 8.12±1.05

a

Fig.16: Effect of different concentration of diethyl phthalate on the mean cell volume

3.8. Mean Cell Haemoglobin (MCH) in Various Groups

The mean volume of the control fishes showed mean values of 18.55, 18.32, and

19.56 Pg. The fishes exposed to the toxicant diethyl phthalate showed the mean values of

*Means within the same column followed by different letters are significantly different (P

≤ 0.05).Means within the same column followed by the same letters are not significantly

different (P ≥ 0.05).

50

MCH as 21.6, 22.4, 24.2 and 20.28 pg for 30 µg/l, 40 µg/l, 60 µg/l and 80 µg/l of the

toxicant concentration at day 0, 14.3, 17.5, 16.51 and 14.4 Pg for 30 µg/l, 40 µg/l, 60

µg/l and 80 µg/l treatment at day 15 and 17.32,18.8, 14.8 and 17.9 Pg 10-12

at 30 µg/l, 40

µg/l,60 µg/l and 80 µg/l respectively as shown in Table 24 and Fig.17. The means were

not statistically significant P > 0.05.

Table 24. Changes in the mean cell haemoglobin (Pg) of various groups exposed to

diethyl phthalate

Duration of exposure (days)

GROUPS 0 15 30

GROUP A 30 µg/l 21.60±4.93a 14.31±5.41

a 17.32±2.42

a

GROUP B 40 µg/l 22.42±4.94a 17.46±4.58

a 18.76±1.67

a

GROUP C 60 µg/l 24.20±2.32a 16.51±1.79

a 14.76±7.76

a

GROUP D 80 µg/l 20.28±9.45a 14.44±8.15

a 17.86±9.43

a

Control 18.56±5.09a 18.32±4.95

a 19.56±1.45

a

*Means within the same column followed by different letters are significantly different (P

≤ 0.05).Means within the same column followed by the same letters are not significantly

different (P ≥ 0.05).

51


haemoglobin.

3.9. Mean Cell Haemoglobin Concentration (MCHC) in Various Groups.

The control fishes showed mean .244, .27, and .241 g/dl for mean cell haemoglobin

concentration. The fish were exposed to the chronic concentrations of diethyl phthalate

showed the MCHC’s mean value of .24, .30, .274 and .240 g/dl MCHC at 30 µg/l, 40

µg/l, 60 µg/l and 80 µg/l treatment at day 0; 2.01, .192, .27 and .26 g/dl MCHC at 30

µg/l, 40 µg/l, 60 µg/l and 80 µg/l treatment respectively at day 15 and .25, .26, .29 and

0.3 g/dl MCHC at 30 µg/l, 40 µg/l, 60 µg/l and 80 µg/l at day 30 as shown in Table 25,

Fig.18. The means were not statistically significant (P > 0.05).

52

Table 25. Changes in the mean cell haemoglobin concentration (g/dl) of various

groups exposed to diethyl phthalate


GROUPS 0 15 30

GROUP A 30µg/l 0.24±.08a 2.02±3.96

a 0.25±0.06

a

GROUP B 40µg/l 0.30±.08a 0.19±0.07

a 0.26±0.08

a

GROUP C 60µg/l 0.27±.05a 0.27±0.03

a 0.29±0.18

a

GROUP D 80µg/l 0.24±.06a 0.26±0.22

a 0.3±0.11

a

Control 0.24±.07a 0.27±.06

a 0.24±0.03

a


haemoglobin concentration.

Differential Blood Count

The mean values in day 0 for differential counts (lymphocytes, monocytes and

neutrophils) showed that monocytes were significantly different P≤0.05 compared to the




53

control. No significant difference was seen between the lymphocytes and the neutrophils.

In day 15, the monocytes and the lymphocytes showed a significant difference P < 0.05.

Whereas no significant difference was seen in day 30 between the lymphocytes,

monocytes and neutrophils as shown in Table 26, Fig 19

Table 26. Changes in the lymphocyte count in Clarias gariepinus fingerlings exposed



GROUPS 0 15 30

GROUP A 30µg/l 84.00±4.53ab

88.60±3.44b

87.40±3.36a

GROUP B 40µg/l 80.80±4.66a 82.20±6.83

a 84.00±3.74

a

GROUP C 60µg/l 80.60±6.73ab

91.40±2.07b 84.40±3.64

a

GROUP D 80µg/l 88.40±2.96b 89.00±2.24

ab 85.40±3.20

a

Control 85.80±4.98a 86.40±4.22

ab 87.80±2.49

a




Fig.19: Effect of different concentration of diethyl phthalate on the lymphocytes count.

54

The monocyte was significantly different (P < 0.05) in day 0 and 30 as shown in Table 27

and Fig 20. The decrease in monocyte count was concentration dependent and with

increasing duration of treatment.

Table 27. Changes in Monocyte count in Clarias gariepinus fingerlings exposed to

diethyl phthalate


GROUPS 0 15 30

GROUP A 30µg/l 19.00±1.58c 11.0±3.87

abc 15.20±3.11

a

GROUP B 40µg/l 13.20±3.11a 14.80±3.83

c 13.60±3.32

a

GROUP C 60µg/l 13.60±3.29a 8.60±2.07

a 14.00±2.55

a

GROUP D 80µg/l 17.80±1.64bc

9.60±2.07ab

14.60±2.88a

Control 14.40±3.65b 13.40±4.27

bc 11.60±2.07

a




The neutrophils count showed no significant different (P < 0.05) throughout the exposure

period as shown in Table 28 below.

Fig.20: Effect of different concentration of diethyl phthalate on the monocytes

count.

55

Table 28. Changes in the Neutrophil count in Clarias gariepinus fingerlings exposed



GROUPS 0 15 30

GROUP A 30µg/l 2.00±1.414 - 2.00±.

GROUP B 40µg/l 1.00±.000 2.00±. 1.00±.

GROUP C 60µg/l 1.00±. - 1.00±

GROUP D 80µg/l 1.00± 1.00±. 1.00±.

Control 1.00± 1.00±. 1.50±.707




3.10 Histopathology Results

Histopathological changes observed in the gill, liver and kidney of Clarias

gariepinus fingerlings subjected to different concentration of diethyl phthalate and the

control and examined under the light microscope are as follows.

Gills

No recognizable changes were observed in the gills of the control fish. The

control gill consisted of a primary filament and secondary lamellae as shown in Plate 1

At different concentration of diethyl phthalate, there were haemorrhaging of the gill

filament as shown in Plate 3, fatty cells prominent as shown in Plate 4, enlarged filament

as shown in Plate 5, Disjointed lamella as shown in Plates 6 and 7, severe destruction of

lamella as shown in Plate 8 and extensive lamellae fusion as shown in Plate 9 below.

56

Plate 1: Gill section of control fish showing no changes magnification (mag) (H&E)

x40.

Plate 2: Gill section of group A exposed to 30µg/l of diethyl phthalate for 15 days

mag (H&E) x40. Prominent lamellar showing the acidophil cells.

57

Plate 3: Gill section of group A exposed to 30µg/l diethyl phthalate for 30 days mag

(H&E) x40. Haemorrhaging of the gill filament.

Plate 4: Gill section of group B exposed to 40µg/l diethyl phthalate for 15 days mag

(H&E) x40 . Fatty cells prominent.

58

Plate 5: Gill section of group B exposed to 40µg/l diethyl phthalate for 30 days mag

(H&E) x40. Enlarged filament.

Plate 6: Gill section of group C exposed to 60µg/l diethyl phthalate for 15 days mag

(H&E) x40. Filaments are not enlarged, they are narrow with disjointed lamella and

the width is not uniform.

59

Plate 7: Gill section of group C exposed to 60µg/l diethyl phthalate for 30 days mag

(H&E) x40. There is enlargement of the filament and the lamella is disjointed.

Plate 8: Gill section of group D exposed to 80µg/l diethyl phthalate for 15 days mag

(H&E) x40. Severe destruction of the lamella is shown with arrows.

60

Plate 9: Gill section of group D exposed to 80µg/l diethyl phthalate for 30 days mag.

(H&E) x40. Extensive lamellar fusion is shown by the arrows.

KIDNEY

No recognizable changes were observed in the kidney of the control fish as shown

in Plate 10. The kidney tissue from Clarias gariepinus exposed to different

concentrations of diethyl phthalate showed; destruction or fusion of the tubules,

degenerated kidney tubule, pyknosis, condensation of the gromeruli content as shown in

Plates 13, 14, 15, 16 17 and 18 below respectively. No changes were seen in control and

group A treated with 30 µg/l as shown in Plates 10, 11 and 12.

61

Plate 10: No recognizable changes were observed in the kidney of control fish

Plate 11: Kidney section of group A exposed to 30µg/l diethyl phthalate for 15 days

mag (H&E) x40. Tubules are intact.

62

Plate 12: Kidney section of group A exposed to 30µg/l diethyl phthalate for 30 days

mag (H&E) x40. Tubules are intact (CS) and sequensiation of kidney architecture.

Plate 13: Kidney section of group B exposed to 40µg/l diethyl phthalate for 15 days

mag(H&E) x40 . Destruction or fusion of the tubules.

63

Plate 14: Kidney section of group B exposed to 40µg/l diethyl phthalate for 30 days

mag (H&E) x40. Severe destruction of the tubules.

Plate 15: Kidney section of group C exposed to 60µg/l diethyl phthalate for 15 days

mag (H&E) x40. Pyknosis (pyvnotic nuclei present, degenerated kidney tubule

pyknosis.

64

Plate 16: Kidney section of group C exposed to 60µg/l diethyl phthalate for 30 days

mag (H&E) x40. Destruction of tubule, Tubules are not continuous.

Plate 17: Kidney section of group D exposed to 80µg/l diethyl phthalate for 15 days

mag(H&E) x40 . Condensation of the glomeruli content.

65

Plate 18: Kidney section of group D exposed to 80 µg/l diethyl phthalate for 30 days

mag (H&E) x40. Condensation of the glomeruli content.

LIVER

The histology of control fish liver revealed normal typical parenchymatous appearance.

The liver was made up of hepatocytes that were polygonal cells with a central spherical

nucleus and densely stained nucleolus. There were severe cellular proliferation at 40 µg/l

and 30 µg/l at 15 days of exposure as shown in Plates 20, 22 and 23 and congestion at 30

days at the same concentration of 40 µg/l as shown in Plate 23, hepatic necrosis and

sinusoid enlargement were also observed at 60 µg/l at 15 and 30 days as shown in Plates

24 and 25 respectively. There were glycogen degeneration and parenchymatous

degeneration at application of 80 µg/l diethyl phthalate at day 15 and 30 respectively as

shown in Plates 26 and 27.

66

Plate 19: No recognizable changes were observed in the liver of control fish

Plate 20: Liver section of group A exposed to 30 µg/l diethyl phthalate for 30days

mag x40. Cellular proliferation.

CP

67

Plate 21: Liver section of group A exposed to 30 µg/l diethyl phthalate for 15 days

mag x40 a. Liver exhibits normal morphology.

Plate 22: Liver section of group B exposed to 40 µg/l diethyl phthalate for 15 days

mag x40 a. severe cellular proliferation.

68

Plate 23: Liver section of group B exposed to 40 µg/l diethyl phthalate for 30 days

mag x40 a. Congestion.

Plate 24: Liver section of group C exposed to 60 µg/l diethyl phthalate for 15 days

mag x40. Severe necrosis.

N

69

Plate 25: Liver section of group C exposed to 60 µg/l diethyl phthalate for 30 days

mag x40. Sinusoid enlargement.

Plate 26: Liver section of group D exposed to 80 µg/l diethyl phthalate for 15 days

mag x40. Parenchymatous degeneration.

70

Plate 27: Liver section of group D exposed to 80 µg/l diethyl phthalate for 30 days

mag x40. Fatty or glycogen degeneration.

71

CHAPTER FOUR

DISCUSSION

Diethyl phthalate is used in pharmaceutical coating as a fixative in cosmetics, in

the manufacture of celluloid, as solvent for cellulose acetate in the manufacture of

varnishes and ropes in the denaturation of alcohol, perfume binders (Sonde et al., 2000).

Because DEP has being used extensively for various purposes, contamination of the

environment by DEP cannot be ruled out.

Results obtained from this investigation showed that the percentage mortality of C.

gariepinus fingerlings increased with increase in concentration of diethyl phthalate and

was dose dependent. This is in consonance with a similar investigation by Nivedita et al.,

(2002) on the toxic effect of diethyl phthalate on Cirrhina mrigala. These observations

are in agreement with earlier reports by Omoregie et al. (1990), on effect of sublethal

concentration of gammalin 20 and Acetellic 25 EC on Oreochromis niloticus, Ghatak and

Konar, (1991) on the chronic effect of mixture of pesticide, detergent, heavy metal and

petroleum hydrocarbon on various combination of fish and Okoli-Anunobi, et al. (2002)

on detergent, elephant blue on nile tilapia, Oreochromis niloticus. The LD50 reported in

this study is less than the observed field concentration in the water column (0.16 - 3.53

mg/L) and sediment (0.16 - 0.32 mg/L) of DEP in the Venda region of South African

waters (Fatoki et al., 2010) where similar indiscriminate discharge of DEP-laden

effluents and wastes take place as in Nigeria. In Nigeria, there is dearth of information

about the field levels of DEP but it is expected to be higher than the LD50 reported in the

present study. The LD50 of the carbofuran to juvenile fathead chubs was 1.96 mg/L

(Fisher et al., 1999), a data that is comparable to the result in this study. Since DEP binds

to the sediment and remains in the water column, it is possible that it could pose serious

threat to fish and other aquatic life.

72

The rapid opercula movement, erratic swimming and loss of balance observed in

this study suggested possible nervous disorder. Haemorrhaging of the gill when the test

fish were exposed to 100 μg/L and 150 μg/L of the compound is indicative of toxicity of

the chemical. This is probably due to rupture of blood vessels of the gills and possible

reduction in the haemotological parameters of erythrocyte count, haematocrit and mean

corpuscular volume of the fish. Mgbenka et al. (2005) reported similar toxic effects with

clogging of the gills with mucus of C. gariepinus due to lindane exposure. These

observations are also in agreement with earlier reports by Okoli-Anunobi et al. (2002) on

the lethal effect of the detergent (Elephant blue) on the Nile tilapia. Haemorrhaging has

also been reported in fathead minnow exposed to organic-based insecticide (Buckler et

al., 1981). At higher concentrations of 0.16 mg/L – 4.04 mg/L DEP in the water of some

rivers in the Venda region of South Africa, Fatoki et al. (2010) have suggested that DEP

is toxic and could be carcinogenic to aquatic organisms and man though it is less harmful

than di-(2-ethylhexyl) phthalate (DEHP).

Acetylcholinesterase is of interest because it is the target site for organophosphate

and carbamate pesticides in the central nervous system and its role in cholinergic

synapses is essential for life. It is an enzyme that degrades the neurotransmitter

acetylcholine, producing choline and acetate group. It is mainly found in neuromuscular

junction and cholinergic synapses in CNS, where it terminates the synaptic transmission.

It is also found in red blood cell membranes, where it constitutes the Yt blood group

antigen. Some studies reported evidence that AchE activity may be inhibited by

environmental contaminants other than organophosphate and carbamate compounds,

including some metals, surfactants agents, and combustion hydro carbons (Guilhermino

et al., 1994; Herbert et al., 1995; Pagne et al., 1996, Labrot et al., 1996). It was evident

in this study that the AchE activity in the brain and muscle of diethyl phthalate treated

73

fish was found to be significantly increased and also decreased based on the duration,

indicating that DEP inhibit AchE activity. This could be due to the lipophilic nature of

DEP, which may be taken up faster by the brain tissues. This correlates with the sluggish,

non motile behavior of the DEP treated fish. In previous studies the sensitivity of AchE to

endosulfan was similar to the activity of non-exposed animals. The higher the AchE level

in the tissue, the more susceptible it is to inhibition and low concentration of toxicants

can inhibit AchE, which leads to an accumulation of Acetylcholine at the central

cholinergic synapses and neuromuscular junction which was evident in group B.

Cholinesterase inhibition in brain and muscle produces effect in the movement of fish

because acetylcholinesterase participates in the neuronal and neuromuscular transmission

(Fernadev-Vega et al., 1999). The unexposed fishes (control) showed inhibition in the

control group in day 15 and higher specific activity in the brain compared to the muscle,

this inhibition can be species dependent according to Sancho et al. (1998). Thus, this

enzyme seems not to be sensitive to this chemical, results agree with those obtained by

Inbaraj and Hainder (1988) in Channa punctatus.

It was evident that liver and muscle acid phosphatase level were significantly

increased in DEP treated fish at various concentrations and also decreased significantly at

other groups. This increase is probably due to increased lysosomal activity in the liver

and muscle tissues. This goes in consonance of Nivedita et al. (2002), that ACP is an

inducible enzyme because its activity goes up when there is a toxic impact and the

enzyme begins to counteract the toxic effect. Subsequently, the enzyme may begin to

drop either as a result of having partly or fully encountered the toxin or as a result of cell

damage. In a study of male Sprague-dawley rats treated with 50 ppm (w/v)DEP in

drinking water for four months, there was significant increase in liver ACP (Sonde et al.,

2000). It is apparent that DEP causes increased ACP activity in the liver and muscle by

74

interacting with lysosome. (Lowe et al., 1992) reported that alteration in the membrane

permeability can have severe consequences such as leakage of hydrolytic enzyme

including ACP, which could have detrimental effect on the cell.

Liver AST levels were significantly increased in DEP treated fish though not

statistically significant compared with the control. This indicates that DEP stimulates

glutamate transaminase activity in the liver which could be due to toxic injury caused by

DEP, which may stimulate tissue repair through protein turn over and increased

respiration. AST levels were comparatively lower in DEP treated group indicating that

DEP does affect mitochondrial function. This agrees with Nivedita et al. (2002). This

correlates well with increased AST activity in the liver of DEP treated fish. In this regard,

it can be said that DEP toxicity leads to enhanced AST activity, which is indicative of

high protein turnover and amino acid metabolism. This is in consonance with Nivedita et

al. (2002), Muthuviveganadavel et al. (2007).

Blood parameters are considered pathophysiological indicators of the whole body and

therefore are important in diagnosing the structural and functional status of fish exposed

to toxicants (Adhikari and Sarkar, 2004). In recent years haematological variables have

been used more to determine the sub lethal concentration of pollutants (Wedemeyer and

Yasutake, 1997). The use of immune system parameters to access alterations in fishes

experiencing pollutants exposure and interest in defense mechanisms stem from the need

to develop healthy management tools to support rapidly growing aquaculture industry

(Jones, 2001). Results of the present investigation show that diethyl phthalate treatment

inflicted slight changes in the blood parameters (HB, PCV, RBC, and WBC). The

reduction was dosage dependent to the duration of exposure showing possible anaemia to

the fishes. This is in agreement with Joshi et al. (2002b) that reported effects of toxicants

on blood parameters in fresh water teleost fish Clarias batrachus. Bhatt and Farswan,

75

(1992) also observed that red blood cell (RBC), total white blood cell (TWBC),

haemoglobin (HB), packed cell volume (PCV) decreases with exposure of Barilus

bendalensis(HAM) to plant toxicants. The increase in haematological parameters

observed in control fish not treated with DEP agreed with the findings of Joshi et al.,

(2002b) that survival of fish can be correlated with increase in antibody production which

help in the survival and recovery. Panigrahi and Misra (1978) observed reduction in

haemoglobin percentage and red blood cell (RBC) count of the fish Anabas scandens

treated with mercury. Sampathy et al. (1998) also reported a decrease in hematological

parameters of Oreochromis mossambicus exposed to copper and zinc. Lower

haemoglobin level according to Joshi et al. (2002c) might decrease the ability of fish to

enhance its activity in order to meet occasional demand. Van Vureen (1986) reported that

metasystox caused decreased haemoglobin concentration in Labeo umbratus while

Omoregie et al. (1990) observed decrease in haemoglobin in O. niloticus exposed to

gammalin 20 and actellic 25EC. Other studies (Sarthakumar et al., 1999; Mgbenka et al.,

2003) reported that monocrotophos and acetellic exposures decreased the haemoglobin

concentration in Anabas sp. and C. albopunctatus, respectively. The decreased

haemoglobin concentration observed in this study is an indication of impaired oxygen

delivery to the tissues.

The reduction in erythrocyte count (erythrocytosis) observed in this study represents part

of the overall physiological mechanism to compensate for low oxygen intake in the fish

(Wepener et al., 1992, Smith and Piper, 1972) due to possible damage to the gill

epithelium. Nilson and Groove (1974) had earlier observed that erythrocytosis was due to

adrenergic stimulation of the erythopoeitic tissue to release stored erythrocytes to cope

with the challenge of inadequate oxygen level and increasing oxygen debt. The reduction

in erythrocytes count in C. gariepinus due to DEP is in consonance with the report of

76

Sarthakumar et al. (1999) on Anabas sp. exposed to monocrotophos and in C.

albopunctatus treated with actellic 25 EC (Mgbenka et al., 2003). Similar results were

reported for Oreochromis mossambicus exposed to copper (Nussey et al., 1995). The

general reduction in haemoglobin concentration, erythrocyte count and PCV in the fish

treated with DEP is an indication of anaemia. Thakur and Bais (2000) also reported

erythrocyte count, MCV, MCH, and MCHC decreased in Heteropneutes sp. treated with

insecticides with concomitant decline in oxygen transport. White blood cells play a major

role in the defence mechanism of the fish and consist of granulocyte, monocyte,

lymphocytes and thrombocyte and monocytes function as phagocytes to salvage debris

from injured tissues and lymphocytes produce antibodies (Ellis and Roberts, 1978;

Wedemeyer and Mc leay, 1981). In this study, it was evident that monocytes produced

interleukin 10 which protects the organism from being anaemic. Lymphocytes (B and T

cells are the protective cells) in that they recruit cytokines so that the antibody can help to

fight the toxicant and enhance recovery or stability. The mean corpuscular volume

(MCV) obtained from Clarias gariepinus fingerlings in this study is an indication of

reduction in erythrocyte size. This according to Nussey et al. (1995), was due to the

release of large number of immature erythrocytes into the general circulation. The

increased mean corpuscular haemoglobin concentration (MCHC) found in the group A

day 15 is as a result of physiological adaptation to increase oxygen carrying capacity of

each erythrocyte in the treated fish. However, other treated groups showed no increase in

the mean cell haemoglobin concentration throughout the study period. This agreed with

the report of the effect of mercury in O aureus and stripped bass (Dawson, 1982) as well

as effect of hypoxia on channel cat fish (Scutt and Rodgers, 1981).

In the present investigation, leucocyte concentration showed greater and quite

different pattern of change with the effect of DEP when compared with the erythrocytes

77

levels of the control group. Blood of all experimental groups, contained higher

concentrations of leucocytes than those of controls and based on the duration of exposure

of the toxicants similar leucocytosis was found in fish exposed to heavy metals. An

increase in lymphocytes number may be the compensatory response of lymphoid tissues

to the destruction of circulating lymphocytes (Shah and Altinday, 2005). The increase in

WBC (leucopenia) observed in the present study could be attributed to a stimulation of

the immune system in response to tissue damage caused by DEP. This agrees with Gill

and Pant, (1985) that the stimulation of the immune system causes an increase in

lymphocyte by an injury or tissue damage. Dhanekar and Snivastava (1985) reported

increased in large lymphocytes, reduction in small lymphocytes and thrombocyte

populations as also elevation in monocyte, neutrophil and eosinophil cells in

Hereopneustes fossilis, Channa punctatus, and Mastacebalus punculus on long term

exposure to least effective concentration of mercury chloride.

Exposure to DEP induced variations in differential leucocyte counts showed a

rapid increase to the duration of exposure. Leucocytosis is a usual response of the

vertebrates to conditions or substances that attempt to change their normal physiology.

Thus, the leucocytosis recorded in this study shows that DEP elicited the stimulation of

the immune system of the fish to protect it against infection or secondary effect of DEP to

predispose it to disease. Similar leukocytosis was reported in C. albopunctatus exposed to

gammalin 20 (Mgbenka et al., 2003) and in Indian catfish (Heteropneustes fossilis)

treated with sewage, fertilizer and insecticides (Srivastava and Narain, 1982). Also,

Trivadi et al. (1990) reported a similar trend in C. batrachus exposed to fertilizers.. The

lymphocyte increased rapidly based on the duration of exposure. This implies that the

lymphocytes produced antibodies and chemical substances that will fight against the

pollutant (DEP) or infection that has being accumulated in the fish blood. The monocytes

78

and the neutrophils have a wide variation compared to the duration of exposure to the

graded concentration which has led to monocytosis and the latter neutrophillia. This

agrees with the finding of Kumar and Patri (2004). This shows that DEP caused

immunological impairment in Clarias gariepinus fingerlings, which suggests that DEP

may weaken the immune system and may result in severe physiological problems,

ultimately leading to the death of fish.

The literature on histopathology effects of DEP on fish is still rare .Neskovic et al.

(1996) conducted sub lethal toxicity test (14 days) of sub lethal glyphosate concentration

on histopathological changes of carp organ such as gill, liver and kidneys. In the present

study damages of the gills indicated that the sublethal concentration of DEP caused

impairment in gaseous exchange efficiency of the gills. The major changes were

enlargement of the filament, stunted lamella, extensive lamella fussion and branchial

epithelial hyperplasia. Histopathological changes of gill such as hyperplasia and

hypertrophy ,epithelial lifting, aneurysm and increase in mucus secretion have been

reported after the exposure of fish to a variety of noxious agents in the water, such as

herbicides, phenols and heavy metal (Nowak,1992) .The kidney of Clarias gariepinus

fingerlings exposed to DEP graded concentration showed tubular destruction or fussion

of the tubules, pyknosis, condensation of glomeruli content and accumulation of hyaline

droplets in the tubular epithelial cells. Oulmi et al. (1995) studied the effect of linuron

herbicide on the rainbow trout (Oncorhynchus mykiss).Their results showed small

cytoplasmic vacuoles, nuclear deformation in the epithelium of the first and second

segments of the proximal tubule. The kidney cells (hepatocytes) were observed to have

been massively destroyed. The renal corpuscles of the kidney were scattered resulting in

the disorganization and consequently obstruction to their physiological function. Some of

the kidney cells were found clogging together while they were disintegrated in some

79

tissues of the organ. This also agrees with the findings of Omoniyi et al. (2002) and

Rahman et al. (2002). Lesions in the kidney tissues of fish exposed to deltamethrin in the

epithelial cells of renal tubule, pyknotic nuclei in the hematopoietic tissues, dilution of

glomerular capillaries and degeneration of glomerulus were observed (Elif, 2006). In the

present study, the liver of C. gariepinus exposed to DEP concentration showed

congestion (sinosis), reduction of filament and enlargement of sinusoid and necrosis.

Liver is especially useful organ in assessing the possible impact of pollutant in fish. This

is because chemical tends to concentrate there. This is also a major site for

biotransformation of toxic chemicals which usually makes them less toxic and more

easily excreted. In the study of Risbourg and Bastide (1995), the exposure of fish to

atrazine herbicide increased in the size of lipid droplets and vacuolisation in the liver.

The most frequent encountered types of degenerative changes are those of hydropic

degeneration, cloudy swelling, vacuolization and focal necrosis. This also agrees with

Baba et al. (2007) in the exposure of fish to fenevalerate on the liver tissues of Cirrhina

mrigala, when necrosis of tubular epithelium and pycnotic nuclei in the hematopoietic

tissue occurred. Necrosis of the liver tissues in the study was observed, probably resulted

from the excessive work required by the fish to get rid of the toxicant from its body

during the process of detoxification by the liver. The inability of fish to regenerate new

liver cells may also have led to necrosis.

Conclusion

The environmental hazards that result from the pollution of water bodies by

diethyl phthalate are highly attributed to the non degradable metabolic components that

are made of this compound. In addition, acute and chronic bioassays should be carried

out on Diethyl phthalate (DEP) from other sources in other to harmonize the effect. It can

80

be concluded from this study that DEP is capable of interfering with the metabolic

processes by altering the enzyme activity in vital organs, changes in haematological

parameters and the tissues in an organism, and these may prove detrimental to survival in

nature.

81

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APPENDIX 1

Table 29. Anova of the Effect DEP on Acetylcholinesterase enzyme on Day 0

Sum of

Squares Df Mean Square F Sig.

BRAIN Between

Groups 1.876E8 4 4.689E7 11.555 .000

Within Groups 1.015E8 25 4058121.854

Total 2.890E8 29

MUSCLE Between

Groups 1.927E7 4 4816668.039 5.098 .004

Within Groups 2.362E7 25 944737.976

Total 4.289E7 29

92

Table 30. Anova of the effect of DEP on AchE activity on day 15

Sum of

Squares df Mean Square F Sig.

BRAIN Between

Groups 2.476E9 4 6.190E8 25.919 .000

Within Groups 5.971E8 25 2.388E7

Total 3.073E9 29

MUSCLE Between

Groups 4.402E8 4 1.100E8 24.149 .000

Within Groups 1.139E8 25 4556902.615

Total 5.541E8 29

93

Table 31. Anova of the effect of the DEP on the AchE activity day 30

ANOVA

Sum of


BRAIN Between

Groups 1.876E8 4 4.689E7 11.555 .000

Within Groups 1.015E8 25 4058121.854

Total 2.890E8 29

MUSCLE Between

Groups 1.927E7 4 4816668.039 5.098 .004

Within Groups 2.362E7 25 944737.976

Total 4.289E7 29

94

Table 32. Anova of the effect of DEP on the acid phosphatase activity on day 0

ANOVA

Sum of


LIVER Between

Groups .566 4 .142 2.396 .182

Within Groups .295 5 .059

Total .862 9

MUSCLE Between

Groups 9.623 4 2.406 2.001 .233

Within Groups 6.011 5 1.202

Total 15.634 9

95

Table 33. Anova of the effect of DEP on the ACP enzyme activity on Day 15

ANOVA

Sum of


LIVER Between

Groups 9.342 4 2.336 10.135 .000

Within Groups 3.457 15 .230

Total 12.799 19

MUSCLE Between

Groups 9.459 4 2.365 1.100 .392


Total 41.700 19

96

Table 34. Anova of the effect of DEP on the ACP enzyme activity on day 30

ANOVA

Sum of


LIVER Between Groups 1426.530 4 356.633 2.044 .139

Within Groups 2616.672 15 174.445

Total 4043.202 19

MUSCLE Between Groups 4212.087 4 1053.022 5.427 .007

Within Groups 2910.763 15 194.051

Total 7122.849 19

97

Table 35. Anova of the effect of DEP on the AST enzyme activity on day 0

Sum of


OD Between Groups .000 4 .000 .120 .969


Total .005 9

CONC Between Groups 29.746 4 7.436 .120 .970


Total 340.776 9

Table 36. Anova of the effect of DEP on the AST enzyme activity on Day 15

Sum of


OD Between Groups .006 4 .002 1.405 .353


Total .012 9

CONC Between Groups 462.136 4 115.534 1.404 .354


Total 873.521 9

98

Table 37. Anova of the effect of DEP on the AST enzyme activity on day 30

Sum of




Total .233 9

CONC Between Groups 4616.584 4 1154.146 3.356 .108

Within Groups 1719.300 5 343.860

Total 6335.884 9

Table 38. Anova of the effect of DEP on the ALT enzyme activity on day 0

Sum of


OD Between

Groups .006 4 .001 .623 .667


Total .017 9

CONCENTRATI

ON

Between

Groups 140.526 4 35.132 .591 .685


Total 437.756 9

99


Sum of




Total .234 9

CONCENTRATI

ON

Between Groups 278.116 4 69.529 2.441 .177


Total 420.516 9

100


Sum of




Total .003 9

CONCENTRATI

ON

Between Groups 85.416 4 21.354 6.463 .033


Total 101.936 9

101

Table 41. Anova of the haematological parameters day 0

Sum of


HB Between Groups .386 4 .096 .069 .991


Total 28.230 24

PCV Between Groups 49.840 4 12.460 1.716 .186


Total 195.040 24

RBC Between Groups 1.992 4 .498 1.837 .161


Total 7.415 24

WBC Between Groups 2.377E8 4 5.943E7 2.107 .118


Total 8.018E8 24

MCV Between Groups 13.067 4 3.267 .352 .839


Total 198.425 24

MCH Between Groups 91.296 4 22.824 .674 .618

Within Groups 677.387 20 33.869

Total 768.683 24

MCHC Between Groups .013 4 .003 .854 .508


Total .091 24

102

Table42. Anova of the effect of DEP on the haematological parameters on day 15

Sum of


HB Between Groups 15.614 4 3.904 1.822 .164


Total 58.470 24


Within Groups 304.800 20 15.240

Total 412.000 24



Total 10.284 24


Within Groups 1.697E8 20 8483600.000

Total 2.758E8 24

MCV Between Groups 36.804 4 9.201 3.121 .038


Total 95.768 24


Within Groups 578.168 20 28.908

Total 642.475 24

MCHC Between Groups 12.566 4 3.142 .998 .432


103

Table 43. Anova of the effect of DEP on haematological parameters on day 30

Sum of


HB Between Groups 8.977 4 2.244 1.660 .199


Total 36.020 24



Total 402.586 24



Total 6.131 24



Total 2.883E8 24

MCV Between Groups 25.506 4 6.377 1.632 .205


Total 103.662 24


Within Groups 640.056 20 32.003

Total 706.982 24

MCHC Between Groups .012 4 .003 .263 .898


Total .243 24

104

Table 44. Anova of the effect of DEP on the differential count on day 0

Sum of


N Between Groups 1.556 4 .389 .778 .593


Total 3.556 8

M Between Groups 138.000 4 34.500 4.423 .010


Total 294.000 24

L Between Groups 221.840 4 55.460 2.292 .095

Within Groups 484.000 20 24.200

Total 705.840 24

105

Table 45. Anova of the effect of DEP on the differential count day 15

Sum of


N Between Groups .667 2 .333 . .

Within Groups .000 0 .

Total .667 2

M Between Groups 133.840 4 33.460 2.956 .045

Within Groups 226.400 20 11.320

Total 360.240 24

L Between Groups 239.840 4 59.960 3.502 .025

Within Groups 342.400 20 17.120

Total 582.240 24

106

Table 46. Anova of the effect of DEP on the differential count day 30

Sum of


N Between Groups .833 4 .208 .417 .804


Total 1.333 5

M Between Groups 37.600 4 9.400 1.172 .353


Total 198.000 24

L Between Groups 47.600 4 11.900 1.080 .393

Within Groups 220.400 20 11.020

Total 268.000 24

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