1
PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES IN TILAPIA
(Oreochromis mossambicus) EXPOSED TO CADMIUM AND
CHLORPYRIFOS
PADMANABHA, A., M.F.Sc.
DEPARTMENT OF AQUATIC ENVIRONMENT MANAGEMENT
COLLEGE OF FISHERIES, MANGALORE
KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR.
AUGUST, 2015
2
PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES IN TILAPIA
(Oreochromis mossambicus) EXPOSED TO CADMIUM AND
CHLORPYRIFOS
Thesis submitted to the Karnataka Veterinary, Animal and Fisheries Sciences
University, Bidar in partial fulfillment of the requirements for the award of the
degree of
DOCTOR OF PHILOSOPHY IN FISHERIES SCIENCE
IN
AQUATIC ENVIRONMENT MANAGEMENT
BY
PADMANABHA, A., M.F.Sc.
DEPARTMENT OF AQUATIC ENVIRONMENT MANAGEMENT
COLLEGE OF FISHERIES, MANGALORE
KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR
AUGUST, 2015
3
4
Dedicated
To
My Beloved Parents
and Teachers
5
ACKNOWLEDGEMENTS
I wish to convey my sincere and deep debt of gratitude to my supervising guide,
Dr. H. R. V. Reddy, Professor and Head, Department of Aquatic Environment Management
and Former Director of Research, Karnataka Veterinary, Animal and Fisheries Sciences
University, Bidar, for his elegant and eminent guidance, constant encouragement and
abundant affection evinced during the period of research and preparation of this thesis. I am
very thankful to him for helping to secure financial support for my Ph.D.
I gratefully acknowledge my advisory committee member Dr. K. N. Prabhudeva,
Professor and Head, FRIC, Hebbal, Bangalore, for providing me the laboratory facility and
requirements for the present study and also creating light atmosphere in the stressing times of
my research work and those handy ideas, insightful advice for my research work. I am also
thankful to him for critical assessment and careful scrutiny of the manuscript.
I remain indebted to my advisory committee members Dr. Gangadhara Gowda,
Professor, Department of Aquatic Environment Management, Dr. Shrikrishna Isloor,
Associate Professor, Department of Veterinary Microbiology and Dr. N. B. Shridhar,
Associate Professor, Department of Veterinary Pharmacology and Toxicology, Veterinary
College, Bangalore for their kind counsel and co-operation leading to the completion of this
thesis in a successful way.
I sincerely thank Dr. Yathiraj, Dean, Veterinary College, Bangalore for the facilities
provided to carry out my research work. I record my gratitude to Dr. Lalith Achoth,
Professor and Head, Department of Dairy Economics and Business Management, Dairy
Science College, Bangalore for his immense help during statistical analysis.
6
I extend sincere thanks to Dr. Shivakumar Magada, Professor, Dr. Lakshmipathi M.
T., Associate Professor and Dr. A. T. Ramachandra Naik, Associate Professor, Department
of Aquatic Environment Management for their support during my studies.
I am grateful to staff of FRIC, Hebbal, Bangalore, Mr. Rajanna K. B, Dr. Chethan N.,
Shri. V. Shrinivas, Mrs. Sujatha S, Shivaraj, Bettaswamy L. N., Munikrishnaih (Driver),
Vallamma, Ananda, Munikrishna Reddy, Muniswamy and Devegowda for their help and
support showed during my research work.
I owe my sincere gratitude to Dr. K. S. Venkatesh Murthy, Research Associate,
Department of Aquatic Environment Management for his support during the study period.
My special thanks to Shri. Praveen Attavar, Venugopal Bagambila, Yadav Salian,
Dayanand Navoor who has helped me in every way all through my research work.
I am also thankful to my research colleague Muttappa Khavi for his help during the
experimental tenure of my thesis. My sincere gratitude to Nagendra Babu, Harish
Dhamagaye, Harsha Nayak, Shruthishree, Livi Wilson and Shruthi for their support during
the study period.
My thanks are also due to academic and library staff, College of Fisheries, Mangalore
for their help and co-operation during the study period.
My family has been a great source of encouragement in my academic pursuits. The
prayers and sacrifices of my parents and enthusiasm of my younger brother, sisters,
brothers-in-law, Yajna, Kshama and Karthik have been instrumental in providing me the
courage and fortitude to surmount every obstacle in my path.
Mangalore
August 2015
Padmanabha, A.
7
CONTENTS
CHAPTER
NO.
I
INTRODUCTION
II
REVIEW OF LITERATURE
4
Heavy metals
5
2.1.1
Cadmium
6
2.2
Pesticides
7
Chlorpyrifos
8
2.3
Oreochromis mossambicus
9
2.4
Lethal toxicity
10
2.5
Combined toxicity
11
2.6
Sublethal toxicity
12
Physiological responses
13
2.6.1.1
Behaviour of fish
13
2.6.1.2
Oxygen consumption rate
14
2.6.1.3
Food consumption rate
15
2.6.1.4
Ammonia-N excretion rate
16
2.6.1.5
Oxygen:Nitrogen ratio
17
2.6.1.6
Relative growth rate
17
Biochemical responses
18
2.6.2.1
Test organs
19
2.6.2.2
Cholinesterase enzymes
21
2.1
2.2.1
2.6.1
2.6.2
TITLE
PAGE
NO.
1
8
2.6.2.2.1 Acetylcholinesterase (AChE; E.C. 3.1.1.7)
22
2.6.2.2.2 Butyrylcholinestarase (BChE; E.C. 3.1.1.8)
22
2.6.2.3
Oxidative stress
2.6.2.3.1 Lipid peroxidation (LPx)
2.6.2.4
23
24
Antioxidant defense mechanisms
24
2.6.2.4.1 Superoxide dismutase (SOD; E.C.1.15.1.1)
24
2.6.2.4.2 Catalase (CAT; E.C.1.11.1.6)
25
2.6.2.4.3 Glutathione peroxidase (GPx; EC.1.11.1.9)
25
2.6.2.4.4 Glutathione reductase (GR; E.C.1.6.4.2)
26
2.6.2.4.5 Glutathione-S-transferase (GST; E.C.2.5.1.18)
26
2.6.2.5
Non-enzymatic antioxidants
28
2.6.2.5.1 Total reduced glutathione (GSH)
28
2.6.2.5.2 Ascorbic acid (Vitamin C)
29
2.6.2.6
Total protein
29
31
III
MATERIALS AND METHODS
3.1
31
Test animals
3.2
31
Maintenance of test animals
3.3
31
Laboratory conditioning of test animals
3.4
32
Toxicants
3.4.1
32
Cadmium
3.4.2
33
Chlorpyrifos
3.5
34
Studies on lethal toxicity
3.5.1
34
9
3.5.2
Studies on lethal toxicity of Cadmium
35
Studies on lethal toxicity of Chlorpyrifos
35
3.6.1
Studies on joint toxicity analysis
35
3.6.2
Joint lethal toxicity of Cadmium + Chlorpyrifos
36
3.6.3
Joint lethal toxicity of Chlorpyrifos + Cadmium
36
3.6.4
Categorization of toxicants
36
Joint action toxicity
37
Studies on sublethal toxicity
38
3.7.1.1
Physiological responses
38
3.7.1.2
Behaviour of normal and exposed fish
38
3.7.1.3
Estimation of oxygen consumption rate
39
3.7.1.4
Estimation of food consumption rate
39
3.7.1.5
Estimation of ammonia-N excretion rate
40
3.7.1.6
Estimation of Oxygen:Nitrogen ratio
40
Estimation of relative growth rate
41
3.7.2.1
Biochemical responses
41
3.7.2.2
Sample preparations
42
3.7.2.3.1 Assay of cholinesterase enzymes
42
3.7.2.3.2 Assay of acetylcholinesterase (AChE)
42
3.6
3.7
3.7.1
3.7.2
3.7.2.4
Assay of butyrylcholinesterase (BChE)
42
3.7.2.5
Assay of lipid peroxidation (LPx)
43
3.7.2.5.1 Assays of antioxidant enzymes
43
3.7.2.5.2 Superoxide dismutase (SOD)
43
10
3.7.2.5.3 Catalase (CAT)
44
3.7.2.5.4 Glutathione peroxidase (GPx)
44
3.7.2.5.5 Glutathione-S-transferase (GST)
44
3.7.2.6
Glutathione reductase (GR)
44
3.7.2.6.1 Assays of non-enzymatic antioxidants
45
3.7.2.6.2 Total reduced glutathione (GSH)
45
3.7.2.7
Ascorbic acid (ASA)
45
Estimation of total protein
45
Statistical analysis
47
4.1
RESULTS
47
4.2
Individual lethal toxicity (LC50)
48
4.3
Joint lethal toxicity (LC50)
49
4.3.1
Sublethal toxicity
49
4.3.2
Physiological responses
59
Biochemical responses
127
5.1
DISCUSSION
127
5.2
Lethal toxicity
130
5.2.1
Sublethal toxicity
131
5.2.2
Physiological responses
145
VI
Biochemical responses
175
VII
SUMMARY
180
VIII
BIBLIOGRAPHY
227
3.8
IV
V
ABSTRACT
11
LIST OF TABLES
TABLE
TITLE
1
Concentrations of test toxicants selected for sublethal toxicity studies.
2
The 96 h LC50 for Cadmium in Oreochromis mossambicus.
3
The 96 h LC50 for Chlorpyrifos in Oreochromis mossambicus.
4
The 96 h LC50 for varying concentrations of Cadmium + constant concentration of
Chlorpyrifos (i.e. 1/5th of LC50, 0.0044 ppm) in Oreochromis mossambicus.
5
The 96 h LC50 for varying concentrations of Chlorpyrifos + constant concentration
of Cadmium (i.e. 1/5th of LC50, 34 ppm) in Oreochromis mossambicus.
6
Categorization of toxicants based on the toxic units in Oreochromis mossambicus.
7
Joint action toxicity of Cadmium and Chlorpyrifos in Oreochromis mossambicus
based on the synergistic ratio (S.R.) model.
8
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the oxygen consumption rate in Tilapia (Oreochromis mossambicus)
during 7, 14 and 21 days of exposure.
9a
ANOVA for changes in oxygen consumption rate in Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
9b
Tukey's studentized range (HSD) test for oxygen consumption rate in Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
10
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the food consumption rate in Tilapia (Oreochromis mossambicus)
during 7, 14 and 21 days of exposure.
11a
ANOVA for changes in food consumption rate in Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
11b
Tukey's studentized range (HSD) test for food consumption rate in Tilapia
12
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
12
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ammonia-N excretion rate in Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
13a
ANOVA for changes in ammonia-N excretion rate in Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
13b
Tukey's studentized range (HSD) test for ammonia-N excretion rate in Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
14
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the Oxygen:Nitrogen ratio in Tilapia (Oreochromis mossambicus)
during 7, 14 and 21 days of exposure.
15a
ANOVA for changes in Oxygen:Nitrogen ratio in Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
15b
Tukey's studentized range (HSD) test for Oxygen:Nitrogen ratio in Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
16
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the relative growth rate in Tilapia (Oreochromis mossambicus)
during 7, 14 and 21 days of exposure.
17a
ANOVA for changes in relative growth rate in Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
17b
Tukey's studentized range (HSD) test for relative growth rate in Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
13
18
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the acetylcholinesterase activity in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
19
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the acetylcholinesterase activity in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
20
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the acetylcholinesterase activity in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
21
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the acetylcholinesterase activity in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
22
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the acetylcholinesterase activity in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
23
Percentage change in the acetylcholinesterase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
24a
ANOVA for changes in acetylcholinesterase activity in different organs of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
24b
Tukey's studentized range (HSD) test for acetylcholinesterase activity in different
organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
25
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the butyrylcholinesterase activity in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
26
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the butyrylcholinesterase activity in the liver of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
14
27
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the butyrylcholinesterase activity in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
28
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the butyrylcholinesterase activity in the brain of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
29
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the butyrylcholinesterase activity in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
30
Percentage change in the butyrylcholinesterase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
31a
ANOVA for changes in butyrylcholinesterase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
31b
Tukey's studentized range (HSD) test for butyrylcholinesterase activity in different
organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
32
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the lipid peroxidation level in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
33
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the lipid peroxidation level in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
34
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the lipid peroxidation level in the kidney of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
35
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the lipid peroxidation level in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
15
36
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the lipid peroxidation level in the muscle of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
37
Percentage change in the lipid peroxidation level in different organs of Tilapia
(Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
38a
ANOVA for changes in levels of lipid peroxidation in different organs of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
38b
Tukey's studentized range (HSD) test for levels of lipid peroxidation in different
organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
39
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the superoxide dismutase activity in the gills of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
40
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the superoxide dismutase activity in the liver of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
41
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the superoxide dismutase activity in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
42
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the superoxide dismutase activity in the brain of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
43
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the superoxide dismutase activity in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
44
Percentage change in the superoxide dismutase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
16
45a
ANOVA for changes in superoxide dismutase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
45b
Tukey's studentized range (HSD) test for superoxide dismutase activity in
different organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th
LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 7, 14 and 21 days.
46
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the catalase activity in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
47
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the catalase activity in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
48
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the catalase activity in the kidney of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
49
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the catalase activity in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
50
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the catalase activity in the muscle of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
51
Percentage change in the catalase activity in different organs of Tilapia
(Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
52a
ANOVA for changes in catalase activity in different organs of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
52b
Tukey's studentized range (HSD) test for catalase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
17
21 days.
53
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione peroxidase activity in the gills of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
54
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione peroxidase activity in the liver of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
55
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione peroxidase activity in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
56
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione peroxidase activity in the brain of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
57
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione peroxidase activity in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
58
Percentage change in the glutathione peroxidase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
59a
ANOVA for changes in glutathione peroxidase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
59b
Tukey's studentized range (HSD) test for glutathione peroxidase activity in
different organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th
LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 7, 14 and 21 days.
60
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione S-transferase activity in the gills of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
61
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione S-transferase activity in the liver of Tilapia
18
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
62
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione S-transferase activity in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
63
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione S-transferase activity in the brain of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
64
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione S-transferase activity in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
65
Percentage change in the glutathione S-transferase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
66a
ANOVA for changes in glutathione S-transferase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
66b
Tukey's studentized range (HSD) test for glutathione S-transferase activity in
different organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th
LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 7, 14 and 21 days.
67
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione reductase activity in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
68
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione reductase activity in the liver of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
69
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione reductase activity in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
70
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione reductase activity in the brain of Tilapia
19
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
71
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione reductase activity in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
72
Percentage change in the glutathione reductase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
73a
ANOVA for changes in glutathione reductase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
73b
Tukey's studentized range (HSD) test for glutathione reductase activity in different
organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
74
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total reduced glutathione level in the gills of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
75
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total reduced glutathione level in the liver of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
76
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total reduced glutathione level in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
77
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total reduced glutathione level in the brain of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
78
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total reduced glutathione level in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
79
Percentage change in the total reduced glutathione level in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos,
20
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
80a
ANOVA for changes in levels of total reduced glutathione in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
80b
Tukey's studentized range (HSD) test for levels of total reduced glutathione in
different organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th
LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 7, 14 and 21 days.
81
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ascorbic acid content in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
82
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ascorbic acid content in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
83
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ascorbic acid content in the kidney of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
84
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ascorbic acid content in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
85
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ascorbic acid content in the muscle of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
86
Percentage change in the ascorbic acid content in different organs of Tilapia
(Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
87a
ANOVA for changes in levels of ascorbic acid in different organs of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
87b
Tukey's studentized range (HSD) test for levels of ascorbic acid in different organs
of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
21
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
88
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total protein content in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
89
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total protein content in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
90
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total protein content in the kidney of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
91
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total protein content in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
92
Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total protein content in the muscle of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
93
Percentage change in the total protein content in different organs of Tilapia
(Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
94a
ANOVA for changes in levels of total protein in different organs of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
94b
Tukey's studentized range (HSD) test for levels of total protein in different organs
of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
22
LIST OF FIGURES
FIGURE
TITLE
1
Oxidative stress disturbed balance in the cell (Gupta and Sharma, 1999).
2
Graphical derivation of 96 h LC50 for Cadmium in Oreochromis mossambicus.
3
Graphical derivation of 96 h LC50 for Chlorpyrifos in Oreochromis mossambicus.
4
Graphical derivation of 96 h LC50 for varying concentrations of Cadmium +
constant concentration of Chlorpyrifos in Oreochromis mossambicus.
5
Graphical derivation of 96 h LC50 for varying concentrations of Chlorpyrifos +
constant concentration of Cadmium in Oreochromis mossambicus.
6
Changes in the oxygen consumption rate in Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7
Variation in the rate of oxygen consumption in terms of % decrease over control
in Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
8
Changes in the food consumption rate in Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
9
Variation in the rate of food consumption in terms of % decrease over control in
Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
10
Changes in the ammonia-N excretion rate in Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
11
Variation in the rate of ammonia-N excretion in terms of % decrease over control
in Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
12
Changes in the Oxygen:Nitrogen ratio in Tilapia (Oreochromis mossambicus)
23
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
13
Variation in the ratio of Oxygen:Nitrogen in terms of % increase over control in
Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
14
Changes in the relative growth rate in Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
15
Variation in the rate of relative growth in terms of % decrease over control in
Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
16
Changes in the acetylcholinesterase activity in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
17
Variation in the activity of acetylcholinesterase in terms of % decrease over
control in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
18
Changes in the acetylcholinesterase activity in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
19
Variation in the activity of acetylcholinesterase in terms of % decrease over
control in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
20
Changes in the acetylcholinesterase activity in the kidney of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
21
Variation in the activity of acetylcholinesterase in terms of % decrease over
control in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
24
Chlorpyrifos + Cadmium for 21 days.
22
Changes in the acetylcholinesterase activity in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
23
Variation in the activity of acetylcholinesterase in terms of % decrease over
control in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
24
Changes in the acetylcholinesterase activity in the muscle of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
25
Variation in the activity of acetylcholinesterase in terms of % decrease over
control in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
26
Mean variation in the activity of acetylcholinesterase in terms of % decrease over
control in different organs of Tilapia (Oreochromis mossambicus) exposed to
sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
27
Changes in the butyrylcholinesterase activity in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
28
Variation in the activity of butyrylcholinesterase in terms of % decrease over
control in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
29
Changes in the butyrylcholinesterase activity in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
30
Variation in the activity of butyrylcholinesterase in terms of % decrease over
control in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
25
31
Changes in the butyrylcholinesterase activity in the kidney of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
32
Variation in the activity of butyrylcholinesterase in terms of % decrease over
control in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
33
Changes in the butyrylcholinesterase activity in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
34
Variation in the activity of butyrylcholinesterase in terms of % decrease over
control in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
35
Changes in the butyrylcholinesterase activity in the muscle of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
36
Variation in the activity of butyrylcholinesterase in terms of % decrease over
control in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
37
Mean variation in the activity of butyrylcholinesterase in terms of % decrease
over control in different organs of Tilapia (Oreochromis mossambicus) exposed
to sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
38
Changes in the lipid peroxidation level in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
39
Variation in the level of lipid peroxidation in terms of % increase over control in
the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
26
40
Changes in the lipid peroxidation level in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
41
Variation in the level of lipid peroxidation in terms of % increase over control in
the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
42
Changes in the lipid peroxidation level in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
43
Variation in the level of lipid peroxidation in terms of % increase over control in
the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
44
Changes in the lipid peroxidation level in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
45
Variation in the level of lipid peroxidation in terms of % increase over control in
the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
46
Changes in the lipid peroxidation level in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
47
Variation in the level of lipid peroxidation in terms of % increase over control in
the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
48
Mean variation in the level of lipid peroxidation in terms of % increase over
control in different organs of Tilapia (Oreochromis mossambicus) exposed to
sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
49
Changes in the superoxide dismutase activity in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
27
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
50
Variation in the activity of superoxide dismutase in terms of % increase over
control in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
51
Changes in the superoxide dismutase activity in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
52
Variation in the activity of superoxide dismutase in terms of % increase over
control in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
53
Changes in the superoxide dismutase activity in the kidney of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
54
Variation in the activity of superoxide dismutase in terms of % increase over
control in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
55
Changes in the superoxide dismutase activity in the brain of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
56
Variation in the activity of superoxide dismutase in terms of % increase over
control in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
57
Changes in the superoxide dismutase activity in the muscle of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
58
Variation in the activity of superoxide dismutase in terms of % increase over
control in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
28
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
59
Mean variation in the activity of superoxide dismutase in terms of % increase
over control in different organs of Tilapia (Oreochromis mossambicus) exposed
to sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
60
Changes in the catalase activity in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
61
Variation in the activity of catalase in terms of % increase over control in the gills
of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
62
Changes in the catalase activity in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
63
Variation in the activity of catalase in terms of % increase over control in the
liver of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 21 days.
64
Changes in the catalase activity in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
65
Variation in the activity of catalase in terms of % increase over control in the
kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
66
Changes in the catalase activity in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
67
Variation in the activity of catalase in terms of % increase over control in the
brain of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 21 days.
29
68
Changes in the catalase activity in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
69
Variation in the activity of catalase in terms of % increase over control in the
muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
70
Mean variation in the activity of catalase in terms of % increase over control in
different organs of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
71
Changes in the glutathione peroxidase activity in the gills of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
72
Variation in the activity of glutathione peroxidase in terms of % increase over
control in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
73
Changes in the glutathione peroxidase activity in the liver of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
74
Variation in the activity of glutathione peroxidase in terms of % increase over
control in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
75
Changes in the glutathione peroxidase activity in the kidney of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
76
Variation in the activity of glutathione peroxidase in terms of % increase over
control in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
30
Chlorpyrifos + Cadmium for 21 days.
77
Changes in the glutathione peroxidase activity in the brain of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
78
Variation in the activity of glutathione peroxidase in terms of % increase over
control in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
79
Changes in the glutathione peroxidase activity in the muscle of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
80
Variation in the activity of glutathione peroxidase in terms of % increase over
control in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
81
Mean variation in the activity of glutathione peroxidase in terms of % increase
over control in different organs of Tilapia (Oreochromis mossambicus) exposed
to sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
82
Changes in the glutathione S-transferase activity in the gills of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
83
Variation in the activity of glutathione S-transferase in terms of % increase over
control in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
84
Changes in the glutathione S-transferase activity in the liver of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
85
Variation in the activity of glutathione S-transferase in terms of % increase over
31
control in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
86
Changes in the glutathione S-transferase activity in the kidney of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
87
Variation in the activity of glutathione S-transferase in terms of % increase over
control in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
88
Changes in the glutathione S-transferase activity in the brain of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
89
Variation in the activity of glutathione S-transferase in terms of % increase over
control in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
90
Changes in the glutathione S-transferase activity in the muscle of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
91
Variation in the activity of glutathione S-transferase in terms of % increase over
control in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
92
Mean variation in the activity of glutathione S-transferase in terms of % increase
over control in different organs of Tilapia (Oreochromis mossambicus) exposed
to sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
93
Changes in the glutathione reductase activity in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
32
94
Variation in the activity of glutathione reductase in terms of % increase over
control in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
95
Changes in the glutathione reductase activity in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
96
Variation in the activity of glutathione reductase in terms of % increase over
control in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
97
Changes in the glutathione reductase activity in the kidney of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
98
Variation in the activity of glutathione reductase in terms of % increase over
control in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
99
Changes in the glutathione reductase activity in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
100
Variation in the activity of glutathione reductase in terms of % increase over
control in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
101
Changes in the glutathione reductase activity in the muscle of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
102
Variation in the activity of glutathione reductase in terms of % increase over
control in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
33
103
Mean variation in the activity of glutathione reductase in terms of % increase
over control in different organs of Tilapia (Oreochromis mossambicus) exposed
to sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
104
Changes in the total reduced glutathione level in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
105
Variation in the level of total reduced glutathione in terms of % increase over
control in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
106
Changes in the total reduced glutathione level in the liver of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
107
Variation in the level of total reduced glutathione in terms of % increase over
control in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
108
Changes in the total reduced glutathione level in the kidney of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
109
Variation in the level of total reduced glutathione in terms of % increase over
control in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
110
Changes in the total reduced glutathione level in the brain of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
111
Variation in the level of total reduced glutathione in terms of % increase over
control in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
34
Chlorpyrifos + Cadmium for 21 days.
112
Changes in the total reduced glutathione level in the muscle of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
113
Variation in the level of total reduced glutathione in terms of % increase over
control in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
114
Mean variation in the level of total reduced glutathione in terms of % increase
over control in different organs of Tilapia (Oreochromis mossambicus) exposed
to sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
115
Changes in the ascorbic acid content in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
116
Variation in the ascorbic acid content in terms of % decrease over control in the
gills of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 21 days.
117
Changes in the ascorbic acid content in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
118
Variation in the ascorbic acid content in terms of % decrease over control in the
liver of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 21 days.
119
Changes in the ascorbic acid content in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
120
Variation in the ascorbic acid content in terms of % decrease over control in the
kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
35
121
Changes in the ascorbic acid content in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
122
Variation in the ascorbic acid content in terms of % decrease over control in the
brain of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 21 days.
123
Changes in the ascorbic acid content in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
124
Variation in the ascorbic acid content in terms of % decrease over control in the
muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
125
Mean variation in the ascorbic acid content in terms of % decrease over control in
different organs of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
126
Changes in the total protein content in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
127
Variation in the total protein content in terms of % decrease over control in the
gills of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 21 days.
128
Changes in the total protein content in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
129
Variation in the total protein content in terms of % decrease over control in the
liver of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 21 days.
130
Changes in the total protein content in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
36
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
131
Variation in the total protein content in terms of % decrease over control in the
kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
132
Changes in the total protein content in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
133
Variation in the total protein content in terms of % decrease over control in the
brain of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium for 21 days.
134
Changes in the total protein content in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
135
Variation in the total protein content in terms of % decrease over control in the
muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
136
Mean variation in the total protein content in terms of % decrease over control in
different organs of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
37
LIST OF PLATES
PLATE
TITLE
1
Photograph of Oreochromis mossambicus.
2
Experimental set up of 96 h LC50.
3
Photograph of heavy metal Cadmium and its stock solution.
4
Photograph of pesticide Chlorpyrifos and its stock solution.
5
Photograph showing the behaviour of Tilapia (Oreochromis mossambicus)
in control tanks.
6
Photograph showing the behaviour of Tilapia (Oreochromis mossambicus)
exposed to Cadmium.
7
Photograph showing the behaviour of Tilapia (Oreochromis mossambicus)
exposed to Chlorpyrifos.
38
Abbreviations & Symbols used in the thesis
%
Percentage
µg
Microgram
µl
Microlitre
cm
Centimeter
g
Gram
h
Hour
M
Molar
L
Litre
g mol-1
Gram per mole
mg
Milligram
min
Minute
mM
Millimolar
nm
Nanometer
0
C
Degree Celsius
ppb
Parts per billion
ppm
Parts per million
rpm
Revolutions per minute
U
Units
>
Greater than
<
Less than
Fig.
Figure
39
AChE
Acetylcholinesterase
BChE
Butyrylcholinesterase
ACh
Acetylcholine
BCh
Butyrylcholine
SOD
Superoxide dismutase
CAT
Catalase
GPx/GSHPx
Glutathione peroxidase
GR
Glutathione reductase
GSH
Glutathione (reduced)
GST
Glutathione-S- transferase
LPx
Lipid peroxidation
ASA
Ascorbic acid
MDA
Malondialdehyde
ROS
Reactive oxygen species
TBARS
Thiobarbituric acid reactive substances
CDNB
1-Chloro-2,4-dinitrobenzene
EDTA
Ethylene diamine tetra acetic acid
HCl
Hydrochloric acid
GSSG
Oxidized glutathione
DTT
Dithiothreitol
KCl
Potassium chloride
PMSF
Phenyl methane sulfonyl fluoride
40
SDS
Sodium dodecyl sulfate
BHT
Butylated hydroxyl toluene
NADH
Nicotinamide adenine dinucleotide
MnCl2
Manganese chloride
H2O2
Hydrogen peroxide
NADPH
Nicotinamide adenine dinucleotide phosphate
Cd
Cadmium
CPF
Chlorpyrifos
Cd + CPF
Cadmium + Chlorpyrifos
CPF + Cd
Chlorpyrifos + Cadmium
EC
Emulsifiable concentrate
LC50
Lethal concentration causing 50% mortality
TU
Toxic units
SR
Synergistic ratio
NH3-N
Ammonia-Nitrogen
O:N ratio
Oxygen:Nitrogen ratio
OPs
Organophosphates
TCA
Trichloro acetic acid
DTNB
5, 5′ Dithiobis-2-nitrobenzoic acid
Na2HPO4
Disodium hydrogen phosphate
HSD
Honestly significant difference
41
I. INTRODUCTION
Of late, the aquatic pollution and subsequent degradation of aquatic environment is
receiving worldwide attention. Among the many pollutants, heavy metals and pesticides are of
prime importance because their intense toxicity to the environment and the organisms living in
it. Heavy metals from anthropogenic source reach the aquatic system through direct dumping
of ore tailings, mining, drilling, etc. Similarly, the pesticides from agricultural fields, urban
areas and factory washings also reach the rivers, streams, lakes, etc. Because of increased
concern on the effects of heavy metals and pesticides on the life and activities of aquatic
organisms, efforts are being made to collect information to compare the magnitude of the
toxicity of these pollutants singly and in combinations.
In the present investigation, certain important aspects of Cadmium and Chlorpyrifos
singly and in combination have been studied. Recent studies have shown that when
experimental media contained more than one toxicant, such combinations could conspicuously
influence the toxic reaction of the animal both in terms of quantity and nature. These
responses include behavior, growth, oxygen utilization, feed intake, ammonia-N excretion and
many other biochemical functions like enzymatic activity which are found to be demonstrably
sensitive in the presence of pollutants like heavy metals and pesticides. If it is assumed that the
presence of a single contaminant in higher concentration could affect the life function of
aquatic animals, more than one contaminant in the experimental media should manifest such
effects in a greater scale commonly known as synergism or more than additive. Majority of
heavy metals and pesticides bring about synergistic reaction. Antagonism or less than additive
can also occur as a result of combined action of contaminants but in majority of such instances
one of the contaminants will be a non-essential one. Understanding the effects involves
42
experimentation with well recognized experimental species. Among the fresh water fish
species, Tilapia (Oreochromis mossambicus) is one of the most widely cultured and
commercially important fish in most parts of the world.
The work presented in this thesis comprises lethal and sublethal toxicity of Cadmium
and Chlorpyrifos singly and in combination on fingerlings of Tilapia. Information on
combined toxicity of metals and pesticides with reference to tropical fresh water fishes is
scanty. The present trend in toxicological investigations is to have a multi-factorial approach
on the response of aquatic organisms to the combined stress forced by the presence of more
than one toxicant in natural fresh water. A clear understanding of the cause and effect could be
had if only laboratory experiments are conducted employing sublethal concentration of these
toxicants. Therefore, during the present investigation, sublethal concentration of Cadmium and
Chlorpyrifos singly and in combinations were employed to assess the concentration dependent
effect on behaviour, oxygen consumption, food consumption, ammonia-N excretion,
Oxygen:Nitrogen ratio, growth rate in Tilapia. To further understand the nature and mode of
action of these pollutants at cellular level at sublethal concentrations, studies on cholinesterase
enzymes (acetylcholinesterase and butyrylcholinesterase), oxidative stress response (lipid
peroxidation), enzymatic antioxidant activity (superoxide dismutase, catalase, glutathione
peroxidase, glutathione S-transferase and glutathione reductase), non-enzymatic activity
(reduced glutathione and ascorbic acid) and total protein needs to be carried out with a view to
open up very interesting aspects of toxicology, the understanding of which would help in
delineating the impact of heavy metals and pesticides contamination in inland water bodies.
43
Considering the need for understanding physiological and biochemical responses in
Tilapia (Oreochromis mossambicus) exposed to Cadmium and Chlorpyrifos, the present study
was designed with the following objectives:
1. To study the effect of Cadmium and Chlorpyrifos on the physiological responses in
Tilapia (Oreochromis mossambicus).
2. To determine the effect of Cadmium and Chlorpyrifos on the biochemical responses in
Tilapia (Oreochromis mossambicus).
3. To determine the combined effects of Cadmium and Chlorpyrifos on the physiological
and biochemical responses in Tilapia (Oreochromis mossambicus).
44
II. REVIEW OF LITERATURE
The review of literature on the different aspects of aquatic pollution is a hazardous
task, has tremendous proliferation of printed matter in this aspect in different parts of the
world. Lot of information is available on ecosystem damages, radioactive pollution, pollution
by heavy metals and their compounds, petroleum hydrocarbons, pesticides and technical
organic chemicals, domestic waste and thermal pollution. Lack of proper concepts from the
factors contributing to pollution, differences in literature reporting, utilization of nonconventional strategies, study of cause and effects are a few factors which make a clear
understanding of the available data to plan future strategies. The present investigation has
taken into consideration only a small facet of the whole problem. An endeavor was made to
study the lethal and sublethal effects of heavy metal, Cadmium and pesticide, Chlorpyrifos,
singly and in combination on commercially important and widely cultured fresh water fish,
Tilapia (Oreochromis mossambicus).
Heavy metals and pesticides are some of the most dangerous group of contaminants
encountered in the aquatic ecosystem. Although many of the heavy metals are essential for
well being of the aquatic organisms, above optimum levels, they cause severe damage to the
ecosystem in general and aquatic organism in particular (Canpolat and Calta, 2003). Since
insitu concentrations of trace metals and natural pesticides are very low, the possibilities of
increase in concentrations by anthropogenic activities are high.
Although, numerous reports are available on the relative toxicity of individual metals
and pesticides on aquatic organisms, our present status of knowledge on the effects of metalpesticide mixtures on aquatic organisms is limited. Since metals and pesticides occur as
mixtures rather than alone in aquatic environment, information on their interactions might
45
provide a more realistic assessment of their toxicity to aquatic organisms. Studies have shown
that metal and pesticides may interact synergistically or antagonistically on survival and
development of various aquatic organisms (Gravato et al., 2006).
Freshwater ecosystems like rivers, ponds and lakes receive contaminants from
agriculture, aquaculture, industry and human settlements. However, these ecosystems are
fragile in nature and are cardinal abodes of aquatic organisms of commercial, aesthetic and
ecological importance. The rivers serve as breeding and nursery grounds for many
commercially important finfishes and shellfishes. Therefore, any pollutant material put into
inland water bodies are subjected to alterations and slowly build up contaminants demanding
constant monitoring and surveillance. The contamination of fresh water systems with a wide
range of pollutants has become a matter of concern over the last few decades. Among the
pollutants, heavy metals and pesticides rank top position, since heavy metals and pesticides
comprise the most dangerous group of pollutants.
2.1 Heavy metals
The pollution of aquatic ecosystems with heavy metals is a serious threat to the
environment due to their persistent nature, long distance transport and toxicity to aquatic
organisms. Contamination of aquatic ecosystems (e.g. lakes, rivers, streams etc.) with heavy
metals has been receiving increased worldwide attention (Tekin-Ozan and Kir, 2006). The
adverse effect of heavy metals is due to their non-degradation leading to accumulation in
tissues and interaction with protein or enzyme leading to changes in physiological and
metabolic processes. The metalloids and high concentrations of transitional metals tend to
accumulate in different tissues of body, hence become bioaccumulated (Canpolat and Calta,
46
2003). Heavy metals such as Cadmium (Cd), Lead (Pb), and Chromium (Cr) are of
toxicological importance.
2.1.1 Cadmium
Cadmium (Cd) is a known toxic heavy metal listed as one of the major hazardous
materials (no. 7 of 275) and group 1 carcinogens (Agency for Toxic Substance and Disease
Registry, 2007 and Moulis and Thevenod, 2010). It is a non-essential and non-degradable
metal whose dispersion in the environment has increased over the past decades due to its
widespread use in batteries, metal and mining industry, color pigment in paints, electroplating
and galvanizing, dentistry, etc. Furthermore, it is non-corrosive in nature (Guinee et al., 1999).
It is estimated that annually more than 30,000 tons of Cadmium is released into the
environment with 4000-13,000 tons coming from human activities (ATSDR, 2003b).
Considerable amounts of Cadmium get released through industrial effluents into the soil,
surface and ground water systems and gradually buildup to toxic levels causing damage to the
biota of the aquatic ecosystem. Elevated levels of Cadmium may cause some impairment to
enzymatic processes in fresh water fish. Therefore, the enzymatic activities have the potential
to indicate toxic stress and are sensitive parameters for testing of water for the presence of
toxicants.
Cadmium is a problem of high magnitude and of ecological significance due to its high
toxicity and ability to be accumulated in living organisms. Cadmium gets concentrated in
organs of fish associated with osmoregulation (gills), metal detoxification (liver and kidney),
digestion (intestine), neuro-endocrine regulation (brain and head kidney), locomotion (muscle)
and reproduction (gonads) (Pelgrom et al., 1995). Available reports indicate that the gill, liver
47
and kidney are the critical targets for Cadmium in fishes in which they have been reported to
cause significant metabolic, biochemical and physiological effects (WHO, 1992b).
2.2 Pesticides
With increasing competition to produce more food per unit area, pesticides have
become important tools for plant protection to boost food production. Further, pesticides play
a significant role to keep away many dreadful diseases. Currently, India is the largest producer
of pesticides in Asia and ranks twelth in the world for the use of pesticides. Pesticides are
used to prevent yield losses, enhance market opportunities, facilitate farm work, harvesting
and improve cost/profit ratios.
Although Indian average consumption of pesticide is far lower than many other
developed economies, the problem of pesticide residue is very high in India (Abhilash and
Nandita Singh, 2009). Pesticides used in agriculture are among the most hazardous chemicals.
Many of these chemicals are mutagenic, carcinogenic or cause developmental deficits. Such
chemicals may reach lakes and rivers through rains and wind, affecting many aquatic
organisms including fish. The pesticide concentration of water bodies can reach magnitudes of
hundreds of milligrams per liter. The levels of water pesticide pollution can be ranked as:
cropland water > field ditch water > runoff > pond water > groundwater > river water > deep
groundwater > sea water (Lin et al., 2000). Pesticides are poisons and would be expected to
have adverse effects on any non-target organism, having physiological functions common
with those of the target organisms that are attacked or inhibited by the pesticide. Physical,
chemical and biological processes affect the distribution and fate of these substances in the
environment. Such compounds are fat soluble and are therefore readily taken up from the
48
water, sediment and food sources into the tissues of aquatic organisms (Gil and Pla, 2001 and
Farah et al., 2004).
Organophosphate (OP) pesticides account for a major percentage of pesticides used in
domestic, agricultural and industrial applications throughout the world. They are highly
popular because they are effective, non persistent and relatively less expensive. Due to their
rapid breakdown in water, accompanied by low environmental persistence, organophosphate
pesticides have largely replaced the use of organochlorines in recent years (Zhang and Li,
2002).
2.2.1 Chlorpyrifos
Chlorpyrifos is the organophosphate insecticide used in the present study because it is
considered as one of the highest consumed pesticides in India. Chlorpyrifos (O, O-diethyl-O3, 5, 6-trichlor-2-pyridyl phosphorothioate; CPF) is a broad spectrum insecticide widely used
for the control of foliar insects in agricultural crops (Rusyniak and Nanagas, 2004). It is also
extensively used for the control of cutworms, corn rootworms, cockroaches, grubs, flea
beetles, flies, termites, fire ants, mosquitoes, and lice. It is largely used in paddy, pulses,
sugarcane, cotton, groundnut, mustard, vegetables, fruits and tobacco, as well as on lawns and
ornamental plants. It is the second highest selling organophosphate insecticide and is more
toxic to fish than organochlorine compounds (Tilak et al., 2001). Chlorpyrifos is a nonsystemic insecticide acting as a cholinesterase inhibitor with contact stomach and respiratory
action. It is designed to be effective by direct contact, ingestion and inhalation and which has
become a matter of concern because of its potentiality and hazardous effect (Tomlin, 2006).
Because of its broad spectrum, Chlorpyrifos (CPF) represents a threat to non-target
species including aquatic organisms. The pesticide passes via air drift or surface runoff into
49
natural waters, where it is accumulated in different organisms living in water, especially in
fish, thus making it vulnerable to several prominent effects (Varo et al., 2002). Chlorpyrifos is
known to inhibit acetylcholinesterase and can cause behavioural, neurological, oxidative and
other effects at low doses (Kwong, 2002 and Goel et al., 2005). Chlorpyrifos intoxication is
shown to cause a significant decrease in the reduced glutathione (GSH), catalase (CAT) and
glutathione S-transferase (GST) activities (Goel et al., 2005). Since Chlorpyrifos is
extensively applied in agriculture for pest eradication in India, it is pertinent to study its
hazardous effect on the aquatic system as it is assumed that the residue might affect the
aquatic vertebrates.
Among aquatic vertebrates, fishes are the inhabitants that cannot escape from the
detrimental effects of the pollutants (Olaifa et al., 2004). Easy to capture and fairly easy to
maintain and rear in captivity, fresh water fishes are remarkable indicators of the health status
of aquatic ecosystem (Chellappa et al., 2008). Fish are responding to the toxicant for the
period of acute as well as the chronic stress. The stress not only brings about physiological
changes but also affects the behavioural and the cellular changes in the tissues of the exposed
fish.
2.3 Oreochromis mossambicus
Oreochromis mossambicus, the exotic species known by the common name Tilapia,
forms the experimental animal in the present investigation because of its known tolerance to
environmental stresses. The toxicity levels exhibited by this fish will enable us to predict
hazards posed to other more sensitive species. Tilapia is the most popular fish species which is
economically important for fisheries, aquaculture, game fishing, recreational, aquarium fish
and are also used extensively in biological, physiological and behavioural research (Skelton,
50
2001). They are a good biological organism for toxicological studies (Casas-Solis et al., 2007
and Parthesarathy and Joseph, 2011) and have good tolerance to a wide range of
environmental conditions (Kumar et al, 2011). Particularly, O. mossambicus is widely
distributed in freshwater ecosystems; rivers, lakes, ponds and can tolerate wide fluctuations in
salinity. It is also considered as future fish for aquaculture, and is nicknamed “the aquatic
chicken” due to its ability to grow quickly with poor-quality input.
2.4 Lethal toxicity
Acute aquatic toxicity represents the intrinsic property of a substance to be injurious to
an organism in a short-term exposure to that substance. Static acute toxicity tests provide rapid
and reproducible concentration-response curves for estimating toxic effects of chemicals on
aquatic organisms (Casarez, 2001 and Yuill and Miller, 2008). With the help of these tests, the
relative toxicity of large number of chemicals present in the natural aquatic systems due to
variety of chemical spills can be determined. There is a vigorous documentation of the use of
acute toxicity tests for assessing the potential hazard of chemical contaminants to aquatic
organisms (Brack et al., 2002 and Diez et al., 2002).
Acute toxicity is expressed as the median lethal concentration (LC50) that is the
concentration in water which kills 50% of a test batch of fish within a continuous period of
exposure which must be stated (Amweg and Weston, 2005). The application of the LC 50 has
gained acceptance among toxicologists and is generally the most highly rated test of assessing
potential adverse effects of chemical contaminants to aquatic life (Fagr et al., 2008;
Khayatzadeh and Abbasi, 2010). The use of 96 h, LC50 has been widely recommended as a
preliminary step in toxicological studies on fishes (ASTM, 2002; USEPA, 2004 and APHA,
2005). LC50 is customary to represent the lethality of a toxicant to a test species in terms of
51
lethal concentration (for aquatic animals) and lethal dose (for terrestrial animal). It is always
expressed in terms of ‘g’ or mg/kg body weight of the animal and lethal concentrations (LC)
in terms of parts/million (ppm) or parts/billion (ppb) or milligram/litre (mg/L). The
relationship between the concentration of an environmental toxicant and its lethal effects on
living organisms is often a sigmoid curve. Probit analysis is a parametric statistical procedure
for making the sigmoidal response curve into a straight line so that an LC50 can be calculated
and the associated 95% confidence interval can be arrived at (Finney, 1978 and Hahn and
Soyer, 2008).
2.5 Combined toxicity
Organisms are rarely exposed to individual chemicals in the environment. Research
has shown that many compounds can enter the environment, disperse and persist to a greater
extent. Some compounds, such as pesticides are unintentionally released into the aquatic
environment through their application in agriculture while the heavy metals and others, such
as industrial by-products, are released through regulated and unregulated industrial discharges
to the water resources (Kolpin et al., 2002 and Battaglin and Fairchild, 2002). Aquatic
organisms are often exposed to the mixtures of toxicants because it is believed that regardless
of where the pollution occurs, it will eventually end up in the aquatic environment (Firat et al.,
2011). The mechanisms of action of toxicants are unknown and there is a great need to bridge
the gap between our understanding of the toxic effects of exposure to individual xenobiotics
and those effects from exposure to mixtures of such chemicals (Junghans, 2004; Olmstead and
LeBlanc, 2005). Hence, the present study was undertaken to evaluate the toxicity of individual
and combination of Cadmium and Chlorpyrifos on the fish Oreochromis mossambicus.
52
The interactions between different chemical components in a mixture may result in
either a weaker (antagonistic) or a stronger (synergistic, potentiated) combined effect than the
additive effect that would be expected from knowledge about the toxicity and mode of action
of each individual compound. Interactions may take place in the toxicokinetic phase (i.e.
processes of uptake, distribution, metabolism and excretion) or in the toxicodynamic phase
(i.e. effects of chemicals on the receptor, cellular target or organ). Assessing the cumulative
toxicity of pollutants in mixtures has therefore been an enduring challenge for environmental
health research as well as ecotoxicology for the past several decades (Eggen et al., 2004).
Toxicity studies have examined the effects of mixtures of multiple pesticides following
laboratory exposures (Cuppen et al., 2002 and Boone and James, 2003) and evaluated
pesticides as potential toxic components of more complex mixtures that include other types of
contaminants (Amweg et al., 2006). The mode of action for several groups of toxicants is
different; combination of agricultural pesticides with heavy metals in aquatic environment risk
for additive or even synergistic effects is obvious (Gravato et al., 2006).
2.6 Sublethal toxicity
Recently, extensive research is being conducted on physiological and biochemical
responses of the heavy metals and agricultural pesticides on fish. Biochemical and
physiological biomarkers are frequently used for detecting or diagnosing sublethal effects in
fish exposed to different toxic substances (Lavado et al., 2006). Prominent among these
biomarkers are physiological variables; behaviour, metabolic rate, food consumption, growth
rate and biochemical variables; cholinesterase enzymes, antioxidant enzyme activities are used
as stress indicators (De la Tore et al., 2000 and Orbea et al., 2002). In general, the pollutants
increase the activities of some enzymes and decrease the activities of others, while the
53
activities of a few enzymes remain unchanged in various tissues of fish. The process of
physiological stress response starts from the moment the body realizes the presence of the
stressor, followed by the sending of signals to the brain and to the specific sympathetic and
hormonal responses to eliminate, reduce or cope with the stress. A stressor is a stimulus that
acts on a biological system and a stress response is the animal’s reaction to the stimulus
(Barton, 2002).
2.6.1 Physiological responses
Fish physiology is now becoming an integral part of aquatic toxicology. The pollutants
in the environment at sublethal concentrations are an important variable to which a fish
respond physiologically. It is valuable to understand the mechanisms responsible for the
manifestations of toxicity, i.e. how a toxicant enters the organism, how it interacts with target
molecules, how it exerts its effects and how the organism deals with the exposure as this has
led to a better understanding of fundamental physiological processes (Gregus and Klaassen,
1996).
2.6.1.1 Behaviour of fish
Alterations in the chemical composition of the natural aquatic environment usually
affect behavioural responses of aquatic organisms (Rao et al., 2005). In aquatic toxicology
however, the nexus of behavioural sciences with the study of toxicants has only become
prominent recently. Mortality is obviously not the only end point to consider and there is
growing interest in the development of behavioural markers to assess the lethal effects of
toxicants. Abnormal behaviour is one of the most conspicuous endpoints produced by these
toxicants (Gerhardt, 2007 and Hellou, 2011).
54
Fish are ideal sentinels for behavioural assays of various stressors and toxic chemical
exposure due to their constant, direct contact with the aquatic environment where chemical
exposure occurs over the entire body surface, ecological relevance in any natural systems
(Omitoyin, 2007). Fishes exposed to toxicants undergo stress, which is a state of reestablished homeostasis, a complex suite of mal-adaptive responses. Fishes in a contaminated
environment show some altered behavioural patterns which may include avoidance, locomotor
activity and aggression and these may be attempts by the fish to escape or adjust to the stress
condition (Gormley et al., 2003). Avoidance and attractance behaviour in fish has proven to be
an easy and realistic behavioural endpoint of exposure.
A group of scientists (Allin and Wilson, 2000 and Kwak et al., 2002) have
demonstrated alterations in swimming behaviours due to sublethal exposures to metals and
pesticides. These alterations in the swimming behaviours results in an increase in the
expenditure of energy (Venkata Rathnamma et al., 2008), which may result in hyperactivity.
Similar behaviour had been observed in Channa striatus (Yadav et al., 2007) and in Clarias
gariepinus, Heterobranchus bidorsalis and their hybrid (Ekweozor et al., 2001; Bobmanuel et
al., 2006 and Inodi et al., 2010) exposed to toxicant showed hyperactivity characterized by
linear movement, jumping, opercular and tail beat frequencies, distance movements and
somersaulting depending on the concentrations.
2.6.1.2 Oxygen consumption rate
Oxygen consumption is one of the indicators of the general well being of the fish. It
may also be useful to assess the physiological state of an organism, helps in evaluating the
susceptibility or resistance potentiality and also useful to correlate the behaviour of the animal,
which ultimately serve as predictor of functional disruption of population. Hence, the analysis
55
of oxygen consumption can be used as a biodetectory system to evaluate the basic damage
inflicted on the animal which could either increase or decrease the oxygen uptake.
In the aquatic environment one of the most important manifestation of the toxic action
of a chemical is the over stimulation or depression of respiratory activity. The changes in the
respiratory activity of fish have been used by several investigators as indicators of response to
environmental stress. As aquatic organisms have their outer bodies and important organs such
as gills almost entirely exposed to water, the effect of toxicants on the respiration is more
pronounced. Pesticides enter into the fish mainly through gills and with the onset of symptoms
of poisoning, the rate of oxygen consumption increases (Premdas and Anderson, 1963 and
Ferguson et al., 1966a). Studying the effect of Copper and Cadmium on oxygen consumption
of the juvenile common carp, Cyprinus carpio (L.), Hassan (2011) noticed a negative
correlation between oxygen consumption and metal concentration.
2.6.1.3 Food consumption rate
The food consumption is one of the indicators of the general well being of the fish.
Change in feeding behaviour is considered to be a sensitive indicator to detect pollution due to
heavy metals and pesticides. Feed intake depends on the metabolic rate of fish (Broeck et al.,
1997) and taste receptors (Foster et al., 1966).
Jezierska et al. (2006) studied on the effects of heavy metal exposures on feeding
activity of Common carp larvae (as number of Artemia nauplii consumed within 10 minutes).
The fish were exposed during embryonic or larval development stage to Copper, Cadmium
and mix of both metals (Cu-0.2 mg /l, Cd-0.2 mg /l, Mix-0.1 mg/l of Cu + 0.1 mg/l of Cd).
The results show that at both embryonic and larval stages the feeding activity gets impaired.
Ferrari et al. (2011) observed significant decrease in the food intake of juvenile fish, Cyprinus
56
carpio after a short-term exposure to sublethal water-borne Cadmium. According to
Manoharan et al. (2008), the rate of feeding reduced by 5.94% to 9.02% in Barhus stigma
(Pisces: Cyprinidae) when exposed to different sublethal concentrations of Endosulfan.
2.6.1.4 Ammonia-N excretion rate
Ammonia-N excretion provides important information on the physiological condition
of the organism. It can be used as indicators of fish nitrogen balance and to determine the
effects of environmental and nutritional factors on protein metabolism (Fournier et al., 2003).
Most bony fishes excrete predominantly ammonia, which is energetically advantageous
compared to converting ammonia to urea. However, there are several examples of increased
excretion of ammonia in fish subjected to stressful conditions, such as environmental
contaminants, high concentrations of environmental ammonia, high pH, crowding, etc (Frick
and Wright, 2002). The rate of ammonia release to the water is therefore closely related to the
production of ammonia by the fish.
Barbieri et al. (2009) studied the effect of 2,4-D herbicide (2,4-Dichlorophenoxyacetic
acid) on ammonium excretion in juveniles of a Geophagus brasiliensis (Cichlidae), found that
exposure of fish to 40 mg/l 2,4-D caused 85% reduction in ammonium excretion compared to
the controls. Observing the effects of prolonged exposure to Copper in the marine gulf
Toadfish (Opsanus beta), Grosell et al. (2004) noticed increase in ammonia production due to
metal induced stress together with an impaired ability to excrete ammonia across the gill and
elevated plasma ammonia levels. Increased ammonia production can arise from a general
corticosteroid-mediated stress response that includes increased protein catabolism and
gluconeogenesis.
57
2.6.1.5 Oxygen:Nitrogen ratio
Since most of the nitrogenous end products of freshwater fish originate from protein
catabolism, with ammonia as the principal end product, the contribution of protein catabolism
to the total energy production of the fish can be assessed by determination of the ammonia
quotient. (AQ means mole to mole ratio of ammonia-N excreted to oxygen consumed).
Oxygen consumption and ammonia-N excretion provide a wider view of fish metabolism and
health status.
Santos et al., (2006) carried out a study on the effects of Naphthalene on metabolic rate
and ammonia excretion of juvenile Florida Pompano, Trachinotus carolinus. Fish after acute
exposures; show a tendency to increase oxygen consumption by virtue of Naphthalene
concentrations. After chronic exposures, a decrease was observed at the highest concentration
evidencing a narcotic effect of Naphthalene. Ammonia excretion was reduced significantly, as
compared to that of the controls, in all the exposed organisms. The O:N ratios of fish exposed
to different concentrations of Naphthalene were higher than that of the controls. Chinni et al.
(2000) studied the oxygen consumption, ammonia-nitrogen excretion and metal accumulation
in Penaeus indicus post larvae exposed to Lead. The decrease in oxygen consumption was
significant from 24 h up to 30 days exposure. In control post larvae, the rate of ammonianitrogen excretion increased with increasing time, but the increase was not much in exposed
post larvae. They have noticed a high O:N ratio in post larvae exposed to Lead, when
compared to control in all the exposure days.
2.6.1.6 Relative growth rate
The growth rate is an index associated with stress and is generally used as a sensitive
and reliable end-point in chronic toxicity investigations (Rosas et al., 2001; Benimeli et al.,
58
2003 and Huang and Chen, 2004). Xenobiotics are potentially harmful to fish by inducing
growth retardation (Gad and Sadd, 2008).
Kim and Kang (2004) reported reduced growth rate of Rockfish (Sebartes schlegeli)
due to Cu stress and there was an inverse relationship between growth and Cu exposure. Hayat
et al. (2007) exposed the fingerlings of three major carps viz. Catla catla, Labeo rohita and
Cirrhina mrigala, to sublethal concentrations of Manganese for 30 days. During this exposure
period, all the fish species showed negative growth. Ali et al. (2003) observed reduced growth
of Oreochromis niloticus under different water-borne Cu levels. Moraes et al. (2013) observed
the acute toxicity of pyrethroid-based insecticides in the neotropical freshwater fish Brycon
amazonicus. Exposure to synthetic pyrethroid pesticides is known to decrease growth and
impair swimming performance.
2.6.2 Biochemical responses
Biochemical biomarkers are frequently used for detecting or diagnosing sublethal
effects in fish exposed to toxic substances (Dong et al., 2009). Monitoring of the biomarkers
in living organisms including fish is a valuable approach and serves as early warning of
adverse changes and damage resulting from chemical exposure (Van der Oost et al., 2003).
Following the definition by Hinton and Lauren (1990), biomarkers can be defined as any
contaminant-induced biochemical changes in a not-too-sensitive organism, which leads to the
formation of altered structure (a lesion) in the cells, tissues or organs. It is a well known fact
that any biochemical alteration, if only severe enough and/or protected, will eventually result
in structural modification and vice versa (Sanchez et al., 2008). Contaminant-induced
oxidative stress, being a condition capable of eliciting biochemical alterations, it is highly
likely that histological modifications also occur. As such, fish diseases and pathologies, with a
59
broad range of etiologies, are used as indicators of environmental stress since they provide a
definite biological end-point of historical exposure.
2.6.2.1 Test organs
The route that the toxicant takes during its metabolisation often dictates the choice of
organs for examining the effect of xenobiotics. In this context, gill, liver, kidney and the brain
of teleost are the best suited organ system to analyze the biochemical aberrations due to
xenobiotic stress (Cengiz and Unlu, 2003; Durmaz et al., 2006; Triebskorn et al., 1997 and
Tilak et al., 2005).
Gills
Gills are the first organs which come in contact with environmental pollutants in fish
(Evans et al., 2005), principal sites for exchange of dissolved substances including metals and
main osmoregulatory surface tissue in aquatic animals. Therefore, gills may be the first site
where the sublethal effects of chemicals are observed (Jiraungkoorskul et al., 2007). The gills
of a fish perform essential functions such as respiration, nitrogenous waste excretion,
osmoregulation and acid-base balance. The pillar cells may detoxify or bioactivate foreign
compounds, via cytochrome P- 450 system (Gokosyr and Husoy, 1998).
Liver
Liver is the major site of metal storage and excretion in fish and as a result of its major
role in metabolism and its sensitivity to metals in the environment, particular attention has
been given to liver in toxicological investigations (Parvez et al., 2006). The liver and kidneys
play a crucial role in detoxification and excretion of toxicants mainly through the induction of
metal-binding proteins such as metallothioneins (MTs). The liver is noted as site of multiple
oxidative reactions and maximal free radical generation (Gul et al., 2004; Avci et al., 2005
60
and Atli et al., 2006). Cadmium has been shown to be concentrated in the liver of Sparus
aurata and Solea senegalensis (Kalman et al., 2010). Liver is the major organ of metabolism
and detoxification of Chlorpyrifos (ATSDR, 1997). It plays the role in activation and
detoxification of Chlorpyrifos, by desulfuration to Chlorpyrifos oxon.
Kidney
The key roles of fish kidney in the maintenance of homeostasis, hematopoietic,
immune and endocrine functions and mainly in the elimination and rapid clearance of
xenobiotics, make it prone to damage (Pereira et al., 2010). Despite its relevance on fish
physiology, kidney has been under employed in environmental health assessment and
ecotoxicology studies (Filipovic and Raspor, 2003). Several biological studies have associated
with metals in kidney tissues of different fish species. Cd exposed to Oncorhynchus mykiss
(Zohouri et al., 2001), Cyprinus carpio (Reynders et al., 2006) and Cadmium, Copper and
Zinc in Rainbow trout, Oncorhynchus mykiss (Kamunde and MacPhail, 2011). Cadmium is
accumulated primarily in the kidney and liver, but it may reach high concentrations also in the
gill.
Brain
The brain is relatively a large and important organ which is affected by pollutants. It is
the center of the nervous system in all vertebrate. Although it has no direct contact with the
pesticides dissolved in water, yet it is affected through the blood circulation and various
alterations are caused in it. As an organ in which homeostasis must be strictly maintained,
brain tissue contains large amounts of polyunsaturated fatty acids, which are particularly
vulnerable to free radical attacks (Sahin and Gumuslu, 2004). Reactive oxygen species can
attack multiple cellular constituents, including protein, nucleic acids and lipids leading to
61
disruption of cellular function and integrity (Sturve et al., 2008). Brain plays a regulatory role
in fish physiology and it is the most important organ in fish toxicology especially when
pesticides are involved in their mode of action in the nervous system (Ware, 1983).
Organophosphorous insecticides are systemic poisons and produce toxic effects by contact.
Their toxicity results in the inhibition by acetylcholinestrase (AChE) as a result acetylcholine
accumulates and disrupts the normal functioning of the nervous system giving rise to the
typical cholinergic symptoms associated with poisoning i.e. hyper activity, convulsions,
paralysis and death.
Muscle
Fish muscles, comparing to the other tissues, usually contain the lowest levels of
metals. Skin is an important excretory organ for heavy metals with the mucus. Increases of
heavy metals concentration in muscles may be returned to its accumulation in the lipid layer
under skin and muscle is not an active tissue in accumulating heavy metals (Yilmaz et al.,
2005). The most toxic heavy metals of particular concern to aquatic animals are Cadmium
(Cd), Lead (Pb) and Mercury (Hg) that have the way to fish muscle mainly via gills (Tao et
al., 2000). Accumulation of heavy metals in muscles may be due to its strong binding with
cystine residues of metallothionein (Tayel et al., 2008).
2.6.2.2 Cholinesterase enzymes
Cholinesterases are a class of enzymes composed of acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE), acting in the nervous systems of invertebrates as well as
vertebrates including fish (Fulton and Key, 2001).
62
2.6.2.2.1 Acetylcholinesterase (AChE; E.C. 3.1.1.7)
Acetylcholinesterase is the key enzyme in the nervous system of animals. It can be
found in various parts of the body, notably in neuromuscular junction and brain tissue (Radic
and Taylor, 2006). The main function of AChE is to terminate synaptic signal that was sent in
the form of acetylcholine (ACh). AChE differs from BChE by means of inhibitor reactivity,
substrate specificity, kinetic properties and distribution in tissue (Mehrani, 2004). AChE is
enzymes which preferentially hydrolyze acetyl esters such as ACh or acetyl-ß methylcholine,
while butyrylcholinesterases (BChE) are those which demonstrate a preference for other types
of esters such as butyrylcholine or propionylcholine.
2.6.2.2.2 Butyrylcholinesterase (BChE; E.C. 3.1.1.8)
Butyrylcholinesterase or Pseudocholinesterase is one of the serine hydrolase that
primarily targets butyrylcholine (BCh) (Barbosa et al., 2001). The presence of BChE is
important in the regulation of the cholinergic system. BChE is a non-specific cholinesterase
mainly found in blood plasma and is similar to AChE. BChE is known to be synthesized in
liver and distributed in liver, intestine, heart and kidney (Darvesh et al., 2003). BChE
catalyses the hydrolysis of acetylcholine efficiently and it can compensate the lacking of
AChE to allow the continuation regulation of cholinergic neurotransmission (Greig et al.,
2002). BChE detoxifies those inhibitors and act as endogenous scavengers before these
chemicals reach acetylcholinesterase at physiologically relevant target sites.
Inhibition of cholinesterase enzymes in fish exposed to Diazinon, Dimethoate,
Carbofuran and other anti-cholinesterase pesticides has been reported by Dembele et al., 2000;
Frasco and Guilhermino, 2002 and Hernandez-Moreno et al., 2010. AChE and other ChE of
several fish species have been found to be inhibited by other classes of environmental
63
contaminants such as metals, detergents, pyrethroids etc. (Roche et al., 2002 and Badiou et al.,
2008). According to Fulton and Key, 2001, the mode of action of Chlorpyrifos (CPF) in fish is
on cholinesterase (ChE) inhibition. Both AChE and BChE are inhibited by CPF. Chlorpyrifos
induces irreversible ChE inhibition, triggering constant stimulation of the muscles which leads
to paralysis and death. Assays to measure inhibition of ChE activity are the most common tool
to assess sublethal effects of CPF exposure in fish.
2.6.2.3 Oxidative stress
Aerobic life is inevitably linked with oxygen-dependent oxidative processes with the
concurrent danger of cellular damage by reactive oxygen species (ROS). The term “oxidative
stress” was introduced in biological sciences to denote a disturbance in the pro-oxidant and
antioxidant balance potentially leading to oxidative damage (Azzi et al., 2004). Oxidative
stress is caused by the formation of ROS, e.g. hydrogen peroxide (H2O2), hydroxyl radical
(HO•), and superoxide anion radical (O2−), mainly as byproducts of oxidative metabolism
(Zhang et al., 2008).
Defense system
Reactive oxygen species
Fig. 1 Oxidative stress disturbed balance in the cell (Gupta and Sharma, 1999).
64
2.6.2.3.1 Lipid peroxidation (LPx)
Oxidative lipid damage, termed lipid peroxidation results in the production of lipid
radicals and in the formation of a complex mixture of lipid degradation products
(malondialdehyde and other aldehydes such as alkanals, alkenals, hydroxyalkenals, ketones,
etc). Lipid peroxidation produces a progressive loss of membrane fluidity, reduces membrane
potential and increased permeability to ions such as calcium (Rosa, 2005). The most widely
used assay for lipid peroxidation is the malondialdehyde (MDA) formation, which represents
the secondary lipid peroxidation product with the thiobarbituric acid reactive substances test.
Malondialdehyde (MDA) is the final product of lipid peroxidation. The concentration of MDA
is the direct evidence of toxic processes caused by free radicals (Sieja and Talerczyk, 2004). In
biochemical evaluation of metal toxicity, the MDA level was regarded as a well suited
indicator for the extent of lipid peroxidation (Nogueira et al., 2003).
2.6.2.4 Antioxidant defense mechanisms
The antioxidant system protects tissues from the deleterious effects of free radicals.
Free radicals are eliminated from the body following reactions with other free radicals or with
antioxidants. Various antioxidant mechanisms are in operation leading to scavenging or
detoxification of the reactive oxygen species. Antioxidant enzymes like superoxide dismutase,
catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase are
preventive in nature as they act as a primary defense as endogenous physiological antioxidants
by quelling of oxygen, decomposition of hydrogen peroxide and sequestration of metal ions.
2.6.2.4.1 Superoxide dismutase (SOD; E.C.1.15.1.1)
Superoxide dismutase is a metalloprotein found in both prokaryotic and eukaryotic
cells. The iron containing (Fe-SOD) and the manganese containing (Mn-SOD) enzymes are
65
characteristic of prokaryotes (Sheehan and Power, 1999). In eukaryotic cells, the predominant
forms are the Copper containing enzymes and the Zinc containing enzymes located in the
cytosol. The second type contains Manganese and is found in mitochondrial matrix that
contributes up to 60% of total tissue activity. The superoxide dismutase present in cytoplasm
is manganese independent (Radi et al., 1985).
2O-2 + 2H+
H2O2 + O2
This enzyme catalyzes the dismutation of superoxide anion (a free radical) to hydrogen
peroxide and oxygen (Macmillan-Crow et al., 1998).
2.6.2.4.2 Catalase (CAT; E.C.1.11.1.6)
This high molecular weight enzyme contains a haeme group (Fe (III)-protoporphyrin)
attached to its active site. Located primarily in peroxisomes, the enzyme catalyzes the
decomposition of hydrogen peroxide to water and oxygen.
2H2O2
2H2O + O2
Catalase guards the cell from oxidative damages due to hydrogen peroxide and
hydroxyl radicals.
2.6.2.4.3 Glutathione peroxidase (GPx; EC.1.11.1.9)
Glutathione peroxidases are selenoenzymes concerned with the reduction of
hydroperoxides and hydrogen peroxide at the expense of glutathione. During this process
hydrogen peroxide is reduced to water and organic hydroperoxides are reduced to alcohol.
2GSH + ROOH
GSSG + ROH + H2O
2GSH + H2O2
GSSG + 2H2O
In general, glutathione peroxidases exist in the cytosol and mitochondrial matrix.
Glutathione peroxidases reside in the peroxisomes of fish liver cells (Orbea et al., 2002).
66
These fishes which are more susceptible to oxidative damage have generally higher activities
for glutathione peroxidase (Hassipieler et al., 1994) than other enzymes involved in
glutathione metabolism.
2.6.2.4.4 Glutathione reductase (GR; E.C.1.6.4.2)
Contained in cytosol and mitochondrial compartments; glutathione reductase catalyzes
the reduction of glutathione disulphide (oxidized glutathione) to glutathione (Schirmer and
Kranth-Siegel, 1989).
GSSG + NADPH + H+
2GSH + NADP+
2.6.2.4.5 Glutathione-S-transferase (GST; E.C.2.5.1.18)
It utilizes glutathione (GSH) in conjugation reactions with both exogenous and
endogenous substrates. This group of enzymes is present in high amounts in liver cytosol and
in lower amounts in other tissues. It catalyzes the conjugation of electrophilic xenobiotic such
as certain carcinogens to glutathione.
R + GSH
R – S - G where R is an electrophilic xenobiotic.
Glutathione conjugates are more water soluble and are thought to be metabolized
further by cleavage of the glutamate and glycine residues, followed by acetylation of the
resulted free amino group of the cysteinyl residue to produce the final product, a mercapturic
acid, which is then excreted (Habig et al., 1974).
Investigations on oxidative stress and enzymatic and non enzymatic antioxidants in
fishes and aquatic invertebrates in response to environmental changes and exposure to varied
types of pollutants in the laboratory have been studied extensively. Cadmium and Mercury in
Tilapia nilotica (El-Demerdash and Elagamy, 1999, Wilhem-Filho et al., 2001, Ritola et al.,
2002, Achuba & Osakwe, 2003, Oakes et al., 2004, Martinez- Alvarez et al., 2005, Trendazo
67
et al., 2006, Copper in Esomus danricus (Vutukuru et al., 2006); Cd, Cu, Cr, Zn in
Oreochromis niloticus (Oner et al., 2009 and Atli and Canli, 2010) and Deltamethrin in
Cyprinus carpio (Yonar and Sakin, 2011)).
Fish are frequently subjected to pro-oxidant effects of different pollutants often present
in the aquatic environment. Oxidative stress responses in freshwater teleost, Esomus dandricus
in response to exposure to Copper were so apparent that it can be considered as biomarkers of
oxidative damage (Vutukuru et al., 2006). Increased lipid peroxidation in viscera concomitant
with decrease in the activity of catalase and superoxide dismutase clearly demonstrated the
oxidative stress. Berntssen et al. (2000) studied direct effect of dietary Copper on lipid
peroxidation in the liver, kidney and intestine of Salmo salar and reported that increased lipid
peroxidation was accompanied by a decrease in glutathione peroxidase activity.
Several enzymes from glutathione system constitute a sensitive biochemical indicator
of chemical pollution. Relative changes of glutathione-dependent enzymes in both fish species
suggest a different susceptibility to toxins. Hamed et al. (2003) measured glutathione related
enzyme levels of freshwater fish as bioindicators of pollution. The liver and kidney
glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx)
were higher in Oreochromis niloticus and African catfish, Clarias lazera captured from all the
polluted areas compared to the control.
Various studies have shown devastating effects of pesticides in various biochemical
activities (Ullah et al., 2014c). The changes in antioxidants systems of fish are often tissue
specific. Such changes have been traced in brain, gills, muscles, kidneys and viscera of
different fish with varying results in different organs. Peroxidase activities were found higher
in brain, viscera, gills and muscles of Tilapia but gill was the organ received highest
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disturbance in peroxidase (Ahmad et al., 2005). Similarly, changes in lipid peroxidase have
been observed on account of different pesticides as well as environmental pollutants.
2.6.2.5 Non-enzymatic antioxidants
Non-enzymatic antioxidants are essential for the conversion of reactive oxygen species
to harmless substances and for maintenance of cellular metabolism and function (Zhang et al.,
2008). The second line of defense to oxidative stress is the use of antioxidant substances such
as glutathione, ascorbic acid (vitamin C), uric acid, albumin, bilirubin, vitamin E, carotenoids,
flavonoids and ubiquinol. The first two components are described as they only are considered
in the present study.
2.6.2.5.1 Total reduced glutathione (GSH)
Reduced glutathione (GSH) is an important antioxidant, which is readily oxidized by
ROS to oxidized glutathione, but can also bind metals that might induce oxidative stress
(Hansen et al., 2007). The oxidized glutathione redox status and increased activities of several
antioxidative enzymes in polluted fish confirmed that they were subjected to oxidative stress.
Glutathione levels and glutathione dependent enzyme activities have been employed as
biomarkers for xenobiotic exposure in some species (Almar et al., 1998).
Wilczek et al. (2008) suggested that GSH is presumably an important element of the
defense against toxic effects of heavy metals, either through binding metal ions or through
neutralizing reactive oxygen species generated by metabolic processes and/or exogenic
factors. GSH may play a role in inducing resistance to metals by protecting gill against the
attack by free radicals (Firat and Kargin, 2009). GSH is synthesized in the liver and released
to the blood for transferring to the other organs such as the kidney and muscle (Pena et al.,
2000). Because metal exposures did not alter the levels of GSH in the blood, muscle and gill,
69
it suggests that metals taken up from the gill were immediately transferred (via the blood) to
the liver for the usage in the metabolism or sequestered. Thus, GSH levels in the tissues may
be a good indicator to understand the degree of metal exposure.
2.6.2.5.2 Ascorbic acid (Vitamin C)
Vitamin C is not only a dietary requirement for fish ensuring optimal growth rates as
well as collagen and hormone synthesis but it is also a powerful antioxidant in aqueous media
and the major protective agent against oxidative damage is blood plasma. Ascorbic acid
deficiency may reduce the activity of xenobiotic metabolizing enzymes. Ascorbic acid
ameliorates Copper and Cadmium toxicity and is required for trace element homeostasis. It is
released into the digestive tract, but reabsorbed almost quantitatively, thus conserving the
animal’s ascorbic acid pools (Dabrowski, 1990). Vitamin C can protect host cells against
harmful oxidants released into the extracellular medium. The free metal ion independent
protein oxidation in cells is exclusively prevented by ascorbic acid.
2.6.2.6 Total protein
Proteins are important organic substance required by organisms in tissue building. It
constitutes the building block and the basic molecule for any biochemical reaction. They are
intimately related with almost all physiological processes, which maintain a simple
biochemical system in ‘living condition’ (Joshi and Kulkarni, 2011). Proteins are mainly
involved in the architecture of the cell. Proteins occupy a unique position in the metabolism of
cell because of the proteinaceous nature of all the enzymes which mediate at various
metabolic pathways. The levels of total protein are considered to be major indices of the
health status of teleosts. Both the protein degradation and synthesis are sensitive over a wide
range of conditions and show changes to a variety of physical and chemical modulators.
70
Assessment of protein and enzymes activities can be considered as a diagnostic tool to
determine the physiological status of cells or tissues (Manoj, 1999). The effects of toxicants
on protein content of fish have been observed by a number of investigators. Datta et al. (2007)
in Clarias batrachus after exposure to Arsenic; El-Sayed et al. (2007) in Oreochromis
niloticus after Deltamethrin exposure; Min and Kang (2008) in Oreochromis niloticus after
Benomyl exposure. The storage or mobilization of metabolic substrates such as glucose,
glycogen, lactate, lipid and protein are disrupted by exposure to several trace metals including
Cadmium (Fabbri et al., 2003).
Physiological and biochemical marker studies in fish open a number of research areas
aimed at providing greater knowledge of fish physiology and toxicology. In addition, such
studies would provide more precise information concerning the response of antioxidant
defenses in different species under various circumstances as well as on the regulatory
mechanisms of this response. Such future studies will, no doubt, benefit aspects related to fish
farming and aquaculture practices.
The present investigation therefore aims at elucidating the aquatic toxicity of the
selected heavy metal, Cadmium and largest market-selling and multipurpose insecticide
Chlorpyrifos, on the commonly available and edible aquatic organism, Tilapia (Oreochromis
mossambicus). The study was carried out with special emphasis on physiological and
biochemical responses of the individual and combination of Cadmium and Chlorpyrifos in the
experimental fish.
71
III. MATERIALS AND METHODS
3.1 Test animals
The selection of organisms for toxicity test is mainly based on certain criteria like its
ecological status, position within the food chain, suitability for laboratory studies, genetical
stability and uniform populations and adequate background data on the organism. The cichlid
fish, Oreochromis mossambicus (Tilapia) was selected for the present study (Plate 1) due to its
wide geographical distribution, availability throughout the year and suitability as model for
toxicity testing (Ruparrelia et al., 1986) and also due to sustainability in laboratory conditions.
This fish shows a well adaptive nature with the changing environment.
3.2 Maintenance of test animals
Oreochromis mossambicus fry (2-3 cm) procured from a private fish farm at
Chintamani, Chickaballapur district, Karnataka were transported to the FRIC farm, Hebbal,
Bangalore in well oxygenated polythene bags containing clean pond water. Soon after arrival
at the fish farm, they were released into the pond for proper acclimation. The polythene bags
carrying the Tilapia fry were allowed to float on the pond for one hour. Later the fry were
allowed to enter into the pond voluntarily by opening the bags. Tilapia fry were allowed to
grow in pond till they attained fingerling size (9-10 cm) with artificial feeding.
3.3 Laboratory conditioning of test animals
Tilapia fingerlings collected from the pond were released into the glass aquaria (10
No’s each) containing 50 L freshwater, for proper acclimatization in the laboratory. The glass
aquaria were kept as holding tanks to maintain the experimental animals. Vigorous aeration
was provided in the tanks with natural photoperiod. The fish were fed every 24 h with
commercial floating feed. The walls of the holding tank were thoroughly cleaned periodically
72
to avoid algal growth. The excreta were siphoned off on a daily basis to prevent the build up
of ammonia in the medium. Fish were conditioned in holding tank water for 10 days before
employing them for the experiments.
The tanks were kept in the wet laboratory; the water temperature, dissolved oxygen
level and pH of the water were monitored regularly. Individual fish fingerlings measuring 9.5
± 0.5 cm in total length and weighing 15.0 ± 1.0 g were selected for the present study. A
mixed population was used to exclude the possibilities of influence of sex of the individuals
on the parameters studied. Healthy and vigorous individuals of uniform size, selected from the
bulk collection brought from the field, were used for the experiments.
3.4 Toxicants
Two test compounds were used to assess the toxicity and its impact on the
physiological and biochemical responses in experimental fish. Test compounds belonged to
two categories viz a heavy metal, Cadmium and an organophoshporous pesticide,
Chlorpyrifos. The expiry date of the test substances was checked prior to the initiation of the
treatment and it was found to be suitable for the exposure.
3.4.1 Cadmium
Chemical name: Cadmium chloride, monohydrate
Molecular formula: CdCl2.H2O
Molecular weight: 183.32 g mol−1
Appearance: White solid, hygroscopic
Solubility: Water, Ethanol.
It is usually found as a mineral combined with other elements such as oxygen
(Cadmium oxide), chlorine (Cadmium chloride) or sulfur (Cadmium sulphate /sulphide).
73
Cadmium chloride monohydrate A. R. grade was used for the present study. The stock
solution was prepared having strength of 1000 ppm by adding known quantity of Cadmium
chloride to 1000 ml distilled water. Calculated amount of stock solution was added to test
tanks containing 45 L water and mixed thoroughly to arrive at the desired Cadmium
concentration.
3.4.2 Chlorpyrifos
Chemical name: Chlorpyrifos 20% EC
IUPAC name: O, O-diethyl-O-(3, 5, 6-trichloro-2-Pyridinyl)-phosphorothioate
Brand name: Deviban
Molecular formula: C9H11Cl3NO3PS
Molecular weight: 350.59 g mol−1
Appearance: Cream coloured solid with some liquid separation depending on temperature
Solubility: Water
Mode of Action: Non-systemic insecticide with contact, stomach and respiratory action
Chemical composition: The emulsifiable concentrate containing 20% w/w Chlorpyrifos as its
active ingredient with 80% w/w of emulsifiers and solvents.
Chlorpyrifos 20% EC used in the present study was obtained from the local market of
Bangalore, Karnataka. Chlorpyrifos stock solution was prepared having strength of 1000 ppm
by adding known volume to 1000 ml distilled water. Calculated amount of stock solution was
added to test tank containing 45 L water and mixed thoroughly to arrive at the desired
Chlorpyrifos concentration.
74
3.5 Studies on lethal toxicity
Lethal toxicity study was carried out by following the standard guidelines (APHA,
2005) to determine the lethal (LC50) level of Cadmium and Chlorpyrifos using static and static
renewal system respectively. Laboratory conditioned fishes of uniform size were selected to
assess the lethal concentration of the toxicants. Each experimental container was of 50 L
capacity, made of glass (Plate 2). Ten fish each were accommodated in 45 L of test solution.
Appropriate controls were run for each set of experiment. Three sets of replicates were
performed for each concentration. All the bioassays were carried out under laboratory
condition. The animals were not fed during the experiment and water was not aerated. Care
was taken to leave the animals with minimum disturbance.
The specimen were inspected at every 6 hours and were considered dead if no
opercular beating was noticed and locomotory responses ceased even on mechanical
stimulation. Dead fishes were removed immediately from the test medium to avoid
disintegration and the cumulative percentage mortality was recorded at every 6 h. The
percentage mortality of fishes was taken into account for 96 hours. The 96 h LC50 value for
each exposure concentration, of toxicant, were recorded and tested by probit analysis program
described by Finney (1971).
3.5.1 Lethal toxicity of Cadmium (Cd)
Lethal toxicity of Cadmium was carried out as described in section 3.5, following
static system. The stock solution of Cadmium was prepared having strength of 1000 ppm
using distilled water (Plate 3). There was minimum 10% mortality at lowest concentration
(160 ppm) and 100% mortality at highest concentration (184 ppm) in the range finding tests.
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3.5.2 Lethal toxicity of Chlorpyrifos (CPF)
Lethal toxicity study was carried by static renewal system to determine the LC 50 value
of Chlorpyrifos to Oreochromis mossambicus as described in section 3.5. The water was
changed daily to keep the concentrations of Chlorpyrifos near to the nominal level. The stock
solution of Chlorpyrifos was prepared having strength of 1000 ppm using distilled water (Plate
4). There was minimum 10% mortality at lowest concentration (0.015 ppm) and 100%
mortality at highest concentration (0.030 ppm) in the range finding tests. Hence,
concentrations of the Chlorpyrifos used in short term definitive tests were between 0.015 ppm
to 0.030 ppm.
3.6 Studies on joint toxicity analysis
The concept of prediction of toxicity of mixture of pollutants has received wide
approval in aquatic toxicological studies as it provides scope to study simultaneous effects of
several pollutants in single set of experiments, the results of which can be expressed as a
single number. Experiments were carried out to assess the lethal responses of combinations of
Cadmium and Chlorpyrifos by the test organisms. The experimental conditions and set up are
explained in section 3.5 above.
3.6.1 Joint lethal toxicity of Cadmium + Chlorpyrifos (Cd + CPF)
Joint lethal toxicity of Cadmium (varying concentrations) + Chlorpyrifos (fixed
concentration i.e. 1/5th of its LC50 value, 0.0044 ppm) was carried by following static renewal
system. There was minimum 10% mortality at lowest concentration (80 ppm) and 100%
mortality at highest concentration (110 ppm) in the range finding tests. Hence, concentrations
of the Cadmium + Chlorpyrifos used in short term definitive tests were between 80 ppm to
110 ppm.
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3.6.2 Joint lethal toxicity of Chlorpyrifos + Cadmium (CPF + Cd)
Joint lethal toxicity of Chlorpyrifos (varying concentrations) + Cadmium (fixed
concentration i.e. 1/5th of its LC50 value, 34 ppm) was carried out by following static renewal
system. There was minimum 10% mortality at lowest concentration (0.010 ppm) and 100%
mortality at highest concentration (0.028 ppm) in the range finding tests. Hence,
concentrations of the Chlorpyrifos + Cadmium used in short term definitive tests were
between 0.010 ppm to 0.028 ppm.
3.6.3 Categorization of toxicants
Toxicity data obtained as the 50% mortality end point were converted into toxic units
(TU) by the following formula TU = [1/LC50] × 100 (Michniewicz et al., 2000) and were
characterized according to the categorization proposed by Pablos et al. (2011).
If, TU > 100
: Highly toxic
TU - 10 to 100 : Very toxic
TU - 1 to 10
: Toxic
TU < 1
: Less toxic
3.6.4 Joint action toxicity
Joint action toxicity data were analyzed based on the synergistic ratio model following
the method Hewlett and Plackett (1969) where:
LC50 of toxicant A
S. R index = ------------------------------------LC50 of toxicant A+ toxicant B
If, S.R. = 1: Joint action is additive
S.R. < 1: Joint action is antagonistic
S.R. > 1: Joint action is synergistic
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3.7 Studies on sublethal toxicity
Test animals measuring 9.5 ± 0.5 cm in total length and weighing 15.0 ± 1.0 g were
selected for the present study. In all, 10 fish each were exposed to two sublethal
concentrations of the individual and combinations of Cadmium (Cd) and Chlorpyrifos (CPF)
in a glass aquarium of 50 L capacity to study physiological and biochemical responses of fish
for varying treatment durations of 7, 14 and 21 days. The medium was exchanged at every 24
h to reduce the build-up of metabolic wastes and to keep concentrations of test compounds
near the nominal level. Control was run for each experiment, kept in fresh water without the
addition of test compound. The concentrations of test compounds selected for sublethal
toxicity studies are tabulated below.
Table 1. Concentrations of test toxicants selected for sublethal toxicity studies
Test toxicants
Cadmium (Cd)
Concentrations
1) 34 ppm (1/5th of LC50 value)
2) 17 ppm (1/10th of LC50 value)
Chlorpyrifos (CPF)
1) 0.0044 ppm (1/5th of LC50 value)
2) 0.0022 ppm (1/10th of LC50 value)
Cadmium + Chlorpyrifos 1) 18.4 ppm + 0.00088 ppm (1/5th of LC50 value)
(Cd + CPF)
2) 9.2 ppm + 0.00044 ppm (1/10th of LC50 value)
Chlorpyrifos + Cadmium 1) 0.003 ppm + 6.8 ppm (1/5th of LC50 value)
(CPF + Cd)
2) 0.0015 ppm + 3.4 ppm (1/10th of LC50 value)
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3.7.1 Physiological responses
Experiments were carried out to assess the physiological responses; behaviour of
normal and exposed fish, oxygen consumption rate, food consumption rate, ammonia
excretion rate, Oxygen:Nitrogen ratio and relative growth rate in test organisms exposed to
sublethal concentrations of test toxicants.
3.7.1.1 Behaviour of normal and exposed fish
The experiments on the behaviour of fish were carried out in glass aquarium of
50 L
capacity. The lethal (1, 2, 3 and 4 days) and sublethal concentrations (7, 14 and 21 days) of
test toxicants tabulated above were taken. Ten fish each were accommodated in 45 L of test
solution to study the behavioural pattern.
3.7.1.2 Estimation of oxygen consumption rate
The experiments on the oxygen consumption of the fish were carried out in glass
aquarium of 50 L capacity. The concentrations of test toxicants tabulated above were selected
to study the oxygen consumption rate for 21 days in static renewal system with 7 days
interval. Ten fish each were accommodated in 45 L of test solution. The water layer of the
control and test chamber was covered with a thin film of liquid paraffin, which prevents the
diffusion of atmospheric air into the test medium. The amount of dissolved oxygen in water,
for every 24 h, was estimated by Winkler method (Golterman and Clymo, 1969). The
difference in the dissolved oxygen content between initial and final water samples represents
the amount of oxygen consumed by the fish. No deaths occurred during the oxygen
consumption tests. To minimize the effect of low oxygen concentration and metabolite
accumulation on the metabolism, the experiment duration was regulated so that the oxygen
concentration by the end of experiments was above 70% of its initial concentration.
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The oxygen consumption examination was based on the method as described by
Chinni et al. (2000). In brief, oxygen consumption (QO2, mg O2/l/g/h) was calculated as
follows:
QO2 = PPM ×1/BW ×V × 1/t
Where ‘‘QO2’’ is the amount of oxygen (mg /1) consumed in the interval ‘‘t’’ (h),
‘BW’ is the body weight (g) of the fish,
‘V’ is the volume (litre) of the aquarium tank.
3.7.1.3 Estimation of food consumption rate
The food consumption rate was estimated according to Broek et al. (1997). The
experiments on the food consumption of the fish were carried out in a glass aquarium of 50 L
capacity. The concentrations of test toxicants tabulated above were selected to study the food
consumption rate for 21 days in static renewal system with 7 days interval. Ten fish each were
accommodated in 45 L of test solution. For determining the food consumption rate, fish were
fed once in 24 h with floating type dry feed pellet. After 30 min, the remaining food was
removed. It was dried overnight at 60°C and weighed to compare mean food consumption.
3.7.1.4 Estimation of ammonia excretion rate
The experiments on the ammonia excretion of the fish were carried out in a glass
aquarium of 50 L capacity. The concentrations of test compounds tabulated above were
selected to study the ammonia excretion rate for 21 days in static renewal system with 7 days
interval. The experimental conditions and methods adopted remain the same as given for
estimation of oxygen consumption rate. The amount of ammonium-nitrogen in water for every
24 h was estimated by phenol-hypochlorite method (Solarzano, 1969). Excreted ammonia, by
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the fish, was calculated as the net difference between the initial and final value of the test
period, corrected by the test chamber capacity and body weight of organisms.
3.7.1.5 Estimation of Oxygen:Nitrogen ratio
The Oxygen:Nitrogen (O:N) ratios were calculated as the ratios of atoms of oxygen
consumed to atoms of nitrogen excreted for sublethal concentrations of test toxicants exposure
in Oreochromis mossambicus. The O:N ratio in atomic equivalents estimated according to
Taboada et al. (1998) using the following equation:
O:N = (R  1.428  14)/(16  E)
Where, R is the respiratory rate in millilitres of oxygen per hour
‘E’ is the ammonia excretion in milligrams of NH3- N per hour.
3.7.1.6 Estimation of relative growth rate
The experiments on the growth of the fish were carried out in a glass aquarium of 50 L
capacity. The concentrations of test toxicants tabulated above were selected to study the
relative growth rate for 21 days in static renewal system with 7 days interval. Ten fish each
were accommodated in 45 L of test solution. The medium was exchanged at every 24 h and
the animals were fed with floating type dry feed pellet soon after refilling with aerated fresh
water. The total initial weights of the fish were estimated in each tank before the beginning of
trial and the final weights of the fish were measured after the trial ended. Fish growth was
determined by the total initial weight and the final weight of 10 fish per tank. The following
equation was applied to measure the relative growth rate (RGR):
RGR= ((mt - m0)/m0) × 100%
Where, m0 is the initial weight at the start of the trial
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mt is the final weight at the end of the trial.
3.7.2 Biochemical responses
Ten healthy Tilapia fingerlings each were stocked in 50 L capacity glass aquaria.
Calculated amount of stock solutions were added to test tank containing fresh water and mixed
thoroughly to arrive at the required concentrations. Control was run for each experiment, kept
in fresh water without the addition of test compound.
Duplicates were run for each
concentration. The medium was exchanged at every 24 h to reduce the build-up of metabolic
wastes and to keep concentrations of test compounds near the nominal level. The animals were
fed soon after refilling with aerated fresh water. The medium was then charged with the
required concentration of the toxicants and mixed well. The total period of experiment was 21
days. The specimens were sampled after 7, 14 and 21 days of exposure for biochemical
studies. The fishes were sacrificed by decapitation and the test tissues (gills, liver, kidney,
brain and muscle) were immediately dissected out, the post-mitochondrial fraction from the
pooled tissue samples were washed in ice-cold 1.15% KCl solution, blotted, weighed and
maintained at -800C.
3.7.2.1 Sample preparations
The test tissues (gills, liver, kidney, brain and muscle) were thoroughly washed with
phosphate buffer (50 mM; pH 7.4) and homogenized with homogenizing solution (50 mM
phosphate buffer pH 7.4 containing, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.15 M KCl
and 0.01% (w/v) PMSF). Homogenization was performed at 40C using motor driven
homogenizer and centrifuged at 10,000 rpm for 20 min at 40C using refrigerated centrifuge.
The supernatant was decanted and stored at -200C until biochemical analysis.
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3.7.2.3 Assay of cholinesterase enzymes
3.7.2.3.1 Assay of acetylcholinesterase (AChE)
Acetylcholinesterase activity was determined according to the method of Ellman
et
al. (1961). The reaction mixture was prepared in 100 mM of potassium phosphate buffer pH
7.4 containing acetylthiocholine iodide and 5, 5′ dithiobis-2-nitrobenzoic acid in the final
concentrations of 1 mM and 0.5 mM, respectively. 100 μl of protein supernatant was added to
start the reaction, which was followed spectrophotometrically at 412 nm and 25°C for 15 min.
AChE activity was expressed in nmol of acetylthiocholine iodide hydrolysed/min/mg protein
(extinction coefficient ε412=13,600 M−1cm−1).
3.7.2.3.2 Assay of butyrylcholinesterase (BChE)
Butyrylcholinesterase activity was determined according to the method of Ellman et al.
(1961). The reaction mixture was prepared in 100 mM of potassium phosphate buffer pH 7.4
containing butyrylthiocholine iodide and 5, 5′ dithiobis-2-nitrobenzoic acid in the final
concentrations of 1 mM and 0.5 mM, respectively. 100 μl of protein supernatant was added to
start the reaction, which was followed spectrophotometrically at 412 nm and 25°C for 15 min.
BChE activity was expressed in nmol of butyrylthiocholine iodide hydrolysed/min/mg protein
(extinction coefficient ε412=13,600 M−1cm−1).
3.7.2.4 Assay of lipid peroxidation (LPx)
Lipid peroxidation level was assayed by measuring malondialdehyde (MDA), a
decomposition product of polyunsaturated fatty acids. LPx concentration was determined by
the thiobarbituric acid (TBA) reaction as described by Ohkawa et al. (1979) with minor
modifications. Homogenates of liver, kidney, gill, brain and muscle were prepared in
phosphate buffer (50 mM; pH 7.4). The reaction mixture containing 0.5 ml of sample,
1.5 ml
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of 0.4% aqueous solution of TBA, 1.5 ml of 20% acetic acid (pH 3.5), 0.2 ml of SDS (8.1%),
0.2 ml of double distilled water and 0.1 ml of BHT (0.76%) was heated at 95ºC for 60 min
using glass balls as condensers, then cooled to room temperature and centrifuged at 2,000 rpm
for 10 min. The absorbance of the supernatant was read at 532 nm against an appropriate
blank. The amount of TBARS formed was calculated by using an extinction coefficient of
1.56×105 M-1cm-1 (Wills, 1969) and expressed as nmol of thiobarbituric acid reactive
substances (TBARS) formed/mg protein.
3.7.2.5 Assay of antioxidant enzymes
3.7.2.5.1 Superoxide dismutase (SOD)
Superoxide dismutase activity was measured according to Paoletti et al. (1990). The
assay mixture contained 50 mM phosphate buffer pH 7.4, 8.4 mM NADH, EDTA (1.4 mM)
and MnCl2 (7 mM) solution, sample (100-150 µg protein) and 28 mM β-mercaptoethanol. The
decrease in absorbance was measured at 340 nm. One unit of SOD activity was defined as 50
% inhibition of superoxide driven NADH oxidation process. The SOD activity was expressed
as units/mg protein.
3.7.2.5.2 Catalase (CAT)
Catalase activity was determined according to Aebi (1974). The method was based on
the decomposition rate of H2O2 by the enzyme. The assay mixture contained 50 mM
phosphate buffer pH 7.4, 12 mM H2O2 and 0.1 ml of sample (100-150 µg protein).
Absorbance was measured at 240 nm and CAT activity was expressed as units/mg protein
using a molar extinction coefficient of 0.0436 mM-1cm-1. One unit of catalase activity was
defined as decomposition of 1.0 µmol of H2O2 to oxygen and water per minute at pH 7.4 at
250C at a substrate conc. of 12 mM H2O2.
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3.7.2.5.3 Glutathione peroxidase (GPx)
Glutathione peroxidase activity was measured according to Paglia and Valentine
(1967). The reaction mixture contained 50 mM phosphate buffer pH 7.4, 3 mM GSH,
4.5
mM NADPH, GR (1 Unit), 7.5 mM cumene-hydroperoxide and sample (100-150 µg protein).
The absorbance was recorded at 340 nm. Enzyme activity was expressed as nmol of NADPH
oxidized/min/mg protein using a molar extinction coefficient of 6.22mM-1cm-1.
3.7.2.5.4 Glutathione-S-transferase (GST)
Glutathione-S-transferase activity was measured according to Habig et al. (1974) using
1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate. Assay mixture contained 100 mM
phosphate buffer (pH 6.9), 3 mM GSH, 1.5 mM CDNB and sample (100-150 µg protein). The
change in absorbance was recorded at 340 nm and enzyme activity was expressed as nmol of
CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 mM-1cm-1
3.7.2.5.5 Glutathione reductase (GR)
Glutathione reductase activity was measured according to the method of Massey and
Williams (1965). The assay tubes contained 50 mM phosphate buffer pH 7.4, 120 mM GSSG,
4.5 mM NADPH and 0.1 ml of samples (100-150 µg protein). The absorbance was recorded at
340 nm. Enzyme activity was expressed as nmol of NADPH oxidized/min/mg protein using a
molar extinction coefficient of 6.22 mM-1cm-1.
3.7.2.6 Assays of non-enzymatic antioxidants
Sample preparations
Tissue homogenates of gills, liver, kidney, brain and muscle were precipitated in 5%
(w/ v) TCA in an ice bath and centrifuged at 5000×g for 10 min. The deproteinised
supernatant was used for the estimation of non-enzymatic antioxidants.
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3.7.2.6.1 Total reduced glutathione (GSH)
Reduced glutathione was determined by the method of Moron et al. (1979). The
supernatant (0.1 ml) was made up to 1.0 ml with 0.2 M sodium phosphate buffer (pH 8.0), 2
ml of freshly prepared 0.6 mM DTNB solution was added and the intensity of the yellow
colour developed was measured in a spectrophotometer at 412 nm after 10 minutes. Standards
of GSH were also prepared. The values are expressed as µmol GSH/g sample.
3.7.2.6.2 Ascorbic acid (ASA)
Ascorbic acid content was determined following the stoichiometric reduction of
phosphomolybdate by ascorbic acid (Mitusi and Ohata, 1961). The reaction mixture contained
2 % sodium molybdate, 0.15 N H2SO4, 1.5 mM Na2HPO4 and 0.1 ml of sample. The mixture
was boiled at 900C for 45 min and then centrifuged at 1,000×g for 15 min. The absorbance of
the supernatant was measured at 660 nm. ASA was taken as standard and results were
expressed as µg ASA/ g tissues.
3.7.2.7 Estimation of total protein
Protein concentrations were measured by the method of Lowry et al. (1951) using
bovine serum albumin as standard. Samples were treated with folin-phenol reagent and the
absorbance was measured at 750 nm. Protein concentration was expressed as mg protein/100 g
wet weight of the tissue.
3.8 Statistical analysis
The physiological and biochemical data were subjected to statistical analysis
employing ANOVA to compare variables among controls and treatments, and Tukey's
studentized range (HSD) test at P<0.05 to determine which individual groups were
86
significantly different from control. For all type of analyses SAS 9.1 was utilized. All the data
are expressed as mean ± standard deviation (SD) of a group of 10 fishes.
87
IV. RESULTS
During the present investigation, experiments were conducted to gather data on lethal
toxicity of Cadmium and Chlorpyrifos individually and in combination. Further, sublethal
aspects like physiological and biochemical responses of the fish were also studied. The results
obtained are presented in different sections.
4.1 Individual lethal toxicity (LC50)
4.1.1 Lethal toxicity of Cadmium
The cumulative percentage mortality of Tilapia (Oreochromis mossambicus) exposed
to Cadmium at 96 h is presented in Table 2 and Fig. 2. The mortality of fish increased with
increase in the concentration of Cadmium, depicting a direct correlation between mortality and
concentration. Probit analysis revealed that, the LC50 value of Cadmium for O. mossambicus
as 168.90 ppm (95% confidence limit) within 96 h. The mortality of fish started at low
concentration (160 ppm) and there was 100% mortality at 184 ppm within 96 h.
4.1.2 Lethal toxicity of Chlorpyrifos
Table 3 represents the cumulative percentage mortality of Tilapia (Oreochromis
mossambicus) exposed to different concentrations of Chlorpyrifos. In general, Chlorpyrifos
did not bring in conspicuous mortality of the fish till the concentration reached up to 0.015
ppm and all fishes died (100% mortality) within 96 h at 0.030 ppm. The results reveal that, the
Chlorpyrifos can be rated as highly toxic to fish with LC50 value registered at 0.022 ppm (95%
confidence limit) (Fig. 3).
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4.2 Joint lethal toxicity (LC50)
4.2.1 Lethal toxicity of Cadmium + Chlorpyrifos (Cd + CPF)
In this combined bioassay experiment, the Chlorpyrifos concentration was kept
constant (i.e 1/5th LC50 value, 0.0044 ppm) while the Cadmium concentrations varied and the
details are depicted in Table 4 and Fig. 4. The combination of Cadmium + Chlorprifos proved
lethal at 92.04 ppm (95% confidence limit) within 96 h. A synergistic effect was found in this
combined toxicity (with S. R. ratio 1.84) which resulted in early death of O. mossambicus than
those obtained for individual toxicants (Table 7). The progress of death of fish started at 80
ppm (16%) and there was 100% mortality at 110 ppm within 96 h.
4.2.2 Lethal toxicity of Chlorpyrifos + Cadmium (CPF + Cd)
Table 5 and Fig. 5 provides the details of toxicants used, log curve and trend of
mortality of fish employed for lethal toxicity study of Chlorpyrifos + Cadmium. Fixed
concentration of Cadmium (i.e. 1/5th LC50 value, 34 ppm) and varying concentrations of
Chlorpyrifos were used to study the combined toxicity of these two pollutants. The LC50 value
of Chlorpyrifos + Cadmium to O. mossambicus was found to be 0.016 ppm (95% confidence
limit) within 96 h. Here also, the joint action toxicity was found to be more than additive or
synergistic (with S. R. ratio 1.37) in causing death of O. mossambicus (Table 7). The fish
mortality initiated at concentration as low as 0.01 ppm and 100% mortality recorded at 0.028
ppm within 96 h. It is evident from the results of lethal toxicity that the mortality of fish
increased with increase in the concentration of the toxicants, depicting a direct correlation
between mortality and the concentration.
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4.2.3 Categorization of toxicants based on toxic units
In order to categorize the toxicants according to the results from the toxicity tests, the
values of LC50 were converted into toxic units (TU). The results of toxic units revealed that,
Chlorpyrifos + Cadmium (TU- 6250) and Chlorpyrifos (TU – 4545.45) are highly toxic,
Cadmium + Chlorpyrifos (TU - 1.08) is toxic and Cadmium (TU – 0.58) is less toxic (Table
6).
4.3 Sublethal toxicity
4.3.1 Physiological responses
The physiological responses of Tilapia were studied in relation to exposure of fish to
sublethal concentrations, 1/5th LC50 and 1/10th LC50, Cadmium, Chlorpyrifos, combination of
varying concentrations of Cadmium + constant concentration of Chlorpyrifos and varying
concentrations of Chlorpyrifos + constant concentration of Cadmium for a period of 7, 14 and
21 days.
4.3.1.1 Behaviour of fish under normal and exposed conditions
Experiments were done to assess the behaviour of Tilapia without any toxicant and
with lethal and sublethal concentrations of Cadmium and Chlorpyrifos. In the present study,
the control fish behaved in a natural manner, i.e. fish were actively feeding and were alert to
the slightest disturbance with their well synchronized movements (Plate 5). The behaviour did
not vary significantly between the control groups. Therefore, the results of these non exposure
series were taken as standards for the whole test period.
In Cadmium media, Tilapia showed disrupted shoaling behaviour, localization at the
bottom of the test chamber, and independency (spreading out) in swimming (Plate 6). The
above symptoms followed the loss of coordination among individuals and occupancy of twice
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the area of the control group were the early symptoms of Cadmium exposure in both the
sublethal and lethal concentrations. Subsequently, fish moved to the corners of the test
chambers, which can be viewed as avoidance behaviour of the fish to the Cadmium. A thick
film of mucous on the whole body and gills was observed in almost all test fishes. At the start
of the experiment, the fish exposed to the Cd became alert; however, with the progression of
the experiment, they stopped swimming and remained in static position in response to the
sudden changes in the surrounding environment.
On the other hand, when exposed to Chlorpyrifos, the shoal was observed as disturbed.
Initially fishes were surfaced, followed by vigorous and erratic swimming showing agitation
(Plate 7). Quick opercular and fin movements exhibited by the fish initially became gradually
feeble and often showed gulping of air. Excess secretion of mucus was a prominent
observation. Opercular opening became wider and exhibited respiratory distress. As the period
of exposure advanced, the fishes settled down to bottom and towards the final phase of
exposure, they showed barrel-rolling indicating loss of equilibrium. Swimming with belly
upwards and gradually became lethargic.
During the initial phase of exposure, fishes
responded vigorously to mechanical stimulation but later failed to respond. Fishes were
hanging vertically in water and often hitting to the walls of the test tank, finally sunk to the
bottom just before death.
Some of the behavioural changes recorded when the fish were exposed to a
combination of Chlorpyrifos + Cadmium include opercular movement, dullness, loss of
equilibrium, stop of food intake, erratic and hysteric swimming, swimming at the water
surface, circling movement, and gasping. Prior to death, the fish became less active or
91
generally inactive, remained hanging vertically in the water or lay down on their sides at
higher concentrations.
In the combination of Cadmium + Chlorpyrifos, Tilapia exhibited disrupted normal
behaviour, sluggish, decreased rate of opercular movement, copious mucous secretion and
localization to the bottom of the test chamber. Few of these behavioural activities were also
seen in fishes exposed to Cadmium. The behaviour of fish, during experimentation was
directly influenced by both duration of exposure and concentration of the toxicant. The
behavioural changes were severe in fish exposed to lethal concentrations than that of the
sublethal concentrations of Cadmium and Chlorpyrifos.
4.3.1.2 Oxygen consumption rate
The trend in oxygen consumption in Tilapia exposed to Cadmium and Chlorpyrifos
individually and to combination of Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium are
provided in Table 8. Tilapia showed oxygen consumption rate (mg O2 consumption/l/g/h)
between 0.120 ± 0.0005 on 7th day, 0.104 ± 0.0010 on 14th day and 0.080 ± 0.0012 on 21st day
in 1/5th LC50 of Cadmium and 0.148 ± 0.0015 on 7th day, 0.135 ± 0.0012 on 14th day and 0.112
± 0.0005 on 21st day in 1/10th LC50 of Cadmium. On the other hand, exposure to Chlorpyrifos,
the fish registered different trend and it was between 0.195 ± 0.0003 (7th day), 0.182 ± 0.0010
(14th day) and 0.170 ± 0.0006 (21st day) at 1/5th LC50 concentration and 0.198 ± 0.0009 (7th
day), 0.194 ± 0.0013 (14th day) and 0.185 ± 0.0011 (21st day) at 1/10th LC50 concentration.
However, the oxygen consumption pattern was between 0.134 ± 0.0013 (7th day), 0.115 ±
0.0009 (14th day) and 0.096 ± 0.0007 (21st day) under 1/5th LC50 of Cadmium + Chlorpyrifos
and 0.161 ± 0.0001 (7th day), 0.147 ± 0.0013 (14th day) and 0.120 ± 0.0010 (21st day) under
1/10th LC50 of Cadmium + Chlorpyrifos. But exposure to 1/5th LC50 of Chlorpyrifos +
92
Cadmium (0.168 ± 0.0008 on 7th day, 0.149 ± 0.0007 on 14th day and 0.126 ± 0.0013 on 21st
day) and 1/10th LC50 of Chlorpyrifos + Cadmium (0.170 ± 0.0006 on 7th day, 0.151 ± 0.0003
on 14th day and 0.136 ± 0.0006 on 21st day), fish depicted altogether different trend in oxygen
consumption rate when compared to control group (between 0.203 ± 0.0015 on 7th day, 0.232
± 0.0011 on 14th day and 0.281 ± 0.0016 on 21st day) during 21 days of exposure. The
combined toxicity of Cadmium + Chlorpyrifos was simple additive in nature compared to
individual toxicity of Cadmium while Chlorpyrifos + Cadmium was highly synergistic in
nature compared to individual toxicity of Chlorpyrifos (Table 8).
Reduction in oxygen consumption in relation to control was observed in fish exposed
to 1/5th LC50 and 1/10th LC50 of Cadmium (71.28% and 60.14%), Cadmium + Chlorpyrifos
(65.82% and 57.28%), Chlorpyrifos + Cadmium (55.06% and 51.28%) and Chlorpyrifos
(39.18% and 33.95%) at 21 days (Fig. 7). It is clearly evident from the results that individual
and combination of Cadmium and Chlorpyrifos affect the oxygen consumption rate of Tilapia
under sublethal concentrations.
In experimental groups, there was decrease in oxygen consumption with increase
duration of exposure and lowest oxygen consumption rate was on 21st day. While in control, it
increased with increase in duration of exposure and highest oxygen consumption rate reached
on 21st day (Fig. 6). However, comparison between test concentrations and duration of
exposure on oxygen consumption of Tilapia, highest oxygen consumption rate attained was
during 7th day and 14th day in 1/5th LC50 and 1/10th LC50 of Chlorpyrifos. Further, it reached to
minimum during 21st day in both the sublethal concentrations of Cadmium followed by
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium. One-way ANOVA followed by
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Tukey's studentized range (HSD) test revealed that there was significant (P<0.05) impact of
test toxicants on oxygen consumption rate (Table 9a and 9b).
4.3.1.3 Food consumption rate
The food consumption rate of Tilapia was determined using floating feed for a period
of 7, 14 and 21 days. The Table 10 represents the trend in food consumption in Tilapia
exposed to Cadmium and Chlorpyrifos individually and to combination of Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium. Tilapia exhibited food consumption rate (g feed
consumption/g body wt.) between 0.0040 ± 0.0003 on 7th day, 0.0035 ± 0.0002 on 14th day
and 0.0021 ± 0.0003 on 21st day in 1/5th LC50 of Cadmium and 0.0057 ± 0.0002 on 7th day,
0.0052 ± 0.0001on 14th day and 0.0036 ± 0.0002 on 21st day in 1/10th LC50 of Cadmium. But
exposure to Chlorpyrifos, it was between 0.0106 ± 0.0004 (7th day), 0.0099 ± 0.0005 (14th
day) and 0.0095 ± 0.0002 (21st day) at 1/5th LC50 concentration and 0.0129 ± 0.0005 (7th day),
0.0119 ± 0.0009 (14th day) and 0.0111 ± 0.0006 (21st day) at 1/10th LC50 concentration. On the
other hand, exposure to 1/5th LC50 of Cadmium + Chlorpyrifos (0.0045 ± 0.0004 on 7th day,
0.0040 ± 0.0003 on 14th day and 0.0029 ± 0.0001 on 21st day) and 1/10th LC50 of Cadmium +
Chlorpyrifos (0.0062 ± 0.0005 on 7th day, 0.0055 ± 0.0004 on 14th day and 0.0041 ± 0.0001
on 21st day), the fish registered different trend in oxygen consumption rate. However, the food
consumption pattern was between 0.0083 ± 0.0003 (7th day), 0.0078 ± 0.0002 (14th day) and
0.0064 ± 0.0004 (21st day) in 1/5th LC50 of Chlorpyrifos + Cadmium and 0.0087 ± 0.0002 (7th
day), 0.0080 ± 0.0001 (14th day) and 0.0077 ± 0.0002 (21st day) in 1/10th LC50 of Chlorpyrifos
+ Cadmium when compared to control group (between 0.0143 ± 0.0012 on 7th day, 0.0185 ±
0.0019 on 14th day and 0.0242 ± 0.0016 on 21st day) during 21 days of exposure. The joint
toxicity of Cadmium + Chlorpyrifos was simple additive in nature compared to individual
94
toxicity of Cadmium while Chlorpyrifos + Cadmium was synergistic in nature compared to
individual toxicity of Chlorpyrifos (Table 10).
The food consumption rate in Tilapia was significantly (P<0.05) affected by the test
concentrations (Table 11a and 11b). Fish exposed to sublethal concentrations of test toxicants
showed a sharp decline in food consumption rate during the entire duration compared to the
control (Fig. 8). Reduction in food consumption in relation to control was observed in those
fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (91.30% and 84.91%), Cadmium +
Chlorpyrifos (87.84% and 82.80%), Chlorpyrifos + Cadmium (73.28% and 68.08%) and
Chlorpyrifos (60.78% and 54.10%) at 21 days (Fig. 9).
In general, the food consumption rate was minimum in fish exposed to 1/5th of LC50
than that of 1/10th of LC50 of test toxicants. Similarly, the interaction between test
concentrations and duration of exposure reveal that highest food consumption rate was
attained during 7th day and 14th day at 1/5th LC50 and 1/10th LC50 of Chlorpyrifos. It reached to
minimum during 21st day in both the sublethal concentrations of Cadmium followed by
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium. Thus Cadmium and Chlorpyrifos had
a significant effect on functional activity of Tilapia since an increase in test concentration
induced a decrease in food consumption rate in sublethal concentrations with increased
exposure duration.
4.3.1.4 Ammonia-N excretion rate
The rate of ammonia-N excretion in Tilapia exposed to Cadmium and Chlorpyrifos
individually and to combination of Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium are
documented in Table 12. The ammonia-N excretion rate was decreased sharply in Tilapia
exposed to test concentrations when compared to control. Tilapia showed ammonia-N
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excretion rate (µg-at NH3-N excretion/l/g/h) between 0.0061 ± 0.0004 on 7th day, 0.0052 ±
0.0002 on 14th day and 0.0039 ± 0.0004 on 21st day on exposure to 1/5th LC50 and 0.0080 ±
0.0005 on 7th day, 0.0070 ± 0.0002 on 14th day and 0.0055 ± 0.0007 on 21st day on exposure
to 1/10th LC50 of Cadmium. On the other hand, the ammonia-N excretion pattern was between
0.0136 ± 0.0009 (7th day), 0.0127 ± 0.0001 (14th day) and 0.0109 ± 0.0006 (21st day) under
1/5th LC50 of Chlorpyrifos and 0.0149 ± 0.0005 (7th day), 0.0138 ± 0.0003 (14th day) and
0.0125 ± 0.0007 (21st day) under 1/10th LC50 of Chlorpyrifos. However, exposure to Cadmium
+ Chlorpyrifos, the fish registered different trend and it was between 0.0073 ± 0.0003 (7th
day), 0.0061 ± 0.0004 (14th day) and 0.0050 ± 0.0001 (21st day) at 1/5th LC50 concentration
and 0.0095 ± 0.0008 (7th day), 0.0082 ± 0.0008 (14th day) and 0.0065 ± 0.0005 (21st day) at
1/10th LC50 concentration. But exposure to Chlorpyrifos + Cadmium, ammonia-N excretion
was between 0.0094 ± 0.0004 (7th day), 0.0080 ± 0.0002 (14th day) and 0.0066 ± 0.0007 (21st
day) in 1/5th LC50 concentration and 0.0106 ± 0.0009 (7th day), 0.0090 ± 0.0007 (14th day) and
0.0077 ± 0.0004 (21st day) in 1/10th LC50 concentration when compared to control group
(0.0149 ± 0.0007 on 7th day, 0.0181 ± 0.0005 on 14th day and 0.0235 ± 0.0009 on 21st day)
during 21 days of exposure. The combined toxicity of Cadmium + Chlorpyrifos was simple
additive in nature compared to individual toxicity of Cadmium while Chlorpyrifos + Cadmium
was highly synergistic in nature compared to individual toxicity of Chlorpyrifos (Table 12).
In sublethal concentrations, the ammonia-N excretion reduced with increase in
duration of exposure. Lowest ammonia-N excretion rate reached in 21st day (Fig. 10).
Similarly, the interaction between test concentrations and duration of exposure on ammonia-N
excretion in Tilapia show that highest ammonia-N excretion rate was attained during 7th day
and 14th day in 1/5th LC50 and 1/10th LC50 of Chlorpyrifos. It reached to minimum during 21st
96
day in both the sublethal concentrations of Cadmium followed by Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium.
Depletion in ammonia-N excretion in relation to control was observed in fish exposed
to 1/5th LC50 and 1/10th LC50 of Cadmium (83.25% and 76.23%), Cadmium + Chlorpyrifos
(78.49% and 72.28%), Chlorpyrifos + Cadmium (71.60% and 67.13%) and Chlorpyrifos
(53.57% and 46.47%) at 21 days (Fig. 11). One-way ANOVA followed by Tukey's
studentized range (HSD) test showed that there was significant (P<0.05) impact of test
toxicants on ammonia-N excretion rate (Table 13a and 13b).
4.3.1.5 Oxygen:Nitrogen ratio
The Table 14 depicts the trend in Oxygen:Nitrogen ratio in Tilapia exposed to
Cadmium and Chlorpyrifos individually and to combination of Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium. One-way ANOVA followed by Tukey's studentized range (HSD)
test showed that there was significant (P<0.05) difference in O:N ratio in all the treated
groups compared to control (Table 15a and 15b). Fish exposed to sublethal concentrations of
test compounds showed a gradual increase in O:N ratio in all the exposed duration, when
compared to the control. Tilapia showed O:N ratio between 24.53 ± 0.11 on 7th day, 24.96 ±
0.21 on 14th day and 25.60 ± 0.25 on 21st day in 1/5th LC50 of Cadmium and 23.02 ± 0.18 on
7th day, 24.08 ± 0.13 on 14th day and 25.04 ± 0.20 on 21st day in 1/10th LC50 of Cadmium. But
exposure to 1/5th LC50 of Chlorpyrifos (17.80 ± 0.19 on 7th day, 17.89 ± 0.16 on 14th day and
19.56 ± 0.08 on 21st day) and 1/10th LC50 of Chlorpyrifos (17.18 ± 0.11 on 7th day, 17.55 ±
0.19 on 14th day and 18.42 ± 0.21 on 21st day), fish depicted altogether different trend in O:N
ratio. On the other hand, exposure to Cadmium + Chlorpyrifos, it was between 22.66 ± 0.23
(7th day), 23.25 ± 0.18 (14th day) and 23.72 ± 0.12 (21st day) at 1/5th LC50 concentration and
97
21.13 ± 0.06 (7th day), 22.36 ± 0.12 (14th day) and 23.01 ± 0.19 (21st day) at 1/10th LC50
concentration. However, the O:N ratio pattern was between 22.16 ± 0.15 (7th day), 23.09 ±
0.22 (14th day) and 23.63 ± 0.21 (21st day) under 1/5th LC50 of Chlorpyrifos + Cadmium and
20.03 ± 0.14 (7th day), 21.05 ± 0.17 (14th day) and 22.13 ± 0.13 (21st day) under 1/10th LC50 of
Chlorpyrifos + Cadmium when compared to control (16.98 ± 0.15 on 7th day, 15.99 ± 0.06 on
14th day and 14.93 ± 0.17 on 21st day) during 21 days of exposure. The joint action toxicity of
Cadmium + Chlorpyrifos was simple additive in nature compared to individual toxicity of
Cadmium while Chlorpyrifos + Cadmium was synergistic in nature compared to individual
toxicity of Chlorpyrifos (Table 14).
In sublethal concentrations, the O:N ratio increased with increase in duration of
exposure. Highest O:N ratio was attained during 21st day in both the sublethal concentrations
of Cadmium followed by Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium and
Chlorpyrifos, whereas in control, it was decreased with increase in duration and lowest O:N
ratio was observed in 21st day (Fig. 12).
Elevation in O:N ratio in relation to control was observed in fish exposed to 1/5th LC50
and 1/10th LC50 of Cadmium (71.48% and 67.74%), Cadmium + Chlorpyrifos (58.93% and
54.13%), Chlorpyrifos + Cadmium (58.27% and 48.26%) and Chlorpyrifos (31.02% and
23.42%) at 21 days (Fig. 13). It is clearly evident from the results that individual and
combination of Cadmium and Chlorpyrifos affected the O:N ratio of Tilapia under sublethal
concentrations.
4.3.1.6 Relative growth rate
The growth rate of Tilapia was investigated in relation to sublethal concentrations of
individual and combination of Cadmium and Chlorpyrifos for a period of 7, 14 and 21 days.
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Tilapia exhibited relative growth rate (%) between -6.51 ± 0.40 on 7th day, -12.90 ± 0.86 on
14th day and -19.86 ± 0.95 on 21st day in 1/5th LC50 of Cadmium and -4.26 ± 0.33 on 7th day, 7.69 ± 0.53 on 14th day and -14.53 ± 0.70 on 21st day in 1/10th LC50 of Cadmium (Table 16).
On the other hand, exposure to Chlorpyrifos (-0.57 ± 0.02 (7th day), -1.08 ± 0.08 (14th day)
and -5.00 ± 0.28 (21st day) at 1/5th LC50 concentration and -0.45 ± 0.03 (7th day), -0.86 ± 0.05
(14th day) and -2.20 ± 0.13 (21st day) at 1/10th LC50 concentration), the fish registered different
pattern in growth rate. However, the growth rate trend was between -4.30 ± 0.38 (7th day), 8.75 ± 0.70 (14th day) and -16.45 ± 0.82 (21st day) on exposure to 1/5th LC50 of Cadmium +
Chlorpyrifos and -2.53 ± 0.18 (7th day), -6.00 ± 0.63 (14th day) and -12.03 ± 0.96 (21st day) on
exposure to 1/10th LC50 of Cadmium + Chlorpyrifos. But exposure to Chlorpyrifos +
Cadmium, it was between -1.97 ± 0.06 (7th day), -6.04 ± 0.59 (14th day) and -11.84 ± 0.84
(21st day) at 1/5th LC50 concentration and
-1.92 ± 0.08 (7th day), -4.26 ± 0.48 (14th day) and -
9.93 ± 0.60 (21st day) at 1/10th LC50 concentration when compared to control group (1.53±
0.07 on 7th day, 4.75 ± 0.21 on 14th day and 9.19 ± 0.58 on 21st day) during 21 days of
exposure. The combined toxicity of Cadmium + Chlorpyrifos was moderately antagonistic in
nature compared to individual toxicity of Cadmium while Chlorpyrifos + Cadmium was
moderately synergistic in nature compared to individual toxicity of Chlorpyrifos (Table 16).
Statistical analysis indicated significant (P<0.05) variation in growth rate in all the
treated groups compared to control (Table 17a and 17b). Fish exposed to sublethal
concentrations of test toxicants showed drastic reduction in growth rate in all the exposed
duration, when compared to the control (Fig. 14). The rowth rate was minimum in fish
exposed to 1/5th of LC50 than that of the 1/10th of LC50 of test compounds. Reduction in growth
rate in relation to control was observed in fish exposed to 1/5th LC50 and 1/10th LC50 of
99
Cadmium (291.62% and 270.39%), Cadmium + Chlorpyrifos (283.80% and 216.76%),
Chlorpyrifos + Cadmium (220.11% and 202.79%) and Chlorpyrifos (127.93% and 112.29%)
at 21 days (Fig. 15). Thus, Cadmium and Chlorpyrifos had a significant effect on the
functional activity of Tilapia since an increase in test concentration induces a decrease in
growth rate in sublethal concentrations with increase in duration of exposure.
4.3.2 Biochemical responses
In this study, examination of biochemical markers of cholinesterase enzymes, lipid
peroxidation, antioxidant enzymes and non-enzymatic antioxidants in the gills, liver, kidney,
brain and muscle of freshwater fish Oreochromis mossambicus was carried out. The
biochemical markers were studied in relation to sublethal concentrations of 1/5th LC50 and
1/10th LC50 of Cadmium, Chlorpyrifos, combination of varying concentrations of Cadmium +
constant concentration of Chlorpyrifos and varying concentrations of Chlorpyrifos + constant
concentration of Cadmium for a period of 7, 14 and 21 days.
4.3.2.1 Cholinesterase enzymes
4.3.2.1.1 Acetylcholinesterase (AChE)
An experiment was undertaken to study the effect of sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium on the
functioning of acetylcholinesterase activity (nmol of ASChI hydrolysed/min/mg protein) in
the gills, liver, kidney, brain and muscle in Tilapia fish (Oreochromis mossambicus) during 21
days of exposure. The details are provided in the Tables 18, 19, 20, 21 and 22, respectively.
Gills
The gills of control fish recorded acetylcholinesterase (AChE) activity between 137.49
± 1.45 on 7th day, 139.92 ± 1.10 on 14th day and 140.53 ± 1.39 on 21st day. This activity in the
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gills, was significantly decreased to 94.55 ± 1.72 (7th day), 76.68 ± 1.83 (14th day) and 73.41 ±
0.78 (21st day) when exposed to 1/5th LC50 of Cadmium and 111.43 ± 1.98 (7th day), 97.98 ±
1.32 (14th day) and 89.08 ± 3.87 (21st day) at 1/10th of LC50 of Cadmium during the same
exposure period. However, there was progressive decline in the AChE activity in the gills
(108.76 ± 2.90 on 7th day, 92.69 ± 1.86 on 14th day and 86.26 ± 1.38 on 21st day) of those fish
exposed to 1/5th LC50 of Chlorpyrifos. Similar trend was observed when gills of those fish
exposed to 1/10th LC50 of Chlorpyrifos (123.54 ± 2.46 on 7th day, 112.08 ± 2.18 on 14th day
and 103.25 ± 1.53 on 21st day). On the contrary, fish treated with a combination of Cadmium
+ Chlorpyrifos although registered considerable inhibition in the AChE activity (Table 18), the
joint action toxicity of Cadmium + Chlorpyrifos was moderately antagonistic in nature
compared to individual toxicity of Cadmium. On the other hand, reduced AChE activity in the
gills was noticed when fish was exposed to Chlorpyrifos + Cadmium. Here, contrary to
combined toxicity of Cadmium + Chlorpyrifos, the joint action toxicity of Chlorpyrifos +
Cadmium was moderately synergistic in nature compared to individual toxicity of
Chlorpyrifos (Table 18).
Highest inhibitory activity in the acetylcholinesterase, in relation to control, was
observed in the gills of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (47.76%
and 36.61%) followed by Cadmium + Chlorpyrifos (43.81% and 32.50%), Chlorpyrifos +
Cadmium (41.12% and 30.21%) and least was with Chlorpyrifos (38.62% and 26.53%) during
21 days of experiments (Fig. 17).
Liver
The variation of acetylcholinesterase activity (AChE), in the liver tissue of the fish, in
the control group was between 91.16 ± 0.48 on 7th day, 92.87 ± 1.23 on 14th day and 89.23 ±
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0.79 on 21st day. This AChE activity got decreased, in the liver, to 78.87 ± 2.14 on 7th day,
63.84 ± 1.36 on 14th day and 56.65 ± 1.54 on 21st day in fish treated with 1/5th LC50 of
Cadmium and 84.90 ± 1.58 (7th day), 74.09 ± 1.83 (14th day) and 65.06 ± 2.60 (21st day) at
1/10th LC50 of Cadmium. Similarly, fish treated with 1/5th LC50 of Chlorpyrifos recorded
substantially reduced AChE activity in the liver and the values were 81.08 ± 0.63 (7th day),
68.33 ± 2.84 (14th day) and 59.97 ± 1.91 (21st day). The same trend was noticed in the liver of
those fish exposed to 1/10th LC50 of Chlorpyrifos i.e. AChE activity reduced to 86.39 ± 1.11
(7th day), 77.44 ± 2.39 (14th day) and 68.65 ± 2.10 (21st day). Depleted trend in AChE activity
was continued in the liver of those fish exposed to a combination of Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium (Table 19) and the AChE activity showed significant depletion
at 1/5th LC50 of Chlorpyrifos + Cadmium (73.47 ± 1.73 on 7th day, 58.02 ± 1.87 on 14th day
and 52.68 ± 1.20 on 21st day) compared to control group. The combined toxicity of both the
combination was moderately synergistic in nature. It means to say that the combination of
Cadmium + Chlorpyrifos and or Chlorpyrifos + Cadmium are more toxic than individual
toxicants and act similar in effecting AChE activity in the liver of Tilapia, at the above
concentration during 3 weeks of exposure.
A comparison of intensity of toxicity among toxicants reveal that combination of
Chlorpyrifos + Cadmium (40.96% and 30.39%) was more toxic followed by Cadmium +
Chlorpyrifos (38.62% and 35.68%), Cadmium (36.51% and 27.09%) and Chlorpyrifos
(32.79% and 23.06%) at their 1/5th LC50 and 1/10th LC50 concentrations during 21 days in
bringing down the acetylcholinesterase activity in the liver of fish exposed (Fig. 19).
However, there was moderate deviation in the activity in those fish exposed to 1/10th LC50 of
Chlorpyrifos + Cadmium.
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Kidney
The values of acetylcholinesterase (AChE) activity in the kidney of control fish were
58.81 ± 0.47 on 7th day, 57.02 ± 1.21 on 14th day and 55.95 ± 0.83 on 21st day. However,
exposure of Tilapia fish to 1/5th LC50 of Cadmium caused depletion in the AChE activity in
the kidney and the values were 49.53 ± 0.84 (7th day), 46.42 ± 0.16 (14th day) and 44.24 ± 0.77
(21st day). In the same way, when fish exposed to 1/10th of LC50 of Cadmium, the AChE
activity in the kidney depleted to 52.96 ± 1.30 (7th day), 49.44 ± 1.66 (14th day) and 46.82 ±
0.62 (21st day) compared to control fish. Similarly, reduction of AChE activity in the kidney
of those fish exposed to 1/5th LC50 of Chlorpyrifos was noticed and the values were 50.89 ±
0.28 on 7th day, 48.04 ± 0.79 on 14th day and 45.91 ± 1.03 on 21st day. This reduction trend in
AChE activity in the kidney (53.72 ± 0.54 on 7th day, 50.31 ± 0.64 on 14th day and 47.58 ±
0.41 on 21st day) continued when fish was exposed to 1/10th LC50 of Chlorpyrifos. Further,
inhibition of AChE activity in the kidney was evident in fish exposed to a combination of
Cadmium + Chlorpyrifos and the joint action toxicity at 1/5th LC50 of Cadmium +
Chlorpyrifos was simple additive while 1/10th LC50 of Cadmium + Chlorpyrifos was
moderately synergistic in nature compared to individual toxicity of Cadmium. On the other
hand, fish treated with a combination of Chlorpyrifos + Cadmium (47.04 ± 1.17 on 7th day,
44.80 ± 0.48 on 14th day and 42.33 ± 1.36 on 21st day at 1/5th LC50 concentration) registered
significant decline in AChE activity in the kidney compared to control group, run parallely
with the all the treated group, for 21 days (Table 20). Here, contrary to joint action toxicity of
Cadmium + Chlorpyrifos, the joint action toxicity at 1/5th LC50 of Chlorpyrifos + Cadmium
was moderately synergistic while 1/10th LC50 of Cadmium + Chlorpyrifos was simple additive
in nature compared to individual toxicity of Chlorpyrifos.
103
The AChE activity in the kidney of those fish exposed to 1/5th LC50 and 1/10th LC50 of
Chlorpyrifos + Cadmium (24.34% and 17.59%) was more affected followed by Cadmium +
Chlorpyrifos (22.16% and 19.52%), Cadmium (20.93% and 16.32%) and Chlorpyrifos
(17.94% and 14.96%) during 21 days of exposure (Fig. 21). However, there was slight
deviation in the activity in those fish exposed to 1/10th LC50 of Chlorpyrifos + Cadmium.
Brain
The brain tissue of fish in the control group showed acetylcholinesterase (AChE)
activity between 295.22 ± 4.76 on 7th day, 281.74 ± 1.98 on 14th day and 284.38 ± 2.67 on 21st
day. On the other hand, the AChE activity in the brain tissue decreased drastically to 186.53 ±
2.48 (7th day), 142.68 ± 1.94 (14th day) and 93.56 ± 2.19 (21st day) in those fish treated with
1/5th LC50 of Cadmium. Similar observations were made in the brain of those fish exposed to
1/10th of LC50 of Cadmium (227.06 ± 4.09 on 7th day, 197.92 ± 1.86 on 14th day and 156.79 ±
2.47 on 21st day). Similarly, in fish exposed to 1/5th LC50 of Chlorpyrifos, the AChE activity in
the brain was reduced significantly and the values were 128.60 ± 2.08 on 7th day, 76.07 ± 1.92
on 14th day and 37.94 ± 1.03 on 21st day. On the other hand, in fish exposed to 1/10th LC50 of
Chlorpyrifos, the AChE activity in the brain was also reduced significantly to 212.46 ± 3.89
on 7th day, 174.89 ± 2.67 on 14th day and 138.01 ± 4.83 on 21st day. Highly significant
reduced activity of AChE, in the brain tissue was noticed in those fish exposed to Chlorpyrifos
and this indicates that the pesticide is more toxic to the brain than heavy metal. However,
exposure of fish to a combination of Cadmium + Chlorpyrifos during the same period caused
much more damage to the AChE activity in the brain tissue. It means this combination was
highly synergistic in nature compared to individual toxicity of Cadmium. Further, exposure of
fish to a combination of 1/5th LC50 of Chlorpyrifos + Cadmium although considerably
104
depleted the AChE activity of the brain but it was less than that of individual toxicity of 1/5 th
LC50 of Chlorpyrifos (Table 21). Hence the joint action toxicity at 1/5th LC50 of Chlorpyrifos +
Cadmium was moderately antagonistic in nature. On the other hand, those fish exposed to a
combination of 1/10th LC50 of Chlorpyrifos + Cadmium recorded considerable depletion in the
AChE activity, in the brain, under same duration of exposure (Table 21). In case of 1/10th
LC50 of Chlorpyrifos + Cadmium, the joint action toxicity was highly synergistic in nature
compared to individual toxicity of 1/10th LC50 Chlorpyrifos.
Comparison between control and treatment reveals that exposure of fish to 1/5th LC50
and 1/10th LC50 of Chlorpyrifos (86.66% and 51.47%) was more negatively affected followed
by Chlorpyrifos + Cadmium (81.25% and 68.91%), Cadmium + Chlorpyrifos (73.95% and
62.26%) and Cadmium (67.10% and 44.87%) for 21 days in bringing about decreased
acetylcholinesterase activity in the brain tissue (Fig. 23). However, there was moderate
deviation in the activity in those fish exposed to 1/10th LC50 of Chlorpyrifos only.
Muscle
The acetylcholinesterase (AChE) activity in the muscle tissue of control fish ranged
between 188.28 ± 2.23 on 7th day, 194.71 ± 1.28 on 14th day and 196.53 ± 1.51 on 21st day.
This activity in the muscle progressively decreased to 145.91 ± 2.93 (7th day), 124.54 ± 2.60
(14th day) and 115.96 ± 2.69 (21st day) on exposure to 1/5th LC50 of Cadmium. This decreasing
trend was evident even at 1/10th LC50 of Cadmium and the values were 160.07 ± 2.76 (7th
day), 142.37 ± 1.35 (14th day) and 131.03 ± 1.27 (21st day). Similarly, exposure of fish to 1/5th
LC50 of Chlorpyrifos caused inhibition in the AChE activity in the muscle of Tilapia (130.41 ±
1.82 on 7th day, 106.29 ± 2.05 on 14th day and 95.24 ± 1.58 on 21st day). In the same way,
those fish exposed to 1/10th LC50 concentration, the AChE values were decreased to 167.08
105
±1.80 on 7th day, 148.26 ± 2.48 on 14th day and 137.90 ± 1.92 on 21st day. Further, when fish
was exposed to a combination of Cadmium + Chlorpyrifos and, Chlorpyrifos + Cadmium, it
registered significant depletion of AChE activity in the muscle during 21 days of exposure
(Table 22). Here, the AChE activity showed highly significant variation at 1/5th LC50 of
Cadmium + Chlorpyrifos (114.53 ± 2.01 on 7th day, 84.25 ± 1.50 on 14th day and 65.08 ± 1.39
on 21st day) compared to control group. The combined toxicity at 1/5th LC50 of Chlorpyrifos +
Cadmium was moderately synergistic, 1/10th LC50 of Chlorpyrifos + Cadmium and Cadmium
+ Chlorpyrifos was synergistic and 1/5th LC50 of Cadmium + Chlorpyrifos was highly
synergistic in nature compared to their individual toxicities.
Among the test toxicants, highest inhibitory activity of acetylcholinesterase was
noticed in the muscle of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium +
Chlorpyrifos (66.89% and 36.59%) followed by Chlorpyrifos + Cadmium (57.91% and
47.27%), Chlorpyrifos (51.54% and 29.83%) and Cadmium (41.00% and 33.33%) during 21
days of exposure (Fig. 25). However, there was slight deviation in the activity in those fish
exposed to 1/10th LC50 of Chlorpyrifos and, Chlorpyrifos + Cadmium.
The sublethal studies using Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos
+ Cadmium
demonstrated significant
(P<0.05) inhibitory effect
on
acetylcholinesterase activity in the gills, liver, kidney, brain and muscle tissue of Tilapia
(Oreochromis mossambicus) (Table 24a and 24b). In all the tissues studied, the AChE activity
was decreased progressively on 7th, 14th and 21st day compared to control fishes (Fig. 16, 18,
20, 22 and 24). The interaction between test organs and test toxicants demonstrate that the
lowest acetylcholinesterase activity was experienced in the brain (Chlorpyrifos) followed by
muscle (Cadmium + Chlorpyrifos), gills (Cadmium), liver (Chlorpyrifos + Cadmium) and
106
kidney (Chlorpyrifos + Cadmium) (Table 23 and Fig. 26). The AChE activity was sharply
inhibited in those fish exposed to 1/5th of LC50 than that of 1/10th of LC50 of test toxicants.
4.3.2.1.2 Butyrylcholinesterase (BChE)
The Tables 25, 26, 27, 28 and 29 represent the changes in the butyrylcholinesterase
activity (nmol of BSChI hydrolysed/min/mg protein) in the gills, liver, kidney, brain and
muscle tissue of Tilapia (Oreochromis mossambicus) respectively, exposed to Cadmium and
Chlorpyrifos individually and to a combination of Cadmium + Chlorpyrifos and Chlorpyrifos
+ Cadmium for a period of 7, 14 and 21 days under static renewal conditions.
Gills
The variation of butyrylcholinesterase (BChE) activity in the gill tissue of control fish
was between 32.21 ± 0.22 on 7th day, 32.73 ± 0.46 on 14th day and 32.69 ± 0.17 on 21st day.
This BChE activity in the gills significantly reduced to 24.13 ± 0.18 (7th day), 21.18 ± 0.51
(14th day) and 19.03 ± 0.33 (21st day) on exposure to 1/5th LC50 of Cadmium. This decreasing
trend was evident even at 1/10th LC50 of Cadmium and the values were 26.80 ± 0.89 on 7th
day, 24.11 ± 0.36 on 14th day and 21.76 ± 0.78 on 21st day. Similarly, in fish treated with 1/5th
LC50 of Chlorpyrifos, the BChE activity in the gills declined and the values varied between
26.48 ± 0.56 (7th day), 23.40 ± 0.77 (14th day) and 21.34 ± 0.23 (21st day). In the same way,
those fish exposed to 1/10th LC50 concentration, the BChE values were decreased to 28.33 ±
0.20 on 7th day, 26.89 ± 0.54 on 14th day and 24.30 ± 0.87 on 21st day. Further, depletion in
BChE activity was noticed in the gills of those fish exposed to a combination of Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium (Table 25). Here, the joint action of both the
combination was simple additive in nature. It means to say that the combination of Cadmium
+ Chlorpyrifos and or Chlorpyrifos + Cadmium caused same effect as that of individual
107
toxicants and act similar in effecting BChE activity in the gills of Tilapia, at the above
concentration and exposure period.
In terms of percentage of intensity of toxicity, the order of toxicity was Cadmium
(41.79% and 33.44%), Cadmium + Chlorpyrifos (39.68% and 31.14%), Chlorpyrifos +
Cadmium (37.23% and 26.64%) and Chlorpyrifos (34.72% and 25.67%) respectively at their
1/5th LC50 and 1/10th LC50 concentrations over a period of 21 days in causing depletion in the
butyrylcholinesterase activity in the gills of fish exposed (Fig. 28).
Liver
The values of butyrylcholinesterase (BChE) activity in the liver of control fish were
64.22 ± 1.89 on 7th day, 66.83 ± 1.32 on 14th day and 69.03 ± 1.67 on 21st day. However,
exposure of Tilapia fish to 1/5th LC50 of Cadmium caused considerable reduction in the BChE
activity in the liver and the range was between 46.74 ± 2.28 (7th day), 33.21 ± 2.41 (14th day)
and 23.75 ± 1.39 (21st day). This BChE activity was further declined to 55.90 ± 1.55 (7th day),
47.08 ± 2.80 (14th day) and 37.18 ± 2.17 (21st day) at 1/10th LC50 of Cadmium. Similarly,
marked fluctuation in BChE activity in the liver of those fish treated with 1/5th Chlorpyrifos
was recorded and the values ranged between 52.70 ± 0.85 (7th day), 42.97 ± 1.60 (14th day)
and 30.77 ± 2.35 (21st day). On the other hand, BChE activity recorded was 56.76 ± 1.94 (7th
day), 49.11 ± 0.53 (14th day) and 40.98 ± 1.62 (21st day) when exposed to 1/10th LC50 of
Chlorpyrifos. Further, significant depletion in BChE activity, in the liver, was evident in fish
exposed to a combination of Cadmium + Chlorpyrifos and, Chlorpyrifos + Cadmium (Table
26), but highly significant depletion in BChE activity was observed at 1/5 th of LC50 of
Chlorpyrifos + Cadmium (35.23 ± 1.82 on 7th day, 21.08 ± 1.08 on 14th day and 12.33 ± 1.66
on 21st day) compared to control fish. The combined toxicity of Cadmium + Chlorpyrifos was
108
moderately synergistic, while 1/5th LC50 of Chlorpyrifos + Cadmium was highly synergistic
and 1/10th LC50 of Chlorpyrifos + Cadmium was moderately synergistic in nature compared to
their individual toxicities.
The order of intensity of toxicity, in fish exposed to 1/5th LC50 and 1/10th LC50 was
Chlorpyrifos + Cadmium (82.14% and 53.19%) followed by Cadmium + Chlorpyrifos
(74.11% and 61.87%), Cadmium (65.59% and 46.14%) and Chlorpyrifos (55.43% and
40.63%) in bringing about alterations in butyrylcholinesterase activity in the liver in relation
to control during 21 days of exposure (Fig. 30).
Kidney
The butyrylcholinesterase (BChE) activity in the kidney tissue of control group of fish
varied between 20.44 ± 0.35 on 7th day, 20.09 ± 0.57 on 14th day and 18.88 ± 0.68 on 21st day.
However, the BChE activity in the kidney progressively decreased to 15.45 ± 0.41 on 7th day,
13.75 ± 0.23 on 14th day and 10.64 ± 0.33 on 21st day when exposed to 1/5th LC50 of
Cadmium. This decreasing trend was slightly less at 1/10th LC50 of Cadmium and the values
were 18.74 ± 0.18 on 7th day, 16.13 ± 0.46 on 14th day and 13.52 ± 0.27 on 21st day. Similarly,
those fish exposed to Chlorpyrifos showed BChE activity between 16.35 ± 0.48 (7th day),
14.67 ± 0.79 (14th day) and 12.26 ± 0.35 (21st day) at 1/5th LC50 concentration and, 19.29 ±
0.63 (7th day), 16.94 ± .99 (14th day) and 13.81 ± 0.56 (21st day) at 1/10th LC50 concentration.
On the other hand, exposure of fish to a combination of Cadmium + Chlorpyrifos drastically
affected the BChE activity of the kidney (Table 27) and the joint action toxicity at 1/5 th LC50
of Cadmium + Chlorpyrifos was simple additive while 1/10th LC50 of Cadmium +
Chlorpyrifos was moderately synergistic in nature compared to individual toxicity of
Cadmium. On the other hand, fish treated with a combination of Chlorpyrifos + Cadmium
109
(14.21 ± 1.20 on 7th day, 11.53 ± 0.44 on 14th day and 8.93 ± 0.76 on 21st day at 1/5th LC50
concentration) recorded significant inhibition in BChE activity in the kidney compared to
control group run parallely for 21 days (Table 27). Here, contrary to joint action toxicity of
Cadmium + Chlorpyrifos, the joint action toxicity at 1/5th LC50 of Chlorpyrifos + Cadmium
was moderately synergistic while 1/10th LC50 of Cadmium + Chlorpyrifos was simple additive
in nature compared to individual toxicity of Chlorpyrifos.
The butyrylcholinesterase activity, in relation to control, was more affected in the
kidney of those fish exposed to 1/5th LC50 and 1/10th LC50 of Chlorpyrifos + Cadmium
(52.70% and 34.38%) followed by Cadmium + Chlorpyrifos (46.66% and 41.63%), Cadmium
(43.64% and 28.39%) and Chlorpyrifos (35.06% and 26.85%) during 21 days of exposure
(Fig. 32). However, there was slight deviation in the activity in those fish exposed to 1/10th
LC50 of Cadmium + Chlorpyrifos only.
Brain
The activity of butyrylcholinesterase (BChE) enzyme in the brain tissue of control fish
varied between 42.64 ± 1.38 on 7th day, 40.82 ± 0.89 on 14th day and 41.20 ± 1.54 on 21st day.
This activity, in the brain, was decreased to 35.51 ± 0.61 (7th day), 28.75 ± 0.55 (14th day) and
23.05 ± 0.19 (21st day), in fish treated with 1/5th LC50 of Cadmium. On the other hand,
exposure of fish to 1/10th LC50 of Cadmium also registered declining trend in BChE activity
and the values were 37.82 ± 0.36 (7th day), 33.11 ± 1.27 (14th day) and 28.77 ± 0.66 (21st day).
Similarly, exposure of fish to 1/5th LC50 of Chlorpyrifos brought in significant depletion in
BChE activity in the brain and the values varied between 28.03 ± 1.45 (7th day), 21.33 ± 1.06
(14th day) and 18.09 ± 0.34 (21st day). However, the BChE activity in the brain substantially
decreased to 37.07 ± 0.82 on 7th day, 32.94 ± 1.03 on 14th day and 26.53 ± 0.35 on 21st day
110
when exposed to 1/10th LC50 of Chlorpyrifos. While, fish exposed to a combination of
Cadmium + Chlorpyrifos registered sharp reduction in BChE activity in the brain during the
same period of exposure (Table 28). Here, the joint action toxicity of Cadmium + Chlorpyrifos
was moderately synergistic in nature compared to individual toxicity of Cadmium. Similar
trend was noticed in the BChE activity, in the brain, of those fish exposed to a combination of
1/10th LC50 of Chlorpyrifos + Cadmium. The joint action toxicity at 1/10th LC50 of
Chlorpyrifos + Cadmium was moderately synergistic in nature. Though the BChE activity
reduced considerably in the brain of those fish exposed to a combination of 1/5th LC50 of
Chlorpyrifos + Cadmium (30.10 ± 0.69 on 7th day, 23.07 ± 0.84 on 14th day and 20.14 ± 0.78
on 21st day), the joint action toxicity at 1/5th LC50 of Chlorpyrifos + Cadmium was moderately
antagonistic in nature compared to individual toxicity of 1/5th LC50 of Chlorpyrifos.
Among the test toxicants, the inhibition of butyrylcholinesterase activity was more in
the brain of those fish exposed to 1/5th LC50 and 1/10th LC50 of Chlorpyrifos (56.09% and
35.61%) followed by Chlorpyrifos + Cadmium (51.12% and 48.13%), Cadmium +
Chlorpyrifos (49.61% and 40.27%) and Cadmium (44.05% and 30.17%) during 21 days of
experiments (Fig. 34). However, there was marginal deviation in the BChE activity in those
fish exposed to 1/10th LC50 of Chlorpyrifos.
Muscle
The variation of butyrylcholinesterase (BChE) activity in the muscle tissue of control
fish was between 95.63 ± 2.90 on 7th day, 102.38 ± 3.57 on 14th day and 113.92 ± 3.68 on 21st
day. However, this BChE activity in the muscle of Tilapia fish considerably decreased to
76.66 ± 1.27 on 7th day, 66.58 ± 2.27 on 14th day and 61.00 ± 1.57 on 21st day on exposures to
1/5th LC50 of Cadmium. This BChE activity in the muscle lowered to 81.09 ± 1.49 on 7th day,
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72.74 ± 2.24 on 14th day and 67.86 ± 1.72 on 21st day when exposed to 1/10th LC50 of
Cadmium. Similar observations were made in the muscle of those fish exposed to
Chlorpyrifos (i.e. BChE activity fluctuated to 68.65 ± 2.04 (7th day), 59.03 ± 1.60 (14th day)
and 54.44 ± 2.91 (21st day) at 1/5th LC50 concentration and, 83.86 ± 1.92 (7th day), 75.99 ±
1.59 (14th day) and 69.71 ± 1.13 (21st day) at 1/10th LC50 concentration). On the other hand,
the significant inhibition in BChE activity in the muscle was noticed in those fish treated with
a combination of Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium (Table 29) and
highly significant inhibition in BChE activity was observed at 1/5th of LC50 of Cadmium +
Chlorpyrifos (64.04 ± 1.37 on 7th day, 57.55 ± 1.35 on 14th day and 32.03 ± 4.60 on 21st day)
compared to control group. Here, the combined toxicity of both these toxicant combinations
was synergistic in nature compared to their individual toxicities.
Comparison between control and treatment reveals that exposure of Tilapia fish to 1/5th
LC50 and 1/10th LC50 of toxicants, the butyrylcholinesterase activity was more affected in
Cadmium + Chlorpyrifos (71.88% and 43.64%) followed by Chlorpyrifos + Cadmium
(58.83% and 48.07%), Chlorpyrifos (52.21% and 38.81%) and Cadmium (46.45% and
40.43%) for 21 days in bringing about reduction in BChE activity in the muscle tissue (Fig.
36). However, there was moderate deviation in the BChE activity in those fish exposed to
1/10th LC50 of Chlorpyrifos + Cadmium and Chlorpyrifos.
Significant (P<0.05) difference in the butyrylcholinesterase activity were evident with
respect to concentration, duration of exposure and tissues due to exposure to Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium (Table 31a and 31b).
The interaction between test organs and test toxicants demonstrate that the lowest BChE
activity was noticed in the liver (Chlorpyrifos + Cadmium) followed by muscle (Cadmium +
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Chlorpyrifos), brain (Chlorpyrifos), kidney (Chlorpyrifos + Cadmium) and gills (Cadmium)
(Table 30 and Fig. 37). Butyrylcholinesterase response showed similar trend in the sense that
it registered a continuous decrease in activity at both the concentrations in gills, liver, kidney,
brain and muscle (Fig. 27, 29, 31, 33 & 35) compared to acetylcholinesterase. On the other
hand, there was decrease in BChE activity with increase in duration of exposure and the
lowest BChE activity was recorded at 21st day in all the treatment groups compared to 7th and
14th days of exposure.
4.3.2.2 Oxidative stress
4.3.2.2.1 Lipid peroxidation (LPx)
The results of changes in lipid peroxidation levels (nmol of TBARS formed/mg
protein) in the gills, liver, kidney, brain and muscle tissue of Tilapia (Oreochromis
mossambicus) exposed to sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium have been provided in Tables 32, 33, 34, 35 and
36, respectively.
Gills
The gills of control fish recorded lipid peroxidation (LPx) level between 6.23 ± 0.16
on 7th day, 6.92 ± 0.07 on 14th day and 7.09 ± 0.05 on 21st day. This level of LPx in the gill
tissue of Tilapia fish was significantly increased to 12.75 ± 0.41 (7th day), 16.34 ± 0.39 (14th
day) and 18.27 ± 0.28 (21st day) on exposure to 1/5th LC50 of Cadmium and 8.43 ± 0.21 (7th
day), 13.12 ± 0.32 (14th day) and 16.63 ± 0.26 (21st day) on exposure to 1/10th LC50 of
Cadmium compared to control group run parallely for 21 days. Similar observations were
made in the gills of those fish exposed to 1/5th LC50 of Chlorpyrifos (i.e. LPx level elevated to
7.22 ± 0.17 on 7th day, 8.23 ± 0.09 on 14th day and 10.12 ± 0.18 on 21st day). On the other
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hand, the LPx level enhanced in the gills of those fish exposed to 1/10th of LC50 of
Chlorpyrifos and the values were 7.01 ± 0.07 (7th day), 7.63 ± 0.13 (14th day) and 8.91 ± 0.37
(21st day). Further, the LPx level enhanced substantially in the gills of those fish exposed to a
combination of Cadmium + Chlorpyrifos (Table 32). The joint action toxicity of Cadmium +
Chlorpyrifos was simple additive in nature compared to individual toxicity of Cadmium.
Similarly, increased LPx level in the gills continued in fish treated with a combination of
Chlorpyrifos + Cadmium. Here contrary to combined toxicity of Cadmium + Chlorpyrifos, the
joint action toxicity of Chlorpyrifos + Cadmium was synergistic in nature compared to
individual toxicity of Chlorpyrifos (Table 32).
Highest level in the lipid peroxidation, in relation to control, was observed in the gills
of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (157.69% and 134.56%)
followed by Cadmium + Chlorpyrifos (147.95% and 122.14%), Chlorpyrifos + Cadmium
(145.56% and 98.17%) and lowest was with Chlorpyrifos (42.74% and 25.67%) during 21
days of experiments (Fig. 39).
Liver
The lipid peroxidation (LPx) level in the liver tissue of the experimental fish in the
control group ranged between 6.31 ± 0.24 on 7th day, 6.74 ± 0.16 on 14th day and 6.32 ± 0.10
on 21st day. This value significantly increased to 10.84 ± 0.36 on 7th day, 13.3 ± 0.29 on 1th
day and 14.62 ± 0.41 on 21st day in the liver of those fish treated with 1/5th LC50 of Cadmium.
The same trend was noticed in the liver (9.98 ± 0.31 on 7th day, 11.81 ± 0.18 on 14th day and
13.77 ± 0.39 on 21st day) of those fish treated with 1/10th LC50 of Cadmium during the same
exposure period. On the other hand, the LPx level in the liver showed moderate enhancement
and the value was 6.85 ± 0.20 (7th day), 8.75 ± 0.13 (14th day) and 10.23 ± 0.27 (21st day) in
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fish exposed to 1/5th LC50 of Chlorpyrifos and 6.56 ± 0.12 (7th day), 7.63 ± 0.20 (14th day) and
8.05 ± 0.17 (21st day) on exposure to 1/10th of LC50 of Chlorpyrifos. Further, fish treated with a
combination of Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium registered
considerable increase in LPx level, in the liver, compared to control fish during 21 days of
exposure (Table 33) and those fish exposed to 1/5th LC50 of Cadmium + Chlorpyrifos showed
significant increment in the LPx level (11.49 ± 0.30 on 7th day, 13.53 ± 0.31 on 14th day and
14.71 ± 0.25 on 21st day). Notably, the joint action toxicity of Cadmium + Chlorpyrifos was
simple additive in nature compared to individual toxicity of Cadmium, but the joint action
toxicity of Chlorpyrifos + Cadmium was moderately synergistic in nature compared to
individual toxicity of Chlorpyrifos (Table 33).
High degree of lipid peroxidation level, in relation to control, was observed in the liver
of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium + Chlorpyrifos (132.75% and
96.99%) followed by Cadmium (131.33% and 117.88%), Chlorpyrifos + Cadmium (123.26%
and 88.61%) and Chlorpyrifos (61.87% and 27.37%) during 21 days of exposure (Fig. 41).
However, there was moderate variation in the LPx value in those fish exposed to 1/10 th LC50
of Cadmium + Chlorpyrifos.
Kidney
The normal level of lipid peroxidation (LPx) in the kidney tissue of control fish was in
the range of 2.78 ± 0.14 on 7th day, 3.02 ± 0.06 on 14th day and 3.27 ± 0.09 on 21st day. This
level progressively increased to 4.32 ± 0.13 (7th day), 5.34 ± 0.21 (14th day) and 6.21 ± 0.24
(21st day) in the kidney of those fish treated with 1/5th LC50 of Cadmium. However, this
increasing trend was evident even at 1/10th LC50 of Cadmium and the values were 3.82 ± 0.29
on 7th day, 4.75 ± 0.18 on 14th day and 5.57 ± 0.16 on 21st day. Similarly, the LPx level in the
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kidney was moderately increased to 3.43 ± 0.13 (7th day), 3.68 ± 0.36 (14th day) and 4.52 ±
0.26 (21st day) at 1/5th LC50 of Chlorpyrifos and 3.22 ± 0.18 (7th day), 3.29 ± 0.12 (14th day)
and 4.06 ± 0.31 (21st day) at 1/10th LC50 Chlorpyrifos. In a combination of Cadmium +
Chlorpyrifos (4.57 ± 0.24 on 7th day, 5.89 ± 0.31 on 14th day and 6.98 ± 0.35 on 21st day at
1/5th LC50 concentration), the LPx level in the kidney significantly enhanced during 21 days of
exposure (Table 34). Here, the joint action toxicity of Cadmium + Chlorpyrifos was simple
additive in nature compared to individual toxicity of Cadmium. Further, elevated LPx level in
the kidney was also noticed in fish exposed to a combination of Chlorpyrifos + Cadmium
during the same exposure period (Table 34). The joint action toxicity at 1/5th LC50 of
Chlorpyrifos + Cadmium was moderately synergistic while 1/10th LC50 of Chlorpyrifos +
Cadmium was simple additive in nature compared to individual toxicity of Chlorpyrifos.
A comparison of intensity of toxicity among toxicants reveal that exposure of fish to
1/5th LC50 and 1/10th LC50 of Cadmium + Chlorpyrifos (113.61% and 65.54%) was more
positively affected followed by Cadmium (89.94% and 70.34%), Chlorpyrifos + Cadmium
(78.75% and 49.85%) and Chlorpyrifos (38.50% and 24.40%) for 21 days in bringing about
increased lipid peroxidation values in the kidney tissue (Fig. 43). However, there was
moderate deviation in the activity in those fish exposed to 1/10th LC50 of Cadmium +
Chlorpyrifos.
Brain
The variation of lipid peroxidation (LPx), in the brain tissue of fish, in the control
group was between 3.46 ± 0.18 on 7th day, 4.35 ± 0.09 on 14th day and 4.59 ± 0.13 on 21st day.
However, exposure of Tilapia fish to 1/5th LC50 of Cadmium caused elevation in the LPx level
in the brain and the values were 5.65 ± 0.39 (7th day), 7.36 ± 0.40 (14th day) and 8.45 ± 0.36
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(21st day). In the same way, when fish exposed to 1/10th of LC50 of Cadmium, the LPx level in
the brain elevated to 3.74 ± 0.26 (7th day), 5.31 ± 0.29 (14th day) and 5.63 ± 0.10 (21st day)
compared to control fish. Similarly, those fish exposed to Chlorpyrifos showed LPx level
between 4.47 ± 0.26 (7th day), 5.95 ± 0.18 (14th day) and 6.64 ± 0.20 (21st day) at 1/5th LC50
concentration and, 3.55 ± 0.12 (7th day), 4.43 ± 0.20 (14th day) and 5.21 ± 0.18 (21st day) at
1/10th LC50 concentration. Moreover, the elevated trend in LPx level was continued in the
brain of those fish exposed to a combination of Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium during 3 weeks of exposure (Table 35) and exposure of fish to 1/5th LC50 of
Chlorpyrifos + Cadmium registered significant elevation in LPx level in the brain tissue i.e.
6.78 ± 0.21 (7th day), 8.25 ± 0.26 (14th day) and 9.29 ± 0.32 (21st day). The combined toxicity
at 1/5th LC50 of Cadmium + Chlorpyrifos was simple additive while 1/10th LC50 of Cadmium +
Chlorpyrifos was moderately synergistic in nature compared to individual toxicity of
Cadmium and whereas in case of 1/5th LC50 and 1/10th LC50 of Chlorpyrifos + Cadmium, the
combined toxicity was moderately synergistic in nature compared to individual toxicity of
Chlorpyrifos (Table 35).
The lipid peroxidation level in the brain of those fish exposed to 1/5th LC50 and 1/10th
LC50 of Chlorpyrifos + Cadmium (102.40% and 67.39%) was more affected followed by
Cadmium + Chlorpyrifos (91.29% and 57.45%), Cadmium (84.16% and 22.83%) and
Chlorpyrifos (44.79%and 13.68%) during 21 days of exposure (Fig. 45).
Muscle
The muscle tissue of fish in the control group showed lipid peroxidation (LPx) level
between 0.91 ± 0.02 on 7th day, 0.95 ± 0.01 on 14th day and 0.94 ± 0.03 on 21st day. On the
other hand, the LPx level in the muscle enhanced to 1.42 ± 0.03 on 7th day, 1.46 ± 0.01 on 14th
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day and 1.48 ± 0.02 on 21st day in fish exposed to 1/5th LC50 of Cadmium. Similar trend was
recorded at 1/ 10th of LC50 of Cadmium and the values were 1.19 ± 0.06 (7th day), 1.30 ± 0.01
(14th day) and 1.31 ± 0.01 (21st day) during the same period of exposure. However, exposure
of fish to 1/5th LC50 of Chlorpyrifos caused somewhat increment in the LPx level in the
muscle of Tilapia (0.98 ± 0.04 on 7th day, 1.16 ± 0.03 on 14th day and 1.20 ± 0.02 on 21st day).
In the same way, those fish exposed to 1/10th LC50 concentration, the LPx values were slightly
increased to 0.95 ± 0.01 on 7th day, 1.09 ± 0.05 on 14th day and 1.12 ± 0.06 on 21st day.
Further, increase in LPx level in the muscle tissue was evident in fish exposed to a
combination of Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium compared to control
group run parallely for 21 days (Table 36). The joint action toxicity of both the combination
was simple additive in nature. It means to say that the combination of Cadmium +
Chlorpyrifos and or Chlorpyrifos + Cadmium caused same effect as that of individual
toxicants during 21 days of exposure.
Highest lipid peroxidation value, in relation to control, was registered in the muscle
tissue of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (58.30% and 40.00%)
followed by Cadmium + Chlorpyrifos (46.60% and 38.19%), Chlorpyrifos + Cadmium
(44.57% and 29.04%) and Chlorpyrifos (28.30% and 20.00%) during 21 days of exposure
(Fig. 47).
The lipid peroxidation values, as evidenced by TBARS levels were found to increase
significantly in all the test tissues analyzed under both the experimental concentrations (1/5th
LC50 and 1/10th LC50) of the test toxicants at the end of 21st day. When a comparison was
made between test organs and test toxicants, the highest lipid peroxidation was observed in the
gills (Cadmium) followed by liver (Cadmium + Chlorpyrifos), kidney (Cadmium +
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Chlorpyrifos), brain (Chlorpyrifos + Cadmium) and muscle (Cadmium) of Tilapia fish (Table
37 and Fig. 48). On the other hand, there was increase in LPx level with increase in duration
of exposure and the highest LPx value was recorded at 21st day in all the treatment groups
compared to 7th and 14th days of exposure (Fig. 38, 40, 42, 44 and 46). Two-way ANOVA
followed by Tukey's studentized range (HSD) test revealed that there was significant (P<0.05)
variation in lipid peroxidation level between treatments, days and also between tissues (Table
38a and 38b).
4.3.2.3 Antioxidant enzymes
4.3.2.3.1 Superoxide dismutase (SOD)
A perusal of Tables 39, 40, 41, 42 and 43 reveals the details of changes in superoxide
dismutase activities (Units/mg protein) in the gills, liver, kidney, brain and muscle tissue of
Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium, respectively.
Gills
The superoxide dismutase (SOD) activity in the gill tissue of fish in the control group
varied between 4.22 ± 0.07 on 7th day, 4.14 ± 0.11 on 14th day and 4.37 ± 0.15 on 21st day.
This activity of SOD in the gills enhanced in fish treated with 1/5th LC50 of Cadmium and the
values between 9.10 ± 0.25 on 7th day, 8.34 ± 0.18 on 14th day and 7.62 ± 0.13 on 21st day.
Similarly, exposure of fish to 1/10th LC50 of Cadmium also caused changes in the SOD
activity in the gills and the values varied between 7.29 ± 0.22 (7th day), 5.90 ± 0.10 (14th day)
and 5.79 ± 0.06 (21st day). However, in those fish exposed to 1/5th LC50 of Chlorpyrifos, the
SOD activity in the gills registered lower activity compared to Cadmium and the values were
5.27 ± 0.05 (7th day), 5.58 ± 0.03 (14th day) and 5.93 ± 0.13 (21st day). On the other hand,
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moderate increasing trend in SOD activity (5.12 ± 0.02 on 7th day, 5.15 ± 0.06 on 14th day and
5.78 ± 0.11 on 21st day) was observed when fish was exposed to 1/10th of LC50 of Chlorpyrifos
during the same exposure period. Only a slight altered SOD activity was evident in the gills of
those fish exposed to a combination of Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
during 21 days of exposure (Table 39). The combined toxicity of Cadmium + Chlorpyrifos
was simple additive in nature compared to individual toxicity of Cadmium while Chlorpyrifos
+ Cadmium was moderately synergistic in nature compared to individual toxicity of
Chlorpyrifos (Table 39).
Comparison between control and treatment reveals that exposure of fish to 1/5th LC50
and 1/10th LC50 of Cadmium (74.44% and 32.54%) recorded higher activity of superoxide
dismutase in the gills followed by Cadmium + Chlorpyrifos (42.72% and 26.29%),
Chlorpyrifos + Cadmium (36.68% and 23.73%) and Chlorpyrifos (35.88% and 32.33%)
during 21 days of experiments (Fig. 50). However, there was moderate deviation in the SOD
activity in those fish exposed to 1/10th LC50 of Chlorpyrifos only.
Liver
The superoxide dismutase (SOD) activity in the liver tissue of control fish varied
between 6.32 ± 0.10 on 7th day, 6.43 ± 0.07 on 14th day and 6.76 ± 0.09 on 21st day. This
activity in the liver fluctuated between 13.54 ± 0.47 (7th day), 11.52 ± 0.34 (14th day) and
10.15 ± 0.28 (21st day) in fish treated with 1/5th LC50 of Cadmium. In the same way, those fish
exposed to 1/10th of LC50 of Cadmium, the SOD activity was changed to 11.88 ± 0.35 (7th
day), 10.96 ± 0.26 (14th day) and 9.77 ± 0.20 (21st day). On the other hand, exposure of fish to
1/5th LC50 of Chlorpyrifos brought in enhanced SOD activity in the liver compared to control
fish and the activity was between 8.64 ± 0.18 (7th day), 8.75 ± 0.21 (14th day) and 10.12 ± 0.22
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(21st day). The same trend was also noticed in the liver of those fish exposed to 1/10 th LC50 of
Chlorpyrifos i.e. SOD activity was 8.02 ± 0.08 (7th day), 8.91 ± 0.12 (14th day) and 9.78 ±
0.24 (21st day). Further, fish exposed to a combination of Cadmium + Chlorpyrifos (14.79 ±
0.30 on 7th day, 13.44 ± 0.35 on 14th day and 11.58 ± 0.23 on 21st day at 1/5th LC50
concentration) registered significant deviation in the SOD activity in the liver during the same
period of exposure (Table 40). The combined toxicity at 1/5th LC50 of Cadmium +
Chlorpyrifos was moderately synergistic in nature while 1/10th LC50 of Cadmium +
Chlorpyrifos was simple additive in nature compared to individual toxicity of Cadmium.
Similar differences were observed in the SOD activity in the liver of those fish exposed to a
combination of Chlorpyrifos + Cadmium and here the joint toxicity of this combination was
moderately synergistic in nature compared to individual toxicity of Chlorpyrifos (Table 40).
Among the test toxicants, the highest activity in the superoxide dismutase was noticed
in the liver of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium + Chlorpyrifos
(71.30% and 36.20%) followed by Cadmium (50.15% and 44.59%), Chlorpyrifos (49.70%
and 44.73%) and Chlorpyrifos + Cadmium (45.19% and 30.68%) during 21 days of exposure
(Fig. 52). However, there was moderate deviation in the SOD activity in those fish exposed to
1/10th LC50 of Cadmium + Chlorpyrifos.
Kidney
The variation of superoxide dismutase (SOD) activity, in the kidney tissue of fish in
the control group was between 3.25 ± 0.15 on 7th day, 3.43 ± 0.08 on 14th day and 3.40 ± 0.05
on 21st day. On the contrary, those fish exposed to Cadmium showed deviated SOD activity
and the values varied between 6.65 ± 0.22 (7th day), 5.50 ± 0.16 (14th day) and 4.85 ± 0.18
(21st day) at 1/5th LC50 concentration and 5.63 ± 0.15 (7th day), 5.41 ± 0.11 (14th day) and 4.52
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± 0.12 (21st day) at 1/10th LC50 concentration compared to control group run parallely for 21
days. On the other hand, slight enhancement in the SOD activity was recorded in the kidney of
those fish exposed to 1/5th LC50 of Chlorpyrifos (4.13 ± 0.12 on 7th day, 4.40 ± 0.09 on 14th
day and 4.78 ± 0.16 on 21st day) and 1/10th LC50 of Chlorpyrifos (3.88 ± 0.15 on 7th day, 4.00
± 0.05 on 14th day and 4.24 ± 0.21 on 21st day). Further, the SOD activity varied in the kidney
of those fish treated with a combination of Cadmium + Chlorpyrifos (6.95 ± 0.13 on 7th day,
6.76 ± 0.12 on 14th day and 5.61 ± 0.17 on 21st day at 1/5th LC50 concentration) compared to
control fish during 21 days of exposure. Here, the combined toxicity at 1/5th LC50 of Cadmium
+ Chlorpyrifos was moderately synergistic while 1/10th LC50 of Cadmium + Chlorpyrifos was
simple additive compared to individual toxicity of Cadmium (Table 41). Similarly, altered
SOD activity in the kidney continued even when the fish was exposed to a combination of
Chlorpyrifos + Cadmium. The combined toxicity of Chlorpyrifos + Cadmium was moderately
synergistic in nature compared to individual toxicity of Chlorpyrifos (Table 41).
Highest activity in the superoxide dismutase, in relation to control, was observed in the
kidney of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium + Chlorpyrifos
(65.21% and 27.56%) followed by Cadmium (42.82% and 32.97%), Chlorpyrifos (40.74%
and 24.82%) and Chlorpyrifos + Cadmium (38.94% and 22.06%) during 3 weeks of exposure
(Fig. 54). However, there was marginal variation in the SOD activity in the kidney of those
fish exposed to 1/10th LC50 of Cadmium + Chlorpyrifos.
Brain
The brain tissue of control fish recorded superoxide dismutase (SOD) activity between
2.23 ± 0.06 on 7th day, 2.12 ± 0.12 on 14th day and 2.02 ± 0.05 on 21st day. This activity in the
brain varied between 3.86 ± 0.14 (7th day), 3.67 ± 0.12 (14th day) and 2.90 ± 0.17 (21st day)
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when exposed to 1/5th LC50 of Cadmium and 2.59 ± 0.05 (7th day), 2.31 ± 0.10 (14th day) and
2.27 ± 0.03 (21st day) at 1/10th of LC50 of Cadmium during the same exposure period. On the
other hand, the SOD activity in the brain registered a slight fluctuation between 3.15 ± 0.12
(7th day), 2.54 ± 0.07 (14th day) and 2.45 ± 0.05 (21st day) in fish exposed to 1/5th LC50 of
Chlorpyrifos. Similar trend was recorded when brain of those fish exposed to 1/10th LC50 of
Chlorpyrifos (2.36 ± 0.07 on 7th day, 2.15 ± 0.04 on 14th day and 2.12 ± 0.05 on 21st day).
Further, exposure of fish to a combination of Cadmium + Chlorpyrifos caused considerable
deviation in the SOD activity in the brain during the same period of exposure (Table 42). In
the same way, significantly altered SOD activity in the brain was noticed when fish was
exposed to Chlorpyrifos + Cadmium (4.67 ± 0.16 on 7th day, 4.25 ± 0.13 on 14th day and 3.06
± 0.18 on 21st day at 1/5th LC50 concentration) compared to control fish. The joint action
toxicity at 1/5th LC50 of Cadmium + Chlorpyrifos was simple additive while 1/10th LC50 of
Cadmium + Chlorpyrifos was moderately synergistic compared to individual toxicity of
Cadmium, whereas in case of 1/5th LC50 and 1/10th LC50 of Chlorpyrifos + Cadmium was
moderately synergistic in nature compared to individual toxicity of Chlorpyrifos (Table 42).
In terms of percentage of intensity of toxicity, the order of toxicity was Chlorpyrifos +
Cadmium (51.83% and 38.42%), Cadmium + Chlorpyrifos (47.33% and 24.75%), Cadmium
(43.61% and 12.82%), and Chlorpyrifos (21.58% and 5.20%) respectively at their 1/5th LC50
and 1/10th LC50 concentrations over a period of 21 days in causing variation in the superoxide
dismutase activity in the brain of fish exposed (Fig. 56).
Muscle
The variation of superoxide dismutase activity (SOD), in the muscle tissue of the fish,
in the control group was between 1.52 ± 0.04 on 7th day, 1.59 ± 0.02 on 14th day and 1.61 ±
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0.06 on 21st day. This SOD activity increased to some extent in the muscle i.e. 1.84 ± 0.05 on
7th day, 2.01 ± 0.04 on 14th day and 2.12 ± 0.08 on 21st day in fish treated with 1/5th LC50 of
Cadmium and 1.78 ± 0.05 (7th day), 1.84 ± 0.06 (14th day) and 1.95 ± 0.02 (21st day) at 1/10th
LC50 of Cadmium. While, fish treated with 1/5th LC50 of Chlorpyrifos recorded negligibly
enhancement in SOD activity in the muscle and the values were 1.56 ± 0.08 (7th day), 1.64 ±
0.01 (14th day) and 1.67 ± 0.03 (21st day). The same trend was noticed even in the muscle of
those fish exposed to 1/10th LC50 of Chlorpyrifos i.e. SOD activity was 1.54 ± 0.07 (7th day),
1.61 ± 0.07 (14th day) and 1.64 ± 0.02 (21st day). Elevated trend in SOD activity was
continued in the muscle of those fish exposed to a combination of Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium (Table 43). It was interesting to note that the SOD activities in
the muscle of those fish exposed to these combinations were similar to those recorded in fish
exposed to Cadmium and Chlorpyrifos alone for the same period of exposure. Hence the
combined toxicity of both the combination was simple additive in nature (Table 43).
Among the test toxicants, the higher activity of superoxide dismutase was noticed in
the muscle of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (31.68% and
21.12%) followed by Cadmium + Chlorpyrifos (27.33% and 18.01%), Chlorpyrifos +
Cadmium (22.98% and 14.91%) and Chlorpyrifos (3.73% and 1.86%) during 21 days of
exposure (Fig. 58).
The superoxide dismutase activity in the groups treated with Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium showed statistically significant
(P<0.05) difference between them and also with the control group (Table 45a and 45b). The
enzyme activity in the gills, liver, kidney and brain of test fishes were found to enhance
significantly on 7th day of exposure compared to that of control fishes. In the experimental
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tissues, except muscle, the superoxide dismutase activity registered a continuous decreasing
trend in both at low and high concentrations of Cadmium, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium after 7th day of exposure. The activity further decreased on 21st day
of exposure (Fig. 49, 51, 53 & 55). The muscle registered more or less increased activity at all
the test concentrations during experimental periods showing more activity on 21st day (Fig.
57). When a comparison was made between test organs and test toxicants, the highest SOD
activity was observed in the liver (Cadmium + Chlorpyrifos) followed by gills (Cadmium),
kidney (Cadmium + Chlorpyrifos), brain (Chlorpyrifos + Cadmium) and muscle (Cadmium)
of the fish (Table 44 and Fig. 59).
4.3.2.3.2 Catalase (CAT)
The sublethal effects of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium at their 1/5th and 1/10th LC50 concentrations on catalase activities
(Units/mg protein) in the gills, liver, kidney, brain and muscle tissue of Tilapia (Oreochromis
mossambicus), exposed for a period of 21 days, are provided in Tables 46, 47, 48, 49 and 50,
respectively.
Gills
The values of catalase (CAT) activity in the gills of control fish were 3.11 ± 0.10 on 7th
day, 3.37 ± 0.07 on 14th day and 3.23 ± 0.06 on 21st day. However, exposure of Tilapia fish to
1/5th LC50 of Cadmium caused significant enhancement in the CAT activity in the gills and the
values were 6.15 ± 0.26 (7th day), 7.56 ± 0.18 (14th day) and 6.85 ± 0.26 (21st day). Similarly,
in fish exposed to 1/10th of LC50 of Cadmium, the CAT activity in the gills varied between
5.12 ± 0.11 (7th day), 6.54 ± 0.15 (14th day) and 5.50 ± 0.21 (21st day) compared to control
fish. Enhanced CAT activity in the gills of those fish exposed to 1/5th LC50 of Chlorpyrifos
125
was noticed and the values were 4.68 ± 0.07 on 7th day, 4.67 ± 0.18 on 14th day and 4.98 ±
0.20 on 21st day. This increasing trend in CAT activity in the gills (4.20 ± 0.11 on 7th day, 4.53
± 0.16 on 14th day and 4.76 ± 0.14 on 21st day) continued when fish was exposed to 1/10th
LC50 of Chlorpyrifos. Further, alteration in CAT activity in the gills was evident in fish
exposed to a combination of Cadmium + Chlorpyrifos and the combined toxicity of this
combination was simple additive in nature compared to individual toxicity of Cadmium. On
the other hand, fish treated with a combination of Chlorpyrifos + Cadmium registered
variation in CAT activity in the gills compared to control group for 21 days (Table 46). Here,
the combined toxicity at 1/5th LC50 of Chlorpyrifos + Cadmium was moderately synergistic,
while at 1/10th LC50 of Chlorpyrifos + Cadmium was simple additive in nature compared to
individual toxicity of Chlorpyrifos.
The catalase activity in the gills of those fish exposed to 1/5th LC50 and 1/10th LC50 of
Cadmium (112.29% and 70.43%) was highest followed by Cadmium + Chlorpyrifos (97.40%
and 60.87%), Chlorpyrifos + Cadmium (83.62% and 53.68%) and Chlorpyrifos (54.33% and
47.46%) during 21 days of exposure (Fig. 61).
Liver
The liver tissue of fish in the control group showed varied catalase (CAT) activity
between 3.81 ± 0.13 on 7th day, 4.12 ± 0.17 on 14th day and 4.11 ± 0.12 on 21st day. This
activity in the liver tissue considerably increased to 6.47 ± 0.26 (7th day), 8.25 ± 0.21 (14th
day) and 7.67 ± 0.19 (21st day) in those fish treated with 1/5th LC50 of Cadmium. Similar
observations were made in the liver of those fish exposed to 1/10th of LC50 of Cadmium (5.75
± 0.14 on 7th day, 6.93 ± 0.18 on 14th day and 6.54 ± 0.16 on 21st day). However, in fish
exposed to 1/5th LC50 of Chlorpyrifos, the CAT activity in the liver was moderately elevated
126
and the values were 5.46 ± 0.08 on 7th day, 5.72 ± 0.12 on 14th day and 5.87 ± 0.13 on 21st
day. Interestingly, in fish exposed to 1/10th LC50 of Chlorpyrifos, the CAT activity in the liver
was more or less similar to that recorded at 1/5th LC50 concentration and the values were 5.20
± 0.10 on 7th day, 5.58 ± 0.08 on 14th day and 5.69 ± 0.10 on 21st day. On the other hand,
significant enhancement in CAT activity in the liver was evident in fish exposed to a
combination of Cadmium + Chlorpyrifos (6.76 ± 0.24 on 7th day, 8.65 ± 0.21 on 14th day and
7.92 ± 0.24 on 21st day at 1/5th LC50 concentration) (Table 47). Here the joint action toxicity of
Cadmium + Chlorpyrifos was simple additive in nature. Similar trend was also evident in fish
exposed to a combination of Chlorpyrifos + Cadmium. The joint action toxicity at 1/5 th LC50
of Chlorpyrifos + Cadmium was moderately synergistic, while at 1/10th LC50 of Chlorpyrifos
+ Cadmium was simple additive in nature compared to individual toxicity of Chlorpyrifos.
Highest catalase activity in relation to control was registered in the liver of those fish
exposed to 1/5th LC50 and 1/10th LC50 of Cadmium + Chlorpyrifos (92.90% and 50.32%)
followed by Cadmium (86.81% and 59.32%), Chlorpyrifos + Cadmium (65.94% and 46.84%)
and lowest was with Chlorpyrifos (42.48% and 38.11%) during 21 days of exposure (Fig. 63).
However, there was moderate deviation in the CAT activity in those fish exposed to 1/10th
LC50 of Cadmium + Chlorpyrifos.
Kidney
The catalase (CAT) activity in the kidney tissue of control fish ranged between 2.31 ±
0.11 on 7th day, 2.43 ± 0.08 on 14th day and 2.31 ± 0.07 on 21st day. This activity in the kidney
rose to 4.62 ± 0.18 (7th day), 5.22 ± 0.20 (14th day) and 3.82 ± 0.15 (21st day) on exposure to
1/5th LC50 of Cadmium. This varying trend was evident even at 1/10th LC50 of Cadmium and
the values were 4.16 ± 0.16 (7th day), 4.73 ± 0.12 (14th day) and 3.43 ± 0.13 (21st day). While,
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exposure of fish to 1/5th LC50 of Chlorpyrifos though induced increased CAT activity in the
kidney of Tilapia (3.26 ± 0.06 on 7th day, 3.57 ± 0.06 on 14th day and 3.87 ± 0.13 on 21st day)
but it was less than that effected with Cadmium. In the same way, those fish exposed to 1/10 th
LC50 concentration, the CAT also showed enhanced activity and the values were 3.05 ± 0.07
on 7th day, 3.06 ± 0.14 on 14th day and 3.64 ± 0.17 on 21st day. Further, when fish was treated
with combination of Cadmium + Chlorpyrifos and, Chlorpyrifos + Cadmium, it registered
significant variation of CAT activity in the kidney during 21 days of exposure (Table 48) and
more variation in the CAT activity was observed in those fish exposed to 1/5th LC50 of
Cadmium + Chlorpyrifos (4.77 ± 0.21 on 7th day, 5.49 ± 0.27 on 14th day and 4.06 ± 0.15 on
21st day). Here, the combined toxicity of Cadmium + Chlorpyrifos was simple additive in
nature while the combination of Chlorpyrifos + Cadmium was moderately synergistic in
nature in altering CAT activity compared to their individual toxicities (Table 48).
Among the test toxicants, highest inducement of catalase activity was noticed in the
kidney of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium + Chlorpyrifos
(75.76% and 43.07%) followed by Chlorpyrifos (67.79% and 57.92%), Cadmium (65.63%
and 48.79%) and Chlorpyrifos + Cadmium (54.81% and 42.81%) during 21 days of exposure
(Fig. 65). However, there was moderate alteration in the CAT activity in those fish exposed to
1/10th LC50 of Cadmium + Chlorpyrifos.
Brain
The variation of catalase (CAT) activity in the brain tissue of control fish was between
1.87 ± 0.06 on 7th day, 1.78 ± 0.06 on 14th day and 1.71 ± 0.02 on 21st day. This CAT activity
in the brain deviated to 3.13 ± 0.08 (7th day), 3.30 ± 0.09 (14th day) and 2.34 ± 0.16 (21st day)
on exposure to 1/5th LC50 of Cadmium. This varying trend was recorded even at 1/10th LC50 of
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Cadmium and the values were 2.18 ± 0.08 on 7th day, 2.07 ± 0.06 on 14th day and 2.04 ± 0.03
on 21st day. Similarly, in fish treated with 1/5th LC50 of Chlorpyrifos, the CAT activity in the
brain altered and the values varied between 2.36 ± 0.02 (7th day), 2.42 ± 0.05 (14th day) and
2.12 ± 0.11 (21st day). In the same way, in those fish exposed to 1/10th LC50 concentration, the
CAT values varied between 1.93 ± 0.05 on 7th day, 1.96 ± 0.08 on 14th day and 1.92 ± 0.04 on
21st day. Deviation in CAT activity was also observed in the brain tissue of those fish exposed
to a combination of Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium (Table 49) and
more deviation in the CAT activity was observed in those fish exposed to 1/5th LC50 of
Chlorpyrifos + Cadmium 3.37 ± 0.13 on 7th day, 3.78 ± 0.16 on 14th day and 2.56 ± 0.14 on
21st day). The joint action toxicity of Cadmium + Chlorpyrifos was simple additive, whereas
Chlorpyrifos + Cadmium was moderately synergistic in nature compared to their individual
toxicities.
In terms of percentage of intensity of toxicity, the order of toxicity was Chlorpyrifos +
Cadmium (50.00% and 30.58%), Cadmium + Chlorpyrifos (40.53% and 26.61%), Cadmium
(37.19% and 19.36%) and Chlorpyrifos (24.21% and 12.69%), respectively at their 1/5th LC50
and 1/10th LC50 concentrations over a period of 21 days in causing variation in the catalase
activity in the brain of fish exposed (Fig. 67).
Muscle
The catalase (CAT) activity in the muscle tissue of control fish varied between 0.94 ±
0.02 on 7th day, 0.95 ± 0.07 on 14th day and 0.95 ± 0.05 on 21st day. However, exposure of
Tilapia fish to 1/5th LC50 of Cadmium caused moderate enhancement in the CAT activity and
the range was between 1.49 ± 0.07 (7th day), 1.44 ± 0.03 (14th day) and 1.46 ± 0.01 (21st day).
This CAT activity was 1.15 ± 0.06 (7th day), 1.28 ± 0.02 (14th day) and 1.27 ± 0.02 (21st day)
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at 1/10th LC50 of Cadmium. Similarly, slight increment in CAT activity in the muscle of those
fish treated with 1/5th Chlorpyrifos was recorded and the values were 0.99 ± 0.06 (7th day),
1.21 ± 0.01 (14th day) and 1.25 ± 0.03 (21st day).The CAT activity did not show much
variation at 1/10th LC50 of Chlorpyrifos (0.98 ± 0.04 on 7th day, 1.20 ± 0.05 on 14th day and
1.22 ± 0.02 on 21st day). Only marginal elevation in CAT activity, in the muscle, was evident
in fish exposed to a combination of Cadmium + Chlorpyrifos and, Chlorpyrifos + Cadmium
(Table 50). The joint action toxicity of both these toxicants was simple additive in nature. It
means to say that the combination of Cadmium + Chlorpyrifos and or Chlorpyrifos +
Cadmium caused same effect as that of individual toxicants and act similar in effecting CAT
activity in the muscle of Tilapia, at the above concentration during 3 weeks of exposure (Table
50).
Comparison between control and treatment reveals that exposure of Tilapia fish to 1/5th
LC50 and 1/10th LC50 of toxicants, the catalase activity was more affected in Cadmium
(54.04% and 33.47%) followed by Cadmium + Chlorpyrifos (46.48% and 29.17%),
Chlorpyrifos + Cadmium (39.14% and 32.32%) and Chlorpyrifos (31.58% and 28.33%)
during 21 days in bringing about enhancement in CAT activity in the muscle tissue (Fig. 69).
However, there was marginal deviation in the CAT activity in those fish exposed to 1/10 th
LC50 of Cadmium + Chlorpyrifos.
Two-way ANOVA followed by Tukey's studentized range (HSD) test indicated that
there was significant (P<0.05) variation in catalase activity between treatments, days and also
between tissues (Table 52a and 52b). The catalase activity in the gills, liver, kidney and brain
of the experimental fish, was found to increase significantly on 7th day and 14th day compared
to that of control fishes. This CAT activity in the experimental tissues, except muscle, showed
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decreasing trend in both at low and high concentrations of Cadmium, Cadmium+Chlorpyrifos
and Chlorpyrifos+Cadmium on 21st day of exposure (Fig. 60, 62, 64, 66 & 68). When a
comparison was made between test organs and test toxicants, the highest catalase activity was
observed in the kidney (Cadmium + Chlorpyrifos) followed by gills (Cadmium), liver
(Cadmium + Chlorpyrifos), brain (Chlorpyrifos + Cadmium) and muscle (Cadmium) of the
experimental fish (Table 51 and Fig. 70).
4.3.2.3.3 Glutathione peroxidase (GPx)
The details of changes in glutathione peroxidase activity (nmol of NADPH
oxidized/min/mg protein) in the gills, liver, kidney, brain and muscle tissue of Tilapia
(Oreochromis mossambicus) exposed to sublethal concentrations (1/5th and 1/10th LC50) of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium have been
provided in Tables 53, 54, 55, 56 and 57, respectively.
Gills
The glutathione peroxidase (GPx) activity in the gill tissue of control group of fish
varied between 16.62 ± 0.21 on 7th day, 16.04 ± 0.28 on 14th day and 17.05 ± 0.23 on 21st day.
However, the GPx activity in the gills significantly increased to 32.80 ± 0.78 on 7th day, 36.34
± 0.62 on 14th day and 34.26 ± 0.41 on 21st day when exposed to 1/5th LC50 of Cadmium. This
increasing trend was slightly less at 1/10th LC50 of Cadmium and the values were 26.86 ± 0.61
on 7th day, 32.06 ± 0.50 on 14th day and 28.22 ± 0.49 on 21st day. On the contrary, those fish
exposed to Chlorpyrifos showed GPx activity between 21.21 ± 0.55 (7th day), 24.12 ± 0.39
(14th day) and 25.34 ± 0.22 (21st day) at 1/5th LC50 concentration and similar trend was also
recorded at 1/10th LC50 concentration. Further, exposure of fish to a combination of Cadmium
+ Chlorpyrifos although progressively enhanced the GPx activity of the gills but it was less
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than that of individual toxicity of Cadmium (Table 53). Here the joint action toxicity of this
combination was moderately antagonistic in nature. Similarly, increased GPx activity in the
gills was noticed when fish was exposed to Chlorpyrifos + Cadmium. Here contrary to
combined toxicity of Cadmium + Chlorpyrifos, the joint action toxicity of Chlorpyrifos +
Cadmium was moderately synergistic in nature compared to individual toxicity of
Chlorpyrifos (Table 53).
The glutathione peroxidase activity, in relation to control, was highest in the gills of
those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (100.94% and 65.51%) followed
by Cadmium + Chlorpyrifos (85.22% and 63.52%), Chlorpyrifos + Cadmium (68.62% and
54.31%) and Chlorpyrifos (48.62% and 44.05%) during 21 days of experiments (Fig. 72).
Liver
The glutathione peroxidase activity (GPx) in the liver tissue of control fish varied
between 18.97 ± 0.26 on 7th day and 21.42 ± 0.21 on 21st day. This activity, in the liver, was
considerably elevated to 32.83 ± 0.98 (7th day), 41.34 ± 0.83 (14th day) and 37.78 ± 0.92 (21st
day), in fish treated with 1/5th LC50 of Cadmium. On the other hand, exposure of fish to 1/10th
LC50 of Cadmium also registered increasing trend in GPx activity but of lesser magnitude and
the values were 29.09 ± 0.61 (7th day), 38.28 ± 0.59 (14th day) and 34.67 ± 0.79 (21st day).
Similarly, exposure of fish to 1/5th LC50 of Chlorpyrifos brought in substantial enhancement in
GPx activity in the liver compared to control fish and the values ranged between 25.78 ± 0.42
(7th day) and 28.92 ± 0.30 (21st day). However, the GPx activity in the liver moderately
enhanced to 24.32 ± 0.49 on 7th day and 27.18 ± 0.13 on 21st day when exposed to 1/10th LC50
of Chlorpyrifos. In a combination of 1/5th LC50 of Cadmium + Chlorpyrifos, the GPx activity
in the liver significantly increased between 34.47 ± 0.83 (7th day), 43.06 ± 0.79 (14th day) and
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38.50 ± 0.90 (21st day). While, exposure of fish to a combination of 1/10th LC50 of Cadmium +
Chlorpyrifos also caused variation in GPx activity in the liver during the same period of
exposure (Table 54). Similar trend was noticed in those fish exposed to a combination of
Chlorpyrifos + Cadmium. Notably, the combined toxicity of Cadmium + Chlorpyrifos was
simple additive in nature compared to individual toxicity of Cadmium and whereas
Chlorpyrifos + Cadmium was synergistic in nature compared to individual toxicity of
Chlorpyrifos (Table 54).
Highest activity in the glutathione peroxidase, in relation to control, was registered in
the liver of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium + Chlorpyrifos
(79.74% and 55.70%) followed by Cadmium (76.38% and 61.86%), Chlorpyrifos + Cadmium
(66.01% and 51.68%) and lowest was with Chlorpyrifos (35.01% and 26.89%) during 3 weeks
of exposure (Fig. 74). However, there was slight deviation in the GPx activity in those fish
exposed to 1/10th LC50 of Cadmium + Chlorpyrifos.
Kidney
The variation of glutathione peroxidase (GPx) activity in the kidney tissue of control
fish was between 11.56 ± 0.16 on 7th day, 11.75 ± 0.18 on 14th day and 10.75 ± 0.23 on 21st
day. However, this GPx activity in the kidney of Tilapia fish progressively rose to more than 2
times and the values were 22.31 ± 034 on 7th day, 24.82 ± 0.48 on 14th day and 20.39 ± 0.62
on 21st day on exposures to 1/5th LC50 of Cadmium. This GPx activity in the kidney was
slightly low and the values were to 20.43 ± 0.36 on 7th day, 22.37 ± 0.52 on 14th day and 17.24
± 0.41 on 21st day when exposed to 1/10th LC50 of Cadmium. Similar observations were made
in the kidney of those fish exposed to Chlorpyrifos (i.e. GPx activity was 14.14 ± 0.36 (7th
day), 16.50 ± 0.58 (14th day) and 18.32 ± 0.25 (21st day) at 1/5th LC50 concentration and this
133
trend was slightly less at 1/10th LC50 concentration). On the other hand, the significant
elevation in GPx activity in the kidney was noticed in those fish treated with a combination of
1/5th LC50 of Cadmium + Chlorpyrifos and the values ranged between 23.98 ± 0.62 (7th day),
25.65 ± 0.84 (14th day) and 22.86 ± 0.27 (21st day). Similarly, the GPx activity in the kidney
was moderately elevated between 19.72 ± 0.37 (7th day), 21.35 ± 0.57 (14th day) and 15.27 ±
0.76 (21st day) in fish exposed to 1/10th LC50 of Cadmium + Chlorpyrifos during 3 weeks of
experiments. Altered trend in GPx activity was also noticed in the kidney of those fish
exposed to a combination of Chlorpyrifos + Cadmium compared to control group during the
same period of exposure (Table 55). Here, the joint action toxicity of 1/5th LC50 of Cadmium +
Chlorpyrifos was moderately synergistic in nature while 1/10th LC50 of Cadmium +
Chlorpyrifos was simple additive in nature compared to individual toxicity of Cadmium.
However, the joint action of Chlorpyrifos + Cadmium was moderately synergistic in nature
compared to individual toxicity of Chlorpyrifos (Table 55).
The order of intensity of toxicity, in fish exposed to 1/5th LC50 and 1/10th LC50 was
Cadmium + Chlorpyrifos (112.65% and 42.05%) followed by Cadmium (89.67% and
60.37%), Chlorpyrifos + Cadmium (80.56% and 36.19%) and Chlorpyrifos (70.42% and
60.56%) in bringing about alterations in glutathione peroxidase activity in the kidney in
relation to control during 21 days of exposure (Fig. 76). However, there was moderate
alteration in the GPx activity in those fish exposed to 1/10th LC50 of Cadmium + Chlorpyrifos
and 1/10th LC50 of Chlorpyrifos + Cadmium.
Brain
The glutathione peroxidase (GPx) activity in the brain tissue of fish in the control
group varied between 11.03 ± 0.14 on 7th day, 11.35 ± 0.10 on 14th day and 11.13 ± 0.18 on
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21st day. This GPx activity got enhanced in the brain of those fish treated with 1/5 th LC50 of
Cadmium and the values ranged between 16.87 ± 0.49 on 7th day, 19.74 ± 0.37 on 14th day and
17.34 ± 0.26 on 21st day. Exposure of fish to 1/10th LC50 of Cadmium caused a slight
enhancement in the GPx activity in the brain tissue and the values varied between 12.62 ±
0.20 (7th day), 14.65 ± 0.33 (14th day) and 14.21 ± 0.11 (21st day). However, in those fish
exposed to 1/5th LC50 of Chlorpyrifos, the GPx activity in the brain registered lower activity
compared to 1/5th LC50 of Cadmium and the values were 13.91 ± 0.29 (7th day), 16.34 ± 0.39
(14th day) and 14.97 ± 0.51 (21st day). Similar trend in GPx activity was observed when fish
was exposed to 1/10th of LC50 of Chlorpyrifos. Further, considerable alteration in GPx activity
was evident in the brain of those fish exposed to a combination of Cadmium + Chlorpyrifos
compared to control group run parallely for 21 days (Table 56). In the same way, fish treated
with 1/5th LC50 of Chlorpyrifos + Cadmium registered significant altered activity of GPx in the
brain tissue and values ranged between 18.44 ± 0.40 (7th day), 21.86 ± 0.36 (14th day) and
19.96 ± 0.42 (21st day). This alteration trend in GPx activity was moderately less at 1/10 th
LC50 Chlorpyrifos + Cadmium compared to 1/5th LC50 of Chlorpyrifos + Cadmium during 21
days of exposure. The combined toxicity of both these toxicant combinations was moderately
synergistic in nature compared to their individual toxicities (Table 56).
Among the test toxicants, the altered glutathione peroxidase activity was more in the
brain of those fish exposed to 1/5th LC50 and 1/10th LC50 of Chlorpyrifos + Cadmium (79.34%
and 45.10%) followed by Cadmium + Chlorpyrifos (62.44% and 43.40%), Cadmium (55.80%
and 27.67%), and Chlorpyrifos (34.50% and 22.73%) during 21 days of experiments (Fig.
78).
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Muscle
The glutathione peroxidase (GPx) activity in the muscle tissue of control fish varied
between 4.14 ± 0.14 on 7th day, 4.54 ± 0.19 on 14th day and 4.46 ± 0.11 on 21st day. This
activity in the muscle increased to 5.53 ± 0.29 (7th day), 6.45 ± 0.13 (14th day) and 7.06 ± 0.21
(21st day) in fish treated with 1/5th LC50 of Cadmium. In the same way, those fish exposed to
1/10th of LC50 of Cadmium, the GPx activity was 5.29 ± 0.13 (7th day), 5.65 ± 0.20 (14th day)
and 6.37 ± 0.36 (21st day). On the other hand, exposure of fish to 1/5th LC50 of Chlorpyrifos
brought in enhanced GPx activity in the muscle compared to control fish and the activity was
between 4.66 ± 0.29 (7th day) and 5.75 ± 0.35 (21st day). The same trend was also noticed in
the muscle of those fish exposed to 1/10th LC50 of Chlorpyrifos i.e. GPx activity was 4.38 ±
0.28 (7th day) and 5.27 ± 0.30 (21st day). Further, fish exposed to a combination of Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium registered elevation in the GPx activity in the
muscle during the same period of exposure (Table 57). The combined toxicity of Cadmium +
Chlorpyrifos was simple additive in nature while 1/5th LC50 of Chlorpyrifos + Cadmium was
moderately synergistic and 1/10th LC50 of Chlorpyrifos + Cadmium was simple additive in
nature compared to their individual toxicities.
Comparison between control and treatment reveals that exposure of fish to 1/5th LC50
and 1/10th LC50 of Cadmium (58.31% and 42.92%) recorded higher activity of glutathione
peroxidase in the muscle followed by Cadmium + Chlorpyrifos (52.06% and 34.67%),
Chlorpyrifos + Cadmium (46.97% and 26.78%) and Chlorpyrifos (29.02% and 18.22%)
during 21 days of experiments (Fig. 80).
The changes in the activity of glutathione peroxidase were more or less similar to the
response of catalase activity. In the gills, liver, kidney and brain, the activity of glutathione
136
peroxidase enhanced significantly on 7th day and 14th day compared to that of control fishes.
Though the level of the enzyme activity exhibited an enhancement in test tissues (except
muscle), it decreased after 14th day of exposure reaching the minimal level on 21st day (Fig.
71, 73, 75, 77 & 79). The interaction between test organs and test toxicants reveals that the
highest glutathione peroxidase activity was in the gills (Cadmium) followed by kidney
(Cadmium + Chlorpyrifos), liver (Cadmium + Chlorpyrifos), brain (Chlorpyrifos + Cadmium)
and muscle (Cadmium) of the fish (Table 58 and Fig. 81). Two-way ANOVA followed by
Tukey's studentized range (HSD) test showed that there was significant (P<0.05) difference in
glutathione peroxidase activity between treated groups, days and tissues (Table 59a and 59b).
4.3.2.3.4 Glutathione S-transferase (GST)
The Tables 60, 61, 62, 63 and 64 represent the changes in the glutathione S-transferase
activity (nmol of CDNB conjugate formed/min/mg protein) in the gills, liver, kidney, brain
and muscle tissue of Tilapia (Oreochromis mossambicus) respectively, exposed to Cadmium
and Chlorpyrifos individually and to a combination of Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for a period of 7, 14 and 21 days under static renewal conditions.
Gills
The glutathione S-transferase (GST) activity in the gill tissue of control fish ranged
between 25.44 ± 0.32 on 7th day, 26.86 ± 0.20 on 14th day and 27.06 ± 0.27 on 21st day. This
activity was significantly increased to 44.75 ± 1.05 on 7th day, 51.30 ± 1.10 on 14th day and
56.22 ± 1.28 on 21st day in fish treated with 1/5th LC50 of Cadmium and 40.68 ± 0.44 on 7th
day, 46.74 ± 0.38 on 14th day and 52.71 ± 1.34 on 21st day on exposure to 1/10th LC50 of
Cadmium compared to control group run parallely for 21 days. Similarly, exposure of fish to
1/5th LC50 of Chlorpyrifos caused enhanced GST activity between 34.36 ± 0.35 (7th day),
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37.44 ± 0.41 (14th day) and 42.54 ± 0.37 (21st day) in the gills of Tilapia and those fish
exposed to 1/10th LC50 of Chlorpyrifos also showed enhanced GST activity and the values
varied between 31.41 ± 0.26 (7th day) and 39.76 ± 0.50 (21st day). On the contrary, exposure
of fish to a combination of Cadmium + Chlorpyrifos although progressively enhanced the
GST activity of the gills but it was less than that of individual toxicity of Cadmium (Table 60).
Hence the joint action toxicity of this combination was moderately antagonistic in nature.
Further, when fish was exposed to a combination of Chlorpyrifos + Cadmium, it registered
considerable elevation of GST activity in the gills during 21 days of exposure. The joint action
toxicity of Chlorpyrifos + Cadmium was synergistic in nature compared to individual toxicity
of Chlorpyrifos (Table 60).
Highest activity in the glutathione S-transferase, in relation to control, was recorded in
the gills of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (107.76% and
94.79%) followed by Cadmium + Chlorpyrifos (101.15% and 86.77%), Chlorpyrifos +
Cadmium (96.42% and 78.57%) and lowest was with Chlorpyrifos (57.21% and 46.93%)
during 3 weeks of exposure (Fig. 83).
Liver
The glutathione S-transferase (GST) activity in the liver tissue of the experimental fish
in the control group varied between 35.43 ± 0.38 on 7th day and 38.10 ± 0.40 on 21st day. This
value increased progressively to 52.33 ± 1.31 (7th day) and 69.43 ± 0.95 (21st day) in the liver
of those fish treated with 1/5th LC50 of Cadmium and 48.64 ± 0.39 (7th day) and 66.84 ± 1.77
(21st day) with 1/10th LC50 of Cadmium. Similarly, the GST activity in the liver of those fish
exposed to 1/5th LC50 of Chlorpyrifos also showed increment and the values were 42.75 ± 0.50
(7th day) and 56.15 ± 0.81 (21st day). In the same way, exposure of fish to 1/10th LC50 of
138
Chlorpyrifos also registered same intensity in GST activity in the liver tissue i.e. 40.24 ± 0.60
(7th day) and 54.98 ± 0.71 (21st day). However, fish treated with 1/5th LC50 of Cadmium +
Chlorpyrifos recorded significantly very high GST activity in the liver i.e. 54.86 ± 1.17 on 7th
day, 65.32 ± 0.41 on 14th day and 72.45 ± 1.90 on 21st day. Similar trend was also observed
when liver of those fish exposed to 1/10th LC50 of Cadmium + Chlorpyrifos (46.36 ± 0.78 on
7th day, 59.08 ± 1.29 on 14th day and 66.29 ± 0.82 on 21st day). Interestingly, the combined
toxicity at 1/5th LC50 of Cadmium + Chlorpyrifos was moderately synergistic in nature while
1/10th LC50 of Cadmium + Chlorpyrifos was simple additive in nature compared to individual
toxicity of Cadmium (Table 61).
Moreover, the GST activity in the liver substantially
increased in fish treated with a combination of Chlorpyrifos + Cadmium compared to control
fish during 21 days of exposure. Here the combined toxicity of Chlorpyrifos + Cadmium was
synergistic in nature compared to individual toxicity of Chlorpyrifos (Table 61).
Among the test toxicants, the higher activity of glutathione S-transferase was noticed
in the liver of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium + Chlorpyrifos
(90.16% and 73.99%) followed by Cadmium (82.23% and 75.43%), Chlorpyrifos + Cadmium
(77.32% and 62.78%) and Chlorpyrifos (47.38% and 44.30%) during 21 days of exposure
(Fig. 85). However, there was marginal variation in the activity of those fish exposed to 1/10 th
LC50 of Cadmium + Chlorpyrifos.
Kidney
The variation of glutathione S-transferase (GST) activity, in the kidney tissue of the
fish, in the control group was between 23.16 ± 0.30 on 7th day and 23.78 ± 0.25 on 21st day.
However, this activity enhanced to 35.98 ± 0.62 (7th day) and 43.63 ± 0.70 (21st day) on
exposure to 1/5th LC50 of Cadmium. The GST activity did not show much variation at 1/10th
139
LC50 of Cadmium (33.34 ± 0.41 on 7th day and 42.36 ± 0.66 on 21st day) compared to 1/5th
LC50 concentration. Similar observations were made in the kidney of those fish exposed to
Chlorpyrifos i.e. GST activity elevated to 28.51 ± 0.33 (7th day) and 35.33 ± 0.26 (21st day) at
1/5th LC50 concentration and 26.43 ± 0.18 (7th day) and 32.54 ± 0.38 (21st day) at 1/10th
concentration. Further, the GST activity significantly increased in the kidney and the values
were between 36.65 ± 0.58 on 7th day, 41.46 ± 0.60 on 14th day and 45.91 ± 0.75 on 21st day
in fish exposed to 1/5th LC50 of Cadmium + Chlorpyrifos. This increasing trend was evident
even at 1/10th LC50 of Cadmium + Chlorpyrifos and the values were 32.56 ± 0.37 (7th day),
36.84 ± 0.49 (14th day) and 40.94 ± 0.42 (21st day). Similarly, higher GST activity in the
kidney was also noticed in fish treated with a combination of Chlorpyrifos + Cadmium
compared to control group during 3 weeks of experiments. Here the joint action toxicity at
1/5th LC50 of Cadmium + Chlorpyrifos was moderately synergistic in nature while 1/10th LC50
of Cadmium + Chlorpyrifos was simple additive in nature compared to individual toxicity of
Cadmium, whereas Chlorpyrifos + Cadmium was synergistic in nature compared to individual
toxicity of Chlorpyrifos (Table 62).
Comparison between control and treatment reveals that exposure of fish to 1/5th LC50
and 1/10th LC50 of Cadmium + Chlorpyrifos (93.06% and 72.16%) recorded higher glutathione
S-transferase activity in the kidney followed by Cadmium (83.47% and 78.13%), Chlorpyrifos
+ Cadmium (79.65% and 64.30%) and Chlorpyrifos (48.57% and 36.84%) during 21 days of
exposure (Fig. 87). However, there was moderate alteration in the activity in those fish
exposed to 1/10th LC50 of Cadmium + Chlorpyrifos.
140
Brain
The normal activity of glutathione S-transferase (GST) in the brain tissue of control
fish was in the range of 18.54 ± 0.23 on 7th day and 21.09 ± 0.24 on 21st day. However,
exposure of fish to 1/5th LC50 of Cadmium caused elevation in the GST activity in the brain
and the values were 27.36 ± 0.37 (7th day) and 33.00 ± 0.55 (21st day). This increasing trend
was moderately less at 1/10th LC50 of Cadmium and the values ranged between 21.65 ± 0.21
on 7th day and 26.53 ± 0.38 on 21st day. On the other hand, the GST activity in the brain was
slightly increased in fish exposed to 1/5th LC50 of Chlorpyrifos (23.49 ± 0.30 on 7th day and
29.45 ± 0.27 on 21st day) and 1/10th of LC50 of Chlorpyrifos (20.31 ± 0.15 on 7th day and 25.98
± 0.57 on 21st day) compared to control fish. Further, enhancement of GST activity in the
brain was evident in fish exposed to a combination of Cadmium + Chlorpyrifos (Table 63).
While, fish treated with a combination of 1/5th LC50 of Chlorpyrifos + Cadmium registered
significant increment in GST activity in the brain tissue and the values were 29.53 ± 0.40 (7 th
day), 33.46 ± 0.39 (14th day) and 36.77 ± 0.53 (21st day). In the same way, when fish exposed
to 1/10th LC50 of Chlorpyrifos + Cadmium, the GST activity in the brain elevated to 26.54 ±
0.26 (7th day), 28.27 ± 0.19 (14th day) and 32.63 ± 0.54 (21st day) compared to control group.
The combined toxicity at 1/5th LC50 of Cadmium + Chlorpyrifos was simple additive in nature
while 1/10th LC50 of Cadmium + Chlorpyrifos was synergistic in nature compared to
individual toxicity of Cadmium and whereas in case of 1/5th LC50 and 1/10th LC50 of
Chlorpyrifos + Cadmium, the combined toxicity was synergistic in nature compared to
individual toxicity of Chlorpyrifos (Table 63).
A comparison of intensity of toxicity among toxicants reveal that combination of
Chlorpyrifos + Cadmium (74.35% and 54.72%) was more toxic followed by Cadmium +
141
Chlorpyrifos (61.97% and 49.55%), Cadmium (56.47% and 25.79%) and Chlorpyrifos
(39.64% and 23.19%) at their 1/5th LC50 and 1/10th LC50 concentrations during 21 days in
bringing about enhanced glutathione S-transferase activity in the brain of fish exposed (Fig.
89).
Muscle
The range of glutathione S-transferase (GST) activity, in the muscle tissue of control
fish was between 11.34 ± 0.14 on 7th day and 12.42 ± 0.16 on 21st day. This GST activity
increased in the muscle was between 15.32 ± 0.20 (7th day) and 17.43 ± 0.18 (21st day) in fish
treated with 1/5th LC50 of Cadmium. Similar observations were made in the muscle of those
fish exposed to 1/10th LC50 of Cadmium (13.86 ± 0.23 on 7th day and 16.08 ± 0.21 on 21st
day). However, in fish treated with 1/5th LC50 of Chlorpyrifos, the GST activity in the muscle
moderately enhanced and the values varied between 12.74 ± 0.11 (7th day) and 13.90 ± 0.13
(21st day). The GST activity did not show much variation at 1/10th LC50 of Chlorpyrifos (12.75
± 0.17 on 7th day and 13.31 ± 0.15 on 21st day). Further, the elevated trend in GST activity
was recorded in the muscle of those fish exposed to a combination of Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium during 3 weeks of exposure (Table 64). Here, the
joint action toxicity of both the combination was simple additive in nature. It means to say that
the combination of Cadmium + Chlorpyrifos and or Chlorpyrifos + Cadmium caused same
effect as that of individual toxicants and act similar in effecting GST activity in the muscle
tissue, at the above concentration and exposure period.
The GST activity in the muscle of those fish exposed to 1/5th LC50 and 1/10th LC50 of
Cadmium (40.34% and 29.47%) was more affected followed by Cadmium + Chlorpyrifos
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(39.61% and 26.01%), Chlorpyrifos + Cadmium (35.75% and 17.07%) and Chlorpyrifos
(11.92% and 7.17%) during 21 days of exposure (Fig. 91).
Glutathione S-transferase activity responded in a different manner compared to
glutathione peroxidase in the sense that it registered a continuous increase in its activity at
both the concentrations of 1/5th LC50 and 1/10th LC50 in the gills, liver, kidney, brain and
muscle (Fig. 82, 84, 86, 88 & 90). There was increase in GST activity with increase in
duration of exposure and the highest GST activity was recorded on 21st day in all the treatment
groups compared to 7th and 14th days of exposure. When a comparison was made between test
organs and test toxicants, the highest GST activity was observed in the gills (Cadmium)
followed by liver (Cadmium + Chlorpyrifos), kidney (Cadmium + Chlorpyrifos), brain
(Chlorpyrifos + Cadmium) and muscle (Cadmium) of the fish (Table 65 and Fig. 92). All
these observations are statistically significant at 5% levels (Table 66a and 66b).
4.3.2.3.5 Glutathione reductase (GR)
The Tables 67, 68, 69, 70 and 71 depict the details of glutathione reductase activity
(nmol of NADPH oxidized/min/mg protein) in the gills, liver, kidney, brain and muscle tissue
of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations (1/5th LC50 and
1/10th LC50) of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium, respectively.
Gills
The glutathione reductase (GR) activity in the gill tissue of control fish ranged between
8.43 ± 0.17 on 7th day, 9.29 ± 0.08 on 14th day and 9.12 ± 0.10 on 21st day. However, exposure
of fish to 1/5th LC50 of Cadmium caused significant elevation in the GR activity in the gills
and the range was between 21.35 ± 0.68 (7th day), 19.65 ± 0.34 (14th day) and 15.86 ± 0.36
143
(21st day). This elevating trend was moderately less at 1/10th LC50 of Cadmium and the values
were 17.41 ± 0.49 (7th day), 15.43 ± 0.28 (14th day) and 12.56 ± 0.24 (21st day). Similarly,
enhancement of GR activity in the gills of those fish treated with 1/5th LC50 of Chlorpyrifos
ranged between 9.71 ± 0.16 on 7th day and 13.99 ± 0.24 on 21st day. Only marginal elevation
in GR activity (9.04 ± 0.11 on 7th day and 12.50 ± 0.43 on 21st day), in the gills, was evident in
fish exposed to 1/10th LC50 of Chlorpyrifos. On the other hand, fish treated with a combination
of Cadmium + Chlorpyrifos and, Chlorpyrifos + Cadmium registered different activity of GR
in the gills compared to control group run parallely for 21 days (Table 67). The combined
toxicity of Cadmium + Chlorpyrifos was simple additive in nature while Chlorpyrifos +
Cadmium was synergistic in nature compared to their individual toxicities.
All those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (73.79% and
37.63%), Cadmium + Chlorpyrifos (64.04% and 27.66%), Chlorpyrifos (53.30% and 36.97%)
and Chlorpyrifos + Cadmium (49.46% and 13.08%) registered highest glutathione reductase
activity in the gills in relation to control during 21 days of exposure (Fig. 94). However, there
was slight deviation in GR activity in those fish exposed to 1/10th LC50 of Cadmium +
Chlorpyrifos.
Liver
The glutathione reductase (GR) activity in the liver tissue of the experimental fish in
the control group ranged between 10.65 ± 0.38 on 7th day, 11.54 ± 0.25 on 14th day and 12.30
± 0.55 on 21st day. This GR activity in the liver considerably increased between 26.58 ± 0.90
(7th day), 24.12 ± 0.91 (14th day) and 19.67 ± 0.73 (21st day) in fish treated with 1/5th LC50 of
Cadmium and 24.76 ± 0.63 (7th day), 22.56 ± 0.39 (14th day) and 17.05 ± 0.43 (21st day) with
1/10th LC50 of Cadmium. Similarly, in those fish treated with 1/5th LC50 of Chlorpyrifos, the
144
GR activity in the liver varied between 13.00 ± 0.26 (7th day) and 18.59 ± 0.52 (21st day). In
the same way, those fish exposed to 1/10th LC50 of Chlorpyrifos, the GR activity was to 12.72
± 0.42 on 7th day and 16.65 ± 0.60 on 21st day. However, significant increase in GR activity
between 28.56 ± 1.05 (7th day), 25.43 ± 0.82 (14th day) and 20.86 ± 0.62 (21st day) was
evident in the liver of those fish exposed to a combination of 1/5th LC50 of Cadmium +
Chlorpyrifos compared to control group run parallely for 21 days. Further, elevation in GR
activity was also noticed in the liver of those fish exposed to a combination of 1/10 th LC50 of
Cadmium + Chlorpyrifos and the values varied between 22.08 ± 0.18 (7th day), 21.85 ± 0.20
(14th day) and 16.48 ± 0.37 (21st day). Similar outcome was recorded in fish exposed to a
combination of Chlorpyrifos + Cadmium during 21 days of exposure (Table 68). The joint
action toxicity of Cadmium + Chlorpyrifos was simple additive in nature compared to
individual toxicity of Cadmium, whereas Chlorpyrifos + Cadmium was synergistic in nature
compared to individual toxicity of Chlorpyrifos.
Among the test toxicants, highest increased activity of glutathione reductase was
observed in the liver of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium +
Chlorpyrifos (69.59% and 33.98%) followed by Cadmium (59.92% and 38.62%),
Chlorpyrifos (51.14% and 35.37%) and Chlorpyrifos + Cadmium (50.65% and 23.66%)
during 21 days of exposure (Fig. 96). However, there was moderate alteration in the activity in
those fish exposed to 1/10th LC50 of Cadmium + Chlorpyrifos could be because the combined
toxicity was simple additive in nature.
Kidney
The glutathione reductase (GR) activity in the kidney tissue of control fish varied
between 7.80 ± 0.18 on 7th day, 7.51 ± 0.13 on 14th day and 7.81 ± 0.22 on 21st day. This
145
activity in the kidney was enhanced between 15.76 ± 0.26 (7th day), 14.31 ± 0.10 (14th day)
and 13.14 ± 0.06 (21st day), in fish treated with 1/5th LC50 of Cadmium and 14.52 ± 0.50 (7th
day), 12.69 ± 0.27 (14th day) and 11.09 ± 0.34 (21st day) on exposure to 1/10th of LC50 of
Cadmium. However, exposure of fish to 1/5th LC50 of Chlorpyrifos brought in moderate
elevated GR activity in the kidney and the values were between 9.17 ± 0.25 (7th day) and
12.43 ± 0.28 (21st day) and similar trend was also recorded at 1/10th LC50 of Chlorpyrifos.
Further, significant elevation in GR activity (16.89 ± 0.65 on 7th day, 14.54 ± 0.42 on 14th day
and 13.85 ± 0.30 on 21st day), in the kidney, was evident in fish exposed to a combination of
1/5th LC50 of Cadmium + Chlorpyrifos. On the other hand, fish exposed to a combination of
1/10th LC50 of Cadmium + Chlorpyrifos also registered elevation in GR activity in the kidney
and the range was between 14.27 ± 0.18 (7th day), 12.24 ± 0.23 (14th day) and 11.36 ± 0.14
(21st day). The same trend was noticed in GR activity in the kidney of those fish exposed to a
combination of Chlorpyrifos + Cadmium during the same period of exposure (Table 69). The
combined toxicity of Cadmium + Chlorpyrifos was simple additive in nature while
Chlorpyrifos + Cadmium was synergistic in nature compared to their individual toxicities.
In terms of percentage of intensity of toxicity, the order of toxicity was Cadmium +
Chlorpyrifos (77.27% and 45.40%), Cadmium (68.18% and 41.94%), Chlorpyrifos +
Cadmium (61.53% and 29.27%) and Chlorpyrifos (59.09% and 40.92%) respectively at their
1/5th LC50 and 1/10th LC50 concentrations over a period of 21 days in causing elevation in the
glutathione reductase activity in the kidney of fish exposed (Fig. 98). However, there was
moderate variation in the GR activity in those fish exposed to 1/10th LC50 of Chlorpyrifos +
Cadmium.
146
Brain
The glutathione reductase (GR) activity in the brain of fish varied between 7.12 ± 0.18
(7th day) and 5.85 ± 0.27 (21st day) on exposure to 1/5th LC50 of Cadmium and 5.33 ± 0.20 (7th
day) and 4.27 ± 0.18 (21st day) to 1/10th LC50 of Cadmium compared to control group run
parallely for 21 days. However, the values of GR activity in the brain of control fish were 3.37
± 0.12 on 7th day and 3.92 ± 0.20 on 21st day. Observations made in the brain of those fish
exposed to Chlorpyrifos reveals that the GR activity fluctuated between 5.79 ± 0.24 on 7th day
and 4.73 ± 0.11 on 21st day at 1/5th LC50 of Chlorpyrifos and 5.00 ± 0.17 on 7th day and 4.08 ±
0.16 on 21st day at 1/10th LC50 of Chlorpyrifos. Further, the enhancement of GR activity in the
brain was evident in those fish treated with a combination of 1/5th and 1/10th of LC50 of
Cadmium + Chlorpyrifos, respectively (Table 70). On the contrary, those fish exposed to a
combination of Chlorpyrifos + Cadmium showed significant increase in GR activity and the
values were between 7.68 ± 0.23 (7th day) and 6.31 ± 0.28 (21st day) at 1/5th LC50
concentration and, 6.42 ± 0.16 (7th day) and 5.42 ± 0.22 (21st day) at 1/10th LC50 concentration
compared to control fish during 21 days of experiments. Here the joint action toxicity of
Cadmium + Chlorpyrifos was simple additive while Chlorpyrifos + Cadmium was moderately
synergistic in nature compared to their individual toxicities (Table 118).
The glutathione reductase activity, in relation to control, was more affected in the brain
of those fish exposed to 1/5th LC50 and 1/10th LC50 of Chlorpyrifos + Cadmium (60.90% and
38.34%) followed by Cadmium + Chlorpyrifos (58.17% and 30.26%), Cadmium (49.17% and
9.02%), and Chlorpyrifos (20.67% and 4.13%) during 21 days of exposure (Fig. 100).
147
Muscle
The muscle tissue of the experimental fish in the control group showed the glutathione
reductase (GR) activity values between 2.02 ± 0.12 on 7th day, 2.67 ± 0.17 on 14th day and
3.04 ± 0.20 on 21st day. These values moderately increased between 3.83 ± 0.24 on 7th day,
4.46 ± 0.26 on 14th day and 4.86 ± 0.19 on 21st day in those fish treated with 1/5th LC50 of
Cadmium. Similarly, the GR activity in the muscle tissue registered a slight enhancement
between 3.19 ± 0.14 (7th day), 3.86 ± 0.18 (14th day) and 4.47 ± 0.23 (21st day) in fish exposed
to 1/10th LC50 of Cadmium. However, the GR activity did not show much variation at 1/5th
LC50 of Chlorpyrifos (2.21 ± 0.10 on 7th day, 2.89 ± 0.15 on 14th day and 3.56 ± 0.16 on 21st
day) compared to control group during 21 days of exposure. The same trend was noticed in the
muscle of those fish exposed to 1/10th LC50 of Chlorpyrifos i.e. GR activity varied between
2.14 ± 0.12 (7th day) and 3.37 ± 0.11 (21st day). Further, fish exposed to a combination of
Cadmium + Chlorpyrifos and, Chlorpyrifos + Cadmium registered moderate enhancement in
GR activity in the muscle tissue compared to control fish during the same period of exposure
(Table 71). Interestingly, the combined toxicity of 1/5th and 1/10th LC50 of Cadmium +
Chlorpyrifos was simple additive in nature, whereas 1/5th LC50 of Chlorpyrifos + Cadmium
was moderately synergistic and 1/10th LC50 of Chlorpyrifos + Cadmium was simple additive
in nature.
The order of intensity of toxicity, in fish exposed to 1/5th LC50 and 1/10th LC50 was
Cadmium (60.10% and 47.17%) followed by Cadmium + Chlorpyrifos (56.32% and 42.04%),
Chlorpyrifos + Cadmium (48.22% and 22.40%) and Chlorpyrifos (17.11% and 10.89%) in
bringing about alterations in glutathione reductase activity in the muscle tissue in relation to
control fish during 21 days of experiments (Fig. 102).
148
The glutathione reductase activity in the groups treated with Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium indicated statistically significant
(P<0.05) variation between them and also with the control group (Table 73a and 73b). The
changes recorded in glutathione reductase activity were more or less similar to the response of
superoxide dismutase when exposed to 1/5th LC50 and 1/10th LC50 of test toxicants. The
glutathione reductase activity enhanced significantly in all the test tissues after exposure to the
experimental concentrations of test toxicants compared to that of control group. Comparison
between test organs and test toxicants, reveals that the highest GR activity was observed in the
liver (Cadmium + Chlorpyrifos) followed by brain (Chlorpyrifos + Cadmium), gills
(Cadmium), kidney (Cadmium + Chlorpyrifos), and muscle (Cadmium) of the fish (Table 72
and Fig. 103). In all those tissues studied except muscle, the glutathione reductase activity
registered decreasing trend in both at low and high concentrations of Cadmium,
Cadmium+Chlorpyrifos and Chlorpyrifos+Cadmium after 7th day of exposure and this activity
further decreased on 21st day (Fig. 93, 95, 97 & 99). However, during this period the muscle
showed increasing trend in GR activity at all the test concentrations, showing more activity on
21st day (Fig. 101).
4.3.2.4 Non-enzymatic antioxidants
4.3.2.4.1 Total reduced glutathione (GSH)
The level of total reduced glutathione (µmol of GSH/gm wet weight of the tissue) in
the gills, liver, kidney, brain and muscle tissue of Tilapia (Oreochromis mossambicus)
exposed to Cadmium and Chlorpyrifos individually and to a combination of Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium have been summarized in Tables 74, 75, 76, 77 and
78, respectively.
149
Gills
The gills of control fish recorded reduced glutathione (GSH) level between 20.03 ±
0.21 on 7th day, 21.22 ± 0.14 on 14th day and 20.91 ± 0.14 on 21st day. This level in the gills,
was significantly increased to 36.32 ± 0.73 (7th day), 33.75 ± 0.90 (14th day) and 28.43 ± 0.41
(21st day) when exposed to 1/5th LC50 of Cadmium and 34.24 ± 0.52 (7th day), 30.77 ± 0.85
(14th day) and 25.54 ± 0.77 (21st day) at 1/10th of LC50 of Cadmium. However, there was
progressive elevation in the GSH level in the gills (22.52 ± 0.64 on 7th day, 25.70 ± 0.38 on
14th day and 27.55 ± 0.56 on 21st day) of those fish exposed to 1/5th LC50 of Chlorpyrifos.
Similar trend was observed when gills of those fish exposed to 1/10th LC50 of Chlorpyrifos
(21.16 ± 0.22 on 7th day and 25.08 ± 0.14 on 21st day). On the other hand, exposure of fish to a
combination of Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium caused considerable
enhancement in the GSH level in the gills during the same period of exposure (Table 74).
Here, the joint action toxicity of Cadmium + Chlorpyrifos was simple additive in nature while
Chlorpyrifos + Cadmium were synergistic in nature compared to their individual toxicities.
Comparison of intensity of toxicity among toxicants reveal that exposure of fish to
1/5th LC50 and 1/10th LC50 of Cadmium (35.96% and 22.14%) was more toxic followed by
Chlorpyrifos (31.76% and 19.94%), Cadmium + Chlorpyrifos (27.55% and 18.03%) and
Chlorpyrifos + Cadmium (15.54% and 13.53%) during 21 days in bringing about increased
reduced glutathione values in the gill tissue (Fig. 105).
Liver
The values of reduced glutathione (GSH) level in the liver of control fish were 37.21 ±
0.57 on 7th day, 36.98 ± 0.74 on 14th day and 38.64 ± 0.93 on 21st day. However, exposure of
Tilapia fish to 1/5th LC50 of Cadmium caused significant elevation in the GSH level in the
150
liver and the range was between 72.27 ± 3.52 (7th day), 61.49 ± 3.80 (14th day) and 53.22 ±
2.73 (21st day). This GSH level was also elevated to 62.33 ± 2.27 (7th day), 54.09 ± 1.59 (14th
day) and 46.87 ± 1.84 (21st day) at 1/10th LC50 of Cadmium. Similarly, progressive increase in
GSH level in the liver of those fish treated with 1/5th Chlorpyrifos was recorded and the values
ranged between 39.03 ± 1.23 (7th day), 46.43 ± 1.45 (14th day) and 50.64 ± 1.87 (21st day). On
the other hand, GSH level recorded was 38.37 ± 0.84 (7th day) and 48.56 ± 1.20 (21st day)
when exposed to 1/10th LC50 of Chlorpyrifos. Further, significant enhancement in GSH level,
in the liver, was evident in fish exposed to a combination of Cadmium + Chlorpyrifos and,
Chlorpyrifos + Cadmium (Table 75). The combined toxicity of Cadmium + Chlorpyrifos was
simple additive in nature while Chlorpyrifos + Cadmium was highly synergistic in nature
compared to their individual toxicities.
The above results reveal that exposure of fish to 1/5th LC50 and 1/10th LC50 of
Cadmium (37.73% and 21.30%) recorded higher reduced glutathione level in the liver
followed by Cadmium + Chlorpyrifos (33.67% and 17.86%), Chlorpyrifos (31.06% and
25.67%), and Chlorpyrifos + Cadmium (26.06% and 13.82%) during 21 days of exposure
(Fig. 107). Contrary to this, moderate variation in the reduced glutathione level was observed
in those fish exposed to 1/10th LC50 of Chlorpyrifos only.
Kidney
The reduced glutathione (GSH) level in the kidney tissue of control fish ranged
between 15.03 ± 0.16 on 7th day, 15.36 ± 0.21 on 14th day and 16.10 ± 0.12 on 21st day. This
level in the kidney varied to 24.98 ± 0.58 (7th day), 21.92 ± 0.67 (14th day) and 20.70 ± 0.33
(21st day) on exposure to 1/5th LC50 of Cadmium. This varying trend was evident even at
1/10th LC50 of Cadmium and the values were 20.34 ± 0.51 (7th day), 17.69 ± 0.22 (14th day)
151
and 16.47 ± 0.40 (21st day). While, exposure of fish to 1/5th LC50 of Chlorpyrifos though
induced increased GSH level in the kidney of Tilapia (15.94 ± 0.43 on 7th day, 17.26 ± 0.64 on
14th day and 18.86 ± 0.23 on 21st day) but it was less than that effected with Cadmium. But the
GSH level did not show much variation at 1/10th LC50 of Chlorpyrifos (15.73 ± 0.17on 7th day,
16.34 ± 0.32 on 14th day and 18.31 ± 0.52 on 21st day) compared to 1/5th LC50 concentration.
However, there was no significant difference between these two concentrations of
Chlorpyrifos. Further, when fish was treated with combination of Cadmium + Chlorpyrifos
and, Chlorpyrifos + Cadmium, it registered moderate variation of GSH level in the kidney
during 21 days of exposure. Here, the joint action toxicity of Cadmium + Chlorpyrifos was
simple additive in nature while the combination of Chlorpyrifos + Cadmium was moderately
synergistic in nature in altering GSH level compared to their individual toxicities (Table 76).
Comparison between control and treatment reveals that exposure of fish to 1/5th LC50
and 1/10th LC50 of Cadmium (28.57% and 2.30%) recorded higher level of reduced
glutathione in the kidney followed by Cadmium + Chlorpyrifos (26.34% and 4.29%),
Chlorpyrifos (17.14% and 13.73%) and Chlorpyrifos + Cadmium (10.12% and 4.84%) during
21 days of experiments (Fig. 109). However, considerable deviation in the reduced glutathione
level was observed in those fish exposed to 1/10th LC50 of Cadmium and Cadmium +
Chlorpyrifos.
Brain
The reduced glutathione (GSH) level in the brain tissue of fish in the control group
varied between 30.29 ± 0.47 on 7th day, 31.84 ± 0.20 on 14th day and 32.68 ± 0.28 on 21st day.
This GSH level got enhanced in the brain of those fish treated with 1/5th LC50 of Cadmium
and the values ranged between 48.53 ± 1.22 on 7th day, 43.67 ± 0.72 on 14th day and 36.37 ±
152
1.39 on 21st day. In the same way, exposure of fish to 1/10th LC50 of Cadmium also caused
enhancement in the GSH level in the brain tissue and the values varied between 41.75 ± 0.42
(7th day), 37.95 ± 0.59 (14th day) and 34.80 ± 0.48 (21st day). Similarly, those fish exposed to
1/5th LC50 of Chlorpyrifos, the GSH in the brain registered same level compared to 1/5th LC50
of Cadmium and the values were 47.64 ± 1.30 (7th day), 43.82 ± 0.73 (14th day) and 38.44 ±
0.77 (21st day). On the other hand, the GSH level showed significant variation at 1/10th LC50
of Chlorpyrifos (40.06 ± 0.44 on 7th day, 35.73 ± 0.87 on 14th day and 33.51 ± 0.64 on 21st
day) compared to 1/5th LC50 of Chlorpyrifos. Further, significant alteration in GSH level was
evident in the brain of those fish exposed to a combination of 1/5th LC50 of Cadmium +
Chlorpyrifos compared to control group run parallely for 21 days and values ranged between
54.43 ± 1.75 (7th day), 48.06 ± 1.33 (14th day) and 41.46 ± 0.91 (21st day) (Table 77). In the
same way, fish treated with 1/10th LC50 of Cadmium + Chlorpyrifos registered considerable
altered level of GSH in the brain tissue. This alteration trend in GSH level continued even at
1/5th LC50 and 1/10th LC50 of Chlorpyrifos + Cadmium during 21 days of exposure. The
combined toxicity of both these toxicant combinations was moderately synergistic in nature
compared to their individual toxicities (Table 77).
In terms of percentage of intensity of toxicity, the order of toxicity was Cadmium +
Chlorpyrifos (26.87% and 8.75%), Chlorpyrifos + Cadmium (24.39% and 8.41%),
Chlorpyrifos (17.63% and 2.54%) and Cadmium (11.29% and 6.49%), respectively at their
1/5th LC50 and 1/10th LC50 concentrations over a period of 21 days in causing variation in the
reduced glutathione level in the brain of fish exposed (Fig. 111). However, slight alteration in
the reduced glutathione level was observed in those fish exposed to 1/10th LC50 of
Chlorpyrifos only.
153
Muscle
The muscle tissue of the experimental fish in the control group showed the reduced
glutathione (GSH) level values between 14.34 ± 0.11 on 7th day, 14.79 ± 0.25 on 14th day and
15.87 ± 0.18 on 21st day. These values significantly increased between 18.50 ± 0.44 on 7th
day, 20.85 ± 0.60 on 14th day and 23.06 ± 0.37 on 21st day in those fish treated with 1/5th LC50
of Cadmium. Similarly, the GSH level in the muscle tissue registered progressive
enhancement between 18.33 ± 0.26 (7th day), 19.58 ± 0.54 (14th day) and 21.29 ± 0.26 (21st
day) in fish exposed to 1/10th LC50 of Cadmium. While, exposure of fish to 1/5th LC50 of
Chlorpyrifos though induced elevated GSH level in the muscle of Tilapia (15.36 ± 0.50 on 7th
day, 16.11 ± 0.29 on 14th day and 17.65 ± 0.39 on 21st day) but it was less than that effected
with Cadmium. However, the GSH level did not show much variation at 1/10th LC50 of
Chlorpyrifos (14.66 ± 0.26 on 7th day, 15.03 ± 0.13 on 14th day and 16.72 ± 0.11 on 21st day)
compared to control group during 21 days of exposure. Further, fish exposed to a combination
of Cadmium + Chlorpyrifos and, Chlorpyrifos + Cadmium registered considerable increment
in GSH level in the muscle tissue compared to control fish during the same period of exposure
(Table 78). The joint action toxicity at 1/5th and 1/10th LC50 of Cadmium + Chlorpyrifos was
simple additive compared to individual toxicity of Cadmium whereas 1/5th LC50 and 1/10th
LC50 of Chlorpyrifos + Cadmium was moderately synergistic in nature compared to individual
toxicity of Chlorpyrifos.
The GSH level in the muscle of those fish exposed to 1/5th LC50 and 1/10th LC50 of
Cadmium (45.31% and 34.15%) was more affected followed by Cadmium + Chlorpyrifos
(42.66% and 29.49%), Chlorpyrifos + Cadmium (24.39% and 21.36%) and Chlorpyrifos
(11.22% and 5.36%) during 21 days of exposure (Fig. 113).
154
The changes in the total reduced glutathione level were more or less similar to the
response of glutathione reductase. The GSH values in the gills, liver, kidney and brain of test
fishes were found to increase significantly on 7th day of exposure compared to that of control
fishes. In the experimental tissues, except muscle, the GSH level registered a continuous
reducing trend at 1/5th LC50 and 1/10th LC50 of Cadmium, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium after 7th day of exposure. The GSH level further reduced on 21st day
of exposure (Fig. 104, 106, 108 & 110). The muscle registered more or less enhanced level of
GSH at all the test concentrations during experimental periods showing more activity on 21st
day (Fig. 112). Comparison between test organs and test toxicants reveals that the percentage
increase in total reduced glutathione was more in the liver (Cadmium), brain (Cadmium +
Chlorpyrifos) and gills (Cadmium) compared to kidney (Cadmium) and muscle (Cadmium) of
the Tilapia fish (Table 79 and Fig. 114). Two-way ANOVA followed by Tukey's studentized
range (HSD) test revealed that there was significant (P<0.05) variation in total reduced
glutathione level between treatments, days and also between tissues (Table 80a and 80b).
4.3.2.4.2 Ascorbic acid (ASA)
The sublethal studies of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium on ascorbic acid content (µg ASA/gm wet weight of the tissue) in
the gills, liver, kidney, brain and muscle tissue of Tilapia (Oreochromis mossambicus),
exposed for a period of 21 days are depicted in Tables 81, 82, 83, 84 and 85, respectively.
Gills
The gills of control fish recorded ascorbic acid (ASA) content between 37.43 ± 0.12 on
7th day, 37.33 ± 0.28 on 14th day and 36.21 ± 0.19 on 21st day. However, this level of ASA in
the gill tissue of Tilapia fish was significantly decreased to 27.32 ± 0.45 (7th day), 25.75 ±
155
0.60 (14th day) and 20.85 ± 0.37 (21st day) on exposure to 1/5th LC50 of Cadmium and 30.85 ±
0.81 (7th day), 27.66 ± 0.20 (14th day) and 24.99 ± 0.28 (21st day) on exposure to 1/10th LC50
of Cadmium compared to control group run parallely for 21 days. Similar observations were
made in the gills of those fish exposed to 1/5th LC50 of Chlorpyrifos (i.e. ASA content reduced
to 33.56 ± 0.39 on 7th day and 28.22 ± 0.91 on 21st day). On the other hand, exposure of fish to
1/10th of LC50 of Chlorpyrifos resulted in decline of ASA level in the gills and the values were
33.91 ± 0.30 (7th day) and 30.20 ± 0.17 (21st day). However, there was no significant
difference between these two concentrations of Chlorpyrifos. Though the ASA content
reduced considerably in the gills of those fish exposed to a combination of Cadmium +
Chlorpyrifos, the joint action toxicity at 1/5th LC50 of Cadmium + Chlorpyrifos was
moderately antagonistic while 1/10th LC50 of Cadmium + Chlorpyrifos was simple additive in
nature compared to individual toxicity of Cadmium. Similarly, decreased ASA content in the
gills continued in fish treated with another combination of Chlorpyrifos + Cadmium. Contrary
to combined toxicity of Cadmium + Chlorpyrifos, the joint action toxicity of Chlorpyrifos +
Cadmium was moderately synergistic in nature compared to individual toxicity of
Chlorpyrifos (Table 81).
In terms of percentage of intensity of toxicity, the order of toxicity was Cadmium
(42.42% and 30.99%), Cadmium + Chlorpyrifos (31.98% and 29.77%), Chlorpyrifos +
Cadmium (28.83% and 26.46%) and Chlorpyrifos (22.07% and 16.60%) respectively at their
1/5th LC50 and 1/10th LC50 concentrations over a period of 21 days in causing reduction in the
ascorbic acid content in the gills of fish exposed (Fig. 116).
156
Liver
The ascorbic acid (ASA) content in the liver tissue of the experimental fish in the
control group ranged between 60.35 ± 1.27 on 7th day, 63.64 ± 0.48 on 14th day and 64.26 ±
0.29 on 21st day. This ASA value significantly reduced to 48.34 ± 1.50 on 7th day, 41.85 ±
1.31 on 14th day and 31.57 ± 0.85 on 21st day in the liver of those fish treated with 1/5th LC50
of Cadmium. The same trend was noticed in the liver (53.79 ± 0.74 on 7th day, 46.32 ± 0.48 on
14th day and 43.21 ± 0.92 on 21st day) of those fish treated with 1/10th LC50 of Cadmium
during the same exposure period. However, the ASA content in the liver showed moderate
depletion and the value was 56.09 ± 1.80 (7th day) and 47.24 ± 1.43 (21st day) in fish exposed
to 1/5th LC50 of Chlorpyrifos while exposure to 1/10th of LC50 of Chlorpyrifos resulted in ASA
values 56.87 ± 0.67 (7th day) and 50.12 ± 0.46 (21st day). On the contrary, fish treated with a
combination of Cadmium + Chlorpyrifos although registered considerable decrease in ASA
content (Table 82), the combined toxicity at 1/5th LC50 of Cadmium + Chlorpyrifos was
antagonistic while 1/10th LC50 of Cadmium + Chlorpyrifos was simple additive in nature
compared to individual toxicity of Cadmium. On the other hand, fish exposed to a
combination of Chlorpyrifos + Cadmium registered progressive reduction in ASA content in
the liver and here contrary to combined toxicity of Cadmium + Chlorpyrifos, the joint action
toxicity of Chlorpyrifos + Cadmium was synergistic, compared to individual toxicity of
Chlorpyrifos (Table 82).
Lowest level in the ascorbic acid, in relation to control, was observed in the liver of
those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (50.87% and 32.76%) followed
by Cadmium + Chlorpyrifos (38.13% and 34.30%), Chlorpyrifos + Cadmium (35.36% and
31.70%) and Chlorpyrifos (26.49% and 22.00%) during 21 days of experiments (Fig. 118).
157
However, there was marginal variation in the ASA content in those fish exposed to 1/10th LC50
of Cadmium only.
Kidney
The ascorbic acid (ASA) content in the kidney tissue of control fish was in the range of
48.52 ± 0.37 on 7th day, 48.87 ± 0.22 on 14th day and 47.43 ± 0.53 on 21st day. On the
contrary, this level decreased considerably to 33.66 ± 1.48 (7th day), 24.05 ± 1.27 (14th day)
and 18.47 ± 1.50 (21st day) in the kidney of those fish treated with 1/5th LC50 of Cadmium and
continued even at 1/10th LC50 of Cadmium and the values were 37.32 ± 0.52 on 7th day, 29.12
± 1.10 on 14th day and 24.59 ± 1.35 on 21st day. However, the ASA content in the kidney was
moderately reduced to 42.64 ± 1.87 (7th day) and 35.07 ± 0.47 (21st day) at 1/5th LC50 of
Chlorpyrifos. But the ASA content did not show much variation at 1/10th LC50 of Chlorpyrifos
(43.98 ± 0.56 on 7th day and 36.65 ± 0.39 on 21st day) compared to 1/5th LC50 concentration.
On the other hand, in a combination of Cadmium + Chlorpyrifos, the ASA content in the
kidney although progressively depleted (35.42 ± 0.64 on 7th day and 21.62 ± 0.80 on 21st day
at 1/5th LC50 concentration and 39.86 ± 0.32 on 7th day and 25.27 ± 0.78 on 21st day at 1/10th
LC50 concentration) during 21 days of exposure, the combined toxicity of Cadmium +
Chlorpyrifos was moderately antagonistic in nature compared to individual toxicity of
Cadmium. While, low ASA content in the kidney was also recorded in fish exposed to a
combination of Chlorpyrifos + Cadmium during the same period of exposure (Table 83). The
combined toxicity of this combination was synergistic in nature compared to individual
toxicity of Chlorpyrifos.
The ascorbic acid content in the kidney was more affected in those fish exposed to
1/5th LC50 and 1/10th LC50 of Cadmium (61.06% and 48.16%) followed by Cadmium +
158
Chlorpyrifos (54.42% and 46.72%), Chlorpyrifos + Cadmium (52.52% and 41.22%) and
Chlorpyrifos (26.06% and 22.73%) during 21 days of exposure (Fig. 120).
Brain
The ascorbic acid (ASA) content in the brain of fish varied between 34.78 ± 0.70 (7th
day) and 26.32 ± 1.20 (21st day) on exposure to 1/5th LC50 of Cadmium and 37.65 ± 0.57 (7th
day) and 31.34 ± 0.42 (21st day) to 1/10th LC50 of Cadmium compared to control group run
parallely for 21 days. However, the values of ASA content in the brain of control fish were
41.66 ± 0.42 on 7th day and 41.13 ± 0.34 on 21st day. Similarly, those fish exposed to
Chlorpyrifos registered ASA content between 33.43 ± 1.40 (7th day) and 25.46 ± 0.91 (21st
day) at 1/5th LC50 concentration and, 37.52 ± 0.66 (7th day) and 33.57 ± 1.19 (21st day) at
1/10th LC50 concentration. While, fish treated with a combination of 1/5th LC50 of Cadmium +
Chlorpyrifos recorded significant decline in ASA content in the brain tissue and the values
were 31.24 ± 0.86 (7th day), 25.54 ± 0.63 (14th day) and 22.05 ± 0.37 (21st day). In the same
way, when fish exposed to 1/10th LC50 of Cadmium + Chlorpyrifos, the ASA content in the
brain decreased to 35.12 ± 0.92 (7th day), 32.89 ± 0.76 (14th day) and 28.20 ± 1.26 (21st day)
compared to control group. Further, the declining trend in ASA content was continued in the
brain of those fish exposed to a combination of Chlorpyrifos + Cadmium during 3 weeks of
exposure (Table 84). The combined toxicity of both these toxicant combinations was
moderately synergistic in nature compared to their individual toxicities.
Comparison of intensity of toxicity among toxicants reveal that exposure of fish to
1/5th LC50 and 1/10th LC50 of Cadmium + Chlorpyrifos (46.39% and 31.44%) was more toxic
followed by Chlorpyrifos + Cadmium (42.43% and 28.57%), Chlorpyrifos (38.10% and
18.38%) and Cadmium (36.01% and 23.80%) during 21 days in bringing about decreased
159
ascorbic acid values in the brain tissue (Fig. 122). However, there was moderate deviation in
the ASA content in those fish exposed to 1/10th LC50 of Chlorpyrifos.
Muscle
The muscle tissue of fish in the control group showed ascorbic acid (ASA) content
between 31.07 ± 0.24 on 7th day and 31.22 ± 0.36 on 21st day. On the other hand, the ASA
content in the muscle progressively declined to 23.22 ± 0.48 on 7th day and 20.12 ± 0.15 on
21st day in fish exposed to 1/5th LC50 of Cadmium. Similar trend was recorded even at 1/ 10th
of LC50 of Cadmium and the values were 25.41 ± 0.78 (7th day) and 20.90 ± 0.87 (21st day)
during the same period of exposure. Further, exposure of fish to 1/5th LC50 and 1/10th LC50 of
Chlorpyrifos caused reduction in the ASA content in the muscle of Tilapia (28.33 ± 0.98 on 7th
day and 24.28 ± 0.55 on 21st day at 1/5th LC50 and 28.90 ± 0.24 on 7th day and 26.55 ± 0.78 on
21st day at 1/10th LC50). However, decrease in ASA content in the muscle tissue was evident in
fish exposed to a combination of Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
compared to control group run parallely for 21 days (Table 85). Here, the joint action toxicity
of Cadmium + Chlorpyrifos was simple additive in nature while Chlorpyrifos + Cadmium
were moderately synergistic in nature compared to their individual toxicities.
Among the test toxicants, highest decrease in ascorbic acid content was observed in the
muscle of those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (35.55% and 33.06%)
followed by Cadmium + Chlorpyrifos (33.44% and 30.91%), Chlorpyrifos + Cadmium
(28.09% and 24.02%) and Chlorpyrifos (22.23% and 14.96%) during 21 days of exposure
(Fig. 124).
Significant (P<0.05) changes in the level of ascorbic acid were evident with respect to
concentration, duration of exposure and tissues due to exposure to Cadmium, Chlorpyrifos,
160
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium (Table 87a and 87b). In general, the
ascorbic acid content was below the control values in the tissues at the two sublethal
concentrations of test toxicants and the level of ASA was lower in kidney, liver, brain, gills
and muscle as the tissues showed a lower response on 7th, 14th and 21st day of exposure (Fig.
115, 117, 119, 121 & 123 ). These results reveal that, the reduction in ASA content was
directly proportional to the concentration of the test toxicants and exposure time. When a
comparison was made between test organs and test toxicants, the percentage decrease in
ascorbic acid content was more in the kidney (Cadmium) followed by liver (Cadmium), brain
(Cadmium + Chlorpyrifos), gills (Cadmium) and muscle (Cadmium) of Tilapia fish (Table 86
and Fig. 125).
4.3.2.5 Total protein
The experimental results of changes in the total protein content (mg protein/100gm wet
weight of the tissue) in the gills, liver, kidney, brain and muscle tissue of Tilapia
(Oreochromis mossambicus) exposed to sublethal concentrations (1/5th LC50 and 1/10th LC50)
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium have been
prescribed in Tables 88, 89, 90, 91 and 92, respectively.
Gills
The total protein content in the gill tissue of control fish ranged between 25.06 ±
0.062, 26.14 ± 0.058 and 26.77 ± 0.080 on 7th day, 14th day and 21st day, respectively. This
protein content was significantly reduced to 18.45 ± 0.093 on 7th day, 16.5 ± 0.028 on 14th day
and 13.28 ± 0.049 on 21st day in fish treated with 1/5th LC50 of Cadmium and 22.41 ± 0.061 on
7th day, 21.28 ± 0.058 on 14th day and 19.79 ± 0.038 on 21st day on exposure to 1/10th LC50 of
Cadmium compared to control group run parallely for 21 days. Similarly, exposure of fish to
161
1/5th LC50 of Chlorpyrifos caused depleted protein content between 24.06 ± 0.017 (7th day),
23.64 ± 0.042 (14th day) and 21.52 ± 0.018 (21st day) in the gills of Tilapia and those fish
exposed to 1/10th LC50 of Chlorpyrifos also showed decline in protein content and the values
varied between 24.44 ± 0.193 (7th day) and 22.26 ± 0.061 (21st day). On the other hand, fish
was exposed to a combination of Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium also
registered considerable reduction of protein content in the gills during 21 days of exposure
(Table 88). The combined toxicity of Cadmium + Chlorpyrifos was simple additive in nature
compared to individual toxicity of Cadmium while Chlorpyrifos + Cadmium was moderately
synergistic in nature compared to individual toxicity of Chlorpyrifos (Table 88).
A comparison of intensity of toxicity among the toxicants, reveals that Cadmium
(50.39% and 26.07%) was more toxic followed by Cadmium + Chlorpyrifos (42.85% and
30.44%), Chlorpyrifos + Cadmium (33.47% and 24.69%) and Chlorpyrifos (19.61% and
16.85%) at their 1/5th LC50 and 1/10th LC50 concentrations during 21 days in bringing about
reduction in protein content in the gills of fish exposed (Fig. 127). However, only in those fish
exposed to 1/10th LC50 of Cadmium there was marginal variation in the protein content.
Liver
The total protein content in the liver of Tilapia fish varied considerably between 34.01
± 0.066 (7th day) and 23.09 ± 0.072 (21st day) on exposure to 1/5th LC50 of Cadmium and
36.26 ± 0.067 (7th day) and 26.92 ± 0.087 (21st day) to 1/10th LC50 of Cadmium compared to
control group run parallely for 21 days. The protein content in the liver of control fish were
45.21 ± 0.022 on 7th day and 48.95 ± 0.223 on 21st day. Similarly, the protein content in the
liver of those fish exposed to 1/5th LC50 of Chlorpyrifos also showed depletion and the values
were 41.67 ± 0.055 (7th day) and 36.27 ± 0.153 (21st day). In the same way, exposure of fish to
162
1/10th LC50 of Chlorpyrifos also registered reduction in protein content in the liver tissue i.e.
42.48 ± 0.081 (7th day) and 38.41 ± 0.043 (21st day). Further, fish treated with 1/5th LC50 of
Cadmium + Chlorpyrifos recorded significantly low protein content in the liver i.e. 33.93 ±
0.071 on 7th day, 26.11 ± 0.058 on 14th day and 18.35 ± 0.037 on 21st day and the joint action
toxicity at 1/5th LC50 of Cadmium + Chlorpyrifos was moderately synergistic in nature
compared to individual toxicity of 1/5th LC50 of Cadmium (Table 89). On the contrary, fish
treated with a combination of 1/10th LC50 of Cadmium + Chlorpyrifos although registered
significant decrease in protein content (37.06 ± 0.072 on 7th day, 33.75 ± 0.036 on 14th day
and 29.38 ± 0.092 on 21st day), the combined toxicity at 1/10th LC50 of Cadmium +
Chlorpyrifos was moderately antagonistic in nature compared to individual toxicity of 1/10th
LC50 of Cadmium. On the other hand, the protein content in the liver progressively decreased
in fish treated with a combination of Chlorpyrifos + Cadmium compared to control fish during
21 days of exposure. Here the joint action toxicity of Chlorpyrifos + Cadmium was synergistic
in nature compared to individual toxicity of Chlorpyrifos (Table 89).
Lowest protein content, in relation to control, was recorded in the liver of those fish
exposed to 1/5th LC50 and 1/10th LC50 of Cadmium + Chlorpyrifos (62.51% and 39.98%)
followed by Cadmium (52.83% and 45.01%), Chlorpyrifos + Cadmium (35.18% and 32.20%)
and Chlorpyrifos (25.90% and 21.53%) during 3 weeks of exposure (Fig. 129). But moderate
alteration in the protein content, in those fish exposed to a combination of Cadmium +
Chlorpyrifos with 1/10th LC50 concentration could be because the combined toxicity was
moderately antagonistic in nature.
163
Kidney
The total protein content in the kidney tissue of control fish varied between 22.14 ±
0.037 on 7th day, 22.89 ± 0.081 on 14th day and 22.73 ± 0.067 on 21st day. On the contrary,
this protein content reduced to 16.23 ± 0.153 (7th day) and 13.04 ± 0.075 (21st day) on
exposure to 1/5th LC50 of Cadmium. While, the protein content did not show much variation at
1/10th LC50 of Cadmium (17.58 ± 0.084 on 7th day and 14.66 ± 0.032 on 21st day) compared to
1/5th LC50 concentration. Similar observations were made in fish exposed to Chlorpyrifos i.e.
protein content declined to 20.25 ± 0.019 (7th day) and 17.34 ± 0.102 (21st day) at 1/5th LC50
concentration and 20.83 ± 0.014 (7th day) and 18.46 ± 0.081 (21st day) at 1/10th concentration.
On the other hand, the protein content significantly decreased in the kidney and the values
were between 15.06 ± 0.132 on 7th day, 13.75 ± 0.094 on 14th day and 11.88 ± 0.053 on 21st
day in fish exposed to 1/5th LC50 of Cadmium + Chlorpyrifos. This decreasing trend was
evident even at 1/10th LC50 of Cadmium + Chlorpyrifos and the values were 18.3 ± 0.065 (7th
day), 17.11 ± 0.148 (14th day) and 15.04 ± 0.077 (21st day). However, lower protein content in
the kidney was also noticed in fish treated with a combination of Chlorpyrifos + Cadmium
compared to control group during 21 days of experiments. Here, the joint action toxicity of
both the combination was simple additive in nature. It means to say that the combination of
Cadmium + Chlorpyrifos and or Chlorpyrifos + Cadmium caused same effect as that of
individual toxicants and act similar in effecting protein content in the kidney during 21 days of
experiments (Table 90).
The protein content in the kidney of those fish exposed to 1/5th LC50 and 1/10th LC50 of
Cadmium + Chlorpyrifos (47.73% and 33.83%) was more affected followed by Cadmium
(42.63% and 35.50%), Chlorpyrifos + Cadmium (29.04% and 25.43%) and Chlorpyrifos
164
(23.71% and 18.79%) during 21 days of exposure (Fig. 131). On the other hand, there was
marginal variation in the protein content only in those fish exposed to 1/10th LC50 of Cadmium
+ Chlorpyrifos could be because the joint action toxicity was simple additive in nature.
Brain
The total protein content in the brain tissue of control fish was in the range of 30.41 ±
0.162 on 7th day and 32.77 ± 0.075 on 21st day. However, exposure of fish to 1/5th LC50 of
Cadmium caused reduction in the protein content in the brain and the values were 24.09 ±
0.072 (7th day) and 20.51 ± 0.098 (21st day). This decreasing trend was moderately less at
1/10th LC50 of Cadmium and the values ranged between 27.91 ± 0.033 on 7th day and 24.07 ±
0.081 on 21st day. On the other hand, the protein content in the brain was significantly
decreased in fish exposed to 1/5th LC50 of Chlorpyrifos (22.19 ± 0.059 on 7th day and 18.42 ±
0.033 on 21st day) and but only slight decrease was observed at 1/10th of LC50 of Chlorpyrifos
(28.36 ± 0.059 on 7th day and 25.39 ± 0.025 on 21st day) compared to control fish. Further,
depletion of protein content in the brain was evident in fish exposed to a combination of
Cadmium + Chlorpyrifos (Table 91). While, fish treated with a combination of 1/5th LC50 of
Chlorpyrifos + Cadmium registered significant decrease in protein content in the brain tissue
and the values were 21.03 ± 0.134 (7th day), 19.62 ± 0.037 (14th day) and 14.37 ± 0.078 (21st
day). In the same way, when fish was exposed to 1/10th LC50 of Chlorpyrifos + Cadmium, the
protein content in the brain declined to 25.38 ± 0.041 (7th day), 24.29 ± 0.096 (14th day) and
22.05 ± 0.074 (21st day) compared to control group. The joint action toxicity of Cadmium +
Chlorpyrifos was simple additive in nature compared to individual toxicity of Cadmium while
Chlorpyrifos + Cadmium was moderately synergistic compared to individual toxicity of
Chlorpyrifos (Table 91).
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The above results reveal that exposure of fish to 1/5th LC50 and 1/10th LC50 of
Chlorpyrifos + Cadmium (56.15% and 32.71%) recorded lower protein content in the brain
followed by Chlorpyrifos (43.79% and 22.52%), Cadmium + Chlorpyrifos (41.26% and
27.34%) and Cadmium (37.41% and 26.55%) during 21 days of exposure (Fig. 133). Contrary
to this, moderate variation in the protein content was observed in those fish exposed to 1/10th
LC50 of Chlorpyrifos only.
Muscle
The range of total protein content, in the muscle tissue of control fish was between
37.16 ± 0.089 on 7th day and 39.94 ± 0.298 on 21st day. This protein content significantly
depleted between 23.77 ± 0.077 (7th day), 19.03 ± 0.260 (14th day) and 15.57 ± 0.026 (21st
day) in fish treated with 1/5th LC50 of Cadmium. However, in those fish exposed to 1/10th LC50
of Cadmium, the protein content in the muscle was higher compared to 1/5th LC50 of Cadmium
and the values were 31.04 ± 0.086 (7th day) and 27.64 ± 0.055 (21st day). On the other hand, in
fish treated with 1/5th LC50 of Chlorpyrifos, the protein content in the muscle tissue
moderately reduced and the values varied between 34.64 ± 0.018 (7th day) and 31.28 ± 0.070
(21st day). The protein content did not show much variation at 1/10th LC50 of Chlorpyrifos
(35.88 ± 0.025 on 7th day and 32.73 ± 0.047 on 21st day). Though the protein content reduced
significantly in the muscle tissue of those fish exposed to a combination of 1/5th LC50 of
Cadmium + Chlorpyrifos, the joint action toxicity at 1/5th LC50 of Cadmium + Chlorpyrifos
was moderately antagonistic in nature compared to individual toxicity of 1/5th LC50 of
Cadmium (Table 92). However, reduced trend in protein content was also observed in the
muscle of those fish exposed to a combination of 1/10th LC50 of Cadmium + Chlorpyrifos and
the joint action toxicity at 1/10th LC50 of Cadmium + Chlorpyrifos was moderately synergistic
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in nature compared to individual toxicity of 1/10th LC50 of Cadmium. Further, decreased
protein content in the muscle continued in fish treated with another combination of
Chlorpyrifos + Cadmium. Here, the joint action toxicity at 1/5th LC50 of Chlorpyrifos +
Cadmium was synergistic while 1/10th LC50 of Chlorpyrifos + Cadmium was moderately
synergistic in nature compared to individual toxicity of Chlorpyrifos (Table 92).
Among the test toxicants, the lower protein content was noticed in the muscle of those
fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (61.02% and 30.80%) followed by
Cadmium + Chlorpyrifos (53.61% and 39.66%), Chlorpyrifos + Cadmium (45.12% and
25.49%) and Chlorpyrifos (21.68% and 18.05%) during 21 days of exposure (Fig. 135).
However, there was moderate alteration in the protein content of those fish exposed to 1/10th
LC50 of Cadmium.
Two-way ANOVA followed by Tukey's studentized range (HSD) test showed that
there was significant (P<0.05) variation in total protein content level between treatments,
duration of exposure and also between tissues (Table 94a and 94b). In all the experimental
tissues, the levels of total protein content showed continuous decrease in both at low and high
concentrations of test toxicants after 7th day of exposure and activity was least on 21st day of
exposure (Fig. 126, 128, 130, 132 & 134). Comparison between test organs and test toxicants
reveals that the percentage decrease in total protein content was more in the liver (Cadmium +
Chlorpyrifos) followed by muscle (Cadmium), brain (Chlorpyrifos + Cadmium), kidney
(Cadmium + Chlorpyrifos) and gills (Cadmium) of the fish (Table 93 and Fig. 136).
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V. DISCUSSION
The presence of a xenobiotic compound in an aquatic ecosystem does not, by itself,
indicate injurious effects. Therefore, a relationship must be established between external levels
of exposure, internal levels of tissue contamination and early adverse effects. Many of the
hydrophobic organic compounds and their metabolites, which contaminate aquatic ecosystem,
have yet to be identified and their impact on aquatic life has yet to be determined. It is in this
context, the environmental toxicologists have more interest on the study of the exposure, fate
and effects of chemical contaminants or pollutants in the aquatic ecosystem.
The pollution of aquatic system in general and coastal waters, rivers and streams in
particular, with chemical contaminants, has become one of the most critical environmental
problems of the century. Heavy metals, pesticides and other organic chemicals are the harmful
group of pollutants in aquatic habitat. Aquatic ecosystem is almost incapable of degrading
such foreign compounds though some organisms posses limited capabilities for metabolizing
them. Therefore, anthropogenic organic chemicals released into the freshwater and marine
environments tend to accumulate and cause long term effects (Gupta and Aggarwal, 2007).
Adverse effects at the organismal level include both short-term and long-term lethality
(expressed as mortality or survival) and at suborganismal level include induction or inhibition
of enzymes and/or enzyme systems and their associated functions, changes in behaviour,
metabolic activity, growth, development, reproduction, uptake and detoxification activity and
tissue structure.
5.1 Lethal toxicity (LC50)
Lethal toxicity is expressed as the lethal concentration (LC50), that is the concentration
in water which kills 50% of a test batch of fish within a continuous period of exposure of 96 h
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(Amweg et al., 2005). The application of the LC50 has gained acceptance among toxicologists
and is generally the most highly rated test of assessing potential adverse effects of chemical
contaminants to aquatic life (Gad and Saad, 2008 and Khayatzadeh and Abbasi, 2010). The
use of 96 h, LC50 has been widely recommended as a preliminary step in toxicological studies
on fishes (APHA, 2005; Parrott et al., 2006 and Moreira et al., 2008). Death is usually used as
a criterion of change in the 96 hour test, while the extension of duration can be adopted for
investigation of physiological and biochemical changes at concentrations considerably below
the lethal concentration.
Fish mortality due to heavy metals and pesticide exposure mainly depends upon its
sensitivity to the toxicants, its concentration and duration of exposure time (Kamble et al.,
2011). The evaluation of LC50 concentration of pollutants is an important step before carrying
out further studies on physiological changes in animals. The lethal toxicity data provides
useful information to identify the mode of action of a substance and also help in comparison
of dose response among different chemicals. In the present studies no fish died during the
acclimation period before exposure and in control group during lethal toxicity tests. The 96 h
LC50 tests are conducted to measure the vulnerability and survival potential of fishes to
particular toxic chemical. The probit analysis revealed that, the LC50 values for Cadmium,
Chlorpyrifos were found to be 169.80 ppm and 0.022 ppm respectively.
One of the most important areas in pollution bioassay experiments is the analysis of
combined toxicity of mixture of pollutants, effluents or mixture of chemicals, rather than
single chemical. Pollutants of dissimilar chemical nature interact in a variety of ways in water
of various compositions. Most studies on the effects of environmental pollutants are limited to
reporting the effects of either pesticide treatment or metal exposure individually and only few
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reports are available on the effects of multiple stressors of pesticide contamination and heavy
metal pollution on aquatic organisms. Studying the effects of mixture of contaminants on
organisms is necessary because aquatic organisms are exposed to combinations of toxicants in
their environment. The lethal toxicity of combination of Cadmium (varying concentrations) +
Chlorpyrifos (fixed concentration i.e. 1/5th of its LC50 Value) and Chlorpyrifos (varying
concentrations) + Cadmium (fixed concentration i.e. 1/5th of its LC50 Value) indicated the 96
h LC50 value for the Tilapia fingerlings as 92.04 ppm and 0.016 ppm respectively. These
results indicate that, Cadmium in combination with Chlorpyrifos become more toxic
(synergistic) compared to its individual exposure. Similarly, Chlorpyrifos become more toxic
(synergistic) to experimental fish fingerlings in combination with Cadmium compared to its
individual exposure (Table 7). In order to categorize the toxicants according to the results
obtained from the toxicity tests, the values of LC 50 were converted to toxic units (TU). The
toxicity categorization was established using toxic unit ranges (highly toxic (TU > 100); very
toxic (10 > TU < 100); toxic (1 > TU < 10); and less toxic (TU < 1) (Pablos et al., 2011). The
results of toxic units reveals that, Chlorpyrifos + Cadmium (TU- 6250) and Chlorpyrifos (TU
– 4545.45) are highly toxic, Cadmium + Chlorpyrifos (TU - 1.08) is toxic and Cadmium (TU
– 0.58) is less toxic (Table 6).
Several studies have been conducted in assessing the toxicity of heavy metals and
pesticides to the aquatic biota especially fishes (Vasait and Patil, 2005 and Susan et al., 2010).
The results of the present work are in agreement with previous lethal studies for Cadmium
performed by El-Moselhy (2001) on Mugil seheli, Sobha et al. (2007) on Catla catla, Shukla
et al. (2007) on Channa punctatus. Similarly the results of the present study are in parallel
with previous lethal studies for Chlorpyrifos by Oruc (2010) on Oreochromis niloticus,
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Sharbidre et al. (2011) on Poecilia reticulate, Kavithaa and Venkateshwara (2008) on
Gambusia affinis and Xing et al. (2012) on common carp. Works on the combined toxicity of
heavy metal (Cadmium) and pesticide (Chlorpyrifos) are scanty. Nikam et al. (2011) in their
studies have mentioned that the acute toxicity study is essential to find out the toxicants limit
and safe concentration, so that there will be minimum harm to aquatic fauna. Among the
several aspects of toxicity studies, the bioassay constitutes one of the most commonly used
methods in aquatic environmental studies with suitable organisms. The necessity of
determining the toxicity of substances to commercially aquatic forms at the lower level of the
food chain has been useful and accepted for water quality management.
5.2 Sublethal toxicity
The effects of exposure to sublethal levels of pollutants can be measured in terms of
biochemical, physiological or histological responses of the fish (Mondon et al., 2001). Over
the last decades, biomarkers at suborganismal levels of organization have been considered to
be viable measures of responses to stressors (Huggett et al., 1992). In acute water pollution
incidents, the physiological disturbances of fish are well known, e.g., respiratory distress, loss
of locomotor ability and behavioural alterations. Such responses to environmental stressors
have very little value as biomarkers because they are insensitive endpoints from the ecosystem
perspective and give little information on environmental contamination. In an extreme case,
death is indicative that the lethal threshold has been exceeded. In contrast, when the exposure
is chronic or sublethal, changes in physiological and biochemical parameters within the
natural homeostasis variability associated with biotic and abiotic parameters allow correlating
those changes with the effects of exposure to pollutants (Handy and Depledge, 1999).
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Altered biochemical machinery is a prelude to variation in physiology, serving as
forecasting signals of toxicity. Induction of physiological and biochemical stress in the
experimental fish, Oreochromis mossambicus by exposing them to individual and combination
of heavy metal, Cadmium and organophosphorous pesticide, Chlorpyrifos and estimating it by
physiological responses, cholinesterase and antioxidant enzyme assays is the mainstay of
present study. Measurement of biochemical and physiological parameters is a commonly used
diagnostic tool in aquatic toxicology and biomonitoring. Biochemical parameters assessed in
fish may provide quantitative measurement of metals impact as well as valuable information
of ecological relevance on the effects of metals (Oner et al., 2009). Biochemical and
physiological biomarkers have been used in order to prevent irreversible damage in whole
organisms, communities and ecosystems (Lopez-Barea and Pueyo, 1998).
5.2.1 Physiological responses
In the last few decades, acute toxicity tests based on mortality were widely used as
indication of toxicity. However, in the environment most of the exposures of organisms to
pollutants are at sublethal levels, so these acute toxicity tests have restricted value in practice
(Maltby and Naylor, 1990). Since the 1960s, researchers interested in environmental studies
have used a variety of physiological responses to account for the effect of pollutants on
individual organisms and great efforts have been made to assess the stress indices with
predictive value. It is well recognized that acute exposures to pollutants that kill few
organisms may or may not have ecological impact, where as chronic exposures that cause
delays in development, diseases, reproductive malfunctions or decrease in probability of
survival, might result in ecological consequences (Moriarty, 1988). In this sense, some
behavioural and physiological integrative responses may be advantageous to account for the
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health condition of individual organisms and some of them could provide feasible
explanations of higher organization levels. In general, behavioural and physiological
responses that give relevant information on the environmental impact of chemicals are those
that describe the performance, account for the health of individual organisms and indicate
their chance of survival or long term opportunity to reproduce (Vaughn et al., 1984).
Behaviour of fish
During the experimental period, the control fish behaved in a natural manner i.e., they
were actively feeding and were alert to slightest disturbance with their well synchronized
movements. In Cadmium media, Tilapia exhibited abnormal behaviour in the form of erratic
swimming, enhanced surfacing behaviour, gulping of air, hyperactivity, restlessness, difficulty
in respiration and vertical movement of the fish due to loss of equilibrium. The gulping of air
may help to avoid contact of toxic medium and surfacing phenomenon might be a demand of
higher oxygen level during the exposure period (Katja et al., 2005).Ural and Simsek (2006)
stated that surfacing phenomenon is because of the catastrophic impact posed by the toxicant.
Fish showed disrupted shoaling behaviour, localization to the bottom of the test chamber, and
independency (spreading out) in swimming. At the start of the exposure, the fish exposed to
the Cd became alert; with the progression of the experiment, the fish stopped swimming and
remained in static position in response to the sudden changes in the surrounding environment.
When exposed to Chlorpyrifos, fish exhibited gulping air, rapid opercular movements,
excess secretion of mucus, disrupted shoaling behaviour, irregular, erratic and darting
swimming movements, loss of equilibrium, followed by hanging vertically in water and
hitting to the walls of the test tank before finally sinking to the bottom just before death. These
behaviour changes take place to maintain normal posture and balance with increasing
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exposure time (Pandey et al., 2009). When exposed to lethal concentration, body surface
acquired dark colour before their death which is one of the symptoms of Chlorpyrifos toxicity.
Similar observations were made by Ural and Simsek (2006) and Chebbi and David (2010) in
European catfish fingerlings exposed to Dichlorovos and Common carp to Quinalphos
respectively. Ramesh and Munniswamy (2009) also reported an excess secretion of mucus in
Cyprinus carpio when exposed to Chlorpyrifos. Pesticides in sublethal concentrations present
in the aquatic environment are too low to cause rapid death directly, but may affect the
functioning of the organisms, disrupt normal behaviour and reduce the fitness of natural
population.
In the combinations of Chlorpyrifos + Cadmium; fish swimming at the water surface,
gulping air, dullness, stop of food intake, circling movement, abnormal swimming, loss of
equilibrium, rapid opercular movements and excess secretion of mucus was observed during
exposure period. Prior to death, the fish became less active or generally inactive, remained
hanging vertically in the water or lay down on their sides at higher concentrations. The
behavioural changes were severe in fish, exposed to lethal than that of the sublethal
concentrations of test compounds. A similar observation was also noticed by Dube (2010) in
Labio rohita (Ham) when exposed to lethal, 1/3rd and 1/5th sublethal concentrations of Sodium
cyanide. Altered behavioural changes include erratic swimming, fast jerky movements and
convulsions which is again dose dependent (Scott and Sloman, 2004). These symptoms are
due to inhibition of acetylcholine esterase (AChE) activity leading to accumulation of
acetylcholine in cholinergic synapses ensuring hyperstimulation.
In general, fish poisoned with anticholinesterase insecticides show signs of muscle
paralysis, especially of the fins and respiratory apparatus, hyperactivity and loss of balance
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(Sancho et al., 1997). The organophosphorous pesticide exposed fish exhibited irregular,
erratic and darting movements and loss of equilibrium due to inhibition of AChE activity
leading to accumulation of acetylcholine in cholinergic synapses ending up with
hyperstimulation (Patil V. K and David M. 2008). Dembele et al. (2000) indicated that the
abnormalities in fish behaviour observed in exposure with organophosphorous insecticides
(Chlorfenvinphos and Diazinon) could be related to failure of energy production or the release
of stored metabolic energy, which may cause severe stress, leading to the death of the fish.
Behavioural ecotoxicology provides an approach that clearly links disturbances at the
biochemical level to effects at the population level either in a direct or indirect way (AmiardTriquet, 2009). Because behavioural disturbances may be observed in aquatic biota at
concentrations of contaminants that can exist in the field, the sensitivity of these responses can
allow improving environmental risk assessment. Therefore, to use behavioural biomarkers,
associated to biochemical and physiological markers in carefully selected species that are keyspecies in the structure and functioning of ecosystems because impairments of their responses,
used as biomarkers, will reveal a risk of cascading deleterious effects at the community and
ecosystem levels.
Thus, behaviour can be considered as a promising tool in ecotoxicology (Little and
Brewer, 2001). Behaviour is both a sequence of quantifiable actions, operating through the
central and peripheral nervous systems (Gravato and Guilhermino, 2009), and the cumulative
manifestation of genetic, biochemical, and physiologic processes essential to life, such as
feeding, reproduction and predator avoidance (Moreira et al., 2006). It allows an organism to
adjust to external and internal stimuli in order to best meet the challenge of surviving in a
changing environment.
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Oxygen consumption rate
The respiratory potential or oxygen consumption of an animal is one of the indicators
of the general well being of the fish and important physiological parameter to assess the toxic
stress. It may also be useful to assess the physiological state of an organism, helps in
evaluating the susceptibility or resistance potentiality and also useful to correlate the
behaviour of the animal, which ultimately serve as predictors of functional disruptions of
population. Hence, the analysis of oxygen consumption can be used as a biodetectory system
to evaluate the basic damage inflicted on the animal which could either increase or decrease
the oxygen uptake.
In the present study, oxygen consumption rate per hour was lower at both sublethal
concentrations (1/5th of LC50 and1/10th of LC50) when compared to control during 7, 14 and 21
days of exposure to Cadmium and Chlorpyrifos. Reduction in oxygen consumption was
observed in fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (71.28% and 60.14%),
Cadmium + Chlorpyrifos (65.82% and 57.28%), Chlorpyrifos + Cadmium (55.06% and
51.28%) and Chlorpyrifos (39.18% and 33.95%). Lowest oxygen consumption rates were
attained during 21st day in 1/5th LC50 of Cadmium, Cadmium + Chlorpyrifos, Chlorpyrifos +
Cadmium and Chlorpyrifos. Highest oxygen was consumption rate were attained during 21 st
day in control. It is clearly evident from the results that test compounds affected the oxygen
consumption rate of fish under sublethal concentrations. A decrease in the respiratory rate in
both the sublethal concentrations due to toxicants induced stress, avoidance and
biotransformation. If gills or membrane functions are destroyed due to xenobiotic chemicals or
the membrane functions are disturbed by a change in permeability the oxygen uptake rate
would rapidly decrease (Grinwis et al., 1998 and Hartl et al., 2001).
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According to Tilak and Satyavardhan, (2002), the decrease in oxygen consumption at
sublethal concentration of the toxicant appears to be lowering of energy requirements, in such
a case, because of highly toxic action maintenance of energy requirement is considered and
the decrease in oxygen consumption is going to cause pronounced haematological changes.
The fluctuated response in respiration may be attributed to reduction in gill permeability
causing a drop in oxygen consumption for which the fish compensates by increasing the
ventilation volume as observed by Kalavathy et al. (2001).
Barbieri et al. (2009) found that exposure of juveniles of Geophagus brasiliensis
(Osteichthyes, Cichlidae) to 40 ppm 2,4-D herbicide (2,4-Dichlorophenoxyacetic acid) caused
reduction in oxygen consumption of 59%, in relation to the controls. In present study, the
oxygen consumption reduced in sublethal concentration of Cadmium and Chlorpyrifos. Tilak
and Swarna kumari, (2009) stated that, the depletion of the oxygen consumption is due to the
disorganization of the respiratory action caused by rupture in the respiratory epithelium of the
gill tissue. The decrease in oxygen consumption uptake from pesticide water is mainly due to
shrinkage of the respiratory epithelium since there is swelling of the secondary lamellar tips
(Natarajan, 1981). Chebbi et al. (2010) recorded that Quinalphos exposed fish, Cyprinus
carpio showed a significant decrease in the whole animal oxygen consumption in sublethal
doses for 1, 2, 3 and 4 days. Similar observation was also recorded in the present study. If
gills are destroyed due to xenobiotic chemicals or the membrane functions are disturbed by a
changed permeability, the oxygen uptake rate would rapidly decrease; altered respiratory rate
can be correlated with the altered opening and closing of opercular coverings and mouth
(Chebbi et al., 2010).
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As aquatic organisms have their outer bodies and important organs such as gills almost
entirely exposed to water, the effect of toxicants on the respiration is more pronounced.
Toxicants enter into the fish mainly through gills and with the onset of symptoms of
poisoning, the rate of oxygen consumption may increases or decreases. Holden (1973)
observed that one of the earliest symptoms of acute pesticide poisoning is respiratory distress.
This serves not only as a tool in evaluating the susceptibility or resistance potentiality of the
animal, but also useful to correlate the behaviour of the animal. Oxygen consumption of
aquatic animals is a very sensitive physiological process and therefore, alteration in the
respiratory activity is considered as an indicator of stress of animals exposed to heavy metals
and pesticides.
Food consumption rate
Change in feeding behaviour is considered to be a sensitive indicator to detect
pollution due to heavy metals and pesticides. The results revealed that the food consumption
rate decreased significantly in the fishes exposed to Cadmium and Chlorpyrifos when
compared to the control for 21 days. The fishes in control continued to consume the feed
throughout the experiment and remained in a good condition. The control fish had
significantly higher average feed intake than that of treated fish. Decrease in food
consumption was observed in those fish exposed to 1/5th LC50 and 1/10th LC50 of Cadmium
(91.30% and 84.91%), Cadmium + Chlorpyrifos (87.84% and 82.80%), Chlorpyrifos +
Cadmium (73.28% and 68.08%) and Chlorpyrifos (60.78% and 54.10%). The highest food
consumption was attained during 21st day in control and lower food consumption was attained
during 21st day in 1/5th LC50 of Cadmium, Cadmium + Chlorpyrifos, Chlorpyrifos + Cadmium
and Chlorpyrifos. The food consumption was very less in Cadmium and combination of
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Cadmiun + Chlorpyrifos at 21 days. The decreased food intake may be due to damage caused
to taste receptors as reported by Foster et al. (1966). In this context, Heath (1987) is of the
opinion that the fish subjected to long term exposure to pollutants exhibited reduction in the
appetite. The mechanism for this has not been determined, but probably is in part caused by
hormonal changes. These hormonal changes could cause a direct inhibition of feeding.
Cadmium and Chlorpyrifos had a significant effect on functional activity of Tilapia
since an increase in test concentrations induced a decrease in food consumption rate in all the
concentration when compared to control. Oreochromis mossambicus exposed to sublethal
level of Phosphamidon and Methyl parathion significantly affected the rates of feeding,
absorption, metabolism and conversion (Singh et al., 2010). Fish experiencing acute exposure
to sublethal concentrations of the insecticide exhibited significant feeding impairment with
potentially severe consequences for their ecological fitness (Floyd et al., 2008) presumably,
the decrease in food intake may be due to increase in metabolic rate associated with tissue
repair and development of defense and copper excreting metabolisms (Broeck et al., 1997).
According to Jezierska et al. (2006), embryonic and larval exposures of Common carp
to Copper, Cadmium, or mix of both metals (Cu 0.2 mg /l, Cd 0.2 mg /l, Mix – 0.1 ppm of Cu
+ 0.1 ppm of Cd) considerably impaired feeding activity. Similarly, in present work the
Tilapia fingerling exposed to heavy metal and pesticide consumed less food when compared to
the control. Sarnowski (2004) revealed that the heavy metals might have disturbed the
development of locomotors abilities that might also adversely affect the feeding abilities.
According to Ali et al. (2003), Oreochromis niloticus refused to accept the feed immediately
after exposure to sublethal concentrations of Copper (0, 0.15, 0.3 and 0.5 ppm) and only began
taking it up after about 4-5 h as compared with the control. Exposure of the fish to different
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Copper concentrations in water significantly reduced their feed consumption as compared with
the control. These results are similar to the results of the present work. The lowered food
intake of fish could be due to the effects of Cu on the central nervous system (Ali et al., 2003).
Ammonia-N excretion rate
Ammonia is the main excretion product of aquatic organisms and results from the
degradation of proteins, by which the amino acids are transformed into energy useful to the
cell. Due to its solubility and low molecular weight, ammonia diffuses very rapidly and can
largely be lost through the surface that is in contact with the water without the need of being
excreted by the kidney. In teleost fish most of the nitrogen is lost as ammonia through the
gills. Most bony fishes excrete predominantly ammonia, which is energetically advantageous
compared to converting ammonia to urea. However, there are several examples of increased or
decreased excretion of ammonia in fish subjected to stressful conditions, such as
environmental contaminants, high concentrations of environmental ammonia, high pH, air
exposure, or crowding (Frick and Wright, 2002). The rate of ammonia release to the water is
therefore closely related to the production of ammonia by the fish.
In the present study, ammonia-N excretion rate per hour was decreased with increased
exposure duration and lowest ammonia-N excretion rate was reached during 21st day in 1/5th
LC50 of Cadmium and combination of Cadmium + Chlorpyrifos, whereas in control, it was
increased with increased duration and highest ammonia-N excretion rate was reached in 21st
day. Depletion in ammonia-N excretion was observed in fish exposed to 1/5th LC50 and 1/10th
LC50 of Cadmium (83.25% and 76.23%), Cadmium + Chlorpyrifos (78.49% and 72.28%),
Chlorpyrifos + Cadmium (71.60% and 67.13%) and Chlorpyrifos (53.57% and 46.47%). A
decrease in the ammonia-N excretion rate in both the sub lethal concentrations due to
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toxicants induced stress, avoidance and biotransformation. According to Grosell et al. (2004),
a decrease in ammonia excretion rate as a result of metal induced stress together with an
impaired ability to excrete ammonia across the gill is the typical response to metal exposure in
freshwater fish. This indicates that test compounds affected the ammonia-N excretion rate of
fish under sub lethal concentrations.
Barbieri et al. (2009) found that exposure of juveniles of Geophagus brasiliensis to 40
mg/l 2,4-D herbicide (2,4-Dichlorophenoxyacetic acid) caused 85% reduction in ammonium
excretion compared to the controls. Chinni et al. (2000 and 2002) observed that ammonium
excretion was inhibited in P. indicus post larvae exposed to sublethal concentrations of Lead.
It is assumed that the decrease in ammonia-nitrogen excretion by P. indicus post larvae in the
presence of toxicants can be attributed to a reduction in the metabolic rate or to an interaction
of Lead with pathways for the production of ammonia-nitrogen.
As ammonia is highly toxic, it must either be excreted or be converted to less toxic end
products, such as urea or uric acid. Under adverse environmental conditions where ammonia
excretion is reduced, some fishes can reduce the rate of ammonia production from amino acid
catabolism to slow down the buildup of ammonia internally (Ip et al., 2004a and b). Many
fishes are aminotelic but some species can detoxify ammonia to glutamine or urea. Certain
fish species can accumulate high levels of ammonia in the brain or defense against ammonia
toxicity by enhancing the effectiveness of ammonia excretion through active NH4+ transport,
manipulation of ambient pH, or reduction in ammonia permeability through the branchial and
cutaneous epithelia (Chew et al., 2006b). Ammonia excretion provides important information
on the physiological condition of the organism. It can be used as indicators of fish nitrogen
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balance and to determine the effects of environmental and nutritional factors on protein
metabolism (Fournier et al., 2003).
Oxygen:Nitrogen ratio
Oxygen consumption and ammonia excretion provide a wider view of fish metabolism.
As most of the nitrogenous end products of freshwater fish originate from protein catabolism,
with ammonia as the principal end product, the contribution of protein catabolism to the total
energy production of the fish can be assessed by determination of the Oxygen:Nitrogen ratio
(means mole to mole ratio of oxygen consumed to ammonia excreted).
In the present study, elevation in O:N ratio in relation to control was observed in fish
exposed to 1/5th LC50 and 1/10th LC50 of Cadmium (71.48% and 67.74%), Cadmium +
Chlorpyrifos (58.93% and 54.13%), Chlorpyrifos + Cadmium (58.27% and 48.26%) and
Chlorpyrifos (31.02% and 23.42%). In sublethal concentrations the O:N ratio was increased
with increased exposure duration and highest O:N ratio was attained during 21st day in 1/5th
LC50 of Cadmium and combination of Cadmium + Chlorpyrifos, whereas in control, it was
decreased with increased duration and lowest O:N ratio was observed in 21st day. Fish
exposed to sublethal concentrations of test compounds showed a gradual increase in O:N ratio
in all the exposed duration, when compared to the control. Results showed that Cadmium,
Chlorpyrifos and combinations of Cadmium + Chlorpyrifos affected the O:N ratio of Tilapia
under sublethal concentrations. Inhibition of oxygen consumption and increase ammonium
excretion by Cd has been reported in Litopenaeus vannamei (Wu and Chen, 2004) and
Litopenaeus shmitti (Barbieri, 2007) and has been attributed to mucus production because it
reduces the efficiency of gaseous exchange.
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Santos et al. (2006) noticed that juvenile Florida Pompano, Trachinotus carolinus after
acute exposures showed a tendency to increase oxygen consumption by virtue of Naphthalene
concentrations. After chronic exposures, a decrease was observed at the highest concentration
evidencing a narcotic effect of Naphthalene. Ammonia excretion was reduced significantly, as
compared to that of the controls, in all the exposed organisms. The O:N ratios of fish exposed
to different concentrations of Naphthalene were higher than that of the controls. In the present
study similar observation was made, where fish exposed to sublethal concentrations of test
toxicants showed an increase in O:N ratio when compared to the control. Chinni et al. (2000)
recorded a high O:N ratio in Penaeus indicus post larvae exposed to Lead, when compared to
control in all exposure days. Amin
et al. (2010) observed significantly higher O:N ratio in
commercial crab, Lithodes santolla (Decapoda: Anomura) larvae when exposed to sublethal
concentrations of Copper (40, 80 and 160 μg L-1) than in the controls for 96 h. Similar
observation was made in present study, where the Oxygen:Nitrogen ratio showed significantly
higher in sublethal concentrations of Cadmium and Chlorpyrifos when compared to control.
Nitrogenous waste excretion in aquatic species is accomplished largely at the gills.
Because these species have very large convective water volume requirements in order to
extract oxygen (owing to the low concentration of oxygen in water relative to air), aquatic
species are typically ‘hyperventilated’ with respect to waste gases such as CO2 and NH3. Thus,
in many cases no additional ventilator energy needs to be invested by water breathers to
effectively excrete nitrogenous waste to the water, beyond that used to take up oxygen from
the water. Oxygen:Nitrogen ratio provides a wider view of physiological status of the
organism. It can be used to determine the effects of environmental conditions on fish
metabolism.
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Relative growth rate
The growth rate is an index associated with stress and is generally used as a sensitive
and reliable end-point in chronic toxicity investigations (Rosas et al., 2001; Benimeli et al.,
2003 and Huang and Chen, 2004). In the present study, the growth rate was significantly
affected by the test concentrations. Fish exposed to sublethal concentrations of test toxicants
showed a drastic decrease in growth rate in all the exposed duration, when compared to the
control. Reduction in growth rate in relation to control was observed in fish exposed to 1/5th
LC50 and 1/10th LC50 of Cadmium (291.62% and 270.39%), Cadmium + Chlorpyrifos
(283.80% and 216.76%), Chlorpyrifos + Cadmium (220.11% and 202.79%) and Chlorpyrifos
(127.93% and 112.29%) at 21 days. In sublethal concentrations the growth was decreased with
increased exposure duration and lowest growth rate was reached during 21 st day in 1/5th LC50
of Cadmium and combination of Cadmium + Chlorpyrifos, whereas in control, it was
increased with increased duration and highest growth rate was reached in 21st day.
Xenobiotics are potentially harmful to fish by inducing growth retardation (Gad and Sadd,
2008). The decrease in the feed intake and increase in energy utilized for respiration are the
major reasons for decrease in the growth (Verslycke et al., 2004).
According to Kasthuri and Chandran (1997), food intake has been identified as a prime
factor influencing growth by developing appetite, is found to be affected by toxicants. Growth
decreases when toxicant-exposed fish spend more energy sustaining their normal metabolism,
leaving less energy available for growth (De Boeck et al., 1995). This suggests that present
study on Cadmium and Chlorpyrifos induced the growth rate of fish under sublethal
concentrations. Sapana and Gupta (2014) revealed the significant reduction in growth and
feeding in Anabas testudineus exposed to sublethal concentrations of Deltamethrin and
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Permethrin pesticides. Exposed fish exhibited erratic swimming behavior that is likely to have
impaired their ability to feed, thereby affecting growth.
Ferrari et al. (2011) found a significant decrease in the growth of juvenile Cyprinus
carpio exposed to sublethal concentration of Cd (0.15 mg l-1) when compared to control for
two weeks. Similar results were observed in the present study, the Tilapia exposed to sublethal
concentrations of Cadmium and Chlorpyrifos showed a significant decrease in the growth
compared to control. De Boeck et al. (1995) observed that growth was decreased in sublethal
levels of Copper-exposed common carp, Cyprinus carpio spent more energy sustaining their
normal metabolism, the cessation of feeding, accompanied by the catabolic effects of the
catecholamines and corticosteroids on the energy reserves stored in the body tissues, resulted
in reduced growth in stressed fish.
Growth of an organism is generally used as a sensitive and reliable endpoint in chronic
toxicological investigations. Sublethal levels of a wide variety of toxicants have been found to
slow the growth of fish larvae or juveniles. This can be due to a reduced food intake, but also
due to increased metabolic expenditure for detoxification and maintenance of the normal body
functions. The rate of growth is a fundamental measure of physiological fitness/performance
and provides one of the most sensitive measures of stress in organisms.
Aquatic organisms are subjected to long-term stresses from exposure to sublethal
concentrations of heavy metals and pesticides cause deleterious effects as lethal concentrations
(Ramasamy et al., 2007), because sublethal effects on aquatic organisms may change
physiological responses by altering their behavioural pattern, metabolic rate, the excretion of
ions (e.g., ammonium), respiration, food consumption, and growth rates (Alves et al., 2006
and Hashemi et al., 2008). Gills comprise a large part of the fish body in contact with the
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external environment and play a key role in gas, ion, and water exchanges between the
extracellular fluids and the surroundings (Evans et al., 2005). They also represent an important
site of uptake of heavy metals (Ossana et al., 2009), which can be accumulated at levels
several orders of magnitude above those found in the environment causing lesions that impair
gas and ion exchange (Ferrari et al., 2009 and Witeska et al., 2006). The changes in rate of
oxygen consumption, food consumption, ammonia-N excretion, Oxygen:Nitrogen ratio and
growth rate can be used as a biodetector in monitoring the physiological effects of heavy
metals and pesticides and the oxygen consumption pattern will indicate the possible mapping
of metabolic pathways influenced by the pollution stress.
5.2.2 Biochemical responses
Biochemical markers have been used as a research tool in toxicology, ecotoxicology
and pharmacology (Gohil, 2012). Biochemical techniques are a rapid, sensitive and reliable
tool for the assessment of stress responses to xenobiotics. Evaluation of biochemical
manifestations provides insight into the degree of stress, susceptibility and adaptive capability
of the stressed organism (Sreenivasan et al., 2011). Biochemical markers or biomarkers are
measurable responses to the exposure of an organism to xenobiotics. They usually respond to
the mechanisms of toxic activity and not to the presence of specific xenobiotic and therefore
may react to a group of either similar or heterogenous xenobiotics. Biochemical markers
detect the type of toxicity; in some of them, the magnitude of their response correlates with the
level of pollution (Haluzova et al., 2011). The present study was carried out to investigate the
biochemical markers of Cadmium (Cd) and Chlorpyrifos (CPF) in gill, liver, kidney, brain and
muscle tissues of freshwater fish Oreochromis mossambicus, by exposing it to sublethal
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concentrations of the individual and combinations of heavy metal and pesticide for varying
treatment durations of 7, 14 and 21 days.
Cholinesterase enzymes
Fish cholinesterases (ChE) have been widely used as biomarkers of effect to
organophosphate pesticides and heavy metals; the toxicity of these chemicals is due to the
inhibition of acetylcholinesterase (AChE), a key enzyme of the nervous system.
Butyrylcholinesterase (BChE) is another cholinesterase that has also been used as a biomarker.
BChE is found in plasma, liver, muscle and other tissues, and in some cases it has shown to be
more sensitive to pesticides than AChE (Chambers, Boone, Chambers, & Straus, 2002). Some
authors have proposed using muscle and other non nervous tissues for the assessment of ChE,
because its sensitivity, ease of collection and availability of larger quantity of material.
Acetylcholinesterase (AChE)
Acetylcholinesterase activities in the gills, liver, kidney, brain and muscle tissue of
Tilapia (Oreochromis mossambicus) were gradually inhibited with increase in the
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium during 7, 14 and 21 days of exposure. Heavy metal and pesticide had a significant
effect on AChE activity of Tilapia. It simply means that the inhibitory activity of AChE is
directly related to increase in concentration of toxicant and period of exposure. This can be
explained on the basis of the action of heavy metal and organophosphate insecticide on the
enzyme and the impaired the synthesis of new molecules of enzyme, by the damaged cells.
Extensive studies in a variety of vertebrate and invertebrate species have conclusively shown
that the acute toxic response noted with insecticidal organophosphate esters is due to the rapid
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inhibition of acetylcholinesterase, an essential component of nerve conduction in the central
and peripheral nervous system.
The changes observed in the study were generally tissue specific and dose-dependent.
The higher the concentration of the test toxicants and exposure time, the greater the negative
impact was observed. Maximum inhibition of AChE activity was determined in the brain
(67.06%) followed by the muscle (45.54%), gills (37.14%), liver (33.14%) and kidney
(19.22%) during 21 days of exposure to test toxicants. AChE levels are found to be
significantly lowered in brain tissue. The brain cells might have suffered from neurotoxicity of
organophosphorous pesticide and heavy metal. However, inhibition of enzyme might have
adversely affected to bring the brain AChE activity to lower level in 1/5th LC50 of
Chlorpyrifos, Chlorpyrifos + Cadmium, Cadmium + Chlorpyrifos and Cadmium. This might
be due to the sufficient higher sublethal concentration of test toxicants capable to induce a
toxic response. Chlorpyrifos was found to have the greatest impact on AChE activity in the
brain tissue followed by Cadmium + Chlorpyrifos (muscle), Cadmium (gills) and Chlorpyrifos
+ Cadmium (liver and kidney). Highly significant reduced activity of AChE, in the brain
tissue in those fish exposed to Chlorpyrifos indicates that the pesticide is more toxic to the
brain than heavy metal. Several reports suggested that various organophosphorous pesticides
at concentrations close to their LC50 values can induce a decrease in the enzyme level to 6020% of their normal physiological activity in fish. However, exposure of fish to a combination
of Cadmium + Chlorpyrifos during the same period also caused more damage to the AChE
activity in the brain tissue. It means this combination was highly synergistic in nature
compared to individual toxicity of Cadmium.
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Inhibition of AChE activity in the brain appears to be an early process in response to
test toxicants and could be a more sensitive biomarker than inhibition of AChE activity in the
muscle, gill, liver and kidney (Anandhan et al., 2012). The data also suggested that muscle
was the second non target tissue after brain. The depression of AChE on muscle tissues may
cause distoration in the cell organelles and inhibit various enzymes which disturb the
physiological state of fish (Yadav et al., 2009). The accumulation of heavy metals (Tripathi
and Verma., 2004 and Anandhan and Hemalatha., 2009) from industry and pesticides (Singha
et al., 2003; Singha and Sharma., 2005 and Scholz et al., 2006) from agricultural run-off may
cause AChE depression in muscle tissue of fish. AChE inhibition in the field population is one
of the warning signals for aquatic pollution (Gaitonde et al., 2006).
The inhibition observed in the activity of AChE, is in agreement with the findings of
other workers (Das & Mukherjee, 2003; Rao, 2006; Crestani et al., 2007 and Joseph & Raj,
2011). Exposure of Oncorhynchus mykiss to Carbaryl at 250 and 500 µg/l inhibited brain
AChE activities by 61% and 75%, respectively (Beauvais et al., 2001). Rao (2006) also
observed a 37% inhibition of brain AChE after exposure of Channa punctatus to Carbaryl at
250 µg/l. Reduction of brain AChE activity by 20% or more in fish or invertebrates indicates
exposure to organophosphates or carbamate pesticides, and a 50% or greater reduction is
indicative of a life-threatening situation (Wright and Welbourn 2002). When AChE activity
decreases, ACh is not broken and accumulates within synapses and cannot function in a
normal way (Dutta & Arends, 2003). Hence, the altered locomotor behaviour of fish could be
attributed to the accumulation of acetylcholine which interrupted coordination between the
nervous and muscular junctions (Rao et al., 2005 and Rao, 2006). Considering the role of
AChE in neurotransmission in both central nervous system and at neuromuscular junctions,
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the inhibition of AChE activity could be correlated to behavioural changes observed in Clarias
gariepinus exposed to organophosphate (OP) and carbamate (CB) pesticides (Mdegela et al.,
2010).
From the test toxicants, a clear difference in potency of AChE inhibition was observed.
The Chlorpyrifos exposed fish exhibited irregular, erratic and darting movements, loss of
equilibrium followed by hanging vertically in water and hitting to the walls of the test tank
before finally sinking to the bottom just before death due to inhibition of AChE activity in the
brain. Inhibition of AChE enzyme produce symptoms of acute poisoning and cause ACh
accumulation which leads to hyperexcitation, convulsions and death (Tong et al., 2013).
AChE is the target for many insecticides, such as organophosphates and carbamates due to its
critical function in the nervous system (Shelton et al., 2012). The Clarias gariepinus exposed
to Chlorfenvinphos demonstrated the greatest inhibition of AChE activities in plasma,
compared to eye and brain homogenates, Carbaryl exposure demonstrated a greater reduction
of AChE activities in eye than in brain homogenates and plasma. The inhibition of AChE
consequently leads to excessive Ach accumulation at the synapses and neuromuscular
junctions, resulting in overstimulation of ACh receptors (Gupta, 1994).
Acetylcholinesterase activity is a biomarker extremely used in aquatic ecotoxicology
studies and is a fairly sensitive enzyme to low environmental concentrations of neurotoxic
compounds (Kirby et al., 2000). It is extremely active, acting within microseconds.
Biomarkers of AChE provide a dynamic and powerful approach to understand the spectrum of
neurotoxicity. Due to its sensitivity towards the organophosphate and carbamate groups,
AChE has been manipulated to be used in biomonitoring and bioassay for those contaminants
and has been recently used in various species of fish (Dembele et al., 2000; Matozzo et al.,
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2005 and Narbonne et al., 2005). It has been discovered that AChE from Pangasius pangasius
is useful on detecting heavy metals and its sensitivity towards heavy metals such as Copper,
Silver and Chromium (Tham et al., 2009). AChE activity in invertebrates such as shellfish
(Brown et al., 2004 and Lehtonen et al., 2006) and crustaceans (Bocquene et al., 1997) has
been proved to be useful in biomonitoring programme.
Butyrylcholinesterase (BChE)
Butyrylcholinesterase are crucial for different parts of the immune system. Though its
physiological functions are not well defined, BChE is considered as one of the core
detoxifying enzymes (Assis et al., 2010). Some of the investigations hypothesize that BChE
protect AChE against xenobiotics like pesticides (Whitaker, 1986). Butyrylcholinesterase, in
the absence of AChE helps in regulating cholinergic transmission by the scavenging process
of OP and CB inhibitors comes from esteratic activity of BChE. Excess substrate will inhibit
AChE while BChE exhibits the substrate activation in excess substrate which distinguishes
AChE from BChE (Cokugras, 2003).
Butyrylcholinesterase response showed similar trend in the sense that it registered a
continuous decrease in activity at both the concentrations of Chlorpyrifos, Chlorpyrifos +
Cadmium, Cadmium + Chlorpyrifos and Cadmium in gills, liver, kidney, brain and muscle
compared to acetylcholinesterase. It is reported that accumulation of acetylcholine (ACh)
inhibits acetylcholinesterase and butyryrlcholinesteras (Salles et al., 2006). BChE scavenges
acetylcholine (ACh) from tissue and is reported to protect organophosphorous pesticide
toxicity in target cells by binding to phosphate before reaching biochemical target. On a shortterm and long term exposure to test toxicants, butyrylcholinesterase was found to be
significantly decreased in all the test tissues and there was decrease in BChE activity with
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increase in duration of exposure and the lowest BChE activity was recorded at 21st day in all
the treatment groups compared to 7th and 14th days of exposure. This indicates even a
relatively low concentration of heavy metal and pesticide is capable of causing considerable
inhibition in BChE activity in fish on prolonged period of exposure. A decrease in BChE
activity in aquatic organisms followed by the assay on the analysed tissues is capable of
indicating the presence of contaminant in the aquatic environment (Assis et al., 2012).
Maximum inhibition of BChE activity was noticed in the liver (59.89%) followed by
muscle (50.84%), brain (44.38%), kidney (38.67%) and gills (33.79%) during 21 days of
exposure. The higher degree of BChE inhibition in liver when compared with muscle, brain,
kidney and gills of exposed fish may reflect higher Chlorpyrifos and Cadmium concentrations
in this tissue due to the hepatic activation of test toxicants and hepatic tissues might have
affected by exposure to organophosphorous pesticide and heavy metal. BChE was known to
be synthesized in liver (Darvesh et al., 2003).
Combination of Chlorpyrifos + Cadmium (CPF+Cd), Cadmium + Chlorpyrifos
(Cd+CPF) appeared to most detrimental for test tissues. Chlorpyrifos + Cadmium caused more
impact on BChE activity in the liver and kidney followed by Cadmium + Chlorpyrifos
(muscle), Chlorpyrifos (brain) and Cadmium (gills). Highly significant decreased activity of
BChE, in the liver was noticed in those fish exposed to combinations of Chlorpyrifos +
Cadmium and Cadmium + Chlorpyrifos means that these combinations were highly
synergistic in nature compared to individual toxicity of Chlorpyrifos and Cadmium. Hence,
combination of test toxicants proved to be the most potent inhibitors of butyrylcholinesterase
as compared to individual toxicants thus appearing to be more toxic for fishes. The inhibition
observed in the activity of liver BChE, is in agreement with Wogram et al., 2001, when
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following the exposure of stickleback (Gasterosteus aculeatus) to 1 µg/L Parathion, the
decrease of BChE activity in the liver (~60%) was more pronounced than in the gills and the
axial muscle (30%, significant only for muscle).
The use of in vitro assay for the detection of insecticides was normally done using
AChE but BChE have also been proved by in several studies. Wogram et al. (2001) observed
effects of Parathion on acetylcholinesterase, butyrylcholinesterase, and carboxylesterase in
three-spined stickleback (Gasterosteus aculeatus) following short-term exposure. Chuiko et
al. (2003) studied acetylcholinesterase and butyrylcholinesterase activities in brain and plasma
of freshwater teleosts: cross-species and cross-family differences. Jung et al. (2007) evaluated
characterization of cholinesterases in marbled sole, Limanda yokohamae and their inhibition
in vitro by the fungicide Iprobenfos. Aker et al. (2008) investigated comparison of the relative
toxicity of Malathion and Malaoxon in Blue catfish (Ictalurus furcatus).
In the present study, reduction of muscle BChE activity was also found to be more
when compared to brain, gills and kidney. Rodriguez-Fuentes et al. (2008) observed that
BChE contributed 37% of the total ChE activities in the muscle of flat fish. The muscle ChEs
represents the largest pool of ChE in the body. It is also important to control the muscular
function since the loss of muscular control can have many problems for fish such as loss of
swimming control and blockage of opercular movement. According to Fulton and Key, 2001,
the mode of action of Chlorpyrifos (CPF) in fish is on cholinesterase (ChE) inhibition. Both
AChE and BChE are inhibited by CPF. Chlorpyrifos induces irreversible ChE inhibition,
triggering constant stimulation of the muscles which leads to paralysis and death. Assays to
measure inhibition of ChE activity are the most common tool to assess sublethal effects of
CPF exposure in fish. Studies proved that BChE can act as detoxification enzyme against
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anticholinesterase agents, such as OP pesticides (Cokugras, 2003). In vivo and in vitro
biomarker of pesticides can be made using BChE. The exposure of living specimens toward
the analyzed substance for a period of time has been indicated, as in vivo approach followed
by tissue sample analysis through the dissection on specimen, while in vitro approach makes
use of the extracted tissues from the specimen and directly exposes them with the analyzed
substance. A decrease in BChE activity in aquatic organisms followed by the assay on the
analyzed tissues is capable of indicating the presence of contaminant in the aquatic
environment. Thus, the pollution level of aquatic environment can be determined (Assis et al.,
2012).
As repeated exposures appear to bring about increased depression of ChE activity in
brain and liver tissues of aquatic organisms, intermittent anticholinesterase pesticide
applications, especially organophosphorous insecticides, to the agricultural lands during
cultivation seasons may manifest a threat to the fish populations inhabiting water bodies
adjacent to these lands. In fish living in natural waters, even a relatively low concentration of
organic phosphoric acid esters is capable of causing considerable AChE inhibition. This has
been attributed to the enhanced accumulation of chemical pollutants in fish. Cholinesterases
(ChEs) of aquatic animals appear to have varying sensitivities to organophosphates
(Kozlovskaya, 1993). ChE activities of fish have been recognized a potential biochemical
indicator for toxic effects of insecticides. With repeated inputs of anticholinesterase chemicals
to the aquatic environments, fish may be exposed to acutely lethal to sublethal concentrations
(Chandrasekera, 2005).
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Oxidative stress
Toxicant-induced oxidative stress is the final manifestation of a multi-step pathway,
resulting in an imbalance between pro-oxidant and antioxidant defense mechanisms (Banerjee
et al., 2001). Oxidative stress refers to the cytological consequences of a mismatch between
the production of free radicals and the ability of the cell to defend them. Oxidative stress can
thus occur when the production of free radicals increases, scavenging of free radicals or repair
of oxidatively modified macromolecules decreases, or both. This imbalance results in a buildup of oxidatively modified molecules that can cause cellular dysfunction leading to cell death.
Lipid peroxidation (LPx)
Lipid peroxidation, considered a complex process with self-propagating and high
destruction, increases the rigidity (decreases the fluidity) of cellular membranes. Lipid
peroxidation is one of the major mechanisms involved in the cellular membrane damage by
toxicants. Lipoperoxidation is a free radical-mediated chain reaction, since it is selfperpetuating. The length of the propagation depends upon the chain breaking antioxidant, such
as the enzymes SOD and GPx (Papas, 1996). Tissue and cell membrane alterations promoted
by ROS were considered to be proportional to lipoperoxide contents and cell membrane
damage was tested by lipoperoxide concentration (Oteiza et al., 1997).
The tissues of Oreochromis mossambicus recorded oxidative stress resulting from
exposure to sublethal concentrations of Cadmium and Chlorpyrifos as evidenced by the high
degree of lipid peroxidation levels in the tissues. The present investigation showed that the
highest lipid peroxidation level in different tissues of Cadmium and Chlorpyrifos exposed
fishes are as follow gills (109.31%) > liver (97.51%) > kidney (66.36%) > brain (60.50%) >
muscle (38.13%). Increased TBARS formation observed in this study accords with the results
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of the study carried out by Hai et al. (1997) in C. carpio and Ictalurus nebulosus following the
exposures to Dichlorvos. Elevation of lipid peroxidation in gill suggests toxicants-mediated
free radical production can be the cause of oxidative stress in this tissue. According to Fatima
et al. (2000) gill is the most sensitive tissue to the lipid peroxidation induced by xenobiotics
and its antioxidant potential is also weak compared to that of other tissues. Gills are the first
organs which come in contact with environmental pollutants. Paradoxically, they are highly
vulnerable to toxic chemicals because firstly, their large surface area facilitates greater
toxicant interaction and absorption and secondly, their detoxification system is not as robust as
that of liver. Additionally, absorption of toxic chemicals through gills is rapid and therefore
toxic response in gills is also rapid. Gills have frequently been used in the assessment of
impact of aquatic pollutants in marine as well as freshwater habitats (Athikesavan et al., 2006
and Craig et al., 2007).
On a short-term (7 days) and long term (21 days) exposure to Cadmium (Cd), lipid
peroxidation level was found to be significantly increased in all the test tissues. Cadmium was
found to induce highest lipid peroxidation in the gills and muscle, followed by Cadmium +
Chlorpyrifos in the liver and kidney, Chlorpyrifos + Cadmium in the brain tissue. High degree
of lipid peroxidation observed in test tissues of the fish might also be due to the direct
interaction of the Chlorpyrifos and Cadmium with cellular plasma membrane, as
organophosphorous compounds and heavy metals were found to possess such a potential
(Durmaz et al., 2006 and Atli et al., 2006). Cadmium accumulation causes an increase in
highly reactive oxygen species leading to an oxidative stress in aquatic organisms (Atli et al.,
2006). It is known that in the interaction with living organisms, one of the first effects of Cd is
alteration of enzyme activities and membrane transport mechanisms, which in turn are
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responsible for physiological and metabolic alterations in the whole organism. Accumulation
of heavy metals including Cd in fish could stimulate the production of reactive oxygen species
(ROS) such as superoxide anion radical (O2•−), hydrogen peroxide (H2O2), hydroxyl radical
(OH•), lipid hydroperoxide (LOOH), alkoxyl radical (RO•) and singlet oxygen (1O2) (Ruas et
al., 2008 and Firat et al., 2009). The oxidative stress-inducing effect, Cd2+ itself is unable to
generate free radicals directly (Nemmiche et al., 2007), but an indirect generation of
superoxide radicals and hydroxyl radicals has been reported (Galan et al., 2001). The
importance of oxidative stress response as potential biomarkers of environmental pollution has
been addressed by different experimental approaches (Orbea et al., 2002 and Ferreira et al.,
2005). Oxidative stress induced by Cd2+ can negatively affect DNA, RNA, and ribosome
synthesis, and consequentially inactivate enzyme systems (Stohs et al., 2000). Moreover, most
data on Cd-induced oxidative stress in aquatic organisms originate are from research on
numerous invertebrate and freshwater fish species (Atli et al., 2006). Accordingly, fish would
develop various enzymatic and non-enzymatic defense mechanisms to counteract oxidative
stress due to Cd exposure. Therefore, oxidative stress response is frequently investigated as a
useful biomarker to assess Cd status of the aquatic environment (Asagba et al., 2008 and
Padmini and Rani, 2009).
The combined toxicity of Cadmium + Chlorpyrifos was simple additive in nature
compared to individual toxicity of Cadmium, but the combined toxicity of Chlorpyrifos +
Cadmium was synergistic in nature compared to individual toxicity of Chlorpyrifos in causing
lipid peroxidation in different tissues (except muscle) of Tilapia. In muscle tissue, the
combined toxicity of both the combination was simple additive in nature. It means to say that
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the combination of Cadmium + Chlorpyrifos and or Chlorpyrifos + Cadmium caused same
effect as that of individual toxicants during 21 days of exposure.
Different types of xenobiotics including polycyclic aromatic hydrocarbons (PAHs),
pesticides and other synthetic chemicals and heavy metals are capable of triggering oxidative
stress (Durmaz et al., 2006; Oner et al., 2009; Atli and Canli, 2010; Barse et al., 2010; Yonar
and Sakin, 2011 and Ullah et al., 2014c.). Current knowledge on various processes leading to
contaminant-stimulated reactive oxygen species production and resulting oxidative damage is
summarized by Livingstone (2003). A background on the general aspect of prooxidant
antioxidant and oxidative damage processes in animals and a review of the information known
for aquatic organisms in relation to pollution and the use of prooxidant chemicals in
aquaculture and information on damages due to chemical bioaccumulation in fishes and
warning of undesirable changes in them is available (Van der Oost et al., 2003). The cytotoxic
reactive oxygen species are continuously produced by contaminant-stimulated phagocytes and
excessive reactive oxygen species have shown their role in the development of local tissue
damage in freshwater fish (Fatima et al., 2000 and Ahmad et al., 2000 & 2003).
Increased lipid peroxidation and oxidative stress can affect the activities of protective
enzymatic antioxidants that have been shown to be sensitive indicators of increased oxidative
stress. Since the typical reaction during oxidative stress is peroxidative damage to unsaturated
fatty acids, the oxidative stress response could conveniently be used as biomarkers of effect of
oxidative stress inducing heavy metals and pesticides. The study of biochemical responses in
aquatic animals comprises an area of vigorous investigation within ecotoxicology for a
number of reasons, including the need for highly sensitive biomarkers useful for
biomonitoring in aquatic settings and the observations of elevated rates of structural changes
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in some aquatic species (Narra et al., 2012). Because of the role of reactive oxygen
intermediates as cytotoxic mediators, the generation of free radicals and subsequent oxidative
stress in animals like fish is of particular concern to environmental toxicologists.
Antioxidant enzymes
An important aspect of free-radical mediated toxicity is that it is moderated in the host
by several antioxidant cellular defense mechanisms including enzymatic systems (i.e.
superoxide dismutase (SOD), catalase (CAT), , glutathione peroxidase (GPx), glutathione-Stranferase (GST), glutathione reductase (GR) etc.) as well as non-enzymatic systems (i.e.
glutathione (GSH) and ascorbic acid (Vitamin C) etc.). Antioxidants allow the organism to
protect itself against xenobiotic oxidants. In addition, these enzymes enable reactive oxygen
intermediate-producing cells and adjacent tissues to withstand the oxidative stress exerted by
endogenously generated active oxygen species. Therefore, efficient elimination of reactive
oxygen intermediates constitutes an essential component of the defense systems used by
impacted species exposed to toxicants (Blumberg, 2004).
The SOD-CAT system provides the first defense against oxygen toxicity. SOD
catalyzes the dismutation of the superoxide anion radical to water and hydrogen peroxide,
which detoxified by the CAT activity. Usually a simultaneous induction response in the
activities of SOD and CAT is observed when exposed to pollutants (Dimitrova et al., 1994).
GPx constitutes a family of enzymes, which are capable of reducing a variety of organic and
inorganic hydroperoxides to the corresponding hydroxy compounds, utilizing GSH and/or
other reducing equivalents. GPx serves as the most important peroxidase for hydroperoxide
detoxification, and CAT eliminates hydrogen peroxide, which can penetrate through all
biological membranes and inactive several enzymes (Vutukuru et al., 2006).
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The tissues of Oreochromis mossambicus were found to cause oxidative stress
resulting from exposure to Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium as the tissues experienced high activity of superoxide dismutase,
catalase and the three glutathione associated enzymes (glutathione peroxidase, glutathione-Stransferase and glutathione reductase). Significant elevation of lipid peroxidation (P<0.05) at
both the experimental test concentrations (1/5th LC50 and 1/10th LC50) seemed to impart affect
on the activity of these enzymes.
Superoxide dismutase (SOD) and Catalase (CAT)
The primary enzymatic antioxidant machinery of the gills, liver, kidney and brain of
the fish comprising superoxide dismutase and catalase showed contrasting activities exhibiting
a declension dependent on duration and concentration. The decreased activity of these primary
antioxidative enzymes after 7th, 14th and 21st day exposure signifies the inability of the enzyme
in counteracting the toxic effects of the Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium. It has also been reported that the long-term treatment with
organophosphorous leads to a gradual exhaustion of SOD, GPx and GST or brings about an
increase of antioxidative defense systems (Gultekin et al., 2000). The muscle tissue of Tilapia
fish was found to defend the oxidative stress resulting from exposure to Cadmium and
Chlorpyrifos as the tissue experienced stable activities of superoxide dismutase and catalase at
both the test concentrations during experimental periods. However, the activities of superoxide
dismutase and catalase at high concentrations (1/5th LC50) of the test toxicants showed a
significant (P<0.05) increase when compared to that at low (1/10th LC50) concentrations.
The increased SOD activity was recorded in the liver (81.02%) followed by the gills
(67.93%), kidney (65.41%), brain (58.64%) and muscle (11.91%) of the fish while increased
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CAT activity in experimental fishes are as follows kidney (84.38%) > gills (78.99%) > liver
(69.60%) > brain (59.65%) > muscle (35.88%). Hepatic SOD showed more activity than gills,
kidney, brain and muscle SOD, which may be due to the effective role of liver in xenobiotic
detoxification (Goering et al., 1995). Moreover, kidney seems to be the main site for CAT; in
our study it showed a maximum elevation of CAT under 21st day exposure. Combination of
Cadmium + Chlorpyrifos was found to have more impact on SOD and CAT activities in the
liver and kidney followed by Cadmium (gills and muscle) and Chlorpyrifos + Cadmium
(brain). The liver and kidneys play a crucial role in detoxification and excretion of toxicants
mainly through the induction of metal-binding proteins such as metallothioneins (MTs).
Cadmium has been shown to be concentrated in the liver of Sparus aurata and Solea
senegalensis (Kalman et al., 2010). The liver is noted as site of multiple oxidative reactions
and maximal free radical generation (Gul et al., 2004; Avci et al., 2005 and Atli et al., 2006)
and major organ of metabolism and detoxification of Chlorpyrifos (ATSDR, 1997).
Past studies have measured detoxification and antioxidant enzyme activity in fish as a
means of evaluating the toxic effects of pollutants such as organic chemicals (Cazenave et al.,
2006), heavy metals (Atli et al., 2006), and co-exposure to both types of chemicals (Ahmad et
al., 2005 and Gravato et al., 2006), as well as a means of assessing the condition of the aquatic
environment (Pandey et al., 2003 and Amado et al., 2006). Vinodhini et al. (2009) observed
biochemical changes of antioxidant enzymes in Common carp (Cyprinus carpio L.) after
heavy metal exposure. The analytical results indicated that heavy metal toxicity in fish organs
gradually increases during the exposure period and slightly decreases at the 32nd day. The
activity of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), glutathione
peroxidase (GPx) and glutathione-S-transferase (GST) in the fish were increased.
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Glutathione peroxidase (GPx) and Glutathione-S-transferase (GST)
The changes in the activity of glutathione peroxidase were more or less similar to the
response of catalase activity. In the gills, liver, kidney and brain, the activity of glutathione
peroxidase enhanced significantly on 7th day and 14th day compared to that of control fishes.
Though the level of the enzyme activity exhibited an enhancement in test tissues (except
muscle), it decreased after 14th day of exposure reaching the minimal level on 21st day. Low
activities of GPx after 21st day exposure demonstrate the inefficiency of tissues (except
muscle) in neutralizing the impact of peroxides. It is conceivable that substrate competition
between GPx and CAT might be the cause of the reduction in GPx (Cheung et al., 2004). As
the activity of glutathione peroxidase showed declension at both the concentrations,
superoxide dismutase and catalase failed to cope up with the increasing oxidative stress (as
evidenced by enhanced level of lipid peroxidation) and the accumulating superoxide radicals
might have resulted in tissue injury. The muscle tissue was found to defend the oxidative
stress as the tissue experienced increased activity of glutathione peroxidase at both the
sublethal concentrations of Cadmium and Chlorpyrifos throughout the exposure period.
It was interesting to note that the activities of different enzymes acting differently
(GPx and GST) in fish exposed to the toxicants individually and in combinations in the
present experiment. Here the GPx activity showed increasing trend in the first week of
exposure but from second week onwards it got registered a decreasing trend. However, the
GST activity was directly proportional to the period of exposure to all the toxicants at both
1/5th LC50 and 1/10th LC50 values. There was increase in GST activity with increase in duration
of exposure and the highest GST activity was recorded on 21st day in all the treatment groups
compared to 7th and 14th day of exposure. This indicates that the glutathione conjugation of the
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heavy metal (Cadmium) and pesticide (Chlorpyrifos) or its metabolites due to the activity of
glutathione-S-transferase facilitating the conjugation excretion. Cadmium was appeared to
have more detrimental on GPx and GST activities in the gills and muscle followed by
Combination of Cadmium + Chlorpyrifos (liver and kidney) and Chlorpyrifos + Cadmium
(brain).
The increased GPx activity was recorded in the gills (89.13%) followed by the kidney
(80.34%), liver (70.76%), brain (58.12%) and muscle (34.69%) of the fish while increased
GST activity in experimental fishes are as follow gills (83.70%) > kidney (69.52%) > liver
(69.20%) > brain (48.21%) > muscle (25.92%). The gill showed the highest changes in GPx
and GST activity among the tissues. The highest antioxidant enzyme activity stimulated by
pesticides was measured in C. carpio. Vig and Nemcsok (1989) stated that as a result of
Paraquat treatment, the changes in SOD, GPx and GST activity were highest in the gill,
considerable in the liver and kidney. Since the gill is in direct contact with the medium, the
damaging effect of Paraquat in this tissue is extreme, whereas its effects are limited in liver
and kidney. Various studies have shown devastating effects of pesticides in various
biochemical activities (Ullah et al., 2014c). The changes in antioxidants systems of fish are
often tissue specific. Such changes have been traced in brain, gills, muscles, kidneys and
viscera of different fish with varying results in different organs. Peroxidase activities were
found higher in brain, viscera, gills, and muscles of Tilapia but gills was the organ received
highest disturbance in peroxidase (Ahmad et al., 2005). Similarly, changes in lipid peroxidase
have been observed on account of different pesticides as well as environmental pollutants.
GST-mediated conjugation may be an important mechanism for detoxifying
peroxidised lipid breakdown products which have a number of adverse biological effects when
203
present in high amounts. The changes in the activity of GST show the role of this enzyme in
protection against the lipid peroxidation (Leaver and George, 1998). GSTs catalyze the
conjugation of GSH with electrophilic metabolites, which are involved in the detoxification of
both reactive intermediates and oxygen radicals. Detoxification enzymes, especially
glutathione-S-transferase (GST) helps in eliminating reactive compounds by forming their
conjugates with glutathione and subsequently eliminating them as mercapturic acid, thereby
protecting cells against ROS induced damage (Rodriguez-Ariza et al., 1993).The increase in
the activity of glutathione-S-transferase
with corresponding increase in the activity of
glutathione reductase at high (1/5th LC50) as well as low concentrations (1/10th LC50) of the
test toxicants vividly indicates the glutathione conjugation of the heavy metal and pesticide or
its metabolites due to the activity of glutathione-S-transferase facilitating the conjugation
excretion. An increase in GST activity has also been observed in studies with Salmo trutta
after Propiconazole exposure (Egaas et al., 1999). Increased activities of GST are known to
serve as protective responses to eliminate xenobiotics (Smith and Litwack, 1980). Elevated
GST activity may reflect the possibility of better protection against pesticide toxicity and it is
used as a biomarker for environmental biomonitoring (Oruc et al., 2004). When GST activity
is inhibited, accumulation of lipid peroxidation products occurs. GSTs play a primary
important role in cellular detoxification of toxic aldehydes.
Glutathuione reductase (GR)
In living organisms, oxidation of GSH to GSSG occurs due to many biochemical
reactions. GR converts GSSG to its reduced form GSH, which is important both as a substrate
for peroxide scavenging enzymes (i.e. GPx and GST) and as direct scavenger of oxy-radicals.
In this study, a relatively higher value (P<0.05) of glutathione reductase was observed in gills,
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liver, kidney, brain and muscle in fish at both the experimental test concentrations of
Cadmium and Chlorpyrifos suggests its active role in recycling pathways. Stephensen et al.
(2002) demonstrated that fishes from polluted sites have high GR activity due to higher
peroxidative components in the polluted aquatic site.
The highest GR activity was noticed in the liver (103.64%) followed by the brain
(87.78%), gills (86.78%), kidney (72.05%), and muscle (40.22%) of the fish. Hepatic GR
showed more activity than gills, kidney, brain and muscle GR, which may be due to the
effective role of liver in xenobiotic detoxification (Goering et al., 1995). Hamed et al. (2003)
measured glutathione related enzyme levels of freshwater fish as bioindicators of pollution.
The liver and kidney glutathione-S-transferase (GST), glutathione reductase (GR) and
glutathione peroxidase (GPx) were higher in Oreochromis niloticus and African catfish,
Clarias lazera captured from all the polluted areas compared to the control. Comparison
between test toxicants and test organs reveals that combination of Cadmium + Chlorpyrifos
caused greatest impact on GR activity in the liver and kidney followed by Chlorpyrifos +
Cadmium (brain) and Cadmium (gills and muscle). The liver and kidneys play the role in
activation and detoxification of Chlorpyrifos, by desulfuration to Chlorpyrifos oxon and
Cadmium mainly through the induction of metal-binding proteins such as metallothioneins
(MTs).
The joint action toxicity of Cadmium + Chlorpyrifos was simple additive while
Chlorpyrifos + Cadmium was synergistic in nature in altering the activities of different
antioxidant enzymes in the gills, liver, kidney and brain of Tilapia compared to their
individual toxicities. However, it was interesting to note that the exposure of fish to 1/5th LC50
and 1/10th LC50 of combination of Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
205
brought in simple additive in causing changes in the activities of different antioxidant enzymes
in the muscle of Tilapia. It means a combination of metal + pesticide and pesticide + metal act
neither synergistically nor antagonistically in causing SOD, CAT, GPx, GST and GR activity
in Tilapia muscle.
There have been several hypotheses proposed to explain Cd and CPF induced
tolerance. It was also hypothesized that Cd2+ pretreatment alters the organ distribution of Cd2+,
with more Cd2+ distributing to gills, liver, kidney and less to brain and muscle (WHO, 1992b).
In fact, liver accumulated substantial amounts of Cd2+ after both acute and chronic exposures
(Goering et al., 1995 and Usha Rani, 2000). Chronic Cd2+ administration resulted in a gradual
rise in hepatic as well as renal antioxidant defense (Shaik et al., 1999). O. mossambicus under
sublethal Cd2+ exposure underwent as elevation of antioxidant enzymes in the two major
tissues. The induction of elevated levels of SOD, CAT and GPx, with a simultaneous increase
in the levels of GST and GR, in liver and kidney shows a possible shift toward a detoxification
mechanism under long term exposure to the heavy metal Cd2+. Similar activity levels were
reported in laboratory animals under Dieldrin and Copper toxicity (Dedrajaset et al., 1996).
Pesticide-induced oxidative stress has also been a focus of toxicological research
during the last decade. Study of pesticide induced effects on various antioxidants in fish and
other aquatic organisms can provide the information about the ecotoxicological consequences
of pesticide use. Organisms have evolved a variety of responses that help to compensate the
physiological impact of environmental contaminants. The antioxidant defense mechanism
forms the crux of this whole system. There has been a increasing efforts to employ
antioxidants as a biomarker of toxic responses. Fish are particularly sensitive to environmental
contamination of water and pollutants may significantly damage biochemical processes when
206
they enter the tissues of fish (Lang et al., 1997). Because metabolism of toxicants at
extrahepatic sites is likely to be involved in systemic effects on reproduction, immune
function and other cellular functions. It was shown that exposure to Chlorpyrifos (CPF) causes
oxidative stress in fish (Kavitha and Rao, 2008). Chlorpyrifos can cause oxidative effects at
low doses in brain tissue (Kwong, 2002 and Goel et al., 2005). Although brain has no direct
contact with the pesticides dissolved in water, yet it is affected through the blood circulation
and various alterations are caused in it. It has also been hypothesized that inducing oxidative
stress might be the primary mode of toxicity of CPF in some instances, regardless of ChE
inhibition (Saulsbury et al., 2009). However, data on CPF-induced oxidative stress in fish
species remain scarce. CPF can trigger a response from the detoxifying and antioxidant
enzymes glutathione-S-transferases (GST) in animals. As a consequence, GST activity
generally increases upon exposure to contaminants in order to eliminate the xenobiotics and
has been widely used to measure xenobiotics toxicity in aquatic organisms (Hayes et al., 2005;
Antognelli et al., 2006 and Wang et al., 2009).
Antioxidant systems can be considered as non-specific biomarkers of exposure to
pollutants and also as an indicator of toxicity. The induction of levels of primary antioxidant
defenses preventing cell damage can be regarded as an adaptative response to an altered
environment; in contrast an inhibition can lead to cell damage and toxicity of bioavailable
pollutant in a dose-dependent manner. Since induction of antioxidants represents a cellular
defense mechanism to counteract toxicity of ROS, they have been extensively used in several
field studies to assess the extent of pollution in rivers, lakes and coastal waters (Goksoyr,
1995).
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Non-enzymatic antioxidants
Reduced glutathione (GSH) and Ascorbic acid (Vitamin C) are the most important
antioxidants found in the extracellular fluid. It is water soluble and scavenges a number of
different ROS including thiol radicals generated during xenobiotic reduction. The scavenging
and enzymatic antioxidants are linked in many ways. Often if one is deficient, others increase
to compensate (Puangkaew et al., 2004).
Reduced glutathione (GSH)
Glutathione (L-γ-glutamyl-cysteinyl-glyceine) is an ubiquitous non-protein thiol that is
mainly present in cells in its reduced form (GSH), which basically acts as an intracellular
reductant and nucleophile. It intervenes directly or indirectly in many important physiological
functions. These include the synthesis of proteins and DNA, amino acid transport,
maintenance of the thiol disulfide status (acting as a redox buffer), free radical scavenging
(acting synergistically with ascorbate (vitamin C) and vitamin E (tocopherols and
tocotrienols)), signal transduction, as an essential cofactor of several enzymes, as a non-toxic
storage form of cysteine, and as a defense against oxidizing molecules and potentially harmful
xenobiotics (Cooper and Kristal, 1997). However, it is also present in its oxidized form
(GSSG) where there is a disulfide bond between two molecules of GSH.
The level of reduced glutathione enhanced significantly in all the test tissues subjected
to high concentrations (1/5th LC50) and low concentrations (1/10th LC50) of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium when compared to that
of control group. In the experimental tissues, except muscle, the GSH level registered a
continuous reducing trend after 7th day of exposure. The GSH level further reduced on 21st day
of exposure. The decreased level of GSH in the gills, liver, kidney and brain of Oreochromis
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mossambicus after 7th day of exposure signifies the inability of the antioxidant to cope up with
the increasing oxidative stress induced by Cadmium and Chlorpyrifos toxicity (as evidenced
by enhanced level of lipid peroxidation). Reduced glutathione can also bind metals that might
induce oxidative stress (Hansen et al., 2007). Chlorpyrifos intoxication is shown to cause a
significant decrease in the reduced glutathione level (Goel et al., 2005). Lipid peroxidation is
known to occur as a result of GSH depletion. Glutathione protects from lipid peroxidation and
there exists a close correlation between low level of GSH and accumulation of the
malondialdehydes. The muscle registered more or less enhanced level of GSH at all the test
concentrations during experimental periods showing more activity on 21st day.
The percentage increase in total reduced glutathione was more in the liver (54.47%),
brain (53.75%) and gills (52.50%) compared to kidney (37.43%) and muscle (26.74%) of the
Tilapia fish. The liver was found to possess high amount of glutathione in comparison to the
levels maintained in gills, brain, kidney and muscle. GSH is synthesized in the liver and
released to the blood for transferring to the other organs such as the kidney and muscle (Pena
et al., 2000). Because metal exposures did not alter the levels of GSH in the blood and muscle,
it suggests that metals taken up from the gill were immediately transferred (via the blood) to
the liver for the usage in the metabolism or sequestered. Cadmium was found to have more
effect on reduced glutathione levels in the gills, liver, kidney and muscle compared to
Cadmium + Chlorpyrifos (brain), Chlorpyrifos + Cadmium and Chlorpyrifos. GSH may play a
role in inducing resistance to metals by protecting gill against the attack by free radicals (Firat
and Kargin, 2009). Wilczek et al. (2008) suggested that GSH is presumably an important
element of the defense against toxic effects of heavy metals, either through binding metal ions
or through neutralizing reactive oxygen species generated by metabolic processes and/or
209
exogenic factors. Thus, GSH levels in the tissues may be a good indicator to understand the
degree of metal exposure. The combined toxicity of Cadmium + Chlorpyrifos was simple
additive while Chlorpyrifos + Cadmium was synergistic in nature in altering the levels of
reduced glutathione in different tissues (except brain) of Tilapia compared to their individual
toxicities. In brain tissue, the combined toxicity of both the combination was synergistic in
nature. It means to say that the combination of Cadmium + Chlorpyrifos and or Chlorpyrifos +
Cadmium caused more effect as that of individual toxicants during 21 days of exposure.
Glutathione plays an important role in the detoxification and excretion of xenobiotics.
Glutathione (GSH) is an efficient antioxidant and also serves as a cofactor and substrate for
many redox enzymes, particularly a large class of glutathione-dependent peroxidases and
transferases. GSH is an important tripeptide molecule containing -SH groups basically acts as
an intracellular reductant and nucleophile against numerous toxic substances including most
inorganic pollutants, through the -SH group. Hence, GSH is considered a first line of cellular
defense against metals by chelating and detoxifying them, scavenging oxyradicals and
participating in detoxification reactions catalyzed by glutathione peroxidases (Sies, 1999).
Ascorbic acid (Vitamin C)
Vitamin C is an important water soluble antioxidant in biological fluids and an
essential micronutrient required for normal metabolic functioning of the body. It acts as a
biological reducing agent for hydrogen transport. It neutralizes reactive oxygen metabolites
(ROMs) and reduces oxidative DNA damage and genetic mutations (Ray and Hussain, 2002).
It acts as a co-antioxidant by regenerating α-tocopherol from the α-tocopheroxyl radical
produced during scavenging of ROMs (Shiau and Lin, 2006). Ascorbic acid acts as detoxifier
210
and may reduce the effect of toxicant through its antioxidant property. Its role in detoxification
and immune system may protect the body from various toxic pollutants in environment.
The level of ascorbic acid content revealed significant changes with respect to
concentration and duration of exposure due to exposure to Cadmium and Chlorpyrifos.
Ascorbic acid content was below control values in the tissues at the two experimental
concentrations of test toxicants. The considerable decline in the tissue level of ASA during
exposure to Chlorpyrifos and Cadmium may be due to an increased utilization of ASA. The
ASA depletion in test tissues seems to enhance the risk of oxidative stress due to reduced cell
protection ability. The percentage decrease in ascorbic acid content was more in the kidney
(44.11%) followed by the liver (33.95%), brain (33.14%), gills (28.64%) and muscle (27.78%)
of Tilapia fish. Kidney and liver showed a maximum depletion of ASA under 21st day
exposure. Gradual decrease in the level of liver ascorbic acid due to liver glucose breakdown
was observed by Desai Himadri Sekhar et al. (2002) in freshwater fish Channa punctatus
(Bloch), under the stress of Nickel (NiSO4.6H2O). Tolerance to environmental stressors as
well as regulation of collagen synthesis also is known to demand ascorbic acid by organisms.
Consumption of oxidative products depletes antioxidant within the body such as vitamin C
and E, selenium and carotenoids. These exogenous dietary antioxidants aid in preventing lipid
peroxidation through their association with the cellular lipid membranes (Gapasin et al., 1998
and Kumari and Sahoo, 2005). Vitamin C protects against cell death triggered by various
stimuli and a major property of this protection has been linked with its antioxidant ability
(Valko et al., 2006).
Cadmium was appeared to have more detrimental on the levels of ascorbic acid in the
gills, liver, kidney and muscle compared to Cadmium + Chlorpyrifos (brain), Chlorpyrifos +
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Cadmium and Chlorpyrifos. Ascorbic acid deficiency may reduce the activity of xenobiotic
metabolizing enzymes. Ascorbic acid ameliorates copper and cadmium toxicity and is
required for trace element homeostasis. The joint action toxicity of Cadmium + Chlorpyrifos
was antagonistic while Chlorpyrifos + Cadmium was synergistic in nature in altering the
levels of ascorbic acid in gills, liver and kidney of Tilapia compared to their individual
toxicities. In brain tissue, the joint action toxicity of both the combination was synergistic in
nature whereas in muscle tissue, the joint action toxicity of Cadmium + Chlorpyrifos was
simple additive while Chlorpyrifos + Cadmium was synergistic in nature compared to their
individual toxicities. Studies have shown that metal and pesticides may interact synergistically
or antagonistically on survival and development of various aquatic organisms (Gravato et al.,
2006).
The author has elucidated the chemistry and mechanism of free radical protection by
ascorbate in biological systems. It is proposed that ascorbate radical can function as a marker
of free radical mediated oxidative stress. The immunostimulatory effect ascorbic acid may be
related to its antioxidant activity as a free radical scavenger, protecting cells from autooxidation and maintain their integrity for an optimal functioning of the immune system
(Kumari and Sahoo, 2005). Majority of in vivo studies showed a reduction in markers of
oxidative DNA, lipid and protein damage after supplementation with vitamin C.
Total protein
The protein is the major intake of energy source (Agrahari and Gopal, 2009), precisely
reflecting the condition of the organism and the changes happening to it under influence of
internal and external factors (Hadi et al., 2009). The influence of toxicants on the total protein
212
concentration of fish has been taken into consideration in evaluating the response to stressors
and consequently the increasing demand for energy (Osman et al., 2010).
A significant difference was observed in the protein content of gill, liver, kidney, brain
and muscle tissues in Oreochromis mossambicus on exposure to both the sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium. Depletion in protein content indicated the requirement of large amounts of protein
under a toxic stress during heavy metal and pesticide exposure to compensate the energy
demand and the metabolic requirements. The ketoacids might have been used to synthesize
glucose by gluconeogenesis or the protein pools might have been depleted to meet the caloric
requirement for the biological functions. The protein pools might have deteriorated due to
structural changes and tried to involve in repair mechanisms. But the disrupted homeostasis
and altered sterical conformations, by the binding of the heavy metal and pesticide, may not be
allowing the protein synthetic apparatus to synthesize new proteins. Thus the protein showed a
decrease in its content.
The present investigation showed protein content in different tissues of Cadmium and
Chlorpyrifos exposed fishes in the decreasing order: Liver (39.39%) > Muscle (36.93%) >
Brain (35.97%) > Kidney (32.08%) > Gills (30.55%). Gradual decrease in the level of liver
protein due to proteolysis and reduced metabolic activity in the test tissue induced by
Cadmium and Chlorpyrifos was observed. There was also gradual decrease in the brain
protein level showing significant alterations. Neurotoxicity on toxic exposure to Chlorpyrifos
+ Cadmium and Chlorpyrifos might be the reason for the observation that brain showed
significant change in protein content. During stress conditions fish need more energy to
detoxify the toxicant and to overcome stress. Since fish have fewer amounts of carbohydrates
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so next alternative source of energy is protein to meet the increased energy demand (Singh et
al., 2010).
The present observation is in agreement with the investigations of Datta et al. (2007) in
Clarias batrachus after exposure to Arsenic, El-Sayed et al. (2007) in Oreochromis niloticus
after Deltamethrin exposure, Min and Kang (2008) in Oreochromis niloticus after Benomyl
exposure. The storage or mobilization of metabolic substrates such as glucose, glycogen,
lactate, lipid and protein are disrupted by exposure to several trace metals, including Cadmium
(Fabbri et al., 2003). Sastry and Siddiqui (1984) reported that the protein content was found to
be decreased in liver, muscle, kidney, intestine, brain and gill in Channa punctatus when
treated with Quinalphos. Yeragi et a1. (2000) observed the decreased levels of proteins in
gills, testis, ovaries and muscles of marine crab Uca marionis exposed to acute and chronic
levels of Malathion. Study by Aruna Khare et al. (2000) reported that the sublethal
concentrations of Malathion showed a significant increase in the protein content which
showed a gradual decrease on prolonged exposure to Malathion. Also regarding brain, the
observation from the present study is in consonance with the report by Praveena et al. (1994).
According to the study, exposure to organophosphorous causes delayed neuropathy resulting
in major changes in the concentration of protein and degeneration changes in the central
nervous system, somatic and visceral sensitive system. Gradual decrease in the levels of liver
and brain protein was observed by Desai Himadri Sekhar et al. (2002) in freshwater fish
Channa punctatus (Bloch), under the stress of Nickel (NiSO4 6H2O). A study by Svobodova et
al. (1994) reported that the level of total proteins in the blood plasma had a tendency to
decrease in fish after a long-term exposure to pollutants.
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On a long-term exposure, combination of Cadmium + Chlorpyrifos caused more
impact on the levels total protein content in the liver and kidney followed by Cadmium (gills
and muscle) and Chlorpyrifos + Cadmium (brain). The combined toxicity of Cadmium +
Chlorpyrifos was simple additive while Chlorpyrifos + Cadmium was synergistic in nature in
altering the levels of protein content in gills and brain of Tilapia compared to their individual
toxicities. In liver and muscle, the combined toxicity of both the combination was synergistic
in nature whereas in kidney, the combined toxicity of both the combination was simple
additive in nature compared to their individual toxicities.
Cadmium was found to interfere with many protein and carbohydrate metabolisms by
inhibiting the enzymes involved in the processes (Sobha et al., 2007). In addition to enzymatic
alterations, changes in other proteins also occur in fish exposed to various types of
environmental stresses including pesticides. It has been suggested that the environmental
perturbations including stresses caused by the pesticides may suppress the expression of some
genes and activate the others to produce specific mRNAs which may subsequently be
translated into specific proteins, the stress proteins. Varieties of pollutants have proved to alter
the protein metabolism in fish (Osman et al., 2010).
Stress-borne biochemical and sub-cellular dysfunctions tend to lead to cell damage.
Chronically stressed organisms are often prone to structural and biochemical changes which
would ultimately affect their physiology and well being.
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VI. SUMMARY
The present investigation mainly centers around analyzing the physiological and
biochemical biomarkers to assess the toxic effects of Cadmium and Chlorpyrifos individually
and in combinations in Tilapia, Oreochromis mossambicus. It is a euryhaline fish belongs to
cichlid family, known to tolerate to wide range of environmental stresses and highly suitable
for toxicity testing as model organism. The toxicity levels exhibited by the fish will enable one
to predict hazards posed even to other more sensitive fish species.
The chapter on Introduction describes the aquatic ecosystems as the receptacle
pollutants of anthropogenic origin; heavy metals and pesticides and its toxicity, mode of
action in aquatic organisms.
The chapter on Review of Literature provides comprehensive information derived
from various investigations in the area of physiological and biochemical biomarker research in
fishes. This section also includes studies concerned with the behaviour of fish, oxygen
consumption, ammonia-N excretion, food consumption, growth rate of fish, cholinesterase
enzymes, oxidative stress, circumstances leading to oxidative stress in aquatic organisms,
major enzymatic antioxidant defense mechanisms in fishes, the specific physiological roles
played by the test organs (gills, liver, kidney, brain and muscle) with special reference to
xenobiotic absorption and /or biotransformation, localization of antioxidant enzymes in cells
and their course of action, introduction of the concepts of bioaccumulation, bioconcentration,
biomagnifications and biomarkers, antioxidant machinery in fishes.
The third chapter on Materials and Methods deals with experimental animal,
maintenance in the laboratory, toxicants used, preparation of test solutions, procedures
adopted for lethal toxicity, joint toxicity, general protocols adopted for assessment of sublethal
216
toxicity and physiological responses. Apart from this, sample preparations, assay of
cholinesterase
enzymes,
lipid
peroxidation,
antioxidative
enzymes,
non-enzymatic
antioxidants, total protein and statistical analysis of data are detailed here. In the present study,
the gill was chosen as the major respiratory tissue and major site of uptake of xenobiotic
chemicals while liver was chosen the major site of accumulation, biotransformation and
excretion of xenobiotic compounds. On the other hand, kidney was considered as the major
route for elimination and rapid clearance of xenobiotic compounds. Brain plays a regulatory
role in the physiology and it is the most important organ in fish toxicology especially when
pesticides are involved in their mode of action in the nervous system. Muscle tissue was also
identified as the route for accumulation of heavy metals due to its strong binding with cystine
residues of metallothionein.
The findings of the present study have been documented in the chapter on Results.
 Lethal toxicity studies, i.e. 96 h LC50 assessment was carried out for Cadmium and
Chlorpyrifos singly and in combinations in the test animal. The 96 h LC50 values
determined for Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium were 168.90 ppm, 0.022 ppm, 92.04 ppm and 0.016 ppm respectively.
Chlorpyrifos, even in microgram levels can induce toxicity in fishes and can be rated as
highly toxic to fish. The joint action toxicity was more than additive or synergistic in
bringing out early death of O. mossambicus, than those obtained for individual toxicant.
 The behavioural responses of the fishes to Cadmium included disrupted shoaling
behaviour, localization at the bottom of the test chamber, and independency (spreading
out) in swimming. Exposure of fish to Chlorpyrifos exhibited irregular, erratic, and darting
swimming movements, loss of equilibrium, hanging vertically in water and hyperactivity
217
before collapsing. Excess secretion of mucus, exhaustion and increased opercular rate
were the prominent observations on exposure to the pesticide.
 Cadmium and Chlorpyrifos singly and in combinations had significant effect on functional
activity of Tilapia since increase in test concentrations and duration of exposure induced
decrease in oxygen consumption, food consumption, ammonia-N excretion and growth
rate in both the sublethal concentrations. Fish exposed to sublethal concentrations of test
toxicants showed a gradual increase in O:N ratio in all the exposed duration and toxicant
combinations.
 Significant inhibitory effect of test toxicants as a functioning of duration of exposure was
noticed on acetylcholinesterase activity in the experimental tissues of the fish. Maximum
inhibition of AChE activity was in the brain (67.06%) followed by the muscle (45.54%),
gills (37.14%), liver (33.14%) and kidney (19.22%) during 21 days of exposure to test
toxicants. Inhibition of acetylcholinesterase enzyme in the brain appears to be an early
process in response to Chlorpyrifos and combination of Chlorpyrifos + Cadmium and
could be a more sensitive biomarker than inhibition of AChE activity in the muscle, gill,
liver and kidney indicating that the pesticide is more toxic to the brain than heavy metal.
 Butyrylcholinesterase response showed similar trend, a continuous decrease in activity at
both the concentrations in test tissues compared to acetylcholinesterase. Maximum
inhibition of BChE activity was noticed in the liver (59.89%) followed by muscle
(50.84%), brain (44.38%), kidney (38.67%) and gills (33.79%) during 21 days of
exposure. The higher degree of BChE inhibition in liver compared to muscle, brain,
kidney and gills of exposed fish may reflect higher Chlorpyrifos and Cadmium
concentrations in this tissue due to the hepatic activation of test toxicants.
218
 Highest lipid peroxidation level in different tissues of fishes exposed to Cadmium and
Chlorpyrifos are as follow gills (Cadmium) > liver (Cadmium + Chlorpyrifos) > kidney
(Cadmium + Chlorpyrifos) > brain (Chlorpyrifos + Cadmium) > muscle (Cadmium).
 The heavy metal and pesticide evoked different degrees of activities in the liver, the gill,
the kidney and the brain. The activity of primary antioxidant enzymes (SOD, CAT, GPx
and GR) and non-enzymatic antioxidants (GSH and ASA) decreased drastically on
exposure to sublethal concentrations of test toxicants. The muscle tissue was found to
defend the oxidative stress with stable activities at both the concentrations throughout the
period of exposure. Elevated glutathione-S-transferase (GST) activity in all the test tissues
reflect the possibility of better protection against lipid peroxidation induced by heavy
metal and pesticide toxicity.
 The present investigation showed protein content in different tissues of fishes in the
decreasing order: Liver (Cadmium + Chlorpyrifos) > Muscle (Cadmium) > Brain
(Chlorpyrifos + Cadmium) > Kidney (Cadmium + Chlorpyrifos) > Gills (Cadmium).
The chapter on Discussion enlightens the results obtained in the light of the available
literature. The dose-dependent toxicity in case of pesticide exposure was evident by greater
mortality rate and deviations from the normal behavioural pattern of fish. Highly significant
altered physiological and biochemical responses noticed in fish exposed to a combinations of
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium indicate that the heavy metal in
combination with pesticide and pesticide in combination with heavy metal becomes more
toxic (synergistic) to fish at sublethal concentrations. The oxidative stress resulting from
exposure of fish to low concentration of toxicants is defended more or less successfully by
219
antioxidant enzymes and non-enzymatic antioxidants and the mechanism often failed, when
the animal encountered long-term exposure to heavy metal and pesticide.
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under the chapter on Bibliography.
220
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267
VIII. ABSTRACT
Physiological and Biochemical responses in Tilapia (Oreochromis mossambicus) exposed
to Cadmium and Chlorpyrifos
Increasing input of environmental contaminants such as heavy metals and pesticides to
aquatic ecosystems has generated the need to understand and evaluate the biological effects of
xenobiotics on aquatic biota. The use of physiological and biochemical markers in fishes has
been widely used for both marine environments and for continental waters. Prominent among
these markers are physiologic variables; behaviour, oxygen consumption, food consumption,
ammonia excretion, Oxygen:Nitrogen ratio, growth rate and biochemical variables;
cholinesterase enzymes, antioxidant enzymes, non-enzymatic antioxidants and total protein
levels are used as stress indicators. The toxicological assessment of water contamination
through sublethal bioassays becomes relevant because they allow early detection of adverse
effects on particular test organisms.
The present study aimed at detecting the variation of physiological and biochemical
responses in Tilapia (Oreochromis mossambicus) caused by individual and combination of
heavy metal, Cadmium and organophoshorous pesticide, Chlorpyrifos in response to shortterm and long-term exposures to sublethal concentrations (1/5th and 1/10th of LC50 value) of
test toxicants for 7, 14 and 21 days. Individual lethal toxicity of Cadmium (Cd) and
Chlorpyrifos (CPF) indicated that the 96 h LC50 value for fish were found to be 169.80 ppm
and 0.022 ppm respectively, combined lethal toxicity of varying Cadmium + constant
Chlorpyrifos was 92.04 ppm and combination of varying Chlorpyrifos + constant Cadmium
was 0.016 ppm. These results indicate that, Cadmium in combination with Chlorpyrifos
becomes more toxic (synergistic) compared to its individual exposure. Similarly, Chlorpyrifos
268
become more toxic (synergistic) to Tilapia fish fingerlings in the presence of Cadmium
compared to its individual exposure.
The effects of test toxicants on the physiological and biochemical responses were
remarkable. Oxygen consumption rate, food consumption rate, ammonia excretion rate,
Oxygen:Nitrogen ratio and growth rate of the fishes was affected to sublethal concentrations
of test toxicants. Variations in physiological responses are the consequence of impaired
oxidative metabolism. Alteration in the antioxidant enzymes, glutathione system,
non-enzymatic antioxidants and induction of lipid peroxidation reflects the oxidative stress of
test toxicants in the experimental fish. The decreased activity of cholinesterase enzymes
signifies the inability of the enzymes in countering the toxic effects of the Cadmium and
Chlorpyrifos. Depletion in protein content in the gills, liver, kidney, brain and muscle indicate
the utilization of large amounts of protein under a toxic stress. Hence, the present investigation
recommends the utility of physiological and biochemical responses as diagnostic tools to
assess toxic effects in fishes. All the results were statistically significant at P<0.05.
KEYWORDS: Cadmium, Chlorpyrifos, Combined toxicity, Physiological responses,
Biochemical responses and Oreochromis mossambicus.
269
Tables and Figures
Table 2. The 96 h LC50 for Cadmium in Oreochromis mossambicus.
No. of fish died
Conc.
(ppm)
T- 3
No. of
fish used
Mean %
Mortality
Conc.
×100
Log
Conc.
Probit
Value
T- 1
T- 2
160
2
1
2
10
16
16000
4.204
4.01
164
3
2
4
10
30
16400
4.214
4.48
168
3
4
5
10
43
16800
4.225
4.82
172
6
5
6
10
56
17200
4.235
5.25
176
9
7
8
10
80
17600
4.245
5.84
180
10
9
10
10
96
18000
4.255
6.75
184
10
10
10
10
100
18400
4.264
8.09
270
Fig. 2. Graphical derivation of 96 h LC50 for Cadmium in Oreochromis mossambicus.
Table 3. The 96 h LC50 for Chlorpyrifos in Oreochromis mossambicus.
No. of fish died
T- 3
No. of
fish used
Mean %
Mortality
Conc.
×100
Log
Conc.
Probit
Value
2
2
10
16
1.5
0.176
4.01
3
2
3
10
26
1.8
0.255
4.36
0.021
4
4
4
10
40
2.1
0.322
4.75
0.024
6
6
7
10
63
2.4
0.38
5.33
0.027
8
9
8
10
83
2.7
0.431
5.85
0.030
10
10
10
10
100
3
0.477
8.09
Conc.
(ppm)
T- 1
T- 2
0.015
1
0.018
271
Fig. 3. Graphical derivation of 96 h LC50 for Chlorpyrifos in Oreochromis mossambicus.
Table 4. The 96 h LC50 for varying concentrations of Cadmium + constant concentration
of Chlorpyrifos (i.e. 1/5th of LC50, 0.0044 ppm) in Oreochromis mossambicus.
No. of fish died
T- 1
T- 2
T- 3
No. of
fish used
80
1
2
2
10
16
800
2.903
4.01
85
3
3
2
10
26
850
2.929
4.36
90
4
5
4
10
43
900
2.954
4.82
95
6
6
5
10
56
950
2.977
5.15
100
8
9
7
10
80
1000
2.999
5.84
Conc.
(ppm)
Mean %
Mortality
Conc.
×10
Log
Conc.
Probit
Value
272
105
10
10
9
10
96
1050
3.021
6.75
110
10
10
10
10
100
1100
3.041
8.09
Fig. 4. Graphical derivation of 96 h LC50 for varying concentrations of Cadmium +
constant concentration of Chlorpyrifos in Oreochromis mossambicus.
Table 5. The 96h LC50 for varying concentrations of Chlorpyrifos + constant concentration
of Cadmium (i.e. 1/5th of LC50, 34 ppm) in Oreochromis mossambicus.
No. of fish died
T- 3
No. of
fish used
Mean %
Mortality
Conc.
×1000
Log
Conc.
Probit
Value
1
2
10
16
10
1
4.01
3
2
4
10
30
13
1.11
4.48
0.016
5
4
6
10
50
16
1.2
5
0.019
7
6
7
10
66
19
1.27
5.41
Conc.
(ppm)
T- 1
T- 2
0.01
2
0.013
273
0.022
9
8
8
10
83
22
1.34
5.95
0.025
10
10
9
10
96
25
1.39
6.75
0.028
10
10
10
10
100
28
1.44
8.09
Fig. 5. Graphical derivation of 96 h LC50 for varying concentrations of Chlorpyrifos +
constant concentration of Cadmium in Oreochromis mossambicus.
Table 6. Categorization of toxicants based on the toxic units in Oreochromis mossambicus.
Toxicant
LC50 value
Toxic units
Category
Chlorpyrifos + Cadmium
0.016 ppm
6250
Highly toxic
Chlorpyrifos
0.022 ppm
4545.45
Highly toxic
Cadmium + Chlorpyrifos
92.04 ppm
1.08
Toxic
Cadmium
169.80 ppm
0.58
Less toxic
274
If, TU > 100
: Highly toxic
TU - 10 to 100 : Very toxic
TU - 1 to 10
: Toxic
TU < 1
: Less toxic
Table 7. Joint action toxicity of Cadmium and Chlorpyrifos in Oreochromis mossambicus
based on the synergistic ratio (S.R.) model.
Joint Toxicity
LC50 value
S.R. Index
Joint action
Cadmium + Chlorpyrifos
92.04 ppm
1.84
Synergistic
Chlorpyrifos + Cadmium
0.016 ppm
1.37
Synergistic
If, S.R. = 1: Joint action is additive
S.R. < 1: Joint action is antagonistic
S.R. > 1: Joint action is synergistic
Table 8. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the oxygen consumption rate in Tilapia (Oreochromis mossambicus)
during 7, 14 and 21 days of exposure.
Oxygen consumption rate (mg O2 consumption/l/g/h)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
0.203 ± 0.0015
0.120 ± 0.0005
0.148 ± 0.0015
275
14
0.232 ± 0.0011
0.104 ± 0.0010
0.135 ± 0.0012
21
0.281 ± 0.0016
0.080 ± 0.0012
0.112 ± 0.0005
Chlorpyrifos
7
0.203 ± 0.0015
0.195 ± 0.0003
0.198 ± 0.0009
14
0.232 ± 0.0011
0.182 ± 0.0010
0.194 ± 0.0013
21
0.281 ± 0.0016
0.170 ± 0.0006
0.185 ± 0.0011
Cadmium + Chlorpyrifos*
7
0.203 ± 0.0015
0.134 ± 0.0013
(Simple additive)
0.161 ± 0.0001
(Simple additive)
14
0.232 ± 0.0011
0.115 ± 0.0009
(Simple additive)
0.147 ± 0.0013
(Simple additive)
21
0.281 ± 0.0016
0.096 ± 0.0007
(Simple additive)
0.120 ± 0.0010
(Simple additive)
Chlorpyrifos + Cadmium**
7
0.203 ± 0.0015
0.168 ± 0.0008
(Synergistic)
0.170 ± 0.0006
(Synergistic)
14
0.232 ± 0.0011
0.149 ± 0.0007
(Highly synergistic)
0.151 ± 0.0003
(Highly synergistic)
21
0.281 ± 0.0016
0.126 ± 0.0013
(Highly synergistic)
0.136 ± 0.0006
(Highly synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 9a. ANOVA for changes in oxygen consumption rate in Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
Source
Days
Df
Sum Sq
Mean Sq
F value
P value
2
0.00647
0.003234
12.59
<0.05
276
8
Treatment
0.13543
0.016929
65.88
<0.05
Table 9b. Tukey's studentized range (HSD) test for oxygen consumption rate in Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
Tukey Grouping
Mean
Days
A
0.16761
7
B
0.15683
14
B
0.14570
21
Tukey Grouping
Mean
Treatment
A
0.24427
Control
B
0.19177
CPF2
B
0.18131
CPF1
C
0.15233
CPF+Cd2
C
0.14778
CPF+Cd1
D
0.14344
Cd+CPF2
D
0.13152
Cd2
E
0.11501
Cd+CPF1
E
0.10300
Cd1
A
C
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Table 10. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the food consumption rate in Tilapia (Oreochromis mossambicus)
during 7, 14 and 21 days of exposure.
Food consumption rate (g feed consumption/g body wt.)
277
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
0.0143 ± 0.0012
0.0040 ± 0.0003
0.0057 ± 0.0002
14
0.0185 ± 0.0019
0.0035 ± 0.0002
0.0052 ± 0.0001
21
0.0242 ± 0.0016
0.0021 ± 0.0003
0.0036 ± 0.0002
Chlorpyrifos
7
0.0143 ± 0.0012
0.0106 ± 0.0004
0.0129 ± 0.0005
14
0.0185 ± 0.0019
0.0099 ± 0.0005
0.0119 ± 0.0009
21
0.0242 ± 0.0016
0.0095 ± 0.0002
0.0111 ± 0.0006
Cadmium + Chlorpyrifos*
7
0.0143 ± 0.0012
0.0045 ± 0.0004
(Simple additive)
0.0062 ± 0.0005
(Simple additive)
14
0.0185 ± 0.0019
0.0040 ± 0.0003
(Simple additive)
0.0055 ± 0.0004
(Simple additive)
21
0.0242 ± 0.0016
0.0029 ± 0.0001
(Simple additive)
0.0041 ± 0.0001
(Simple additive)
Chlorpyrifos + Cadmium**
7
0.0143 ± 0.0012
0.0083 ± 0.0003
(Synergistic)
0.0087 ± 0.0002
(Synergistic)
14
0.0185 ± 0.0019
0.0078 ± 0.0002
(Synergistic)
0.0080 ± 0.0001
(Synergistic)
21
0.0242 ± 0.0016
0.0064 ± 0.0004
(Synergistic)
0.0077 ± 0.0002
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
278
Table 11a. ANOVA for changes in food consumption rate in Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
Days
2
0.0000035
1.740e-06
0.579
0.563
Treatment
8
0.0018011
2.251e-04
75.074
<0.05
Table 11b. Tukey's studentized range (HSD) test for food consumption rate in Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
Tukey Grouping
Mean
Days
A
0.00845
7
A
0.00825
14
A
0.00795
21
Tukey Grouping
Mean
Treatment
A
0.01919
Control
B
0.01196
CPF2
C
0.00974
CPF1
C
0.00814
CPF+Cd2
0.00762
CPF+Cd1
E
0.00533
Cd+CPF2
F
E
0.00482
Cd2
F
E
0.00384
Cd+CPF1
0.00330
Cd1
D
D
F
Means with the same letter are not significantly different.
279
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Table 12. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ammonia-N excretion rate in Tilapia (Oreochromis mossambicus)
during 7, 14 and 21 days of exposure.
Ammonia excretion rate (µg-at NH3-N excreta/l/g/h)
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
0.0149 ± 0.0007
0.0061 ± 0.0004
0.0080 ± 0.0005
14
0.0181 ± 0.0005
0.0052 ± 0.0002
0.0070 ± 0.0002
21
0.0235 ± 0.0009
0.0039 ± 0.0004
0.0055 ± 0.0007
Chlorpyrifos
7
0.0149 ± 0.0007
0.0136 ± 0.0009
0.0149 ± 0.0005
14
0.0181 ± 0.0005
0.0127 ± 0.0001
0.0138 ± 0.0003
21
0.0235 ± 0.0009
0.0109 ± 0.0006
0.0125 ± 0.0007
Cadmium + Chlorpyrifos*
7
0.0149 ± 0.0007
0.0073 ± 0.0003
(Simple additive)
0.0095 ± 0.0008
(Simple additive)
14
0.0181 ± 0.0005
0.0061 ± 0.0004
(Simple additive)
0.0082 ± 0.0008
(Simple additive)
21
0.0235 ± 0.0009
0.0050 ± 0.0001
(Simple additive)
0.0065 ± 0.0005
(Simple additive)
Chlorpyrifos + Cadmium**
7
0.0149 ± 0.0007
0.0094 ± 0.0004
(Synergistic)
0.0106 ± 0.0009
(Synergistic)
14
0.0181 ± 0.0005
0.0080 ± 0.0002
(Highly synergistic)
0.0090 ± 0.0007
(Highly synergistic)
280
0.0235 ± 0.0009
21
0.0066 ± 0.0007
(Highly synergistic)
0.0077 ± 0.0004
(Highly synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 13a. ANOVA for changes in ammonia-N excretion rate in Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
Days
2
0.0000249
1.247e-05
4.445
<0.05
Treatment
8
0.0014055
1.757e-04
62.604
<0.05
Table 13b. Tukey's studentized range (HSD) test for ammonia-N excretion rate in Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
Tukey Grouping
Mean
Days
A
0.01050
7
B
0.00983
14
B
0.00914
21
Tukey Grouping
Mean
Treatment
A
0.01896
Control
B
0.01374
CPF2
B
0.01230
CPF1
C
0.00911
CPF+Cd2
D
C
0.00810
CPF+Cd1
D
C
0.00804
Cd+CPF2
A
281
D
E
0.00687
Cd2
F E
0.00615
Cd+CPF1
F
0.00509
Cd1
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Table 14. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the Oxygen:Nitrogen ratio in Tilapia (Oreochromis mossambicus)
during 7, 14 and 21 days of exposure.
Oxygen:Nitrogen ratio
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
16.98 ± 0.15
24.53 ± 0.11
23.02 ± 0.18
14
15.99 ± 0.06
24.96 ± 0.21
24.08 ± 0.13
21
14.93 ± 0.17
25.60 ± 0.25
25.04 ± 0.20
Chlorpyrifos
7
16.98 ± 0.15
17.80 ± 0.19
17.18 ± 0.11
14
15.99 ± 0.06
17.89 ± 0.16
17.55 ± 0.19
21
14.93 ± 0.17
19.56 ± 0.08
18.42 ± 0.21
Cadmium + Chlorpyrifos*
7
16.98 ± 0.15
22.66 ± 0.23
(Simple additive)
21.13 ± 0.06
(Simple additive)
14
15.99 ± 0.06
23.25 ± 0.18
(Simple additive)
22.36 ± 0.12
(Simple additive)
282
23.72 ± 0.12
(Simple additive)
14.93 ± 0.17
21
23.01 ± 0.19
(Simple additive)
Chlorpyrifos + Cadmium**
7
16.98 ± 0.15
22.16 ± 0.15
(Synergistic)
20.03 ± 0.14
(Synergistic)
14
15.99 ± 0.06
23.09 ± 0.22
(Synergistic)
21.05 ± 0.17
(Synergistic)
21
14.93 ± 0.17
23.63 ± 0.21
(Synergistic)
22.13 ± 0.13
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 15a. ANOVA for changes in Oxygen:Nitrogen ratio in Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
Days
2
20.5
10.24
22.04
<0.05
Treatment
8
748.4
93.55
201.43
<0.05
Table 15b. Tukey's studentized range (HSD) test for Oxygen:Nitrogen ratio in Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
Tukey Grouping
Mean
Days
A
21.7837
21
B
21.1105
14
B
20.5541
7
Tukey Grouping
Mean
Treatment
283
A
25.0013
Cd1
B
24.3323
Cd2
C
23.0391
CPF+Cd1
C
22.9976
Cd+CPF1
D
22.2238
Cd+CPF2
E
21.1847
CPF+Cd2
F
18.1325
CPF1
F
17.6249
CPF2
G
15.8085
Control
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Table 16. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the relative growth rate in Tilapia (Oreochromis mossambicus) during
7, 14 and 21 days of exposure.
Relative growth rate (%)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
1.53± 0.07
-6.51 ± 0.40
-4.26 ± 0.33
14
4.75 ± 0.21
-12.90 ± 0.86
-7.69 ± 0.53
21
9.19 ± 0.58
-19.86 ± 0.95
-14.53 ± 0.70
Chlorpyrifos
7
1.53± 0.07
-0.57 ± 0.02
-0.45 ± 0.03
14
4.75 ± 0.21
-1.08 ± 0.08
-0.86 ± 0.05
21
9.19 ± 0.58
-5.00 ± 0.28
-2.20 ± 0.13
Cadmium + Chlorpyrifos*
7
1.53± 0.07
-4.30 ± 0.38
-2.53 ± 0.18
284
(Moderately antagonistic)
(Moderately antagonistic)
14
4.75 ± 0.21
-8.75 ± 0.70
(Moderately antagonistic)
-6.00 ± 0.63
(Simple additive)
21
9.19 ± 0.58
-16.45 ± 0.82
(Moderately antagonistic)
-12.03 ± 0.96
(Simple additive)
Chlorpyrifos + Cadmium**
7
1.53± 0.07
-1.97 ± 0.06
(Simple additive)
-1.92 ± 0.08
(Simple additive)
14
4.75 ± 0.21
-6.04 ± 0.59
(Moderately synergistic)
-4.26 ± 0.48
(Moderately synergistic)
21
9.19 ± 0.58
-11.84 ± 0.84
(Synergistic)
-9.93 ± 0.60
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 17a. ANOVA for changes in relative growth rate in Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
Days
2
651.1
325.6
42.51
<0.05
Treatment
8
2082.8
260.4
33.99
<0.05
Table 17b. Tukey's studentized range (HSD) test for relative growth rate in Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
Tukey Grouping
Mean
Days
285
A
-2.3099
7
B
-4.9077
14
C
-9.1865
21
Tukey Grouping
Mean
Treatment
A
5.1777
Control
B
-1.2742
CPF2
B
-2.3245
CPF1
C
-5.5957
CPF+Cd2
D
C
-6.5397
CPF+Cd1
D
C
-6.7717
Cd+ CPF2
D
E
-9.0035
Cd2
E
-10.0430
Cd+CPF1
F
-12.8379
Cd1
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Table 18. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the acetylcholinesterase activity in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Acetylcholinesterase (nmol of ASChI hydrolysed/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
137.49 ± 1.45
94.55 ± 1.72
111.43 ± 1.98
14
139.92 ± 1.10
76.68 ± 1.83
97.98 ± 1.32
21
140.53 ± 1.39
73.41 ± 0.78
89.08 ± 3.87
Chlorpyrifos
7
137.49 ± 1.45
108.76 ± 2.90
123.54 ± 2.46
286
14
139.92 ± 1.10
92.69 ± 1.86
112.08 ± 2.18
21
140.53 ± 1.39
86.26 ± 1.38
103.25 ± 1.53
Cadmium + Chlorpyrifos*
7
137.49 ± 1.45
97.06 ± 1.57
(Moderately antagonistic)
116.35 ± 3.64
(Moderately antagonistic)
14
139.92 ± 1.10
80.90 ± 0.89
(Moderately antagonistic)
102.65 ± 2.98
(Moderately antagonistic)
21
140.53 ± 1.39
78.97 ± 1.06
(Moderately antagonistic)
94.86 ± 3.04
(Moderately antagonistic)
Chlorpyrifos + Cadmium**
7
137.49 ± 1.45
102.28 ± 2.11
(Moderately synergistic)
120.09 ± 2.31
(Moderately synergistic)
14
139.92 ± 1.10
86.42 ± 1.20
(Moderately synergistic)
105.04 ± 3.09
(Moderately synergistic)
21
140.53 ± 1.39
82.75 ± 1.32
(Moderately synergistic)
98.07 ± 2.40
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 19. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the acetylcholinesterase activity in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Acetylcholinesterase (nmol of ASChI hydrolysed/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
91.16 ± 0.48
78.87 ± 2.14
84.90 ± 1.58
14
92.87 ± 1.23
63.84 ± 1.36
74.09 ± 1.83
21
89.23 ± 0.79
56.65 ± 1.54
65.06 ± 2.60
287
Chlorpyrifos
7
91.16 ± 0.48
81.08 ± 0.63
86.39 ± 1.11
14
92.87 ± 1.23
68.33 ± 2.84
77.44 ± 2.39
21
89.23 ± 0.79
59.97 ± 1.91
68.65 ± 2.10
Cadmium + Chlorpyrifos*
7
91.16 ± 0.48
75.08 ± 2.34
(Moderately synergistic)
80.04 ± 1.28
(Moderately synergistic)
14
92.87 ± 1.23
61.93 ± 1.08
(Moderately synergistic)
65.01 ± 1.99
(Moderately synergistic)
21
89.23 ± 0.79
54.77 ± 1.76
(Moderately synergistic)
57.39 ± 1.21
(Moderately synergistic)
Chlorpyrifos + Cadmium**
7
91.16 ± 0.48
73.47 ± 1.73
(Synergistic)
82.43 ± 1.02
(Moderately synergistic)
14
92.87 ± 1.23
58.02 ± 1.87
(Synergistic)
71.35 ± 2.39
(Moderately synergistic)
21
89.23 ± 0.79
52.68 ± 1.20
(Synergistic)
62.11 ± 1.43
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 20. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the acetylcholinesterase activity in the kidney of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Acetylcholinesterase (nmol of ASChI hydrolysed/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
58.81 ± 0.47
49.53 ± 0.84
52.96 ± 1.30
288
14
57.02 ± 1.21
46.42 ± 0.16
49.44 ± 1.66
21
55.95 ± 0.83
44.24 ± 0.77
46.82 ± 0.62
Chlorpyrifos
7
58.81 ± 0.47
50.89 ± 0.28
53.72 ± 0.54
14
57.02 ± 1.21
48.04 ± 0.79
50.31 ± 0.64
21
55.95 ± 0.83
45.91 ± 1.03
47.58 ± 0.41
Cadmium + Chlorpyrifos*
7
58.81 ± 0.47
48.67 ± 1.15
(Simple additive)
50.05 ± 0.88
(Moderately synergistic)
14
57.02 ± 1.21
45.94 ± 0.37
(Simple additive)
47.32 ± 1.09
(Moderately synergistic)
21
55.95 ± 0.83
43.55 ± 0.65
(Simple additive)
45.03 ± 0.25
(Moderately synergistic)
Chlorpyrifos + Cadmium**
7
58.81 ± 0.47
47.04 ± 1.17
(Moderately synergistic)
51.64 ± 1.18
(Moderately synergistic)
14
57.02 ± 1.21
44.80 ± 0.48
(Moderately synergistic)
48.83 ± 0.72
(Simple additive)
21
55.95 ± 0.83
42.33 ± 1.36
(Moderately synergistic)
46.11 ± 0.94
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 21. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the acetylcholinesterase activity in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Acetylcholinesterase (nmol of ASChI hydrolysed/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
289
Cadmium
7
295.22 ± 4.76
186.53 ± 2.48
227.06 ± 4.09
14
281.74 ± 1.98
142.68 ± 1.94
197.92 ± 1.86
21
284.38 ± 2.67
93.56 ± 2.19
156.79 ± 2.47
Chlorpyrifos
7
295.22 ± 4.76
128.60 ± 2.08
212.46 ± 3.89
14
281.74 ± 1.98
76.07 ± 1.92
174.89 ± 2.67
21
284.38 ± 2.67
37.94 ± 1.03
138.01 ± 4.83
Cadmium + Chlorpyrifos*
7
295.22 ± 4.76
158.43 ± 1.59
(Synergistic)
197.11 ± 3.28
(Synergistic)
14
281.74 ± 1.98
109.88 ± 2.33
(Synergistic)
158.05 ± 1.89
(Highly Synergistic)
21
284.38 ± 2.67
74.09 ± 1.25
(Synergistic)
107.32 ± 1.90
(Highly synergistic)
Chlorpyrifos + Cadmium**
7
295.22 ± 4.76
144.37 ± 1.75
(Antagonistic)
171.76 ± 3.00
(Highly synergistic)
14
281.74 ± 1.98
94.95 ± 2.42
(Antagonistic)
123.01 ± 1.67
(Highly synergistic)
21
284.38 ± 2.67
53.33 ± 1.35
(Antagonistic)
88.42 ± 1.19
(Highly synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 22. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the acetylcholinesterase activity in the muscle of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Acetylcholinesterase (nmol of ASChI hydrolysed/min/mg protein)
290
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
188.28 ± 2.23
145.91 ± 2.93
160.07 ± 2.76
14
194.71 ± 1.28
124.54 ± 2.60
142.37 ± 1.35
21
196.53 ± 1.51
115.96 ± 2.69
131.03 ± 1.27
Chlorpyrifos
7
188.28 ± 2.23
130.41 ± 1.82
167.08 ±1.80
14
194.71 ± 1.28
106.29 ± 2.05
148.26 ± 2.48
21
196.53 ± 1.51
95.24 ± 1.58
137.90 ± 1.92
Cadmium + Chlorpyrifos*
7
188.28 ± 2.23
114.53 ± 2.01
(Synergistic)
151.67 ± 2.74
(Synergistic)
14
194.71 ± 1.28
84.25 ± 1.50
(Highly synergistic)
135.43 ± 1.80
(Synergistic)
21
196.53 ± 1.51
65.08 ± 1.39
(Highly synergistic)
124.61 ± 1.34
(Synergistic)
Chlorpyrifos + Cadmium**
7
188.28 ± 2.23
121.47 ± 1.06
(Moderately synergistic)
138.72 ± 2.66
(Synergistic)
14
194.71 ± 1.28
98.28 ± 1.48
(Moderately synergistic)
117.03 ± 3.27
(Synergistic)
21
196.53 ± 1.51
82.72 ± 1.87
(Moderately synergistic)
103.64 ± 2.60
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
291
Table 23. Percentage change in the acetylcholinesterase activity in different organs of Tilapia
(Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% change in acetylcholinesterase activity
Duration
Test organ
7 Days
14 Days
21 Days
Gills
-20.53
-32.60
-37.14
Liver
-11.93
-27.32
-33.14
Kidney
-14.02
-16.45
-19.22
Brain
-39.61
-52.20
-67.06
Muscle
-24.99
-38.60
-45.54
Table 24a. ANOVA for changes in acetylcholinesterase activity in different organs of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
Days
2
58199.26
29099.63
50.20
<0.05
Organ
4
586198.55
146549.63
252.82
<0.05
Treatment
8
200468.18
25058.52
43.23
<0.05
292
Table 24b. Tukey's studentized range (HSD) test for acetylcholinesterase activity in different
organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
Tukey Grouping
Mean
Days
A
116.491
7
B
99.703
14
C
87.233
21
Tukey Grouping
Mean
Organ
A
153.382
Brain
B
130.635
Muscle
C
101.429
Gills
D
71.849
Liver
E
48.416
Kidney
Tukey Grouping
Mean
Treatment
A
153.331
Control
B
113.300
CPF2
B
112.576
Cd2
C
B
102.910
Cd+CPF2
C
D
95.728
CPF+Cd2
C
D
E
92.496
Cd1
D
E
80.956
CPF1
D
E
79.911
Cd+CPF1
E
79.071
CPF+Cd1
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
293
Table 25. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the butyrylcholinesterase activity in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Butyrylcholinesterase (nmol of BSChI hydrolysed/min/mg protein)
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
32.21 ± 0.22
24.13 ± 0.18
26.80 ± 0.89
14
32.73 ± 0.46
21.18 ± 0.51
24.11 ± 0.36
21
32.69 ± 0.17
19.03 ± 0.33
21.76 ± 0.78
Chlorpyrifos
7
32.21 ± 0.22
26.48 ± 0.56
28.33 ± 0.20
14
32.73 ± 0.46
23.40 ± 0.77
26.89 ± 0.54
21
32.69 ± 0.17
21.34 ± 0.23
24.30 ± 0.87
Cadmium + Chlorpyrifos*
7
32.21 ± 0.22
24.69 ± 0.46
(Simple additive)
27.23 ± 0.78
(Simple additive)
14
32.73 ± 0.46
22.03 ± 1.04
(Simple additive)
24.95 ± 0.69
(Simple additive)
21
32.69 ± 0.17
19.72 ± 0.84
(Simple additive)
22.51 ± 1.27
(Simple additive)
Chlorpyrifos + Cadmium**
7
32.21 ± 0.22
25.92 ± 0.45
(Simple additive)
28.08 ± 0.47
(Simple additive)
14
32.73 ± 0.46
22.81 ± 0.24
(Simple additive)
26.17 ± 0.65
(Simple additive)
21
32.69 ± 0.17
20.52 ± 0.45
23.98 ± 0.74
294
(Simple additive)
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 26. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the butyrylcholinesterase activity in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Butyrylcholinesterase (nmol of BSChI hydrolysed/min/mg protein)
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
64.22 ± 1.89
46.74 ± 2.28
55.90 ± 1.55
14
66.83 ± 1.32
33.21 ± 2.41
47.08 ± 2.80
21
69.03 ± 1.67
23.75 ± 1.39
37.18 ± 2.17
Chlorpyrifos
7
64.22 ± 1.89
52.70 ± 0.85
56.76 ± 1.94
14
66.83 ± 1.32
42.97 ± 1.60
49.11 ± 0.53
21
69.03 ± 1.67
30.77 ± 2.35
40.98 ± 1.62
Cadmium + Chlorpyrifos*
7
64.22 ± 1.89
40.14 ± 2.05
(Moderately synergistic)
49.08 ± 1.46
(Moderately synergistic)
14
66.83 ± 1.32
27.75 ± 2.45
(Moderately synergistic)
38.56 ± 2.05
(Moderately synergistic)
21
69.03 ± 1.67
17.87 ± 1.86
(Moderately synergistic)
26.32 ± 2.34
(Moderately synergistic)
Chlorpyrifos + Cadmium**
7
64.22 ± 1.89
35.23 ± 1.82
(Highly synergistic)
54.01 ± 1.20
(Moderately synergistic)
295
14
66.83 ± 1.32
21.08 ± 1.08
(Highly synergistic)
44.43 ± 1.68
(Moderately synergistic)
21
69.03 ± 1.67
12.33 ± 1.66
(Highly synergistic)
32.31 ± 1.43
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 27. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the butyrylcholinesterase activity in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Butyrylcholinesterase (nmol of BSChI hydrolysed/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
20.44 ± 0.35
15.45 ± 0.41
18.74 ± 0.18
14
20.09 ± 0.57
13.75 ± 0.23
16.13 ± 0.46
21
18.88 ± 0.68
10.64 ± 0.33
13.52 ± 0.27
Chlorpyrifos
7
20.44 ± 0.35
16.35 ± 0.48
19.29 ± 0.63
14
20.09 ± 0.57
14.67 ± 0.79
16.94 ± 0.99
21
18.88 ± 0.68
12.26 ± 0.35
13.81 ± 0.56
Cadmium + Chlorpyrifos*
7
20.44 ± 0.35
14.62 ± 1.16
(Simple additive)
16.88 ± 0.85
(Moderately synergistic)
14
20.09 ± 0.57
12.38 ± 0.35
(Simple additive)
13.23 ± 0.46
(Moderately synergistic)
21
18.88 ± 0.68
10.07 ± 0.21
(Simple additive)
11.02 ± 0.18
(Moderately synergistic)
296
Chlorpyrifos + Cadmium**
7
20.44 ± 0.35
14.21 ± 1.20
(Moderately synergistic)
17.55 ± 1.29
(Simple additive)
14
20.09 ± 0.57
11.53 ± 0.44
(Moderately synergistic)
15.27 ± 0.62
(Simple additive)
21
18.88 ± 0.68
8.93 ± 0.76
(Moderately synergistic)
12.39 ± 0.35
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 28. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the butyrylcholinesterase activity in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Butyrylcholinesterase (nmol of BSChI hydrolysed/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
42.64 ± 1.38
35.51 ± 0.61
37.82 ± 0.36
14
40.82 ± 0.89
28.75 ± 0.55
33.11 ± 1.27
21
41.20 ± 1.54
23.05 ± 0.19
28.77 ± 0.66
Chlorpyrifos
7
42.64 ± 1.38
28.03 ± 1.45
37.07 ± 0.82
14
40.82 ± 0.89
21.33 ± 1.06
32.94 ± 1.03
21
41.20 ± 1.54
18.09 ± 0.34
26.53 ± 0.35
Cadmium + Chlorpyrifos*
7
42.64 ± 1.38
32.47 ± 0.87
(Moderately synergistic)
36.27 ± 0.40
(Simple additive)
14
40.82 ± 0.89
24.81 ± 0.48
(Moderately synergistic)
30.29 ± 1.74
(Moderately synergistic)
297
20.76 ± 0.25
(Moderately synergistic)
41.20 ± 1.54
21
24.61 ± 0.52
(Moderately synergistic)
Chlorpyrifos + Cadmium**
7
42.64 ± 1.38
30.10 ± 0.69
(Moderately antagonistic)
33.01 ± 1.30
(Moderately synergistic)
14
40.82 ± 0.89
23.07 ± 0.84
(Moderately antagonistic)
27.44 ± 1.12
(Moderately synergistic)
21
41.20 ± 1.54
20.14 ± 0.78
(Moderately antagonistic)
21.37 ± 0.95
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 29. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the butyrylcholinesterase activity in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Butyrylcholinesterase (nmol of BSChI hydrolysed/min/mg protein)
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
95.63 ± 2.90
76.66 ± 1.27
81.09 ± 1.49
14
102.38 ± 3.57
66.58 ± 2.27
72.74 ± 2.24
21
113.92 ± 3.68
61.00 ± 1.57
67.86 ± 1.72
Chlorpyrifos
7
95.63 ± 2.90
68.65 ± 2.04
83.86 ± 1.92
14
102.38 ± 3.57
59.03 ± 1.60
75.99 ± 1.59
21
113.92 ± 3.68
54.44 ± 2.91
69.71 ± 1.13
Cadmium + Chlorpyrifos*
7
95.63 ± 2.90
64.04 ± 1.37
78.21 ± 2.85
298
(Synergistic)
(Moderately synergistic)
14
102.38 ± 3.57
57.55 ± 1.35
(Synergistic)
69.22 ± 1.64
(Moderately synergistic)
21
113.92 ± 3.68
32.03 ± 4.60
(Highly synergistic)
64.21 ± 1.89
(Moderately synergistic)
Chlorpyrifos + Cadmium**
7
95.63 ± 2.90
70.75 ± 1.50
(Moderately antagonistic)
73.32 ± 1.76
(Synergistic)
14
102.38 ± 3.57
62.87 ± 1.05
(Moderately antagonistic)
64.04 ± 1.82
(Synergistic)
21
113.92 ± 3.68
46.90 ± 2.76
(Moderately synergistic)
59.16 ± 3.40
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 30. Percentage change in the butyrylcholinesterase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% change in butyrylcholinesterase activity
Duration
Test organ
7 Days
14 Days
21 Days
Gills
-17.86
-26.85
-33.79
Liver
-23.98
-43.10
-59.89
Kidney
-18.61
-29.13
-38.67
Brain
-20.77
-32.10
-44.38
Muscle
-22.02
-35.53
-50.04
Table 31a. ANOVA for changes in butyrylcholinesterase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
299
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
Days
2
64974.71
32487.35
175.59
<0.05
Organ
4
33416.61
8354.15
45.15
<0.05
Treatment
8
20447.56
2555.94
13.81
<0.05
Table 31b. Tukey's studentized range (HSD) test for butyrylcholinesterase activity in
different organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and
1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
Tukey Grouping
Mean
Days
A
52.762
21
B
32.994
7
C
22.169
14
Tukey Grouping
Mean
Organ
A
46.228
Muscle
A
44.120
Brain
B
38.009
Kidney
C
29.513
Gills
D
22.004
Liver
300
Tukey Grouping
Mean
Treatment
A
52.873
Control
B
39.856
CPF2
B
38.662
Cd2
C
B
35.416
CPF+Cd2
C
B
35.302
Cd+CPF2
C
B
32.972
Cd1
C
B
32.544
CPF1
C
28.332
CPF+Cd1
C
27.815
Cd+CPF1
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Table 32. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the lipid peroxidation level in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Lipid peroxidation level (nmol of TBARS formed/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
6.23 ± 0.16
12.75 ± 0.41
8.43 ± 0.21
14
6.92 ± 0.07
16.34 ± 0.39
13.12 ± 0.32
21
7.09 ± 0.05
18.27 ± 0.28
16.63 ± 0.26
Chlorpyrifos
301
7
6.23 ± 0.16
7.22 ± 0.17
7.01 ± 0.07
14
6.92 ± 0.07
8.23 ± 0.09
7.63 ± 0.13
21
7.09 ± 0.05
10.12 ± 0.18
8.91 ± 0.37
Cadmium + Chlorpyrifos*
7
6.23 ± 0.16
10.56 ± 0.25
(Moderately antagonistic)
7.99 ± 0.20
(Simple additive)
14
6.92 ± 0.07
15.64 ± 0.19
(Simple additive)
11.31 ± 0.31
(Moderately antagonistic)
21
7.09 ± 0.05
17.58 ± 0.12
(Simple additive)
15.75 ± 0.28
(Simple additive)
Chlorpyrifos + Cadmium**
7
6.23 ± 0.16
9.89 ± 0.48
(Moderately synergistic)
7.42 ± 0.23
(Simple additive)
14
6.92 ± 0.07
13.69 ± 0.39
(Synergistic)
10.78 ± 0.18
(Moderately synergistic)
21
7.09 ± 0.05
17.41 ± 0.51
(Synergistic)
14.05 ± 0.30
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 33. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the lipid peroxidation level in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Lipid peroxidation level (nmol of TBARS formed/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
6.31 ± 0.24
10.84 ± 0.36
9.98 ± 0.31
14
6.74 ± 0.16
13.30 ± 0.29
11.81 ± 0.18
21
6.32 ± 0.10
14.62 ± 0.41
13.77 ± 0.39
302
Chlorpyrifos
7
6.31 ± 0.24
6.85 ± 0.20
6.56 ± 0.12
14
6.74 ± 0.16
8.75 ± 0.13
7.63 ± 0.20
21
6.32 ± 0.10
10.23 ± 0.27
8.05 ± 0.17
Cadmium + Chlorpyrifos*
7
6.31 ± 0.24
11.49 ± 0.30
(Simple additive)
9.03 ± 0.21
(Simple additive)
14
6.74 ± 0.16
13.53 ± 0.31
(Simple additive)
10.90 ± 0.33
(Simple additive)
21
6.32 ± 0.10
14.71 ± 0.25
(Simple additive)
12.45 ± 0.35
(Simple additive)
Chlorpyrifos + Cadmium**
7
6.31 ± 0.24
10.35 ± 0.30
(Moderately synergistic)
8.76 ± 0.28
(Moderately synergistic)
14
6.74 ± 0.16
12.67 ± 0.36
(Moderately synergistic)
10.47 ± 0.39
(Moderately synergistic)
21
6.32 ± 0.10
14.11 ± 0.41
(Moderately synergistic)
11.92 ± 0.20
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 34. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the lipid peroxidation level in the kidney of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Lipid peroxidation level (nmol of TBARS formed/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
2.78 ± 0.14
4.32 ± 0.13
3.82 ± 0.29
303
14
3.02 ± 0.06
5.34 ± 0.21
4.75 ± 0.18
21
3.27 ± 0.09
6.21 ± 0.24
5.57 ± 0.16
Chlorpyrifos
7
2.78 ± 0.14
3.43 ± 0.13
3.22 ± 0.18
14
3.02 ± 0.06
3.68 ± 0.36
3.29 ± 0.12
21
3.27 ± 0.09
4.52 ± 0.26
4.06 ± 0.31
Cadmium + Chlorpyrifos*
7
2.78 ± 0.14
4.57 ± 0.24
(Simple additive)
3.52 ± 0.18
(Simple additive)
14
3.02 ± 0.06
5.89 ± 0.31
(Simple additive)
4.47 ± 0.26
(Simple additive)
21
3.27 ± 0.09
6.98 ± 0.35
(Simple additive)
5.41 ± 0.31
(Simple additive)
Chlorpyrifos + Cadmium**
7
2.78 ± 0.14
4.24 ± 0.35
(Moderately synergistic)
3.28 ± 0.16
(Simple additive)
14
3.02 ± 0.06
4.88 ± 0.24
(Moderately synergistic)
3.94 ± 0.20
(Simple additive)
21
3.27 ± 0.09
5.84 ± 0.30
(Moderately synergistic)
4.9 ± 0.36
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 35. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the lipid peroxidation level in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Lipid peroxidation level (nmol of TBARS formed/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
304
Cadmium
7
3.46 ± 0.18
5.65 ± 0.39
3.74 ± 0.26
14
4.35 ± 0.09
7.36 ± 0.40
5.31 ± 0.29
21
4.59 ± 0.13
8.45 ± 0.36
5.63 ± 0.10
Chlorpyrifos
7
3.46 ± 0.18
4.47 ± 0.26
3.55 ± 0.12
14
4.35 ± 0.09
5.95 ± 0.18
4.43 ± 0.20
21
4.59 ± 0.13
6.64 ± 0.20
5.21 ± 0.18
Cadmium + Chlorpyrifos*
7
3.46 ± 0.18
6.33 ± 0.16
(Simple additive)
4.56 ± 0.26
(Simple additive)
14
4.35 ± 0.09
7.81 ± 0.24
(Simple additive)
6.79 ± 0.31
(Moderately synergistic)
21
4.59 ± 0.13
8.78 ± 0.11
(Simple additive)
7.22 ± 0.27
(Moderately synergistic)
Chlorpyrifos + Cadmium**
7
3.46 ± 0.18
6.78 ± 0.21
(Moderately synergistic)
4.89 ± 0.23
(Moderately synergistic)
14
4.35 ± 0.09
8.25 ± 0.26
(Moderately synergistic)
7.14 ± 0.06
(Moderately synergistic)
21
4.59 ± 0.13
9.29 ± 0.32
(Moderately synergistic)
7.68 ± 0.17
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 36. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the lipid peroxidation level in the muscle of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Lipid peroxidation level (nmol of TBARS formed/mg protein)
Duration
Exposure concentration
305
(days)
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
0.91 ± 0.02
1.42 ± 0.03
1.19 ± 0.06
14
0.95 ± 0.01
1.46 ± 0.01
1.30 ± 0.01
21
0.94 ± 0.03
1.48 ± 0.02
1.31 ± 0.01
Chlorpyrifos
7
0.91 ± 0.02
0.98 ± 0.04
0.95 ± 0.01
14
0.95 ± 0.01
1.16 ± 0.03
1.09 ± 0.05
21
0.94 ± 0.03
1.20 ± 0.02
1.12 ± 0.06
Cadmium + Chlorpyrifos*
7
0.91 ± 0.02
1.40 ± 0.03
(Simple additive)
1.15 ± 0.07
(Simple additive)
14
0.95 ± 0.01
1.42 ± 0.02
(Simple additive)
1.27 ± 0.02
(Simple additive)
21
0.94 ± 0.03
1.37 ± 0.05
(Simple additive)
1.29 ± 0.03
(Simple additive)
Chlorpyrifos + Cadmium**
7
0.91 ± 0.02
1.23 ± 0.04
(Simple additive)
1.01 ± 0.01
(Simple additive)
14
0.95 ± 0.01
1.31 ± 0.02
(Simple additive)
1.19 ± 0.01
(Simple additive)
21
0.94 ± 0.03
1.35 ± 0.02
(Simple additive)
1.21 ± 0.01
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 37. Percentage change in the lipid peroxidation level in different organs of Tilapia
(Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
306
% change in lipid peroxidation level
Duration
Test organ
7 Days
14 Days
21 Days
Gills
+43.03
+74.76
+109.31
Liver
+46.32
+65.17
+97.51
Kidney
+36.82
+50.18
+66.36
Brain
+44.40
+52.41
+60.50
Muscle
+28.38
+34.71
+38.13
Table 38a. ANOVA for changes in levels of lipid peroxidation in different organs of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
Days
2
3657.74
1828.87
261.69
<0.05
Organ
4
820.40
205.10
29.35
<0.05
Treatment
8
876.05
109.50
15.67
<0.05
Table 38b. Tukey's studentized range (HSD) test for levels of lipid peroxidation in different
organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
307
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
Tukey Grouping
Mean
Days
A
10.7233
7
B
6.0533
14
C
3.4604
21
Tukey Grouping
Mean
Organ
A
9.0936
Gills
B
7.4253
Liver
C
B
6.5528
Kidney
C
D
5.5504
Muscle
D
5.1062
Brain
Tukey Grouping
Mean
Treatment
A
8.5707
Cd1
A
8.5249
Cd+CPF1
B A
8.1838
CPF+Cd1
B A
C
7.0762
Cd2
B A
C
6.8689
Cd+CPF2
B
C
6.7029
CPF+Cd2
C
5.6033
CPF1
D
4.8896
CPF2
D
4.2907
Control
D
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
308
Table 39. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the superoxide dismutase activity in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Superoxide dismutase activity (Units/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
4.22 ± 0.07
9.10 ± 0.25
7.29 ± 0.22
14
4.14 ± 0.11
8.34 ± 0.18
5.90 ± 0.10
21
4.37 ± 0.15
7.62 ± 0.13
5.79 ± 0.06
Chlorpyrifos
7
4.22 ± 0.07
5.27 ± 0.05
5.12 ± 0.02
14
4.14 ± 0.11
5.58 ± 0.03
5.15 ± 0.06
21
4.37 ± 0.15
5.93 ± 0.13
5.78 ± 0.11
Cadmium + Chlorpyrifos*
7
4.22 ± 0.07
8.75 ± 0.25
(Simple additive)
6.82 ± 0.19
(Simple additive)
14
4.14 ± 0.11
7.68 ± 0.21
(Simple additive)
5.88 ± 0.21
(Simple additive)
21
4.37 ± 0.15
6.23 ± 0.18
(Moderately antagonistic)
5.51 ± 0.10
(Simple additive)
Chlorpyrifos + Cadmium**
7
4.22 ± 0.07
8.04 ± 0.31
(Moderately synergistic)
6.26 ± 0.24
(Moderately synergistic)
14
4.14 ± 0.11
6.75 ± 0.16
(Moderately synergistic)
5.53 ± 0.08
(Simple additive)
21
4.37 ± 0.15
5.97 ± 0.18
(Simple additive)
5.40 ± 0.07
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
309
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 40. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the superoxide dismutase activity in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Superoxide dismutase activity (Units/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
6.32 ± 0.10
13.54 ± 0.47
11.88 ± 0.35
14
6.43 ± 0.07
11.52 ± 0.34
10.96 ± 0.26
21
6.76 ± 0.09
10.15 ± 0.28
9.77 ± 0.20
Chlorpyrifos
7
6.32 ± 0.10
8.64 ± 0.18
8.02 ± 0.08
14
6.43 ± 0.07
8.75 ± 0.21
8.91 ± 0.12
21
6.76 ± 0.09
10.12 ± 0.22
9.78 ± 0.24
Cadmium + Chlorpyrifos*
7
6.32 ± 0.10
14.79 ± 0.30
(Moderately synergistic)
11.63 ± 0.24
(Simple additive)
14
6.43 ± 0.07
13.44 ± 0.35
(Moderately synergistic)
9.28 ± 0.07
(Moderately antagonistic)
21
6.76 ± 0.09
11.58 ± 0.23
(Moderately synergistic)
9.20 ± 0.11
(Simple additive)
Chlorpyrifos + Cadmium**
7
6.32 ± 0.10
12.31 ± 0.34
(Moderately synergistic)
10.70 ± 0.27
(Moderately synergistic)
14
6.43 ± 0.07
10.08 ± 0.15
(Moderately synergistic)
9.541 ± 0.22
(Simple additive)
21
6.76 ± 0.09
9.81 ± 0.20
(Simple additive)
8.83 ± 0.15
(Simple additive)
310
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 41. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the superoxide dismutase activity in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Superoxide dismutase activity (Units/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
3.25 ± 0.15
6.65 ± 0.22
5.63 ± 0.15
14
3.43 ± 0.08
5.50 ± 0.16
5.41 ± 0.11
21
3.40 ± 0.05
4.85 ± 0.18
4.52 ± 0.12
Chlorpyrifos
7
3.25 ± 0.15
4.13 ± 0.12
3.88 ± 0.15
14
3.43 ± 0.08
4.40 ± 0.09
4.00 ± 0.05
21
3.40 ± 0.05
4.78 ± 0.16
4.24 ± 0.21
Cadmium + Chlorpyrifos*
7
3.25 ± 0.15
6.95 ± 0.13
(Simple additive)
5.07 ± 0.09
(Simple additive)
14
3.43 ± 0.08
6.76 ± 0.12
(Moderately synergistic)
4.88 ± 0.05
(Simple additive)
21
3.40 ± 0.05
5.61 ± 0.17
(Moderately synergistic)
4.33 ± 0.10
(Simple additive)
Chlorpyrifos + Cadmium**
7
3.25 ± 0.15
5.76 ± 0.19
(Moderately synergistic)
4.92 ± 0.06
(Moderately synergistic)
14
3.43 ± 0.08
5.12 ± 0.16
(Simple additive)
4.69 ± 0.11
(Simple additive)
311
3.40 ± 0.05
21
4.72 ± 0.14
(Simple additive)
4.15 ± 0.08
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 42. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the superoxide dismutase activity in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Superoxide dismutase activity (Units/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
2.23 ± 0.06
3.86 ± 0.14
2.59 ± 0.05
14
2.12 ± 0.12
3.67 ± 0.12
2.31 ± 0.10
21
2.02 ± 0.05
2.90 ± 0.17
2.27 ± 0.03
Chlorpyrifos
7
2.23 ± 0.06
3.15 ± 0.12
2.36 ± 0.07
14
2.12 ± 0.12
2.54 ± 0.07
2.15 ± 0.04
21
2.02 ± 0.05
2.45 ± 0.05
2.12 ± 0.05
Cadmium + Chlorpyrifos*
7
2.23 ± 0.06
4.41 ± 0.12
(Simple additive)
3.43 ± 0.15
(Moderately synergistic)
14
2.12 ± 0.12
4.19 ± 0.08
(Simple additive)
3.27 ± 0.07
(Moderately synergistic)
21
2.02 ± 0.05
2.97 ± 0.15
(Simple additive)
2.52 ± 0.05
(Simple additive)
Chlorpyrifos + Cadmium**
7
2.23 ± 0.06
4.67 ± 0.16
(Moderately synergistic)
3.79 ± 0.18
(Moderately synergistic)
312
14
2.12 ± 0.12
4.25 ± 0.13
(Moderately synergistic)
3.48 ± 0.13
(Moderately synergistic)
21
2.02 ± 0.05
3.06 ± 0.18
(Simple additive)
2.79 ± 0.09
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 43. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the superoxide dismutase activity in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Superoxide dismutase activity (Units/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
1.52 ± 0.04
1.84 ± 0.05
1.78 ± 0.05
14
1.59 ± 0.02
2.01 ± 0.04
1.84 ± 0.06
21
1.61 ± 0.06
2.12 ± 0.08
1.95 ± 0.02
Chlorpyrifos
7
1.52 ± 0.04
1.56 ± 0.08
1.54 ± 0.07
14
1.59 ± 0.02
1.64 ± 0.01
1.61 ± 0.07
21
1.61 ± 0.06
1.67 ± 0.03
1.64 ± 0.02
Cadmium + Chlorpyrifos*
7
1.52 ± 0.04
1.80 ± 0.06
(Simple additive)
1.67 ± 0.01
(Simple additive)
14
1.59 ± 0.02
1.94 ± 0.06
(Simple additive)
1.78 ± 0.04
(Simple additive)
21
1.61 ± 0.06
2.05 ± 0.08
(Simple additive)
1.90 ± 0.02
(Simple additive)
313
Chlorpyrifos + Cadmium**
7
1.52 ± 0.04
1.76 ± 0.07
(Simple additive)
1.62 ± 0.09
(Simple additive)
14
1.59 ± 0.02
1.90 ± 0.03
(Simple additive)
1.66 ± 0.01
(Simple additive)
21
1.61 ± 0.06
1.98 ± 0.05
(Simple additive)
1.85 ± 0.08
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 44. Percentage change in the superoxide dismutase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% change in superoxide dismutase activity
Duration
Test organ
7 Days
14 Days
21 Days
Gills
+67.93
+53.58
+38.08
Liver
+81.02
+60.34
+46.57
Kidney
+65.41
+48.56
+36.89
Brain
+58.64
+53.27
+30.69
Muscle
+11.91
+13.29
+18.03
Table 45a. ANOVA for changes in superoxide dismutase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
314
Days
2
2229.90
1114.95
354.95
<0.05
Organ
4
280.25
70.06
22.31
<0.05
Treatment
8
318.29
39.78
12.67
<0.05
Table 45b. Tukey's studentized range (HSD) test for superoxide dismutase activity in
different organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and
1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
Tukey Grouping
Mean
Days
A
7.9431
7
B
5.4970
14
C
2.2157
21
Tukey Grouping
Mean
Organ
A
6.4852
Gills
B A
5.7777
Brain
B C
5.0790
Muscle
D
4.6016
Liver
4.1495
Kidney
Mean
Treatment
C
D
Tukey Grouping
315
A
6.6573
Cd+CPF1
B A
6.2760
Cd1
B A
C
5.7731
CPF+Cd1
B D
C
5.3451
Cd2
B D
C
5.1413
Cd+CPF2
D
C
5.0202
CPF+Cd2
E
D C
4.7249
CPF1
E
D
4.4469
CPF2
3.5824
Control
E
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Table 46. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the catalase activity in the gills of Tilapia (Oreochromis mossambicus)
during 7, 14 and 21 days of exposure.
Catalase activity (Units/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
3.11 ± 0.10
6.15 ± 0.26
5.12 ± 0.11
14
3.37 ± 0.07
7.56 ± 0.18
6.54 ± 0.15
21
3.23 ± 0.06
6.85 ± 0.26
5.50 ± 0.21
Chlorpyrifos
7
3.11 ± 0.10
4.68 ± 0.07
4.20 ± 0.11
14
3.37 ± 0.07
4.67 ± 0.18
4.53 ± 0.16
316
3.23 ± 0.06
21
4.98 ± 0.20
4.76 ± 0.14
Cadmium + Chlorpyrifos*
7
3.11 ± 0.10
5.74 ± 0.16
(Simple additive)
5.05 ± 0.09
(Simple additive)
14
3.37 ± 0.07
7.07 ± 0.19
(Simple additive)
5.85 ± 0.12
(Simple additive)
21
3.23 ± 0.06
6.37 ± 0.06
(Simple additive)
5.19 ± 0.14
(Simple additive)
Chlorpyrifos + Cadmium**
7
3.11 ± 0.10
5.39 ± 0.17
(Simple additive)
4.83 ± 0.12
(Simple additive)
14
3.37 ± 0.07
6.72 ± 0.16
(Moderately synergistic)
5.29 ± 0.12
(Simple additive)
21
3.23 ± 0.06
5.93 ± 0.22
(Moderately synergistic)
4.96 ± 0.15
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 47. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the catalase activity in the liver of Tilapia (Oreochromis mossambicus)
during 7, 14 and 21 days of exposure.
Catalase activity (Units/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
3.81 ± 0.13
6.47 ± 0.26
5.75 ± 0.14
14
4.12 ± 0.17
8.25 ± 0.21
6.93 ± 0.18
21
4.11 ± 0.12
7.67 ± 0.19
6.54 ± 0.16
Chlorpyrifos
7
3.81 ± 0.13
5.46 ± 0.08
5.20 ± 0.10
317
14
4.12 ± 0.17
5.72 ± 0.12
5.58 ± 0.08
21
4.11 ± 0.12
5.87 ± 0.13
5.69 ± 0.10
Cadmium + Chlorpyrifos*
7
3.81 ± 0.13
6.76 ± 0.24
(Simple additive)
5.69 ± 0.14
(Simple additive)
14
4.12 ± 0.17
8.65 ± 0.21
(Simple additive)
6.76 ± 0.23
(Simple additive)
21
4.11 ± 0.12
7.92 ± 0.24
(Simple additive)
6.17 ± 0.17
(Simple additive)
Chlorpyrifos + Cadmium**
7
3.81 ± 0.13
6.08 ± 0.11
(Simple additive)
5.54 ± 0.09
(Simple additive)
14
4.12 ± 0.17
7.41 ± 0.32
(Moderately synergistic)
6.34 ± 0.17
(Simple additive)
21
4.11 ± 0.12
6.82 ± 0.24
(Moderately synergistic)
6.03 ± 0.05
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 48. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the catalase activity in the kidney of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Catalase activity (Units/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
2.31 ± 0.11
4.62 ± 0.18
4.16 ± 0.16
14
2.43 ± 0.08
5.22 ± 0.20
4.73 ± 0.12
21
2.31 ± 0.07
3.82 ± 0.15
3.43 ± 0.13
318
Chlorpyrifos
7
2.31 ± 0.11
3.26 ± 0.06
3.05 ± 0.07
14
2.43 ± 0.08
3.57 ± 0.06
3.06 ± 0.14
21
2.31 ± 0.07
3.87 ± 0.13
3.64 ± 0.17
Cadmium + Chlorpyrifos*
7
2.31 ± 0.11
4.77 ± 0.21
(Simple additive)
3.94 ± 0.23
(Simple additive)
14
2.43 ± 0.08
5.49 ± 0.27
(Simple additive)
4.58 ± 0.28
(Simple additive)
21
2.31 ± 0.07
4.06 ± 0.15
(Simple additive)
3.30 ± 0.20
(Simple additive)
Chlorpyrifos + Cadmium**
7
2.31 ± 0.11
4.38 ± 0.25
(Moderately synergistic)
3.68 ± 0.17
(Simple additive)
14
2.43 ± 0.08
4.92 ± 0.31
(Moderately synergistic)
4.25 ± 0.16
(Moderately synergistic)
21
2.31 ± 0.07
3.57 ± 0.17
(Simple additive)
3.29 ± 0.24
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 49. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the catalase activity in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Catalase activity (Units/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
1.87 ± 0.06
3.13 ± 0.08
2.18 ± 0.08
319
14
1.78 ± 0.06
3.30 ± 0.09
2.07 ± 0.06
21
1.71 ± 0.02
2.34 ± 0.16
2.04 ± 0.03
Chlorpyrifos
7
1.87 ± 0.06
2.36 ± 0.02
1.93 ± 0.05
14
1.78 ± 0.06
2.42 ± 0.05
1.96 ± 0.08
21
1.71 ± 0.02
2.12 ± 0.11
1.92 ± 0.04
Cadmium + Chlorpyrifos*
7
1.87 ± 0.06
3.25 ± 0.12
(Simple additive)
2.61 ± 0.07
(Simple additive)
14
1.78 ± 0.06
3.55 ± 0.08
(Simple additive)
2.73 ± 0.09
(Simple additive)
21
1.71 ± 0.02
2.40 ± 0.13
(Simple additive)
2.16 ± 0.05
(Simple additive)
Chlorpyrifos + Cadmium**
7
1.87 ± 0.06
3.37 ± 0.13
(Moderately synergistic)
2.84 ± 0.04
(Moderately synergistic)
14
1.78 ± 0.06
3.78 ± 0.16
(Moderately synergistic)
2.88 ± 0.02
(Moderately synergistic)
21
1.71 ± 0.02
2.56 ± 0.14
(Simple additive)
2.23 ± 0.07
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 50. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the catalase activity in the muscle of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Catalase activity (Units/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
320
Cadmium
7
0.94 ± 0.02
1.49 ± 0.07
1.15 ± 0.06
14
0.95 ± 0.07
1.44 ± 0.03
1.28 ± 0.02
21
0.95 ± 0.05
1.46 ± 0.01
1.27 ± 0.02
Chlorpyrifos
7
0.94 ± 0.02
0.99 ± 0.06
0.98 ± 0.04
14
0.95 ± 0.07
1.21 ± 0.01
1.20 ± 0.05
21
0.95 ± 0.05
1.25 ± 0.03
1.22 ± 0.02
Cadmium + Chlorpyrifos*
7
0.94 ± 0.02
1.40 ± 0.04
(Simple additive)
1.07 ± 0.08
(Simple additive)
14
0.95 ± 0.07
1.38 ± 0.03
(Simple additive)
1.26 ± 0.04
(Simple additive)
21
0.95 ± 0.05
1.39 ± 0.03
(Simple additive)
1.23 ± 0.03
(Simple additive)
Chlorpyrifos + Cadmium**
7
0.94 ± 0.02
1.24 ± 0.02
(Simple additive)
1.03 ± 0.04
(Simple additive)
14
0.95 ± 0.07
1.27 ± 0.05
(Simple additive)
1.24 ± 0.02
(Simple additive)
21
0.95 ± 0.05
1.32 ± 0.06
(Simple additive)
1.26 ± 0.02
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 51. Percentage change in the catalase activity in different organs of Tilapia
(Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% change in catalase activity
321
Duration
Test organ
7 Days
14 Days
21 Days
Gills
+65.61
+78.99
+72.51
Liver
+54.04
+69.60
+59.67
Kidney
+72.45
+84.38
+57.07
Brain
+45.07
+59.65
+30.15
Muscle
+24.79
+35.88
+36.82
Table 52a. ANOVA for changes in catalase activity in different organs of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
Days
2
1064.97
532.48
686.61
<0.05
Organ
4
81.31
20.32
26.21
<0.05
Treatment
8
179.82
22.47
28.98
<0.05
Table 52b. Tukey's studentized range (HSD) test for catalase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
322
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
Tukey Grouping
Mean
Days
A
5.6599
7
B
4.0968
14
C
1.7160
21
Tukey Grouping
Mean
Organ
A
4.6665
Gills
B
3.9025
Liver
B
3.5714
Kidney
C
3.5177
Muscle
C
3.4632
Brain
Tukey Grouping
Mean
Treatment
A
4.6909
Cd+CPF1
A
4.6578
Cd1
B A
4.3296
CPF+Cd1
B C
3.9162
Cd2
C
B C
D
3.8513
Cd+CPF2
C
D
3.7313
CPF+Cd2
C
D
3.5122
CPF1
D
3.2751
CPF2
2.4538
Control
E
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
323
Table 53. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione peroxidase activity in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Glutathione peroxidase (nmol of NADPH oxidized/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
16.62 ± 0.21
32.80 ± 0.78
26.86 ± 0.61
14
16.04 ± 0.28
36.34 ± 0.62
32.06 ± 0.50
21
17.05 ± 0.23
34.26 ± 0.41
28.22 ± 0.49
Chlorpyrifos
7
16.62 ± 0.21
21.21 ± 0.55
19.76 ± 0.18
14
16.04 ± 0.28
24.12 ± 0.39
23.00 ± 0.15
21
17.05 ± 0.23
25.34 ± 0.22
24.56 ± 0.20
Cadmium + Chlorpyrifos*
30.45 ± 0.58
25.12 ± 0.46
(Moderately antagonistic) (Moderately antagonistic)
7
16.62 ± 0.21
14
16.04 ± 0.28
35.87 ± 0.61
(Simple additive)
30.64 ± 0.39
(Moderately antagonistic)
21
17.05 ± 0.23
31.58 ± 0.54
(Moderately antagonistic)
27.88 ± 0.73
(Simple additive)
Chlorpyrifos + Cadmium**
7
16.62 ± 0.21
29.90 ± 0.48
(Synergistic)
23.36 ± 0.72
(Moderately synergistic)
14
16.04 ± 0.28
33.23 ± 0.52
(Synergistic)
27.43 ± 0.26
(Moderately synergistic)
21
17.05 ± 0.23
28.75 ± 0.55
(Moderately synergistic)
26.31 ± 0.35
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
324
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 54. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione peroxidase activity in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Glutathione peroxidase (nmol of NADPH oxidized/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
18.97 ± 0.26
32.83 ± 0.98
29.09 ± 0.61
14
21.18 ± 0.10
41.34 ± 0.83
38.28 ± 0.59
21
21.42 ± 0.21
37.78 ± 0.92
34.67 ± 0.79
Chlorpyrifos
7
18.97 ± 0.26
25.78 ± 0.42
24.32 ± 0.49
14
21.18 ± 0.10
29.10 ± 0.27
26.04 ± 0.34
21
21.42 ± 0.21
28.92 ± 0.30
27.18 ± 0.13
Cadmium + Chlorpyrifos*
7
18.97 ± 0.26
34.47 ± 0.83
(Simple additive)
28.22 ± 0.51
(Simple additive)
14
21.18 ± 0.10
43.06 ± 0.79
(Simple additive)
36.13 ± 0.47
(Simple additive)
21
21.42 ± 0.21
38.50 ± 0.90
(Simple additive)
33.35 ± 0.58
(Simple additive)
Chlorpyrifos + Cadmium**
7
18.97 ± 0.26
30.61 ± 0.74
(Synergistic)
27.46 ± 0.40
(Moderately synergistic)
14
21.18 ± 0.10
39.82 ± 0.95
(Synergistic)
35.57 ± 0.51
(Synergistic)
21
21.42 ± 0.21
35.56 ± 0.69
(Synergistic)
32.49 ± 0.24
(Synergistic)
325
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 55. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione peroxidase activity in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Glutathione peroxidase (nmol of NADPH oxidized/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
11.56 ± 0.16
22.31 ± 034
20.43 ± 0.36
14
11.75 ± 0.18
24.82 ± 0.48
22.37 ± 0.52
21
10.75 ± 0.23
20.39 ± 0.62
17.24 ± 0.41
Chlorpyrifos
7
11.56 ± 0.16
14.14 ± 0.36
13.83 ± 0.74
14
11.75 ± 0.18
16.50 ± 0.58
15.06 ± 0.69
21
10.75 ± 0.23
18.32 ± 0.25
17.26 ± 0.29
Cadmium + Chlorpyrifos*
7
11.56 ± 0.16
23.98 ± 0.62
(Simple additive)
19.72 ± 0.37
(Simple additive)
14
11.75 ± 0.18
25.65 ± 0.84
(Simple additive)
21.35 ± 0.57
(Simple additive)
21
10.75 ± 0.23
22.86 ± 0.27
(Moderately synergistic)
15.27 ± 0.76
(Moderately antagonistic)
Chlorpyrifos + Cadmium**
7
11.56 ± 0.16
21.86 ± 0.19
(Synergistic)
18.25 ± 0.23
(Moderately synergistic)
14
11.75 ± 0.18
23.10 ± 0.52
(Synergistic)
20.67 ± 0.20
(Moderately synergistic)
326
19.41 ± 0.49
(Moderately synergistic)
10.75 ± 0.23
21
14.64 ± 0.60
(Moderately antagonistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 56. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione peroxidase activity in the brain of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Glutathione peroxidase (nmol of NADPH oxidized/min/mg protein)
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
11.03 ± 0.14
16.87 ± 0.49
12.62 ± 0.20
14
11.35 ± 0.10
19.74 ± 0.37
14.65 ± 0.33
21
11.13 ± 0.18
17.34 ± 0.26
14.21 ± 0.11
Chlorpyrifos
7
11.03 ± 0.14
13.91 ± 0.29
11.79 ± 0.25
14
11.35 ± 0.10
16.34 ± 0.39
13.78 ± 0.12
21
11.13 ± 0.18
14.97 ± 0.51
13.66 ± 0.16
Cadmium + Chlorpyrifos*
7
11.03 ± 0.14
17.32 ± 0.24
(Simple additive)
14.76 ± 0.51
(Moderately synergistic)
14
11.35 ± 0.10
20.45 ± 0.31
(Simple additive)
17.86 ± 0.48
(Moderately synergistic)
21
11.13 ± 0.18
18.08 ± 0.27
(Simple additive)
15.96 ± 0.42
(Moderately synergistic)
Chlorpyrifos + Cadmium**
7
11.03 ± 0.14
18.44 ± 0.40
15.25 ± 0.18
327
(Moderately synergistic)
(Moderately synergistic)
14
11.35 ± 0.10
21.86 ± 0.36
(Moderately synergistic)
18.89 ± 0.47
(Moderately synergistic)
21
11.13 ± 0.18
19.96 ± 0.42
(Moderately synergistic)
16.15 ± 0.26
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 57. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione peroxidase activity in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Glutathione peroxidase (nmol of NADPH oxidized/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
4.14 ± 0.14
5.53 ± 0.29
5.29 ± 0.13
14
4.54 ± 0.19
6.45 ± 0.13
5.65 ± 0.20
21
4.46 ± 0.11
7.06 ± 0.21
6.37 ± 0.36
Chlorpyrifos
7
4.14 ± 0.14
4.66 ± 0.29
4.38 ± 0.28
14
4.54 ± 0.19
4.85 ± 0.14
4.67 ± 0.16
21
4.46 ± 0.11
5.75 ± 0.35
5.27 ± 0.30
Cadmium + Chlorpyrifos*
7
4.14 ± 0.14
5.38 ± 0.22
(Simple additive)
5.15 ± 0.17
(Simple additive)
14
4.54 ± 0.19
6.16 ± 0.27
(Simple additive)
5.33 ± 0.10
(Simple additive)
21
4.46 ± 0.11
6.78 ± 0.18
(Simple additive)
6.00 ± 0.14
(Simple additive)
328
Chlorpyrifos + Cadmium**
7
4.14 ± 0.14
5.35 ± 0.20
(Simple additive)
5.01 ± 0.13
(Simple additive)
14
4.54 ± 0.19
5.92 ± 0.19
(Moderately synergistic)
5.14 ± 0.26
(Simple additive)
21
4.46 ± 0.11
6.55 ± 0.20
(Moderately synergistic)
5.65 ± 0.22
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 58. Percentage change in the glutathione peroxidase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% change in glutathione peroxidase activity
Duration
Test organ
7 Days
14 Days
21 Days
Gills
+57.54
+89.13
+66.35
Liver
+53.39
+70.76
+56.66
Kidney
+67.08
+80.34
+69.06
Brain
+37.08
+58.12
+46.37
Muscle
+23.02
+34.69
+38.62
Table 59a. ANOVA for changes in glutathione peroxidase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
329
Days
2
24168.55
12084.27
524.02
<0.05
Organ
4
3525.95
881.48
38.22
<0.05
Treatment
8
4874.25
609.28
26.42
<0.05
Table 59b. Tukey's studentized range (HSD) test for glutathione peroxidase activity in
different organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and
1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos
+ Cadmium for 7, 14 and 21 days.
Tukey Grouping
Mean
Days
A
28.7342
7
B
20.5797
14
C
9.8696
21
Tukey Grouping
Mean
Organ
A
24.8952
Gills
B
21.0267
Liver
C
18.3867
Muscle
C
17.4894
Kidney
C
16.8412
Brain
Tukey Grouping
Mean
Treatment
330
A
24.158
Cd+CPF1
B A
23.668
Cd1
B A
C
22.631
CPF+Cd1
B D
C
20.553
Cd2
D
C
20.192
Cd+CPF2
D
C
19.529
CPF+Cd2
17.692
CPF1
16.343
CPF2
12.785
Control
E
D
E
F
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Table 60. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione S-transferase activity in the gills of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Glutathione S-transferase (nmol of CDNB conjugate formed/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
25.44 ± 0.32
44.75 ± 1.05
40.68 ± 0.44
14
26.86 ± 0.20
51.30 ± 1.10
46.74 ± 0.38
21
27.06 ± 0.27
56.22 ± 1.28
52.71 ± 1.34
Chlorpyrifos
7
25.44 ± 0.32
34.36 ± 0.35
31.41 ± 0.26
14
26.86 ± 0.20
37.44 ± 0.41
35.38 ± 0.47
331
27.06 ± 0.27
21
42.54 ± 0.37
39.76 ± 0.50
Cadmium + Chlorpyrifos*
7
25.44 ± 0.32
42.87 ± 0.59
(Moderately antagonistic)
39.19 ± 0.46
(Moderately antagonistic)
14
26.86 ± 0.20
48.65 ± 1.33
(Moderately antagonistic)
44.91 ± 0.26
(Moderately antagonistic)
21
27.06 ± 0.27
54.43 ± 1.89
(Moderately antagonistic)
50.54 ± 1.09
(Moderately antagonistic)
Chlorpyrifos + Cadmium**
7
25.44 ± 0.32
41.24 ± 0.82
(Synergistic)
37.00 ± 0.35
(Synergistic)
14
26.86 ± 0.20
47.32 ± 0.59
(Synergistic)
42.67 ± 0.60
(Synergistic)
21
27.06 ± 0.27
53.15 ± 0.78
(Synergistic)
48.32 ± 1.13
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 61. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione S-transferase activity in the liver of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Glutathione S-transferase (nmol of CDNB conjugate formed/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
35.43 ± 0.38
52.33 ± 1.31
48.64 ± 0.39
14
37.76 ± 0.29
63.23 ± 1.02
60.75 ± 0.82
21
38.10 ± 0.40
69.43 ± 0.95
66.84 ± 1.77
Chlorpyrifos
7
35.43 ± 0.38
42.75 ± 0.50
40.24 ± 0.60
332
14
37.76 ± 0.29
51.09 ± 0.36
48.59 ± 0.53
21
38.10 ± 0.40
56.15 ± 0.81
54.98 ± 0.71
Cadmium + Chlorpyrifos*
7
35.43 ± 0.38
54.86 ± 1.17
(Moderately synergistic)
46.36 ± 0.78
(Moderately antagonistic)
14
37.76 ± 0.29
65.32 ± 0.41
(Moderately synergistic)
59.08 ± 1.29
(Simple additive)
21
38.10 ± 0.40
72.45 ± 1.90
(Moderately synergistic)
66.29 ± 0.82
(Simple additive)
Chlorpyrifos + Cadmium**
7
35.43 ± 0.38
50.04 ± 0.50
(Synergistic)
45.21 ± 0.42
(Moderately synergistic)
14
37.76 ± 0.29
61.34 ± 0.94
(Synergistic)
57.71 ± 0.57
(Synergistic)
21
38.10 ± 0.40
67.56 ± 1.28
(Synergistic)
62.02 ± 0.86
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 62. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione S-transferase activity in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Glutathione S-transferase (nmol of CDNB conjugate formed/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
23.16 ± 0.30
35.98 ± 0.62
33.34 ± 0.41
14
22.65 ± 0.23
39.32 ± 0.53
38.87 ± 0.50
21
23.78 ± 0.25
43.63 ± 0.70
42.36 ± 0.66
333
Chlorpyrifos
7
23.16 ± 0.30
28.51 ± 0.33
26.43 ± 0.18
14
22.65 ± 0.23
31.76 ± 0.29
29.58 ± 0.37
21
23.78 ± 0.25
35.33 ± 0.26
32.54 ± 0.38
Cadmium + Chlorpyrifos*
7
23.16 ± 0.30
36.65 ± 0.58
(Simple additive)
32.56 ± 0.37
(Simple additive)
14
22.65 ± 0.23
41.46 ± 0.60
(Moderately synergistic)
36.84 ± 0.49
(Moderately antagonistic)
21
23.78 ± 0.25
45.91 ± 0.75
(Moderately synergistic)
40.94 ± 0.42
(Simple additive)
Chlorpyrifos + Cadmium**
7
23.16 ± 0.30
34.07 ± 0.24
(Synergistic)
31.43 ± 0.30
(Synergistic)
14
22.65 ± 0.23
37.50 ± 0.56
(Synergistic)
34.65 ± 0.50
(Synergistic)
21
23.78 ± 0.25
42.72 ± 0.63
(Synergistic)
39.07 ± 0.33
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 63. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione S-transferase activity in the brain of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Glutathione S-transferase (nmol of CDNB conjugate formed/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
18.54 ± 0.23
27.36 ± 0.37
21.65 ± 0.21
334
14
19.48 ± 0.30
29.54 ± 0.21
23.34 ± 0.36
21
21.09 ± 0.24
33.00 ± 0.55
26.53 ± 0.38
Chlorpyrifos
7
18.54 ± 0.23
23.49 ± 0.30
20.31 ± 0.15
14
19.48 ± 0.30
26.15 ± 0.44
22.56 ± 0.39
21
21.09 ± 0.24
29.45 ± 0.27
25.98 ± 0.57
Cadmium + Chlorpyrifos*
7
18.54 ± 0.23
28.05 ± 0.36
(Simple additive)
24.98 ± 0.24
(Moderately synergistic)
14
19.48 ± 0.30
30.49 ± 0.48
(Simple additive)
27.08 ± 0.41
(Moderately synergistic)
21
21.09 ± 0.24
34.16 ± 0.61
(Simple additive)
31.54 ± 0.60
(Synergistic)
Chlorpyrifos + Cadmium**
7
18.54 ± 0.23
29.53 ± 0.40
(Synergistic)
26.54 ± 0.26
(Synergistic)
14
19.48 ± 0.30
33.46 ± 0.39
(Synergistic)
28.27 ± 0.19
(Synergistic)
21
21.09 ± 0.24
36.77 ± 0.53
(Synergistic)
32.63 ± 0.54
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 64. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione S-transferase activity in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Glutathione S-transferase (nmol of CDNB conjugate formed/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
Cadmium
1/10th of LC50
335
7
11.34 ± 0.14
15.32 ± 0.20
13.86 ± 0.23
14
11.67 ± 0.20
16.04 ± 0.24
15.13 ± 0.19
21
12.42 ± 0.16
17.43 ± 0.18
16.08 ± 0.21
Chlorpyrifos
7
11.34 ± 0.14
12.74 ± 0.11
12.75 ± 0.17
14
11.67 ± 0.20
13.15 ± 0.16
13.23 ± 0.11
21
12.42 ± 0.16
13.90 ± 0.13
13.31 ± 0.15
Cadmium + Chlorpyrifos*
7
11.34 ± 0.14
14.53 ± 0.26
(Simple additive)
13.21 ± 0.15
(Simple additive)
14
11.67 ± 0.20
16.26 ± 0.31
(Simple additive)
14.39 ± 0.20
(Simple additive)
21
12.42 ± 0.16
17.34 ± 0.29
(Simple additive)
15.65 ± 0.21
(Simple additive)
Chlorpyrifos + Cadmium**
7
11.34 ± 0.14
13.05 ± 0.10
(Simple additive)
12.47 ± 0.17
(Simple additive)
14
11.67 ± 0.20
14.64 ± 0.29
(Simple additive)
13.90 ± 0.18
(Simple additive)
21
12.42 ± 0.16
16.86 ± 0.37
(Moderately synergistic)
14.54 ± 0.13
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 65. Percentage change in the glutathione S-transferase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% change in glutathione S-transferase activity
Duration
Test organ
7 Days
14 Days
21 Days
336
Gills
+53.06
+64.93
+83.70
Liver
+34.22
+54.63
+69.20
Kidney
+39.77
+60.03
+69.52
Brain
+36.13
+41.74
+48.21
Muscle
+18.97
+25.04
+25.92
Table 66a. ANOVA for changes in glutathione S-transferase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
Days
2
47930.48
23965.24
290.61
<0.05
Organ
4
5580.14
1395.03
16.92
<0.05
Treatment
8
10371.73
1296.46
15.72
<0.05
Table 66b. Tukey's studentized range (HSD) test for glutathione S-transferase activity in
different organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and
1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos
+ Cadmium for 7, 14 and 21 days.
337
Tukey Grouping
Mean
Days
A
46.020
7
B
38.230
14
C
20.056
21
Tukey Grouping
Mean
Organ
A
41.912
Gills
B
34.752
Liver
B
33.169
Brain
B
32.131
Muscle
B
31.880
Kidney
Tukey Grouping
Mean
Treatment
A
40.444
Cd+CPF1
A
39.652
Cd1
A
38.822
CPF+Cd1
B A
36.497
Cd2
B A
36.119
Cd+CPF2
B A
C
35.425
CPF+Cd2
B
C
32.375
CPF1
C
29.937
CPF2
23.648
Control
D
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
338
Table 67. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione reductase activity in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Glutathione reductase (nmol of NADPH oxidized/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
8.43 ± 0.17
21.35 ± 0.68
17.41 ± 0.49
14
9.29 ± 0.08
19.65 ± 0.34
15.43 ± 0.28
21
9.12 ± 0.10
15.86 ± 0.36
12.56 ± 0.24
Chlorpyrifos
7
8.43 ± 0.17
9.71 ± 0.16
9.04 ± 0.11
14
9.29 ± 0.08
12.69 ± 0.29
11.50 ± 0.18
21
9.12 ± 0.10
13.99 ± 0.24
12.50 ± 0.43
Cadmium + Chlorpyrifos *
7
8.43 ± 0.17
19.68 ± 0.62
(Moderately antagonistic)
16.09 ± 0.44
(Simple additive)
14
9.29 ± 0.08
17.93 ± 0.47
(Moderately antagonistic)
14.81 ± 0.33
(Simple additive)
21
9.12 ± 0.10
14.97 ± 0.50
(Simple additive)
11.65 ± 0.40
(Simple additive)
Chlorpyrifos + Cadmium**
7
8.43 ± 0.17
18.43 ± 0.34
(Highly synergistic)
14.26 ± 0.26
(Synergistic)
14
9.29 ± 0.08
16.72 ± 0.27
(Synergistic)
13.08 ± 0.15
(Moderately synergistic)
21
9.12 ± 0.10
13.64 ± 0.32
(Simple additive)
10.32 ± 0.20
(Moderately antagonistic)
Values are expressed as mean ± SD of a group of 10 fishes
339
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 68. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione reductase activity in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Glutathione reductase (nmol of NADPH oxidized/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
10.65 ± 0.38
26.58 ± 0.90
24.76 ± 0.63
14
11.54 ± 0.25
24.12 ± 0.91
22.56 ± 0.39
21
12.30 ± 0.55
19.67 ± 0.73
17.05 ± 0.43
Chlorpyrifos
7
10.65 ± 0.38
13.00 ± 0.26
12.72 ± 0.42
14
11.54 ± 0.25
15.38 ± 0.57
14.70 ± 0.38
21
12.30 ± 0.55
18.59 ± 0.52
16.65 ± 0.60
Cadmium + Chlorpyrifos*
7
10.65 ± 0.38
28.56 ± 1.05
(Moderately synergistic)
22.08 ± 0.18
(Moderately antagonistic)
14
11.54 ± 0.25
25.43 ± 0.82
(Simple additive)
21.85 ± 0.20
(Simple additive)
21
12.30 ± 0.55
20.86 ± 0.62
(Simple additive)
16.48 ± 0.37
(Simple additive)
Chlorpyrifos + Cadmium**
7
10.65 ± 0.38
25.43 ± 1.16
(Highly synergistic)
20.37 ± 0.25
(Synergistic)
14
11.54 ± 0.25
23.97 ± 0.69
(Synergistic)
17.41 ± 0.30
(Moderately synergistic)
21
12.30 ± 0.55
18.53 ± 0.54
(Simple additive)
15.21 ± 0.22
(Simple additive)
340
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 69. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione reductase activity in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Glutathione reductase (nmol of NADPH oxidized/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
7.80 ± 0.18
15.76 ± 0.26
14.52 ± 0.50
14
7.51 ± 0.13
14.31 ± 0.10
12.69 ± 0.27
21
7.81 ± 0.22
13.14 ± 0.06
11.09 ± 0.34
Chlorpyrifos
7
7.80 ± 0.18
9.17 ± 0.25
8.33 ± 0.13
14
7.51 ± 0.13
10.58 ± 0.33
10.21 ± 0.20
21
7.81 ± 0.22
12.43 ± 0.28
11.01 ± 0.11
Cadmium + Chlorpyrifos*
7
7.80 ± 0.18
16.89 ± 0.65
(Simple additive)
14.27 ± 0.18
(Simple additive)
14
7.51 ± 0.13
14.54 ± 0.42
(Simple additive)
12.24 ± 0.23
(Simple additive)
21
7.81 ± 0.22
13.85 ± 0.30
(Simple additive)
11.36 ± 0.14
(Simple additive)
Chlorpyrifos + Cadmium**
7
7.80 ± 0.18
14.93 ± 0.25
(Synergistic)
13.48 ± 0.52
(Synergistic)
14
7.51 ± 0.13
12.96 ± 0.32
(Moderately synergistic)
11.64 ± 0.36
(Simple additive)
341
7.81 ± 0.22
21
12.62 ± 0.21
(Simple additive)
10.10 ± 0.20
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 70. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione reductase activity in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Glutathione reductase (nmol of NADPH oxidized/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
3.37 ± 0.12
7.12 ± 0.18
5.33 ± 0.20
14
3.65 ± 0.15
6.75 ± 0.23
4.89 ± 0.13
21
3.92 ± 0.20
5.85 ± 0.27
4.27 ± 0.18
Chlorpyrifos
7
3.37 ± 0.12
5.79 ± 0.24
5.00 ± 0.17
14
3.65 ± 0.15
5.22 ± 0.24
4.64 ± 0.24
21
3.92 ± 0.20
4.73 ± 0.11
4.08 ± 0.16
Cadmium + Chlorpyrifos*
7
3.37 ± 0.12
7.38 ± 0.25
(Simple additive)
5.98 ± 0.20
(Simple additive)
14
3.65 ± 0.15
7.05 ± 0.14
(Simple additive)
5.53 ± 0.14
(Simple additive)
21
3.92 ± 0.20
6.20 ± 0.27
(Simple additive)
5.11 ± 0.12
(Simple additive)
Chlorpyrifos + Cadmium**
7
3.37 ± 0.12
7.68 ± 0.23
(Moderately synergistic)
6.42 ± 0.16
342
(Moderately synergistic)
14
3.65 ± 0.15
7.34 ± 0.30
(Moderately synergistic)
6.24 ± 0.10
(Moderately synergistic)
21
3.92 ± 0.20
6.31 ± 0.28
(Moderately synergistic)
5.42 ± 0.22
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 71. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the glutathione reductase activity in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Glutathione reductase (nmol of NADPH oxidized/min/mg protein)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
2.02 ± 0.12
3.83 ± 0.24
3.19 ± 0.14
14
2.67 ± 0.17
4.46 ± 0.26
3.86 ± 0.18
21
3.04 ± 0.20
4.86 ± 0.19
4.47 ± 0.23
Chlorpyrifos
7
2.02 ± 0.12
2.21 ± 0.10
2.14 ± 0.12
14
2.67 ± 0.17
2.89 ± 0.15
2.86 ± 0.17
21
3.04 ± 0.20
3.56 ± 0.16
3.37 ± 0.11
Cadmium + Chlorpyrifos*
7
2.02 ± 0.12
3.52 ± 0.24
(Simple additive)
2.49 ± 0.13
(Simple additive)
14
2.67 ± 0.17
4.24 ± 0.21
(Simple additive)
3.77 ± 0.19
(Simple additive)
21
3.04 ± 0.20
4.75 ± 0.19
(Simple additive)
4.31 ± 0.11
(Simple additive)
343
Chlorpyrifos + Cadmium**
7
2.02 ± 0.12
3.04 ± 0.14
(Moderately synergistic)
2.25 ± 0.09
(Simple additive)
14
2.67 ± 0.17
4.07 ± 0.22
(Moderately synergistic)
2.93 ± 0.13
(Simple additive)
21
3.04 ± 0.20
4.50 ± 0.18
(Moderately synergistic)
3.72 ± 0.25
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 72. Percentage change in the glutathione reductase activity in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% change in glutathione reductase activity
Duration
Test organ
7 Days
14 Days
21 Days
Gills
+86.59
+63.83
+44.49
Liver
+103.64
+79.18
+45.37
Kidney
+72.05
+65.00
+52.95
Brain
+87.78
+63.22
+33.83
Muscle
+40.22
+36.31
+38.03
Table 73a. ANOVA for changes in glutathione reductase activity in different organs of
Tilapia exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
Source
Days
Df
Sum Sq
Mean Sq
F value
P value
2
10212.74
5106.37
491.88
<0.05
344
Organ
4
647.54
161.88
15.59
<0.05
Treatment
8
1930.37
241.29
23.24
<0.05
Table 73b. Tukey's studentized range (HSD) test for glutathione reductase activity in
different organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and
1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos
+ Cadmium for 7, 14 and 21 days.
Tukey Grouping
Mean
Days
A
16.4119
7
B
11.8491
14
C
4.2381
21
Tukey Grouping
Mean
Organ
A
12.5702
Gills
B A
11.8742
Brain
B C
10.7288
Liver
D
9.8873
Muscle
9.1047
Kidney
Mean
Treatment
C
D
Tukey Grouping
345
A
13.5796
Cd1
A
13.5418
Cd+CPF1
B A
12.6471
CPF+Cd1
B A
C
11.6160
Cd2
B D
C
11.1987
Cd+CPF2
E
D C
10.2367
CPF+Cd2
E
D
9.3184
CPF1
E
F
8.5282
CPF2
F
6.8309
Control
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Table 74. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total reduced glutathione level in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Total reduced glutathione (µmol of GSH/gm wet weight of the tissue)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
20.03 ± 0.21
36.32 ± 0.73
34.24 ± 0.52
14
21.22 ± 0.14
33.75 ± 0.90
30.77 ± 0.85
21
20.91 ± 0.14
28.43 ± 0.41
25.54 ± 0.77
Chlorpyrifos
7
20.03 ± 0.21
22.52 ± 0.64
21.16 ± 0.22
14
21.22 ± 0.14
25.70 ± 0.38
25.13 ± 0.49
346
20.91 ± 0.14
21
27.55 ± 0.56
25.08 ± 0.14
Cadmium + Chlorpyrifos*
7
20.03 ± 0.21
34.61 ± 0.35
(Simple additive)
32.68 ± 0.80
(Simple additive)
14
21.22 ± 0.14
32.94 ± 0.61
(Simple additive)
30.32 ± 0.37
(Simple additive)
21
20.91 ± 0.14
26.67 ± 0.94
(Simple additive)
24.68 ± 0.49
(Simple additive)
Chlorpyrifos + Cadmium**
7
20.03 ± 0.21
31.53 ± 0.55
(Synergistic)
31.31 ± 0.42
(Synergistic)
14
21.22 ± 0.14
29.74 ± 0.78
(Moderately synergistic)
27.93 ± 0.65
(Moderately synergistic)
21
20.91 ± 0.14
24.16 ± 0.30
(Moderately antagonistic)
23.74 ± 0.26
(Moderately antagonistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 75. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total reduced glutathione level in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Total reduced glutathione (µmol of GSH/gm wet weight of the tissue)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
37.21 ± 0.57
72.27 ± 3.52
62.33 ± 2.27
14
36.98 ± 0.74
61.49 ± 3.80
54.09 ± 1.59
21
38.64 ± 0.93
53.22 ± 2.73
46.87 ± 1.84
Chlorpyrifos
7
37.21 ± 0.57
39.03 ± 1.23
38.37 ± 0.84
347
14
36.98 ± 0.74
46.43 ± 1.45
44.09 ± 1.04
21
38.64 ± 0.93
50.64 ± 1.87
48.56 ± 1.20
Cadmium + Chlorpyrifos*
7
37.21 ± 0.57
67.52 ± 1.85
(Moderately antagonistic)
58.90 ± 1.32
(Moderately antagonistic)
14
36.98 ± 0.74
60.26 ± 2.68
(Simple additive)
52.43 ± 1.66
(Simple additive)
21
38.64 ± 0.93
51.65 ± 2.07
(Simple additive)
45.54 ± 1.14
(Simple additive)
Chlorpyrifos + Cadmium**
7
37.21 ± 0.57
63.98 ± 2.01
(Highly synergistic)
57.43 ± 1.95
(Highly synergistic)
14
36.98 ± 0.74
57.84 ± 2.59
(Synergistic)
49.66 ± 1.80
(Synergistic)
21
38.64 ± 0.93
48.71 ± 1.67
(Moderately antagonistic)
43.98 ± 1.11
(Moderately antagonistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 76. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total reduced glutathione level in the kidney of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Total reduced glutathione (µmol of GSH/gm wet weight of the tissue)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
15.03 ± 0.16
24.98 ± 0.58
20.34 ± 0.51
14
15.36 ± 0.21
21.92 ± 0.67
17.69 ± 0.22
21
16.10 ± 0.12
20.70 ± 0.33
16.47 ± 0.40
Chlorpyrifos
348
7
15.03 ± 0.16
15.94 ± 0.43
15.73 ± 0.17
14
15.36 ± 0.21
17.26 ± 0.64
16.34 ± 0.32
21
16.10 ± 0.12
18.86 ± 0.23
18.31 ± 0.52
Cadmium + Chlorpyrifos*
7
15.03 ± 0.16
23.47 ± 0.35
(Simple additive)
21.30 ± 0.67
(Simple additive)
14
15.36 ± 0.21
21.38 ± 0.25
(Simple additive)
18.61 ± 0.50
(Simple additive)
21
16.10 ± 0.12
20.34 ± 0.20
(Simple additive)
16.79 ± 0.38
(Simple additive)
Chlorpyrifos + Cadmium**
7
15.03 ± 0.16
21.96 ± 0.49
(Moderately synergistic)
21.52 ± 0.56
(Moderately synergistic)
14
15.36 ± 0.21
19.78 ± 0.75
(Moderately synergistic)
19.43 ± 0.22
(Moderately synergistic)
21
16.10 ± 0.12
17.73 ± 0.24
(Simple additive)
16.88 ± 0.61
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 77. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total reduced glutathione level in the brain of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Total reduced glutathione (µmol of GSH/gm wet weight of the tissue)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
30.29 ± 0.47
48.53 ± 1.22
41.75 ± 0.42
14
31.84 ± 0.20
43.67 ± 0.72
37.95 ± 0.59
21
32.68 ± 0.28
36.37 ± 1.39
34.80 ± 0.48
349
Chlorpyrifos
7
30.29 ± 0.47
47.64 ± 1.30
40.06 ± 0.44
14
31.84 ± 0.20
43.82 ± 0.73
35.73 ± 0.87
21
32.68 ± 0.28
38.44 ± 0.77
33.51 ± 0.64
Cadmium + Chlorpyrifos*
7
30.29 ± 0.47
54.43 ± 1.75
(Moderately synergistic)
46.11 ± 0.57
(Moderately synergistic)
14
31.84 ± 0.20
48.06 ± 1.33
(Moderately synergistic)
41.09 ± 0.86
(Moderately synergistic)
21
32.68 ± 0.28
41.46 ± 0.91
(Moderately synergistic)
35.54 ± 0.70
(Moderately synergistic)
Chlorpyrifos + Cadmium**
7
30.29 ± 0.47
49.18 ± 0.62
(Simple additive)
44.86 ± 1.24
(Moderately synergistic)
14
31.84 ± 0.20
44.42 ± 0.97
(Simple additive)
40.27 ± 0.57
(Moderately synergistic)
21
32.68 ± 0.28
40.65 ± 0.96
(Moderately synergistic)
35.43 ± 0.20
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 78. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total reduced glutathione level in the muscle of Tilapia
(Oreochromis mossambicus) during 7, 14 and 21 days of exposure.
Total reduced glutathione (µmol of GSH/gm wet weight of the tissue)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
14.34 ± 0.11
18.50 ± 0.44
18.33 ± 0.26
14
14.79 ± 0.25
20.85 ± 0.60
19.58 ± 0.54
350
15.87 ± 0.18
21
23.06 ± 0.37
21.29 ± 0.26
Chlorpyrifos
7
14.34 ± 0.11
15.36 ± 0.50
14.66 ± 0.26
14
14.79 ± 0.25
16.11 ± 0.29
15.03 ± 0.13
21
15.87 ± 0.18
17.65 ± 0.39
16.72 ± 0.11
Cadmium + Chlorpyrifos*
7
14.34 ± 0.11
18.25 ± 0.34
(Simple additive)
17.45 ± 0.23
(Simple additive)
14
14.79 ± 0.25
20.09 ± 0.55
(Simple additive)
18.90 ± 0.46
(Simple additive)
21
15.87 ± 0.18
22.64 ± 0.68
(Simple additive)
20.55 ± 0.27
(Simple additive)
Chlorpyrifos + Cadmium**
7
14.34 ± 0.11
16.84 ± 0.50
(Moderately synergistic)
16.61 ± 0.18
(Moderately synergistic)
14
14.79 ± 0.25
18.35 ± 0.29
(Moderately synergistic)
17.03 ± 0.43
(Moderately synergistic)
21
15.87 ± 0.18
19.74 ± 0.11
(Moderately synergistic)
19.26 ± 0.26
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 79. Percentage change in the total reduced glutathione level in different organs of
Tilapia (Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% change in total reduced glutathione level
Duration
Test organ
Gills
7 Days
14 Days
21 Days
+52.50
+39.18
+23.06
351
Liver
+54.47
+44.09
+25.90
Kidney
+37.43
+24.03
+13.42
Brain
+53.75
+31.52
+13.30
Muscle
+18.55
+23.34
+26.74
Table 80a. ANOVA for changes in levels of total reduced glutathione in different organs of
Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
Days
2
10028.82
5014.41
43.36
<0.05
Organ
4
20602.90
5150.72
44.54
<0.05
Treatment
8
5762.37
720.29
6.23
<0.05
Table 80b. Tukey's studentized range (HSD) test for levels of total reduced glutathione in
different organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and
1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos
+ Cadmium for 7, 14 and 21 days.
Tukey Grouping
Mean
Days
352
A
38.018
7
B
29.681
14
C
26.149
21
Tukey Grouping
Mean
Organ
A
39.179
Gills
A
38.690
Muscle
B
30.320
Brain
B
28.195
Liver
C
20.028
Kidney
Tukey Grouping
Mean
Treatment
A
36.353
Cd+CPF1
A
36.185
Cd1
B A
33.398
CPF+Cd1
B A
32.066
Cd2
B A
31.944
Cd+CPF2
B A
C
30.868
CPF+Cd2
B A
C
29.459
CPF1
B
C
27.286
CPF2
C
23.982
Control
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Table 81. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ascorbic acid content in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Ascorbic acid content (µg ASA/gm wet weight of the tissue)
353
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
37.43 ± 0.12
27.32 ± 0.45
30.85 ± 0.81
14
37.33 ± 0.28
25.75 ± 0.60
27.66 ± 0.20
21
36.21 ± 0.19
20.85 ± 0.37
24.99 ± 0.28
Chlorpyrifos
7
37.43 ± 0.12
33.56 ± 0.39
33.91 ± 0.30
14
37.33 ± 0.28
31.45 ± 0.72
32.18 ± 0.57
21
36.21 ± 0.19
28.22 ± 0.91
30.20 ± 0.17
Cadmium + Chlorpyrifos*
7
37.43 ± 0.12
28.47 ± 0.62
(Simple additive)
30.41 ± 0.24
(Simple additive)
14
37.33 ± 0.28
25.31 ± 0.47
(Simple additive)
28.19 ± 0.55
(Simple additive)
21
36.21 ± 0.19
24.63 ± 0.73
(Moderately antagonistic)
25.43 ± 0.90
(Simple additive)
Chlorpyrifos + Cadmium**
7
37.43 ± 0.12
31.84 ± 0.46
(Moderately synergistic)
32.92 ± 0.56
(Simple additive)
14
37.33 ± 0.28
28.90 ± 0.41
(Moderately synergistic)
29.74 ± 0.75
(Moderately synergistic)
21
36.21 ± 0.19
25.77 ± 0.64
(Moderately synergistic)
26.63 ± 0.38
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 82. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ascorbic acid content in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
354
Ascorbic acid content (µg ASA/gm wet weight of the tissue)
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
60.35 ± 1.27
48.34 ± 1.50
53.79 ± 0.74
14
63.64 ± 0.48
41.85 ± 1.31
46.32 ± 0.48
21
64.26 ± 0.29
31.57 ± 0.85
43.21 ± 0.92
Chlorpyrifos
7
60.35 ± 1.27
56.09 ± 1.80
56.87 ± 0.67
14
63.64 ± 0.48
51.87 ± 1.05
53.33 ± 0.80
21
64.26 ± 0.29
47.24 ± 1.43
50.12 ± 0.46
Cadmium + Chlorpyrifos*
7
60.35 ± 1.27
50.23 ± 1.16
(Moderately antagonistic)
51.86 ± 0.89
(Moderately synergistic)
14
63.64 ± 0.48
41.41 ± 0.53
(Simple additive)
45.24 ± 0.41
(Simple additive)
21
64.26 ± 0.29
39.76 ± 0.60
(Antagonistic)
42.22 ± 0.62
(Simple additive)
Chlorpyrifos + Cadmium**
7
60.35 ± 1.27
51.54 ± 1.76
(Synergistic)
54.91 ± 0.75
(Moderately synergistic)
14
63.64 ± 0.48
43.68 ± 1.06
(Synergistic)
48.90 ± 1.34
(Synergistic)
21
64.26 ± 0.29
41.54 ± 0.59
(Synergistic)
43.89 ± 1.21
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
355
Table 83. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ascorbic acid content in the kidney of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Ascorbic acid content (µg ASA/gm wet weight of the tissue)
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
48.52 ± 0.37
33.66 ± 1.48
37.32 ± 0.52
14
48.87 ± 0.22
24.05 ± 1.27
29.12 ± 1.10
21
47.43 ± 0.53
18.47 ± 1.50
24.59 ± 1.35
Chlorpyrifos
7
48.52 ± 0.37
42.64 ± 1.87
43.98 ± 0.56
14
48.87 ± 0.22
37.64 ± 0.25
39.81 ± 0.72
21
47.43 ± 0.53
35.07 ± 0.47
36.65 ± 0.39
Cadmium + Chlorpyrifos*
7
48.52 ± 0.37
35.42 ± 0.64
39.86 ± 0.32
(Moderately antagonistic) (Moderately antagonistic)
14
48.87 ± 0.22
26.31 ± 0.88
31.43 ± 0.69
(Moderately antagonistic) (Moderately antagonistic)
21
47.43 ± 0.53
21.62 ± 0.80
(Moderately antagonistic)
25.27 ± 0.78
(Simple additive)
Chlorpyrifos + Cadmium**
7
48.52 ± 0.37
35.71 ± 0.46
(Synergistic)
40.25 ± 0.89
(Moderately synergistic)
14
48.87 ± 0.22
28.74 ± 0.90
(Synergistic)
33.93 ± 1.68
(Synergistic)
21
47.43 ± 0.53
22.52 ± 1.26
(Highly synergistic)
27.88 ± 0.97
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
356
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 84. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ascorbic acid content in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Ascorbic acid content (µg ASA/gm wet weight of the tissue)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
41.66 ± 0.42
34.78 ± 0.70
37.65 ± 0.57
14
40.21 ± 0.16
30.44 ± 0.44
35.78 ± 0.67
21
41.13 ± 0.34
26.32 ± 1.20
31.34 ± 0.42
Chlorpyrifos
7
41.66 ± 0.42
33.43 ± 1.40
37.52 ± 0.66
14
40.21 ± 0.16
28.95 ± 0.52
36.06 ± 1.28
21
41.13 ± 0.34
25.46 ± 0.91
33.57 ± 1.19
Cadmium + Chlorpyrifos*
7
41.66 ± 0.42
31.24 ± 0.86
(Moderately synergistic)
35.12 ± 0.92
(Moderately synergistic)
14
40.21 ± 0.16
25.54 ± 0.63
(Moderately synergistic)
32.89 ± 0.76
(Moderately synergistic)
21
41.13 ± 0.34
22.05 ± 0.37
(Moderately synergistic)
28.20 ± 1.26
(Moderately synergistic)
Chlorpyrifos + Cadmium**
7
41.66 ± 0.42
31.87 ± 0.35
(Simple additive)
35.77 ± 1.03
(Moderately synergistic)
14
40.21 ± 0.16
27.87 ± 1.30
(Simple additive)
33.21 ± 0.38
(Moderately synergistic)
21
41.13 ± 0.34
23.68 ± 0.61
29.38 ± 0.86
357
(Moderately synergistic)
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 85. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the ascorbic acid content in the muscle of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Ascorbic acid content (µg ASA/gm wet weight of the tissue)
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
31.07 ± 0.24
23.22 ± 0.48
25.41 ± 0.78
14
31.29 ± 0.20
21.05 ± 0.31
22.84 ± 0.46
21
31.22 ± 0.36
20.12 ± 0.15
20.90 ± 0.87
Chlorpyrifos
7
31.07 ± 0.24
28.33 ± 0.98
28.90 ± 0.24
14
31.29 ± 0.20
26.75 ± 0.64
27.14 ± 0.74
21
31.22 ± 0.36
24.28 ± 0.55
26.55 ± 0.78
Cadmium + Chlorpyrifos*
7
31.07 ± 0.24
23.65 ± 0.21
(Simple additive)
26.67 ± 0.66
(Simple additive)
14
31.29 ± 0.20
22.36 ± 0.34
(Simple additive)
23.53 ± 0.90
(Simple additive)
21
31.22 ± 0.36
20.78 ± 0.27
(Simple additive)
21.57 ± 0.46
(Simple additive)
Chlorpyrifos + Cadmium**
7
31.07 ± 0.24
26.85 ± 0.28
(Simple additive)
26.87 ± 0.30
(Moderately synergistic)
358
14
31.29 ± 0.20
24.34 ± 0.22
(Moderately synergistic)
25.79 ± 0.49
(Moderately synergistic)
21
31.22 ± 0.36
22.45 ± 0.57
(Moderately synergistic)
23.72 ± 0.17
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 86. Percentage change in the ascorbic acid content in different organs of Tilapia
(Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% change in ascorbic acid content
Duration
Test organ
7 Days
14 Days
21 Days
Gills
-16.75
-23.26
-28.64
Liver
-12.26
-26.81
-33.95
Kidney
-20.43
-35.79
-44.11
Brain
-16.77
-22.05
-33.14
Muscle
-15.55
-22.58
-27.78
Table 87a. ANOVA for changes in levels of ascorbic acid in different organs of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
Source
Days
Df
Sum Sq
Mean Sq
F value
P value
2
3461.36
1730.68
203.38
<0.05
359
Organ
4
26162.38
6540.59
768.62
<0.05
Treatment
8
8341.51
1042.68
122.53
<0.05
Table 87b. Tukey's studentized range (HSD) test for levels of ascorbic acid in different
organs of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
Tukey Grouping
Mean
Days
A
37.6256
7
B
33.7153
14
C
30.4750
21
Tukey Grouping
Mean
Organ
A
48.8553
Liver
B
33.9919
Kidney
C
32.2135
Brain
D
29.5294
Gills
E
25.1032
Muscle
Tukey Grouping
Mean
Treatment
A
44.1816
Control
B
37.8036
CPF2
C
35.4216
CPF1
C
33.9802
CPF+Cd2
32.8149
Cd2
32.5307
Cd+CPF2
D
D
D
E
360
F
E
30.8653
CPF+Cd1
F
G
29.3420
Cd+CPF1
G
28.5080
Cd1
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Table 88. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total protein content in the gills of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Total protein content (mg protein/100gm wet weight of the tissue)
Duration
(days)
Exposure concentration
1/5th of LC50
Control
1/10th of LC50
Cadmium
7
25.06 ± 0.062
18.45 ± 0.093
22.41 ± 0.061
14
26.14 ± 0.058
16.50 ± 0.028
21.28 ± 0.058
21
26.77 ± 0.080
13.28 ± 0.049
19.79 ± 0.038
Chlorpyrifos
7
25.06 ± 0.062
24.06 ± 0.017
24.44 ± 0.193
14
26.14 ± 0.058
23.64 ± 0.042
23.09 ± 0.081
21
26.77 ± 0.080
21.52 ± 0.018
22.26 ± 0.061
Cadmium + Chlorpyrifos*
361
7
25.06 ± 0.062
19.67 ± 0.036
(Simple additive)
21.83 ± 0.057
(Simple additive)
14
26.14 ± 0.058
17.74 ± 0.152
(Simple additive)
20.95 ± 0.071
(Simple additive)
21
26.77 ± 0.080
15.3 ± 0.103
(Moderately antagonistic)
18.62 ± 0.039
(Simple additive)
Chlorpyrifos + Cadmium**
7
25.06 ± 0.062
20.02 ± 0.204
(Moderately synergistic)
23.45 ± 0.072
(Simple additive)
14
26.14 ± 0.058
18.34 ± 0.029
(Moderately synergistic)
22.83 ± 0.049
(Simple additive)
21
26.77 ± 0.080
17.81 ± 0.095
(Moderately synergistic)
20.16 ± 0.043
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 89. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total protein content in the liver of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Total protein content (mg protein/100gm wet weight of the tissue)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
45.21 ± 0.022
34.01 ± 0.066
36.26 ± 0.067
14
46.62 ± 0.062
28.72 ± 0.127
31.33 ± 0.104
21
48.95 ± 0.223
23.09 ± 0.072
26.92 ± 0.087
Chlorpyrifos
7
45.21 ± 0.022
41.67 ± 0.055
42.48 ± 0.081
14
46.62 ± 0.062
39.50 ± 0.060
40.97 ± 0.028
362
48.95 ± 0.223
21
36.27 ± 0.153
38.41 ± 0.043
Cadmium + Chlorpyrifos*
7
45.21 ± 0.022
33.93 ± 0.071
(Simple additive)
37.06 ± 0.072
(Simple additive)
14
46.62 ± 0.062
26.11 ± 0.058
(Moderately synergistic)
33.75 ± 0.036
(Moderately antagonistic)
21
48.95 ± 0.223
18.35 ± 0.037
(Moderately synergistic)
29.38 ± 0.092
(Moderately antagonistic)
Chlorpyrifos + Cadmium**
7
45.21 ± 0.022
39.19 ± 0.091
(Moderately synergistic)
40.81 ± 0.188
(Moderately synergistic)
14
46.62 ± 0.062
35.50 ± 0.058
(Moderately synergistic)
37.93 ± 0.052
(Moderately synergistic)
21
48.95 ± 0.223
31.73 ± 0.026
(Synergistic)
33.19 ± 0.038
(Synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 90. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total protein content in the kidney of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Total protein content (mg protein/100gm wet weight of the tissue)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
22.14 ± 0.037
16.23 ± 0.153
17.58 ± 0.084
14
22.89 ± 0.081
15.21 ± 0.260
16.65 ± 0.241
21
22.73 ± 0.067
13.04 ± 0.075
14.66 ± 0.032
Chlorpyrifos
363
7
22.14 ± 0.037
20.25 ± 0.019
20.83 ± 0.014
14
22.89 ± 0.081
19.22 ± 0.092
19.77 ± 0.062
21
22.73 ± 0.067
17.34 ± 0.102
18.46 ± 0.081
Cadmium + Chlorpyrifos*
7
22.14 ± 0.037
15.06 ± 0.132
(Simple additive)
18.30 ± 0.065
(Simple additive)
14
22.89 ± 0.081
13.75 ± 0.094
(Simple additive)
17.11 ± 0.148
(Simple additive)
21
22.73 ± 0.067
11.88 ± 0.053
(Simple additive)
15.04 ± 0.077
(Simple additive)
Chlorpyrifos + Cadmium**
7
22.14 ± 0.037
19.09 ± 0.080
(Simple additive)
19.78 ± 0.201
(Simple additive)
14
22.89 ± 0.081
17.98 ± 0.055
(Simple additive)
18.54 ± 0.123
(Simple additive)
21
22.73 ± 0.067
16.13 ± 0.188
(Simple additive)
16.95 ± 0.090
(Simple additive)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 91. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total protein content in the brain of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Total protein content (mg protein/100gm wet weight of the tissue)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
30.41 ± 0.162
24.09 ± 0.072
27.91 ±0.033
14
31.16 ± 0.027
23.36 ± 0.084
26.41 ± 0.085
364
32.77 ± 0.075
21
20.51 ± 0.098
24.07 ± 0.081
Chlorpyrifos
7
30.41 ± 0.162
22.19 ± 0.059
28.36 ± 0.059
14
31.16 ± 0.027
20.56 ± 0.098
27.62 ± 0.077
21
32.77 ± 0.075
18.42 ± 0.033
25.39 ± 0.025
Cadmium + Chlorpyrifos*
7
30.41 ± 0.162
23.55 ± 0.204
(Simple additive)
26.57 ± 0.055
(Simple additive)
14
31.16 ± 0.027
22.11 ± 0.176
(Simple additive)
25.74 ± 0.078
(Simple additive)
21
32.77 ± 0.075
19.25 ± 0.085
(Simple additive)
23.81 ± 0.181
(Simple additive)
Chlorpyrifos + Cadmium**
7
30.41 ± 0.162
21.03 ± 0. 134
(Simple additive)
25.38 ± 0.041
(Moderately synergistic)
14
31.16 ± 0.027
19.62 ± 0.037
(Simple additive)
24.29 ± 0.096
(Moderately synergistic)
21
32.77 ± 0.075
14.37 ± 0.078
(Moderately synergistic)
22.05 ± 0.074
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 92. Effect of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos +
Cadmium on the total protein content in the muscle of Tilapia (Oreochromis
mossambicus) during 7, 14 and 21 days of exposure.
Total protein content (mg protein/100gm wet weight of the tissue)
Duration
(days)
Exposure concentration
Control
1/5th of LC50
1/10th of LC50
Cadmium
7
37.16 ± 0.089
23.77 ± 0.077
31.04 ± 0.086
365
14
38.37 ± 0.054
19.03 ± 0.260
29.83 ± 0.026
21
39.94 ± 0.298
15.57 ± 0.026
27.64 ± 0.055
Chlorpyrifos
7
37.16 ± 0.089
34.64 ± 0.018
35.88 ± 0.025
14
38.37 ± 0.054
33.96 ± 0.173
34.65 ± 0.066
21
39.94 ± 0.298
31.28 ± 0.070
32.73 ± 0.047
Cadmium + Chlorpyrifos*
7
37.16 ± 0.089
25.55 ± 0.089
(Moderately antagonistic)
29.19 ± 0.065
(Moderately synergistic)
14
38.37 ± 0.054
22.99 ± 0.072
(Moderately antagonistic)
27.22 ± 0.091
(Moderately synergistic)
21
39.94 ± 0.298
18.53 ± 0.155
(Moderately antagonistic)
24.1 ± 0.139
(Moderately synergistic)
Chlorpyrifos + Cadmium**
7
37.16 ± 0.089
27.52 ± 0.201
(Synergistic)
32.93 ± 0.068
(Moderately synergistic)
14
38.37 ± 0.054
24.07 ± 0.079
(Synergistic)
31.44 ± 0.033
(Moderately synergistic)
21
39.94 ± 0.298
21.92 ± 0.027
(Synergistic)
29.76 ± 0.059
(Moderately synergistic)
Values are expressed as mean ± SD of a group of 10 fishes
*Comparison with individual toxicity of Cadmium
**Comparison with individual toxicity of Chlorpyrifos
Table 93. Percentage change in the total protein content in different organs of Tilapia
(Oreochromis mossambicus) exposed to Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% change in total protein content
Duration
Test organ
7 Days
14 Days
21 Days
366
Gills
-13.04
-21.40
-30.55
Liver
-15.56
-26.58
-39.39
Kidney
-16.94
-24.51
-32.08
Brain
-18.17
-23.90
-35.97
Muscle
-19.09
-27.22
-36.93
Table 94a. ANOVA for changes in levels of total protein in different organs of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14
and 21 days.
Source
Df
Sum Sq
Mean Sq
F value
P value
Days
2
2323.02
1161.51
33.75
<0.05
Organ
4
4095.10
1023.77
29.75
<0.05
Treatment
8
5827.15
728.39
21.16
<0.05
Table 94b. Tukey's studentized range (HSD) test for levels of total protein in different organs
of Tilapia (Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 7, 14 and 21 days.
367
Tukey Grouping
Mean
Days
A
27.5456
7
A
26.6348
14
B
22.0713
21
Tukey Grouping
Mean
Organ
A
29.2406
Muscle
B A
28.0059
Brain
B
26.2893
Gills
C
22.7623
Kidney
C
20.7881
Liver
Tukey Grouping
Mean
Treatment
A
32.824
Control
B A
28.979
CPF2
B C
26.833
CPF1
B C
D
26.801
CPF+Cd2
C
D
25.018
Cd2
C
D
24.617
Cd+CPF2
E
D
22.964
CPF+Cd1
E
20.519
Cd1
E
20.200
Cd+CPF1
Means with the same letter are not significantly different.
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
368
Plates
Plate 1: Oreochromis mossambicus
Plate 2. Experimental set up of 96 h LC50.
369
Plate 3. Photograph of heavymetal Cadmium and its stock solution
Plate 4. Photograph of pesticide Chlorpyrifos and its stock solution.
370
Plate 5. Photograph showing the behaviour of Tilapia (Oreochromis mossambicus) in control
tanks.
Plate 6. Photograph showing the behaviour of Tilapia (Oreochromis mossambicus) exposed to
Cadmium.
371
Plate 7. Photograph showing the behaviour of Tilapia (Oreochromis mossambicus) exposed to
Chlorpyrifos.
372
Figures
Fig. 6. Changes in the oxygen consumption rate in Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
mg O2 consumption/l/g/h
0.3
0.24
0.18
0.12
0.06
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 7. Variation in the rate of oxygen consumption in terms of % decrease over control in
Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 21 days.
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
% decrease in O2 consumption
20
33.95
40
39.18
51.28
60
57.28
60.14
65.82
71.28
80
Test groups
55.06
373
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 8. Changes in the food consumption rate in Tilapia (Oreochromis mossambicus) exposed
to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
g feed consumption/g body wt.
7 Days
14 Days
21 Days
0.025
0.02
0.015
0.01
0.005
0
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% decrease in food consumption
Fig. 9. Variation in the rate of food consumption in terms of % decrease over control in
Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
40
54.10
60
60.78
68.08
73.28
80
84.91
91.30
82.80
87.84
100
Test groups
374
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 10. Changes in the ammonia-N excretion rate in Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
µg-at NH3-N excreta/l/g/h
0.025
0.020
0.015
0.010
0.005
0.000
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 11. Variation in the rate of ammonia-N excretion in terms of % decrease over control in
Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
% decrease in ammonia excretion
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
72.28
71.60
CPF+Cd2
30
45
46.47
53.57
60
67.13
75
76.23
90
78.49
83.25
Test groups
375
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 12. Changes in the Oxygen:Nitrogen ratio in Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
28
O:N ratio
24
20
16
12
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 13. Variation in the ratio of Oxygen:Nitrogen in terms of % increase over control in
Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
376
71.48
% increase in O:N ratio
75
67.74
58.93
58.27
54.13
55
48.26
35
31.02
23.42
15
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 14. Changes in the relative growth rate in Tilapia (Oreochromis mossambicus) exposed
to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
Relative growth rate (%)
10
5
0
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
-5
-10
-15
-20
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 15. Variation in the rate of relative growth in terms of % decrease over control in Tilapia
(Oreochromis mossambicus) exposed to sublethal concentrations of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 21 days.
377
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
216.76
220.11
CPF+Cd2
% decrease in growth rate
80
112.29
127.93
160
202.79
240
270.39
320
283.80
291.62
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
nmol of ASChI hydrolysed/min/mg protein
Fig. 16. Changes in the acetylcholinesterase activity in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
150
120
90
60
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 17. Variation in the activity of acetylcholinesterase in terms of % decrease over control in
the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
378
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
% decrease in Acetylcholinesterase
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
20
26.53
30
30.21
32.50
36.61
40
38.62
41.12
43.81
50
47.76
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 18. Changes in the acetylcholinesterase activity in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
nmol of ASChI hydrolysed/min/mg protein
100
80
60
40
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd2
379
Fig. 19. Variation in the activity of acetylcholinesterase in terms of % decrease over control in
the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
% decrease in Acetylcholinesterase
18
23.06
27
27.09
30.39
32.79
36
35.68
36.51
38.62
40.96
45
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
nmol of ASChI hydrolysed/min/mg protein
Fig. 20. Changes in the acetylcholinesterase activity in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
60
55
50
45
40
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd2
380
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% decrease in Acetylcholinesterase
Fig. 21. Variation in the activity of acetylcholinesterase in terms of % decrease over control in
the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
10
15
14.96
16.32
17.59
17.94
20
19.52
20.93
22.16
25
24.34
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
nmol of ASChI hydrolysed/min/mg protein
Fig. 22. Changes in the acetylcholinesterase activity in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
300
240
180
120
60
0
Control
Cd1
Cd2
CPF1
CPF2
Test groups
Cd+CPF1
CPF+Cd2
381
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 23. Variation in the activity of acetylcholinesterase in terms of % decrease over control in
the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
% decrease in Acetylcholinesterase
25
44.87
50
51.47
62.26
75
67.10
68.91
73.95
81.25
86.66
100
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 24. Changes in the acetylcholinesterase activity in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
nmol of ASCh hydrolysed/min/mg protein
382
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
200
160
120
80
40
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% decrease in Acetylcholinesterase
Fig. 25. Variation in the activity of acetylcholinesterase in terms of % decrease over control in
the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
15
30
29.83
33.33
45
36.59
41.00
47.27
51.54
60
75
57.91
66.89
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 26. Mean variation in the activity of acetylcholinesterase in terms of % decrease over
control in different organs of Tilapia (Oreochromis mossambicus) exposed to
383
sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
% decrease in Acetylcholinesterase
Gills
Liver
Kidney
Brain
14 Days
21 Days
Muscle
0
15
19.22
30
45
37.14
33.14
45.54
60
75
67.06
Test organs
Fig. 27. Changes in the butyrylcholinesterase activity in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
nmol of BSChI hydrolysed/min/mg protein
384
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
35
28
21
14
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 28. Variation in the activity of butyrylcholinesterase in terms of % decrease over control
in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
% decrease in Butyrylcholinesterase
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
18
27
25.67
26.64
31.14
33.44
36
34.72
37.23
39.68
45
41.79
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
385
nmol of BSChI hydrolysed/min/mg protein
Fig. 29. Changes in the butyrylcholinesterase activity in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
70
56
42
28
14
0
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 30. Variation in the activity of butyrylcholinesterase in terms of % decrease over control
in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
% decrease in Butyrylcholinesterase
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
30
40.63
45
46.14
53.19
55.43
60
61.87
65.59
75
74.11
82.14
90
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
386
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
nmol of BSChI hydrolysed/min/mg protein
Fig. 31. Changes in the butyrylcholinesterase activity in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
24
18
12
6
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 32. Variation in the activity of butyrylcholinesterase in terms of % decrease over control
in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
% decrease in Butyrylcholinesterase
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
15
30
26.85
28.39
34.38
35.06
45
41.63
43.64
46.66
52.70
60
Test groups
387
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
nmol of BSChI hydrolysed/min/mg protein
Fig. 33. Changes in the butyrylcholinesterase activity in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
45
36
27
18
9
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 34. Variation in the activity of butyrylcholinesterase in terms of % decrease over control
in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
388
% decrease in Butyrylcholinesterase
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
20
30
30.17
35.61
40
40.27
44.05
50
48.13
49.61
51.12
56.09
60
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
nmol of BSChI hydrolysed/min/mg protein
Fig. 35. Changes in the butyrylcholinesterase activity in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
120
100
80
60
40
20
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 36. Variation in the activity of butyrylcholinesterase in terms of % decrease over control
in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
389
% decrease in Butyrylcholinesterase
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
15
30
45
38.81
40.43
43.64
46.45
48.07
52.21
60
58.83
75
71.88
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 37. Mean variation in the activity of butyrylcholinesterase in terms of % decrease over
control in different organs of Tilapia (Oreochromis mossambicus) exposed to
sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% decrease in Butyrylcholinesterase
7 Days
Gills
Liver
Kidney
Brain
14 Days
21 Days
Muscle
0
15
30
33.79
38.67
45
44.38
50.04
60
59.89
Test organs
390
Fig. 38. Changes in the lipid peroxidation level in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
nmol of TBARS formed/mg protein
7 Days
14 Days
21 Days
19
15
11
7
3
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 39. Variation in the level of lipid peroxidation in terms of % increase over control in the
gills of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
% increase in lipid peroxidation
391
160
157.69
147.95
145.56
134.56
122.14
120
98.17
80
42.74
40
25.67
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 40. Changes in the lipid peroxidation level in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
nmol of TBARS formed/mg protein
7 Days
14 Days
21 Days
15
12
9
6
3
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 41. Variation in the level of lipid peroxidation in terms of % increase over control in the
liver of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
392
% increase in lipid peroxidation
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
150
132.75
131.33
123.26
117.88
125
96.99
100
75
88.61
61.87
50
27.37
25
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
nmol of TBARS formed/mg protein
Fig. 42. Changes in the lipid peroxidation level in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
7.5
6
4.5
3
1.5
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd2
393
% increase in lipid peroxidation
Fig. 43. Variation in the level of lipid peroxidation in terms of % increase over control in the
kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
125
100
113.61
89.94
78.75
70.34
75
65.54
49.85
50
38.50
24.40
25
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
nmol of TBARS formed/mg protein
Fig. 44. Changes in the lipid peroxidation level in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
10
8
6
4
2
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd2
394
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% increase in lipid peroxidation
Fig. 45. Variation in the level of lipid peroxidation in terms of % increase over control in the
brain of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
120
102.40
91.29
84.16
90
67.39
57.45
60
44.79
30
22.83
13.68
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
nmol of TBARS formed/mg protein
Fig. 46. Changes in the lipid peroxidation level in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
1.6
1.4
1.2
1
0.8
0.6
Control
Cd1
Cd2
CPF1
CPF2
Test groups
Cd+CPF1
CPF+Cd2
395
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% increase in lipid peroxidation
Fig. 47. Variation in the level of lipid peroxidation in terms of % increase over control in the
muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
63
58.30
46.60
49
40.00
35
44.57
38.19
29.04
28.30
20.00
21
7
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 48. Mean variation in the level of lipid peroxidation in terms of % increase over control in
different organs of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
396
% increase in lipid peroxidation
7 Days
120
14 Days
21 Days
109.31
97.51
90
66.36
60.50
60
38.13
30
0
Gills
Liver
Kidney
Brain
Muscle
Test organs
Fig. 49. Changes in the superoxide dismutase activity in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
397
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
Units/mg protein
10
8
6
4
2
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 50. Variation in the activity of superoxide dismutase in terms of % increase over control
in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
% increase in SOD
80
74.44
60
42.72
40
32.54
35.88
36.68
32.33
26.29
23.73
20
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd1
CPF+Cd2
398
Fig. 51. Changes in the superoxide dismutase activity in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
Units/mg protein
15
12
9
6
3
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 52. Variation in the activity of superoxide dismutase in terms of % increase over control
in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
80
% increase in SOD
71.30
60
50.15
49.70
45.19
44.73
44.59
40
36.20
30.68
20
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd1
CPF+Cd2
399
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 53. Changes in the superoxide dismutase activity in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
Units/mg protein
7.5
6
4.5
3
1.5
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 54. Variation in the activity of superoxide dismutase in terms of % increase over control
in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
75
% increase in SOD
65.21
60
45
42.82
40.74
38.94
32.97
30
27.56
24.82
22.06
15
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd1
CPF+Cd2
400
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 55. Changes in the superoxide dismutase activity in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
Units/mg protein
5
4
3
2
1
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 56. Variation in the activity of superoxide dismutase in terms of % increase over control
in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
60
51.83
% increase in SOD
47.33
45
43.61
38.42
30
24.75
21.58
12.82
15
5.20
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Test groups
Cd+CPF2
CPF+Cd1
CPF+Cd2
401
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 57. Changes in the superoxide dismutase activity in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
Units/mg protein
2.2
2
1.8
1.6
1.4
1.2
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 58. Variation in the activity of superoxide dismutase in terms of % increase over control
in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
402
40
% increase in SOD
32.11
27.64
30
23.35
21.61
18.39
20
15.16
10
3.79
2.17
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 59. Mean variation in the activity of superoxide dismutase in terms of % increase over
control in different organs of Tilapia (Oreochromis mossambicus) exposed to
sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
21 Days
81.02
85
67.93
65.41
68
% increase in SOD
14 Days
58.64
51
34
17
11.91
0
Gills
Liver
Kidney
Test organs
Brain
Muscle
403
Fig. 60. Changes in the catalase activity in the gills of Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
Units/mg protein
8
6.4
4.8
3.2
1.6
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 61. Variation in the activity of catalase in terms of % increase over control in the gills of
Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
404
% increase inCAT
120
112.29
97.40
100
83.62
80
70.43
60.87
54.33
60
53.68
47.46
40
20
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 62. Changes in the catalase activity in the liver of Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
Units/mg protein
9.2
7.4
5.6
3.8
2
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 63. Variation in the activity of catalase in terms of % increase over control in the liver of
Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
405
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
100
92.90
% increase in CAT
86.81
75
65.94
59.32
50.32
50
46.84
42.48
38.11
25
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 64. Changes in the catalase activity in the kidney of Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
Units/mg protein
5.8
4.6
3.4
2.2
1
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd1
CPF+Cd2
406
Fig. 65. Variation in the activity of catalase in terms of % increase over control in the kidney
of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
80
75.76
67.79
% increase in CAT
65.63
57.92
60
54.81
48.79
43.07
42.81
40
20
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 66. Changes in the catalase activity in the brain of Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos
and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
Units/mg protein
3.8
3.2
2.6
2
1.4
0.8
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd1
CPF+Cd2
407
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 67. Variation in the activity of catalase in terms of % increase over control in the brain of
Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
50.00
50
% increase in CAT
40.53
40
37.19
30.58
30
26.61
24.21
19.36
20
12.69
10
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 68. Changes in the catalase activity in the muscle of Tilapia (Oreochromis mossambicus)
exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
Units/mg protein
1.5
1.2
0.9
0.6
Control
Cd1
Cd2
CPF1
CPF2
Test groups
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
408
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 69. Variation in the activity of catalase in terms of % increase over control in the muscle
of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
60
% increase in CAT
54.04
46.48
45
39.14
33.47
32.32
31.58
29.17
28.33
30
15
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 70. Mean variation in the activity of catalase in terms of % increase over control in
different organs of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
409
7 Days
14 Days
21 Days
% increase in Catalase
100
84.38
78.99
69.60
75
59.65
50
35.88
25
0
Gills
Liver
Kidney
Brain
Muscle
Test organs
Fig. 71. Changes in the glutathione peroxidase activity in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
410
nmol of NADPH oxidized/min/mg protein
7 Days
14 Days
21 Days
40
32
24
16
8
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% increase in glutathione peroxidase
Fig. 72. Variation in the activity of glutathione peroxidase in terms of % increase over control
in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
110
100.94
85.22
80
65.51
63.52
68.62
54.31
48.62
50
44.05
20
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd1
CPF+Cd2
411
Fig. 73. Changes in the glutathione peroxidase activity in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
nmol of NADPH oxidized/min/mg protein
7 Days
14 Days
21 Days
45
36
27
18
9
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% increase in glutathioone peroxidase
Fig. 74. Variation in the activity of glutathione peroxidase in terms of % increase over control
in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
90
79.74
76.38
66.01
61.86
65
55.70
40
51.68
35.01
26.89
15
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd2
412
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 75. Changes in the glutathione peroxidase activity in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
nmol of NADPH oxidized/min/mg protein
7 Days
14 Days
21 Days
26
21
16
11
6
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% increase in glutathione peroxidase
Fig. 76. Variation in the activity of glutathione peroxidase in terms of % increase over control
in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
125
100
112.65
89.67
80.56
70.42
75
60.56
60.37
42.05
50
36.19
25
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Test groups
Cd+CPF2
CPF+Cd1
CPF+Cd2
413
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 77. Changes in the glutathione peroxidase activity in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
nmol of NADPH oxidized/min/mg protein
7 Days
14 Days
21 Days
22
18
14
10
6
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 78. Variation in the activity of glutathione peroxidase in terms of % increase over control
in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
% increase in glutathione peroxidase
414
90
79.34
62.44
55.80
60
45.10
43.40
34.50
27.67
30
22.73
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 79. Changes in the glutathione peroxidase activity in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
nmol of NADPH oxidized/min/mg protein
7 Days
14 Days
21 Days
7.4
6.3
5.2
4.1
3
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 80. Variation in the activity of glutathione peroxidase in terms of % increase over control
in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
415
% increase in glutathione peroxidase
concentrations of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos + Cadmium for 21 days.
Chlorpyrifos and
58.31
60
52.06
46.97
42.92
45
34.67
29.02
30
26.78
18.22
15
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 81. Mean variation in the activity of glutathione peroxidase in terms of % increase over
control in different organs of Tilapia (Oreochromis mossambicus) exposed to
sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% increase in Glutathione Peroxidase
7 Days
14 Days
21 Days
100
89.13
80.34
70.76
75
58.12
50
34.69
25
0
Gills
Liver
Kidney
Test organs
Brain
Muscle
416
Fig. 82. Changes in the glutathione S-transferase activity in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
nmol of CDNB conjugate
formed/min/mg protein
63
51
39
27
15
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 83. Variation in the activity of glutathione S-transferase in terms of % increase over
control in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
% increase in glutathione S-transferase
417
120
107.76
101.15
96.42
94.79
86.77
90
78.57
57.21
60
46.93
30
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 84. Changes in the glutathione S-transferase activity in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
21 Days
nmol of CDNB conjugate
formed/min/mg protein
75
60
45
30
15
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 85. Variation in the activity of glutathione S-transferase in terms of % increase over
control in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
418
% increase in glutathione S-transferase
concentrations of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos + Cadmium for 21 days.
Chlorpyrifos and
100
90.16
82.23
75.43
73.99
75
77.32
62.78
47.38
50
44.30
25
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 86. Changes in the glutathione S-transferase activity in the kidney of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
nmol of CDNB conjugate
formed/min/mg protein
50
40
30
20
10
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd2
419
% increase in glutathion S-transferase
Fig. 87. Variation in the activity of glutathione S-transferase in terms of % increase over
control in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
100
93.06
83.47
79.65
78.13
72.16
75
64.30
48.57
50
36.84
25
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 88. Changes in the glutathione S-transferase activity in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
nmol of CDNB conjugate
formed/min/mg protein
40
34
28
22
16
10
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd2
420
% increase in glutathione S-transferase
Fig. 89. Variation in the activity of glutathione S-transferase in terms of % increase over
control in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
74.35
75
61.97
56.47
54.72
55
49.55
39.64
35
25.79
23.19
15
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 90. Changes in the glutathione S-transferase activity in the muscle of Tilapia
(Oreochromis mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium,
Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and
21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
nmol of CDNB conjugate
formed/min/mg protein
18
16
14
12
10
8
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd2
421
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% increase in glutathione S-transferase
Fig. 91. Variation in the activity of glutathione S-transferase in terms of % increase over
control in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
50
40.34
39.61
40
35.75
29.47
30
26.01
17.07
20
11.92
7.17
10
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 92. Mean variation in the activity of glutathione S-transferase in terms of % increase over
control in different organs of Tilapia (Oreochromis mossambicus) exposed to
sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% increase in glutathione S-transferase
7 Days
14 Days
21 Days
100
83.70
69.52
69.20
75
48.21
50
25.92
25
0
Gills
Liver
Kidney
Test organs
Brain
Muscle
422
nmol of NADPH oxidized/min/mg protein
Fig. 93. Changes in the glutathione reductase activity in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
23
18
13
8
3
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 94. Variation in the activity of glutathione reductase in terms of % increase over control
in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
% increase in Glutathione Reductase
423
80
73.79
64.04
60
53.30
49.46
37.63
40
36.97
27.66
20
13.08
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
nmol of NADPH oxidized/min/mg protein
Fig. 95. Changes in the glutathione reductase activity in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
29
24
19
14
9
4
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 96. Variation in the activity of glutathione reductase in terms of % increase over control
in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
424
% increase in glutathione reductase
concentrations of Cadmium, Chlorpyrifos, Cadmium +
Chlorpyrifos + Cadmium for 21 days.
75
Chlorpyrifos and
69.59
59.92
60
51.14
45
50.65
38.62
35.37
33.98
30
23.66
15
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 97. Changes in the glutathione reductase activity in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
nmol of NADPH oxidized/min/mg protein
7 Days
14 Days
21 Days
18
15
12
9
6
3
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd1
CPF+Cd2
425
% increase in glutathione reductase
Fig. 98. Variation in the activity of glutathione reductase in terms of % increase over control
in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
77.27
80
68.18
61.53
59.09
60
45.40
41.94
40.92
40
29.27
20
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 99. Changes in the glutathione reductase activity in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos, Cadmium
+ Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
nmol of NADPH oxidized/min/mg protein
7 Days
14 Days
21 Days
8
6.5
5
3.5
2
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd1
CPF+Cd2
426
% increase in glutathione reductase
Fig. 100. Variation in the activity of glutathione reductase in terms of % increase over control
in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
65
52
60.90
58.17
49.17
38.34
39
30.26
26
20.67
9.02
13
4.13
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 101. Changes in the glutathione reductase activity in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
nmol of NADPH oxidized/min/mg protein
7 Days
14 Days
21 Days
5
4
3
2
1
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd1
CPF+Cd2
427
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% increase in glutathione reductase
Fig. 102. Variation in the activity of glutathione reductase in terms of % increase over control
in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
65
60.10
52
56.32
48.22
47.17
42.04
39
22.40
26
17.11
10.89
13
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 103. Mean variation in the activity of glutathione reductase in terms of % increase over
control in different organs of Tilapia (Oreochromis mossambicus) exposed to
sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
% increase in Glutathione Reductase
7 Days
14 Days
21 Days
103.64
105
87.78
86.59
84
72.05
63
40.22
42
21
0
Gills
Liver
Kidney
Test organs
Brain
Muscle
428
µmol of GSH/gm wet wt. of the tissue
Fig. 104. Changes in the total reduced glutathione level in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
38
31
24
17
10
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 105. Variation in the level of total reduced glutathione in terms of % increase over control
in the gills of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
429
% increase in reduced glutathione
35.96
36
31.76
27.55
26
22.14
19.94
18.03
15.54
16
13.53
6
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 106. Changes in the total reduced glutathione level in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
µmol of GSH/gm wet wt. of the tissue
7 Days
14 Days
21 Days
73
58
43
28
13
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 107. Variation in the level of total reduced glutathione in terms of % increase over control
in the liver of Tilapia (Oreochromis mossambicus) exposed to sublethal
430
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
% increase in reduced glutathione
40
37.73
33.67
31.06
32
26.06
25.67
24
21.30
17.86
16
13.82
8
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
µmol of GSH/gm wet wt. of the tissue
Fig. 108. Changes in the total reduced glutathione level in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
26
22
18
14
10
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd2
431
% increase in reduced glutathione
Fig. 109. Variation in the level of total reduced glutathione in terms of % increase over control
in the kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
32
28.57
26.34
24
17.14
16
13.73
10.12
8
4.84
4.29
2.30
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
µmol of GSH/gm wet wt. of the tissue
Fig. 110. Changes in the total reduced glutathione level in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
55
48
41
34
27
20
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd2
432
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% increase in reduced glutathione
Fig. 111. Variation in the level of total reduced glutathione in terms of % increase over control
in the brain of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
26.87
27
24.39
17.63
18
11.29
8.75
9
8.41
6.49
2.54
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 112. Changes in the total reduced glutathione level in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
µmol of GSH/gm wet wt. of the tissue
7 Days
14 Days
21 Days
24
20
16
12
8
Control
Cd1
Cd2
CPF1
CPF2
Test groups
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
433
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
% increase in reduced glutathione
Fig. 113. Variation in the level of total reduced glutathione in terms of % increase over control
in the muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 21 days.
50
45.31
40
42.66
34.15
29.49
30
24.39
21.36
20
11.22
10
5.36
0
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 114. Mean variation in the level of total reduced glutathione in terms of % increase over
control in different organs of Tilapia (Oreochromis mossambicus) exposed to
sublethal concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
434
7 Days
% increase in reduced glutathione
60
52.50
54.47
14 Days
21 Days
53.75
45
37.43
30
18.55
15
0
Gills
Liver
Kidney
Brain
Muscle
Test organs
Fig. 115. Changes in the ascorbic acid content in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
435
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
µg ASA/gm wet wt. of the tissue
39
33
27
21
15
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 116. Variation in the ascorbic acid content in terms of % decrease over control in the gills
of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
% decrease in Ascorbic acid
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
9
18
16.60
22.07
27
26.46
30.99
36
45
29.77
31.98
42.42
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
28.83
436
Fig. 117. Changes in the ascorbic acid content in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
µg ASA/gm wet wt. of the tissue
65
56
47
38
29
20
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 118. Variation in the ascorbic acid content in terms of % decrease over control in the liver
of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
% decrease in Ascorbic acid
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
15
22.00
27
26.49
31.70
32.76
39
34.30
38.13
51
50.87
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
35.36
437
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 119. Changes in the ascorbic acid content in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
µg ASA/gm wet wt. of the tissue
50
40
30
20
10
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 120. Variation in the ascorbic acid content in terms of % decrease over control in the
kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
% decrease in Ascorbic acid
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
13
26
22.73
26.06
39
41.22
46.72
48.16
52
54.42
65
61.06
Test groups
52.52
438
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 121. Changes in the ascorbic acid content in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
µg ASA/gm wet wt. of the tissue
42
36
30
24
18
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 122. Variation in the ascorbic acid content in terms of % decrease over control in the
brain of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
439
% decrease in Ascorbic acid
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
10
20
18.38
23.80
30
28.57
31.44
40
36.01
38.10
42.43
46.39
50
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
µg ASA/gm wet wt. of the tissue
Fig. 123. Changes in the ascorbic acid content in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
14 Days
Cd+CPF2
CPF+Cd1
21 Days
32
28
24
20
16
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 124. Variation in the ascorbic acid content in terms of % decrease over control in the
muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
440
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
% decrease in Ascorbic acid
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
10
14.96
20
22.23
24.02
30
28.09
30.91
33.06
33.44
35.55
40
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 125. Mean variation in the ascorbic acid content in terms of % decrease over control in
different organs of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
7 Days
Gills
Liver
Kidney
Brain
14 Days
21 Days
Muscle
% decrease in Ascorbic acid
0
10
20
30
40
50
27.78
28.64
33.14
33.95
44.11
Test organs
441
Fig. 126. Changes in the total protein content in the gills of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
mg protein/100gm wet wt. of the tissue
7 Days
14 Days
21 Days
28
24
20
16
12
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 127. Variation in the total protein content in terms of % decrease over control in the gills
of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
442
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
% decrease in Total protein
10
16.85
19.61
25
24.69
26.07
30.44
33.47
40
42.85
55
50.39
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 128. Changes in the total protein content in the liver of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
mg protien/100gm wet wt. of the tissue
7 Days
14 Days
21 Days
50
40
30
20
10
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 129. Variation in the total protein content in terms of % decrease over control in the liver
of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
443
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
% decrease in Total protein
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
15
21.53
27
25.90
35.18
39
32.20
39.98
45.01
51
52.83
63
62.51
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 130. Changes in the total protein content in the kidney of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
mg protein/100gm wet wt. of the tissue
7 Days
14 Days
21 Days
24
21
18
15
12
9
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd1
CPF+Cd2
444
Fig. 131. Variation in the total protein content in terms of % decrease over control in the
kidney of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
% decrease in Total protein
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
10
18.79
25
23.71
25.43
29.04
33.83
35.50
40
42.63
47.73
55
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 132. Changes in the total protein content in the brain of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
mg protein/100gm wet wt.of the tissue
7 Days
14 Days
21 Days
34
28
22
16
10
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
CPF+Cd1
CPF+Cd2
445
Fig. 133. Variation in the total protein content in terms of % decrease over control in the brain
of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations of
Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for
21 days.
% decrease in Total protein
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
15
22.52
26.55
30
27.34
32.71
37.41
45
41.26
43.79
56.15
60
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 134. Changes in the total protein content in the muscle of Tilapia (Oreochromis
mossambicus) exposed to 1/5th and 1/10th LC50 of Cadmium, Chlorpyrifos,
Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium for 7, 14 and 21 days.
mg protein/100gm wet wt. of the tissue
7 Days
14 Days
21 Days
42
34
26
18
10
Control
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd1
CPF+Cd2
446
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 135. Variation in the total protein content in terms of % decrease over control in the
muscle of Tilapia (Oreochromis mossambicus) exposed to sublethal concentrations
of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and Chlorpyrifos + Cadmium
for 21 days.
% decrease in Total protein
Cd1
Cd2
CPF1
CPF2
Cd+CPF1
Cd+CPF2
CPF+Cd1
CPF+Cd2
10
18.05
23
21.68
25.49
30.80
36
39.66
45.12
49
53.61
62
61.02
Test groups
Cd - Cadmium, CPF - Chlorpyrifos, Cd+CPF - Cadmium+Chlorpyrifos,
CPF+Cd - Chlorpyrifos+Cadmium, 1 - 1/5th of LC50, 2 - 1/10th of LC50
Fig. 136. Mean variation in the total protein content in terms of % decrease over control in
different organs of Tilapia (Oreochromis mossambicus) exposed to sublethal
concentrations of Cadmium, Chlorpyrifos, Cadmium + Chlorpyrifos and
Chlorpyrifos + Cadmium for 7, 14 and 21 days.
447
7 Days
Gills
Liver
Kidney
Brain
14 Days
21 Days
Muscle
% decrease in Total protein
0
10
20
30
30.55
40
32.08
35.97
39.39
Test organs
36.93