Embryo-toxic effects of lead nitrate of the African catfish Clarias gariepinus (Burchell, 1822) [Elektronische Ressource] / von Alaa Gad El-Karim Mahmoud Osman

Embryo-toxic effects of lead nitrate of the African catfish
Clarias gariepinus (Burchell, 1822)

DISSERTATION
Zur Erlangung des akademischen Grades
Doctor rerum agriculturarum
(Dr.rer.agr.)

Eingereicht an der
Landwirtschaftlich-Gärtnerischen Fakultät
der Humboldt-Universität zu Berlin

von
Alaa Gad El-Karim Mahmoud Osman, M.Sc.
geboren am 16. Oktober 1970 in Kina, Ägypten


Präsident der Humboldt- Universität zu Berlin
Prof. Dr. Christoph Markschies
Dekan der Landwirtschaftlich-Gärtnerischen Fakultät
Prof. Dr. Dr. h. c. Otto Kaufmann


Gutachter: 1. Prof. Dr. Frank Kirschbaum
2. Prof. Dr. Imam Mekkawy 3. Prof. Dr. Werner Kloas

Tag der mündlichen Prüfung: 02. 04. 2007 .






I dedicate this thesis
To the memory of my Father,
To my mother,
To my lovely wife Sahar,
And
To my pretty kids, Zeyad and Shaza
Contents .

CONTENTS
Acknowledgements…..……………………………………………………… i
Curriculum Vitae…………………………………………………………….. ii
List of Publications…………………………………………………………... iv
Publications in conjunction with this thesis…………………………………. iv
Other publications…………………………………………………………… v
Conference contributions.…………………………………………………… vi

CHAPTER 1 General introduction
1.1 Water Pollution in Egypt…………………………………………………….. 1
1.1.1 Heavy Metals………………………………………………………………… 1
1.1.2 Lead………………………………………………………………………….. 2
1.2 Fish embryos as test organisms...……………………………………………. 2
1.3 The African catfish Clarias gariepinus………….…………………………... 3
1.4 Outline of the work…………..……………………………………………… 4

CHAPTER 2 Materials and Methods
2.1 Artificial reproduction of the African catfish…...…………………………… 6
2.2 Sampling for embryonic development………………………………………. 6
2.3 Transmission Electron Microscope (TEM)………………………………….. 6
2.4 Scanning Electron Microscope (SEM)………………………………………. 7
2.5 Gel electrophoresis of chorion proteins……………………………............... 7
2.6 Terminology of the chorion………………………………………………….. 7
2.7 Experimental setup and sampling for toxicity tests…..……………………... 8
2.8 Morphological malformations.……………………………………............... 8
2.9 Histology and histopathology……………………………………………….. 8
2.10 Determination of lead concentration………………………………………… 9
2.11 Measurements of enzyme activities…………………………………………. 9
2.12 Comet assay………………………………………………………………….. 10
2.13 Statistical analysis…………………………………………………………… 10

CHAPTER 3 Fertilization and associated morphological and chemical
Contents .
variations of the egg surface and envelopes
3.1 Introduction………………………………………………………………….. 12
3.2 Results ……………...……………………………………………….............. 13
3.2.1 The ripe testes and the sperm structure……………..……………….............. 13
3.2.2 The ripe oocyte…………………...………………………………….............. 15
3.2.3 Egg surface…………………………………………………………………... 15
3.2.4 The micropyle……………………………………………………………….. 17
3.2.5 The chorion structure and fertilization………………………………………. 18
3.3 Discussion…………………………………………………………………… 25
3.3.1 The ripe testes and the sperm structure……………………………………… 25
3.3.2 The ripe oocyte………………………………………………………………. 25
3.3.3 26
3.3.4 The micropyle……………………………………………………………….. 27
3.3.5 The chorion structure and fertilization………………………………………. 29
3.3.6 Chorion proteins and their change during the hardening process…………… 30

CHAPTER 4 Embryonic development
4.1 Introduction………………………………………………………………….. 31
4.2 Results……………………………………………………………………….. 31
4.2.1 Embryonic development - egg stages………………………………………... 31
4.2.1.1 Cleavage phase................................................................................................. 33
4.2.1.2 Blastula ………………………………………………………………............ 33
4.2.1.3 Gastrula ………………………………………………………………........... 33
4.2.1.4 Organogenesis……………………………………………………………...... 33
4.2.1.5 Hatching……………………………………………………………………... 33
4.2.2 Embryonic development - eleutheron-embryonal stages……………………. 34
4.2.2.1 Newly hatched embryo………………………………………………………. 34
4.2.2.2 24 hour post-hatching stage (24h-PHS)……………………………………... 34
4.2.2.3 48h-PHS………………….………………………………………………….. 34
4.2.2.4 72h-PHS………………….………………………………………………….. 34
4.2.2.5 96h-PHS………………….………………………………………………….. 36
4.2.2.6 120h-PHS………………….………………………………………………… 36
4.2.4 Ontogenetic development of tissues and organs………………….................. 36
4.2.4.1 Notochord and somites………………………………………………………. 36
Contents .
4.2.4.2 Eye…………………………………………………………………………… 38
4.2.4.3 Inner ear……………………………………………………………………… 38
4.2.4.4 Heart…………………………………………………………………………. 40
4.2.4.5 Digestive system……………………………………………………….......... 40
4.3 Discussion…………………………………………………………………… 42

CHAPTER 5 Lead induced morphological and histological
malformations during embryogenesis
5.1 Introduction………………………………………………………………….. 47
5.2 Results……………………………………………………………………….. 48
5.2.1 Incubation period and hatching success……………………………………... 48
5.2.2 Morphological aberrations in post-hatching stages………………………….. 48
5.2.2.1 Irregular head shape…………………………………………………………. 48
5.2.2.2 Pericardial oedema…………………………………………………………... 49
5.2.2.3 Yolk sac oedema……………………………………………………………. 49
5.2.2.4 Notochordal defect…………………………………………………………... 49
5.2.2.5 Finfold defects……………………………………………………………….. 52
5.5.5.6 Reduced pigmentation……………………………………………………….. 52
5.2.3 Histopathological changes in post-hatching stages …………………………. 53
5.2.3.1 Malformations of the notochord……………………………………………... 53
5.2.3.2 ations of the gill…………………………………………………….. 54
5.2.3.3 Malformations of the eyes…………………………………………………… 54
5.2.3.4 Skin detached from the underlying muscles……………...…………………. 55
5.2.4 Bioaccumulation of the lead…………………………………………………. 56
5.3 Discussion…………………………………………………………………… 58

CHAPTER 6 Effects of lead nitrate on the activity of the metabolic
enzymes G6PDH, LD and PK
6.1 Introduction………………………………………………………………….. 62
6.2 Results……………………………………………………………………….. 64
6.2.1 The normal ontogenetic variations in enzyme activities…………………….. 64
6.2.2 The enzyme activities after exposure to lead nitrate ………………………... 65
6.3 Discussion…………………………………………………………………… 71
6.3.1 The normal ontogenetic variations in enzyme71
Contents .
6.3.2 The enzyme activities after exposure to lead nitrate………………………… 72

CHAPTER 7 Monitoring of DNA breakage after exposure to lead nitrate
7.1 Introduction………………………………………………………………….. 76
7.2 Results……………………………………………………………………….. 76
7.2.1 Quantitative evaluation of the comet parameters (end points)………………. 77
7.2.2 Genotoxicity in embryos after lead nitrate exposure………………………... 78
7.3 Discussion…………………………………………………………………… 82
7.3.1 met parameters (end points)………………. 82
7.3.2 Genotoxicity in embryos after lead nitrate exposure ……………………...... 82

SUMMARY…………………………………………………………………. 85

ZUSAMMENFASSUNG…………………………………………………... 91

CONCLUSIONS………………………………………………………….... 97

LIST OF ABBREVIATIONS .……...……………………………………. 100

REFERENCES...…………………………………………………………… 101

SELBSTÄNDIGKEITSERKLÄRUNG……………………………………. 128

Acknowledgements i
ACKNOWLEDGEMENTS
My great thank to the God for all gifts he gave me
In the first place I would like to record my sincere gratitude to my supervisor,
Prof. Frank Kirschbaum, who guided this work, for his constant support,
recommendations, and motivations and for many decisive scientific contributions. He
helped whenever I was in need. Without his help, this work would not be possible. I am
indebted to him more than he knows.
I gratefully acknowledge Prof. Imam Mekkawy for his advice, encouragement,
and crucial contributions. I could never have embarked and started all of this without his
prior teaching in Fish Biology and thus opened up unknown area to me. A special
thanks also goes to Prof. Werner Kloas, for his invaluable advice, encouragement and
contributions. I am deeply indebted to Dr. Sven Wuertz, for valuable contributions and
continuous support. Sven, I am grateful in every possible way and hope to keep up our
collaboration in the future. I am very grateful to Dr. Peter Bartsch, for kindly
providing the fluorescence microscope.
I gratefully thank Mrs. Karena Kuntze, Mr. Peter Loeschau, Mr. Marcel
Simon, and Mr. Hans-Jürgen Exner for their continuous help during the preparations