Muhammad Iftikhar Khan Zoology 2019 UoP Peshawar 18.07.2019

FRESHWATER MUSSEL (ANODONTA CYGNEA) AS A
BIOINDICATOR FOR MONITORING OF POLLUTION STATUS IN

RIVER KABUL, PAKISTAN
BY
MUHAMMAD IFTIKHAR KHAN
DEPARTMENT OF ZOOLOGY
UNIVERSITY OF PESHAWAR
PAKISTAN
2012-2017
FRESHWATER MUSSEL (ANODONTA CYGNEA) AS A BIOINDICATOR
FOR MONITORING OF POLLUTION STATUS IN RIVER KABUL,
PAKISTAN
A MANUSCRIPT PRESENTED TO THE DEPARTMENT OF ZOOLOGY, UNIVERSITY OF

PESHAWAR IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
ZOOLOGY
BY
DEPARTMENT OF ZOOLOGY
UNIVERSITY OF PESHAWAR
PAKISTAN
2012-2017
PARENTS
Contents
Acknowledgments...............................................................................................................................................................I
Preface.....................................................................................................................................................................................I
Abstract ................................................................................................................................................................................IV
CHAPTER 1 ...................................................................................................................................................................... 1
1.1 General introduction.................................................................................................................................................... 1
1. 2 Systematic position..................................................................................................................................................... 1
1.3 Geographic range ......................................................................................................................................................... 1
1.4 Population ...................................................................................................................................................................... 2
1.5 Habitat and ecology..................................................................................................................................................... 2
1.6 Reproduction ................................................................................................................................................................. 2
1.7 Functions of freshwater mussels.............................................................................................................................. 3
1.7.1 Regulating services: mussels as biofilters that purify water................................................... ………..3
1.7.2 Supporting services: nutrient cycling and storage...................................................................................4
1.7.3 Supporting services: mussels as habitat and habitat modifiers ..............................................................5
1.7.4 Supporting services: mussels support food webs....................................................................................5
1.7.5 Supporting services: mussels as environmental monitors......................................................................5
1.7.6 Provisioning and cultural services ............................................................................................................6
1.7.7 Losses, restoration and conservation ........................................................................................................6
1.8 Description of study area: .......................................................................................................................................... 7
1.8.1 River Kabul: ................................................................................................................................................7
1.8.2 Human population:.....................................................................................................................................8
1.8.3 Industries along River Kabul:....................................................................................................................8
1.8.4: Industries at Aman Garh industrial zone: ...............................................................................................9
1.8.5 Disposal of sewage and industrial effluents to River Kabul. .................................................................9
1.8.6 Water contamination:.............................................................................................................................. 10
1.8.7 Heavy metals: .......................................................................................................................................... 10
1.8.8 Sources of heavy metals: ........................................................................................................................ 11
1.8.9 Metals in the Environment: .................................................................................................................... 11
1.8.10 Metals in the body of an organism: ..................................................................................................... 11
1.8. 11 Essential and non-essential metals:..................................................................................................... 12
1.8.12 Role of heavy metals in the body of an organism:............................................................................. 12
1.8.12.1 Cadmium: ........................................................................................................................................... 13
1.8.12.2 Chromium:.......................................................................................................................................... 13
1.8.12.3 Copper: ................................................................................................................................................ 14
1.8.12.4 Iron:… ................................................................................................................................................. 15
1.8.12.5 Mercury: .............................................................................................................................................. 16
1.8.12.6 Manganese: ......................................................................................................................................... 16
1.8.12.7 Nickel: ................................................................................................................................................. 17
1.8.12.8 Lead……………………………………………………………………………………....18
1.8.12.9 Zinc……………... ............................................................................................................................ 19
1.9 Heavy metals pollution in river kabul ..................................................................................................................19
1.10 Effects of heavy metals on human beings: .......................................................................................................20
1.11 Aims and objectives:...............................................................................................................................................21
CHAPTER 2 ....................................................................................................................................................................23
PHYSICAL AND CHEMICAL CHARACTERISTICS OF RIVER KABUL ..........................................23
Abstract ................................................................................................................................................................................23
2.1 Introduction..................................................................................................................................................................24
2.2 Physico-chemical parameters:................................................................................................................................24
2.3 Heavy metal parameters: .........................................................................................................................................26
2.4 Materials and methods..............................................................................................................................................27
2.4.1 Study area description ............................................................................................................................. 27
2.4.2 Sampling sites .......................................................................................................................................... 27
2.4.3 Sampling points ....................................................................................................................................... 28
2.4.4. Water sample from Warsak dam .......................................................................................................... 28
2.4.5. Water samples from the main River..................................................................................................... 29
2.4.6. Collection of water samples .................................................................................................................. 29
2.4.7. Preservation of water samples............................................................................................................... 29
2.4.8. Water analysis ......................................................................................................................................... 29
2.4.9. Physico-chemical parameters................................................................................................................ 30
2.4.9.1. pH…………. ...................................................................................................................................... 30
2.4.9.2. Electrical conductivity ........................................................................................................................ 30
2.4.9.3. Total dissolved solid (TDS) ............................................................................................................... 30
2.4.9.4. Total suspended solid (TSS) .............................................................................................................. 30
2.4.9.5. Chloride ................................................................................................................................................ 30
2.4.9.6. Total alkalinity ..................................................................................................................................... 31
2.4.9.7. Sodium and Potassium ....................................................................................................................... 31
2.5. Heavy metals parameters .......................................................................................................................... 31
2.6. Statistical analysis .....................................................................................................................................................32
2.7. Results and discussion .............................................................................................................................................33
2.7.1.Water Analysis of River Kabul.............................................................................................................. 33
2.7.1.1.Michini Bridge water sample from site 1 (Sample A= Reference site) ......................................... 33
2.7.1.2. Water sample from Sardaryab site 2 receiving sewages (Water sample-B)................................. 40
2.7.1.3 Water sample C from River Kabul at Aman Garh (site 3) .............................................................. 46
2.7.1.4 Polluted River Kabul water from Peer Sabaq, site 4 receiving cities sewages (Water sampleD) 52
2.7.1.5 Conclusions and Remarks ................................................................................................................... 58
CHAPTER 3 ....................................................................................................................................................................69
HEAVY METALS CONCENTRATIONS IN SEDIMENTS OF RIVER KABUL. ...............................70
3.1. Introduction.................................................................................................................................................................70
3.2 Materials and methods..............................................................................................................................................74
3.2.1. Study area description ............................................................................................................................ 74
3.2.2. Sampling sites ......................................................................................................................................... 74
3.2.3. Sampling points ...................................................................................................................................... 74
3.2.4. Sediment samples from Michini Bridge below Warsak dam ........................................................... 75
3.2.5. Sediment samples from the main River ............................................................................................... 75
3.2.6. Collection of Sediments samples .......................................................................................................... 76
3.2.7. Preparation of Sediment Samples......................................................................................................... 76
3.2.8. Chemical analysis of sediments ............................................................................................................ 76
3.2.9. Heavy metals parameters in sediment samples ................................................................................... 77
3.2.10. Statistical Analysis ............................................................................................................................... 77
3.3. Result and discussion ...............................................................................................................................................78
3.3.1. Chemical analysis of sediments ............................................................................................................ 78
3.3.1.1. Heavy Metals concentration in Sediment Sample A from site 1, Michini Bridge....................... 78
Below Warsak dam (Sample A= Reference site).......................................................................................... 78
3.3.1.2. Heavy Metals concentration in Sediment Sample B from Sardaryab, site 2 ................................ 81
3.3.1.3. Heavy Metals concentration in Sediment Sample C from Aman Garh, site 3. ............................ 84
3.3.1.4. Heavy Metals concentration in Sediment Sample D from Peer Sabaq, site 4. ............................. 87
CHAPTER 4 ....................................................................................................................................................................95
BIOACCUMULATION OF HEAVY METALS IN TISSUES OF FRESHWATER MUSSELS......95
4.1. Introduction.................................................................................................................................................................95
4.2. Materials and methods.............................................................................................................................................98
4.2.1. Study area ................................................................................................................................................ 98
4.2.2. Sampling sites of freshwater mussels................................................................................................... 99
4.2.3. Collection of Freshwater mussels’ samples......................................................................................... 99
4.2.4. Preservation of tissues of freshwater mussels.................................................................................... 100
4.2.5. Tissue digestion .................................................................................................................................... 100
4.2.6. Determination of heavy metals ........................................................................................................... 101
4.2.7. Statistical Analysis................................................................................................................................ 101
4.3. Result and discussion .............................................................................................................................................102
4.3.1. Bioaccumulation of heavy metals in soft tissues............................................................................... 102
4.3.2. Bioaccumulation of heavy metals in Gills of freshwater mussels................................................... 103
CHAPTER 5 ..................................................................................................................................................................111
HISTOPATHOLOGICAL EFFECTS OF HEAVY METALS IN FRESHWATER MUSSELS ......111
5.1. Introduction...............................................................................................................................................................111
5.2. Methods and materials...........................................................................................................................................115
5.2.1. Study area .............................................................................................................................................. 115
5.2.2. Freshwater mussels sampling sites ..................................................................................................... 115
5.2.3. Collection of freshwater mussel’s samples........................................................................................ 115
5.2.4. Preservation of freshwater mussel’s tissues....................................................................................... 115
5.2.5. Procedure............................................................................................................................................... 115
5.2.6. Preparation of solutions for tissue processing.................................................................................... 115
5.2.7. Preparation of solution of fixation ...................................................................................................... 115
5.2.8. Preparation of PBS ............................................................................................................................... 115
5.2.9. Preparation of 10% NBF ..................................................................................................................... 115
5.2.10. Preparation of different ethanol solutions ........................................................................................ 115
5.2.10.1. 50% ethanol solution ...................................................................................................................... 115
5.2.11. Preparation of alcohol-xylene solution............................................................................................. 116
5.2.12. Preparation of xylene-paraffin solution............................................................................................ 116
5.2.13. Preparation of different solutions for staining ................................................................................. 116
5.2.14. Mayer’s albumin ................................................................................................................................ 116
5.2.15. Harris hematoxyl in stain................................................................................................................... 116
5.2.16. Eosin stain ........................................................................................................................................... 116
5.2.17. Eosin-Y stock solution....................................................................................................................... 116
5.2.18. Phloxine-B stock solution.................................................................................................................. 116
5.2.19. Eosin-phloxine working solution...................................................................................................... 116
5.2.20. 1% acid-alcohol solution ................................................................................................................... 116
5.2.21. 1000mL ammonia solution ............................................................................................................... 116
5.2.22. Tissue processing ............................................................................................................................... 116
5.2.23. Tissues fixation ................................................................................................................................... 116
5.2.24. Tissues dehydration............................................................................................................................ 116
5.2.25. Clearing of tissues .............................................................................................................................. 117
5.2.26. Paraffin infiltration of tissues ............................................................................................................ 117
5.2.27. Embedding of tissues ......................................................................................................................... 117
5.2.28. Sectioning of tissues........................................................................................................................... 117
5.2.29. Staining of tissues ............................................................................................................................... 118
5.2.30. Observation of tissues under microscope ........................................................................................ 118
5.2.31. Statistical analysis............................................................................................................................... 118
5. 3 Results and discussion ...........................................................................................................................................119
5.3.1 Gross and histopathological examination ........................................................................................... 119
5.4. Identification and measurements ........................................................................................................................119
5.5. Gross pathology.......................................................................................................................................................119
5.6. Parasitic infestation.................................................................................................................................................119
5.7. Histopathology.........................................................................................................................................................120
5.7.1. Digestive gland ..................................................................................................................................... 120
5.7.1.1. Structure of normal digestive gland................................................................................................. 120
5.7.1.2. Lesions observed in digestive gland................................................................................................ 120
5.7.1.3 Comparison of digestive gland lesions among the sites................................................................. 121
5.7.2. Gonads ................................................................................................................................................... 122
5.7.2.1. Lesions observed in the gonads ....................................................................................................... 122
5.7.2.2. Comparison of gonadal lesions among the sites ............................................................................ 122
5.7.3. Gills ........................................................................................................................................................ 122
5.7.3.1. Lesions observed in the gills ............................................................................................................ 122
5.7.3.2. Comparison of gill lesions among the sites .................................................................................... 123
5.7.4. Intestine.................................................................................................................................................. 123
5.7.4.1. Lesions observed in the intestine ..................................................................................................... 123
5.7.4.2. Comparison of intestinal lesion among the sites ............................................................................ 123
CHAPTER 6 ..................................................................................................................................................................152
GENOTOXICAL EFFECTS OF HEAVY METALS IN FRESHWATER MUSSELS. ......................152
6.1. Introduction...............................................................................................................................................................152
6.2. Methods and materials...........................................................................................................................................156
6.2.1. Study area .............................................................................................................................................. 156
6.2.3. Collection of the freshwater mussels samples ................................................................................... 156
6.2.4. Collection of haemolymph from freshwater samples....................................................................... 156
6.2.5. Comet assay .......................................................................................................................................... 156
6.2.5.1. Preparation different solutions for comet assay ............................................................................. 156
6.2.5.2. Lysing solution .................................................................................................................................. 156
6.2.5.3. Final lysing solution .......................................................................................................................... 156
6.2.5.4. Phosphate Buffer Saline (PBS)........................................................................................................ 156
6.2.5.5. Preparation of stock solutions .......................................................................................................... 156
6.2.5.6. Electrophoresis buffer ....................................................................................................................... 157
6.2.5.7. Neutralization buffer ......................................................................................................................... 157
6.2.5.8. Staining solution ................................................................................................................................ 157
6.2.5.9. Stock solution..................................................................................................................................... 157
6.2.5.10. Working solution............................................................................................................................. 157
6.2.5.11. Preparation of 1% and 0.5 % LMPA and 1% NMA .................................................................. 157
6.2.5.12. Preparation of base slides ............................................................................................................... 157
6.2.5.13. Layering of cells and LMPA on base slides................................................................................. 157
6.2.5.14. Placing of slides in final lysing solution........................................................................................ 157
6.2.5.15. Electrophoresis of slides ................................................................................................................. 158
6.2.5.16. Neutralization of slides ................................................................................................................... 158
6.2.5.17. Drying of slides ............................................................................................................................... 158
6.2.5.18. Rehydration and staining of slides................................................................................................. 158
6.2.5.19. Scoring of slides and Visualization of DNA damage ................................................................. 158
6.2.5.20. Comet classes .................................................................................................................................. 158
6.2.5.21. Statistical analysis............................................................................................................................ 159
6.3. Results and discussion ...........................................................................................................................................160
6.3.1. TCS and comet classes in Hemolymph ............................................................................................. 160
Conclusions and recommendations ......................................................................................................................164
Conclusions ......................................................................................................................................................................164
Recommendations ..........................................................................................................................................................165
7.1. References................................................................................................................................................................166
LIST OF TABLES
Table 1: Operating data of Atomic absorption spectrophotometer for determination of metals............ 32

Table 2: Physico-chemical characteristics of water sample from Michini bridge during winter and
summer during 2013-2015............................................................................................................................... 35
Table 3: National Environmental Quality Standards (NEQS) for municipal and liquid industrial
effluents of Pakistan.......................................................................................................................................... 36
Table 4: Heavy metals concentration (µg/L) of water sample from Michini Bridge during winter and
summer (2013-2015). ....................................................................................................................................... 38
Table 6: Physico-chemical characteristics of water sample from Sardayab during winter and summer
(2013-2015). ...................................................................................................................................................... 42
Table 8: Heavy metals concentration (µg/L) of water sample from Sardaryab During winter and
summer (2013-2015). ....................................................................................................................................... 45
Table 10: Physico-chemical characteristics of water sample from Aman Garh during winter and
summer (2013-2015). ....................................................................................................................................... 48
Table 12: Heavy metals concentration (µg/L) of water sample from Aman Garh during winter and
summer (2013-2015). ....................................................................................................................................... 51
Table 14: Physico-chemical characteristics of water sample D from peer sabaq during winter and
summer season during 2013-2015. ................................................................................................................. 54
Table 15: Heavy metals concentration of water sample D from Peer sabaq during winter and summer
flows during 2013-2015. .................................................................................................................................. 57
Table 17: Comparison of Heavy metals conc.( µg/L) in water of study sites collected during the
summer and winter seasons ............................................................................................................................. 60
Table 18: Operating data of Atomic absorption spectrophotometer for determination of metals........... 77
Table 19: Heavy metals concentration (mg/kg) of sediment sample A from Michini Bridge during
winter and summer during 2013-2015. .......................................................................................................... 80
Table 20: Heavy metals concentration (mg/kg) of sediment sample B from Sardaryab, site 2, in winter
and summer seasons during 2013-2015. ........................................................................................................ 83
Table 21: Heavy metals concentration (mg/kg) of sediment sample C from Aman Garh ...................... 86
Table 22: Heavy metals concentration (mg/kg) of sediment sample D from Peer Sabaq During winter
and summer (2013-2015)................................................................................................................................. 89
Table 23: The standards used to analyze the variable using atomic absorption spectrophotometer
(Spectra-AA-700). .......................................................................................................................................... 101
Table 24: Length of freshwater mussels expressed in terms of maximum, minimum, mean length, and
standard deviation collected from four different sites. ................................................................................ 105
Table 25: Width of freshwater mussels expressed in terms of maximum, minimum, mean width, and
standard deviation collected from four different sites. ................................................................................ 106
Table 26: Weight (shell+soft tissues) of freshwater mussels expressed in terms of maximum,
minimum, mean weight, and standard deviation collected four from different sites............................... 106
Table 27: Weight (soft tissues) of freshwater mussels expressed in terms of maximum, minimum,
mean weight, and standard deviation collected from four different sites.................................................. 107
Table 28: Heavy metals concentration (µg gm-1) in soft tissues of fresh water Mussel collected from
Four sites of River Kabul. .............................................................................................................................. 107
Table 29: Heavy metals concentration (µg gm-1) in gills of freshwater mussels collected from Four
sites of River Kabul......................................................................................................................................... 108
Table 30: Detailed process of Delafield's hematoxylin and eosin staining.............................................. 125
Table 31: Detailed process of routine histological method ....................................................................... 127
Table 32: Semi-quantitative scale for digestive gland a trophy according to Kim et al., (2004) .......... 127
Table 33: Occurrence of parasites at various sites in the freshwater mussels.......................................... 128
Table 34: Developmental stages (in percentage) of male gonads collected from various sites............. 129
Table 35: Developmental stages (in percentage) of freshwater mussel’s female gonads collected from
various sites. .................................................................................................................................................... 129
Table 36: Frequency (in percentage) of sexes in the freshwater mussels at various sites. ..................... 130
Table 37: Intensity of digestive tubules atrophy in mussels from various sites....................................... 130
Table 38: Intensity of atretic oocytes in female gonads of mussels from various sites. ......................... 131
Table 39: Genotoxicity results in the haemocytes of freshwater mussels................................................ 162
LIST OF FIGURES
Figure 1: Life cycle of freshwater mussel ....................................................................................................... 3
Figure 2: The variety of roles paid by freshwater mussels. ........................................................................... 4
Figure 3: Water sampling site 1(water sample A), site 2 (water sample B), site 3 (Water sample C) and
site 4 (water sample D) at River Kabul........................................................................................................... 28
Figure 4: Comparison of pH water samples from four different sites of River Kabul during summer and
winter.................................................................................................................................................................. 61
Figure 5: Comparison of TSS (Total Suspended Solids milli gram per liter) in water samples from four
different sites of River Kabul during summer and winter. ........................................................................... 61
Figure 6: Comparison of TSS (Total Suspended Solids milli gram per liter) in water samples from four
different sites of River Kabul during summer and Winter. .......................................................................... 62
Figure 7: Comparison of EC (Electric Conductivity) of water samples from four different sites of River
Kabul during summer and winter.................................................................................................................... 62
Figure 8: Comparison of Cl (Chlorine in milli gram per liter) in water samples from four different sites
of River Kabul during summer and winter. ................................................................................................... 63
Figure 9: Comparison of K (Potassium in milli gram per liter) in water samples from four different sites
Figure 10: Comparison of Na (Sodium in milli gram per liter) in water samples from four different sites
Figure 11: Comparison of TA (Total Alkalinity) in water samples from four different sites of River Kabul
during summer and winter. .............................................................................................................................. 64
Figure 12: Comparison of Cd (Cadmium in micro gram per liter) in water samples from four different
sites of River Kabul during summer and winter. ........................................................................................... 65
Figure 13: Comparison of Cr (Chromium in micro gram per liter) in water samples from four different
Figure 14: Comparison of Cu (Copper in micro gram per liter) in water samples from four different sites
Figure 15: Comparison of Fe (Iron in micro gram per liter) in water samples from four different sites of
River Kabul during summer and winter. ........................................................................................................ 66
Figure 16: Comparison of Hg (Mercury in micro gram per liter) in water samples from four different
Figure 17: Comparison of Mn (Menganese in micro gram per liter) in water samples from four different
Figure 18: Comparison of Ni (Nickle in micro gram per liter) in water samples from four different sites
Figure 19: Comparison of Pb (Lead in micro gram per liter) in water samples from four different sites of
Figure 20: Comparison of Zn (Zinc in micro gram per liter) in water samples from four different sites of
Figure 21: Sedimets Sampling Sites; Michini Bridge, Sardaryab, Amangarh (adjacent industrial area of
River Kabul) and Peer Sabaq........................................................................................................................... 75
Figure 22: Comparison of Cd (Cadmium in milli gram per kilogram) in sediment samples from four
different sites of River Kabul during Summer and winter............................................................................ 90
Figure 23: Comparison of Cr (Chromium in milli gram per kilogram) in sediment samples from four
different sites of River Kabul during Summer and winter............................................................................ 90
Figure 24: Comparison of Cu (Copper in milli gram per kilogram) in sediment samples from four
different sites of River Kabul during summer and winter ............................................................................ 91
Figure 25: Comparison of Fe ( Iron in milli gram per kilogram) in sediment samples from four different
sites of River Kabul during summer and winter ............................................................................................ 91
Figure 26: Comparison of Hg (Mercury in milli gram per kilogram) in sediment samples from four
Figure 27: Comparison of Mn (Menganese in milli gram per kilogram) in sediment samples from four
Figure 28: Comparison of Ni (Nickle in milli gram per kilogram) in sediment samples from four different
Figure 29: Comparison of Pb (Lead in milli gram per kilogram) in sediment samples from four different
Figure 30: Comparison of Zn (Zn in milli gram per kilogram) in sediment samples from four different
Figure 31: Heavy metals bioaccumulation in a trophic chain..................................................................... 95
Figure 32: Freshwater mussels sampling sites 1, 2, 3 and 4 at River Kabul. .......................................... 100
Figure 33: Heavy metals concentration (µg gm-1) in soft tissues of freshwater mussels collected from
Four sites of River Kabul. .............................................................................................................................. 108
Figure 34: Heavy metals concentration (µg gm-1) in gills of freshwater mussels collected from Four
sites of River Kabul......................................................................................................................................... 109
Figure 35: Image showing the samples of freshwater mussels................................................................. 110
Figure 36: Image showing the freshwater mussel while measuring the width. ...................................... 110
Figure 37: Parasitic infestation in digestive gland (DG), Gonads (GN) and Gills (GL) of freshwater
mussels from four different sites. .................................................................................................................. 132
Figure 38: Histopathological lesions (percentages) in digestive gland of freshwater mussels collected
from Michini Bridge ....................................................................................................................................... 133
from Sardaryab................................................................................................................................................ 133
from Peer Sabaq .............................................................................................................................................. 134
Figure 41: Histopathological lesions (percentages) in gonads of freshwater mussels collected from
Michini bridge ................................................................................................................................................. 135
Sardaryab ......................................................................................................................................................... 135
Aman Garh ...................................................................................................................................................... 136
Figure 44: Histopathological lesions (percentages) in gonads of freshwater mussels collected from Peer
Sabaq ................................................................................................................................................................ 136
Figure 45: Histopathological lesions (percentages) in Gills of freshwater mussels collected from four
different sites.................................................................................................................................................... 137
Figure 46: Histopathological lesions (percentages) in intestine of freshwater mussels collected from
four different sites............................................................................................................................................ 137
Figure 47: Image showing normal epithelium of digestive tubules ......................................................... 138
Figure 48: Image showing Atrophy of digestive tubular epithelium. ...................................................... 138
Figure 49: Image showing lumen of digestive tubule................................................................................ 139
Figure 50: Image showing necrosis of digestive epithelium..................................................................... 139
Figure 51: Image showing granulocytoma in digestive gland .................................................................. 140
Figure 52: Image showing hydropic vaculation ......................................................................................... 140
Figure 53: Image showing degeneration of digestive epithelium............................................................. 141
Figure 54: Image showing lipofusin pigment and necrotic epithelium ................................................... 141
Figure 55: Image showing Rickettsia like parasite in digestive tubules .................................................. 142
Figure 56: Image showing normal eggs in gonads .................................................................................... 143
Figure 57: Image showing atretic eggs in gonads ...................................................................................... 143
Figure 58: Image showing Rickettsia like parasites and necrosis in gonads ........................................... 144
Figure 59: Image showing abnormal elongated eggs in gonads............................................................... 144
Figure 60: Image showing granulocytoma in gonads................................................................................ 145
Figure 61: Image showing inflammation in gonads .................................................................................. 146
Figure 62: Image showing lipofusin pigments in gonads.......................................................................... 146
Figure 63: Image showing hemocytic infiltration in gonads..................................................................... 146
Figure 66: Image showing normal cilia in gills .......................................................................................... 148
Figure 67: Image showing fusion of cilia in gills ....................................................................................... 148
Figure 68: Image showing haemocytic infiltration in gills........................................................................ 149
Figure 69: Image showing degeneration of cilia in gills............................................................................ 149
Figure 70: Image showing degeneration of cilia in intestine..................................................................... 150
Figure 71: Image showing degeneration of epithelium in intestine ......................................................... 150
Figure 72: Image showing normal cilia in intestine ................................................................................... 151
Figure 73: Images showing different comet classes that are induced as a result of heavy metals ........ 163
ACKNOWLEDGMENTS
All prayers for Almighty Allah, the most merciful and beneficent, without whose consent and
consecration nothing would ever be imaginable. I am absolutely beholden by my Lord’s generosity in
this effort. Praises be to Holy Prophet Mohammad (P.B.U.H) for he is a beacon as I pace on in my life
and work.
First of all I want to acknowledge my supervisor Assist. Prof Dr. Mohammad Khisroon,
Zoology Department, University of Peshawar, Pakistan for his kind support during the entire period of
my Ph.D. Without whom I may have not been able to complete this research thesis.
I like to gratitude my respectable teacher and chairman Prof. Dr. Inayat Ali Shahjahan,
Zoology Department, University of Peshawar, Pakistan for his help and providing research facilities in
completing my thesis. I am thankful to external evaluators and internal viva examiners for their kind
suggestions to improve the quality of research presented in this thesis.
Thanks are also due to my worthy and honorable teachers Prof. Dr. Nahid Ali, Prof. Dr. Abdul
Hamid Jan, Prof. Dr. Syed Akram Shah, Dr Sanaullah Khan (Associate prof.), Mr. Zaigham
Hassan (Assist. Prof.), Dr. Farah Zaidi (Assist. Prof.), Dr. Syed Basit Rasheed (Assist. Prof.) and
Qaiser Jamal (Lecturer) Zoology Department, University of Peshawar, Pakistan for their kind
cooperation, moral support and nice guidance during entire period of my Ph.D program.
I am also thankful to my Ph.D colleagues, Dr. Zafar Ali Shah (Assistant Prof.), Dr.
Mohammad Siraj, Ajmal Khan (Lecturer), Abidullah Dawar (Lecturer), Ahmad Ullah (Lecturer),
Mr. Hafeezullah khan (Assistant prof.), Hameedullah (PhD Scholar), Muhsin-ul-Mulk Siddiqui
(lecturer), Zaheerullah khan (Lecturer) and Dr. Shah Fahad for their kind, encouragement, support
and excellent company during the course of my studies.
I present my profound thanks to Mr. Farhat Ullah, Mr. Ishafq khan, Mr. Fida Khan, Mr.
Zafer Khan and all staff of Zoology Department University of Peshawar for their moral support and co-
operation during my research studies.
My thanks are due to my friends Tahirullah khan (M.Phil Scholar), Mr. Muhammad Kashef
khan (M.Phil Scholar), Ashraf Ali Shah (M.Phil Scholar) and Wisal khan (M.Phil Scholar) for
excellent company, cooperation and moral support during my research work.
I pay my humblest thanks to my loving parents and sisters whose everlasting prayers always
chased my success. It is due to their efforts that I have been able to continue my study.
I present my whole hearted thanks to my great father Mr. Gul Noor Ali Khan (Late) for his
i
patience, sacrifices, effort, encouragement and financial support.
I am also thankful to my brothers Muhammad Saeed Shah, Hadayatullah khan, Mohammad
Shoaib Khan and Maulana Aamir Jamil for their kinds and affection during the course of my studies.
I offer my hearted affection to my loving sons Mohammad Yahya Iftikhar, Mohammad Eisa
Iftikhar, Mohammad Zakariyya Iftikhar and loving daughter Aafia Iftikhar, for their innocent faces.
ii
List of abbreviations
AAC: Air acetylene LMPA: Low melting point agarose

Ag: Silver LN: Liquefactive necrosis
As: Arsenic Mn: Manganese
Ba: Barilium MW: Mega watt
B: Boran NA: Necrotic area
CDC: Complete degeneration of cillia. Na: Sodium
NaCl: Sodium chloride
NaOH: Sodium hydroxide
Co: Cobalt NBF: Neutrally buffered formalin
CW: Constructed wetland NEQS: National and environmental
CGL: Clumping of gills lamellae quality standards
Cl: Chloride Ni: Nickel
CN: Coagulative necrosis
CRL: Centralized Resource Laboratory NMA: Normal melting agarose
Cr: Chromium OD: Optical density
Cu: Copper Pb: Lead
DE: Degeneration of epithelium. PBS: Phosphate buffered saline
DC: Degenerative cillia PCA: Principal component analysis
DMSO: Dimethylsulfoxide pH: Power of hydrogen
DNA: Deoxyribo nucleic acid R: Fuel-rich
EC: Electrical conductivity RDA: Recommended daily dietary allowance
EDTA: Ethylene diemine tetra acitic acid S.S: Stainless steel
ED: Epithelial desquamation Sr: Stroncium
Fe: Iron TA: Total alkalinity
FD: Fibrillar degeneration TCS: Total comet score
GAIE: Gadoon Amazai Industrial Estate TDS: Total dissoled solid
HCl: Hydrochloric acid T.S.S: Total suspended solid
HD: Hydropic degeneration VC: Vacuolation
Hg: Mercury WHO: World health organization
INF: Inflammation
K: Potassium
i
KCl: Potassium chloride
KH2PO4: Potassium phosphate monobasic
KPK: Khyber Pukhtun Khowa WQI: Water quality indices
L: Fuel-lean Zn: Zinc
ii
PREFACE
The main objectives of the present thesis were to study physico-chemical and heavy metal
contaminations in water, sediments, heavy metals accumulation, histopathology and genotoxicity due
to heavy metals in freshwater mussels of River Kabul. To achieve these objectives, the thesis of
research work has been divided into six chapters; each chapter is focused on specific objectives in
details. They are as following;
First Chapter deals with introduction of this study. This chapter introduced the study area along with
aims and objectives and justification of this study. This chapter also describes the physio-chemical
parameters like pH, total suspended solids (TSS), total dissolved solids (TDS), total alkalinity (TA),
chloride (Cl), electrical conductivity (EC), sodium (Na) and potassium (K).
Second Chapter focuses on heavy metals such as cadmium (Cd), chromium (Cr), copper (Cu), iron
(Fe), mercury (Hg), manganese (Mn), nickel (Ni), lead (Pb) and zinc (Zn) in water of River Kabul.
This chapter further used statistical analysis for source apportionment of the contaminations in water.
Third Chapter describes such as cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), mercury
(Hg), manganese (Mn), nickel (Ni), lead (Pb) and zinc (Zn) in the sediments of River Kabul.
Fourth Chapter describes bioaccumulation of heavy metals including cadmium (Cd), chromium
(Cr), copper (Cu), iron (Fe), mercury (Hg), manganese (Mn), nickel (Ni), lead (Pb) and zinc (Zn)
in soft tissues of freshwater mussels.
Fifth Chapter focuses on histopathological impacts of heavy metals in different tissues of freshwater
mussels collected from River Kabul.
Sixth Chapter focuses on genotoxical Impacts of heavy metals in hemolymph of freshwater mussels
of River Kabul. This chapter further determined different degree of DNA damage like total comet
score (TCS), comet class 0, class 1, class 2, class 3 and class 4.
This dissertation includes the conclusions and recommendations based on personal study. References
of all chapters are given at the end.
iii
ABSTRACT
The main objectives of this work were to investigate physico-chemical and heavy metal
contaminations in water and sediments as well as their accumulation, histopathology and genotoxicity
in Freshwater mussels of River Kabul. For this purpose water sample A (reference site 1), water
sample B (polluted site 2), water sample C (polluted site 3) and water sample D (polluted site 4)
upstream and downstream of River Kabul were collected during winter and summer periods and
analyzed for eight physico-chemical parameters (pH, TSS, TDS, TA, Cl, K, EC, Na) and nine heavy
metals (Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Zn) and compared with water sample A and NEQS
recommended limits. All the studied physico-chemical and heavy metal parameters in water samples
A, B, C and D except TSS and Hg were below the NEQS proposed limits, where the values of TSS
and Hg were above the NEQS recommended limits in all the water samples A, B, C and D. Thus the
overall sequence of different water samples was D > C > B > A. This highlights that water sample D
had higher and sample A had lower physico-chemical and heavy metal contaminations. Water
samples A and B had highest TDS and lowest K for low flow and had highest TSS and lowest K for
high flow seasons. Similarly water samples C and D showed highest TDS and lowest pH for low
flow and showed higher TSS and lower K for high flow periods. Among heavy metals water sample
A had highest Zn and lowest Cr for both summer and winter seasons. Water sample B showed higher
Zn and lower Cu for summer and greater Zn and smaller Hg for winter seasons. Similarly water
samples C had greater Zn and smaller Cu for both summer and winter seasons. Similarly water
samples D had higher level of Zn for summer and greater Zn and lower Hg for winter seasons.
Similarly among the studied heavy metals in sediments sample A had highest Fe and lowest Hg for
both summer and winter seasons. Sediments sample B, had highest concentration of Fe and lowest
concentration of Hg for both summer and winter seasons. Similarly sediments sample C had highest
Fe and lowest Hg for both summer and winter seasons. Similarly sediments sample D had highest
concentration of Fe and lowest concentration of Hg for both summer and winter seasons.
This investigation was further aimed to determine bioaccumulation of heavy metals including
Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn in soft tissues of freshwater mussels. Overall order of metals
concentration in different tissues showed that Fe was the highly and Hg was the lowest accumulated
metals.
This study was further meant to investigate histopathological impacts of heavy metals in soft
tissues were degeneration of epithelium, lipofusin pigments, atrophy, inflammation, necrosis and
granulocytoma in digestive gland. In gonads necrosis, inflammation, atresia, lipofusin pigments and
iv
granulocytoma were found. While in gills the degeneration of cilia, fusion of gills lamellae,
haemocytic infiltration and gills swelling was recorded. Similarly in intestine degeneration of
epithelium and its cillia were found. The highest intensity of histopathological lesions was recorded
in site 4 and lowest in site1.
Furthermore the present investigation was conducted to assess genotoxical impacts of heavy
metals in hemolymph of selected freshwater mussels. Therefore degree of DNA damage like TCS
and comet class 0, class 1, class 2, class 3 and class 4 were determined. The order of sites according
to DNA damage was 4 > 3 > 2 > 1, i.e. the highest DNA damage was noticed in site 4 while lowest
in site 1.
Key Words: River Kabul, Physico-chemical parameters, Heavy metals, Bioaccumulation,

Genotoxicity, Histopathology
v
CHAPTER 1
1.1: General Introduction
Freshwater mussels belong to a large and diverse phylum of animals called the Mollusca,
which is second only to the Arthropoda (insects, spiders, and crustaceans) in terms of global diversity.
Within Mollusca, mussels are members of class Bivalvia (or Pelecypoda), commonly referred to as
the bivalves. Several families of bivalves are found almost entirely in freshwater. All freshwater
mussels are in the order Unionoida and superfamily Unionacea (Turgeon et al., 1998). Unionid
mussels are useful in monitoring temporal and spatial trends of a wide range of persistent pollutants
in aquatic environments due to many advantages e.g., a wide geographical distribution, sedentary life
style, long lifespan, tolerance to a wide range of contaminants, poor metabolic capacity for pollutants,
correlation of pollutant content between organism and habitat, adequate tissue for analyses (Tanabe
& Subramanian, 2006).This species is geographically widespread throughout Europe, Russia, parts
of the Middle East and Asia, occurring in a wide variety of different habitats. The freshwater unionid
bivalves are one of the most threatened groups of organisms on earth. The global decline of these
mussel species is probably related to both anthropogenic activities and the complex life history traits
of these mussels (Bogan, 2008).
1. 2 Systematic Position
Kingdom: Animalia
Phylum : Mollusca
Class : Pelecypoda (Bivalvia)
Order : Eulamellibranchia
Family : Unionidae
Genus : Anodonta
Species : cygnea
1.3 Geographic Range
The occurrence of fresh mussels is extensive as it is usually occurred in certain specific areas
of Middle East countries, Europe and all through the Asia. It is very interesting to know that these
mussels exist on each and every continent on the globe except Antarctica. Moreover geographically
its peak level (one third among the total of 297 species) is observed in Nearctic region. The number
1
of species found in Neotropical areas are 179. While that found in Oriental, Palearctic, Australasian
and Afrotropical regions are 121, 92, 74 and 29 respectively (Bogan, 2008). In the entire Europe, it is
frequently found in almost all the countries even in the countries located at extremes like Portugal
(southwest) but its presence in Siberia is not established. (Mozley, 1936) revealed its presence in Asia
with a tendency towards northern regions. Studies also showed its presence in Iran’s northern regions.
In the European portion of USSR, it is extensively observed. Its occurrence is limited to an area of 50
Km radius where it is distributed in three lagoons. Its extinction from Spain is reported which is caused
by Mediterranean sea-mediated current constructions in certain areas (Araujo pers comm.). Anodonta
cygnea has been recently recorded in Algeria (Khalloufi and Boumaïza, 2005).
1.4 Population
In spite of its established prevalence, still this very species is rarely assessed for size and
structure of the population. Pollution mediated destruction of habitats has brought this species at a
declining position from the last one hundred years in Poland. River scouring activities under the
project of river management strategies may result in eradication of 20% of its sub population in the
United Kingdom (Bogan, 2008).
1.5 Habitat and Ecology
Usually this species live in standing as well as the tiny water reservoirs but still it may also
be observed in lakes and rivers with tedious water flow. Canals, Dams as well as the drain lines may
also be regarded as its possible habitats (Watters, 1999). The member of this species usually loves
those water bodies that show huge extent of dissolved oxygen, lack floating appearance and have fertile
sediments at the bottom Due to having weak patience level to the lean environmental circumstances,
this species may be used as a bio-indicator for assessment of clean water. Wherever it is present, it
serves as the lonely species of mussels in that particular area (Bogan, 2008).
1.6 Reproduction
This species usually spawn following broadcast technique where sperms are ejaculated out
to the water by males that fertilize the eggs present inside the bodies of female (Wachtler et al., 2001).
The unique larval stage (glochidia) that are originated from post fertilization embryos inside the bodies
of gravid females and are moved out of its specialized “marsupial” gills is regarded as a distinguished
attribute of Unionids (Thorp & Covich, 2009). Studies reveal that a single female is able to produce
about 4 million plus glochidia, with their at once release and synchronization(Bauer, 1987). If these
2
glochidia founds an appropriate fish as a host, they fix their selves with the host’s gills that hold these
glochidia for a handsome period up till they achieve a maturity level and become able to live
independently in the bottoms of water. Due to the shorter life, huge weights, non-motility and weak
resistance to tides, fixing of glochidia with host fishes is vital for retention of Unionid populations
(Strayer et al., 2004). Certain tissues, gills and various other attributes of a lure use to take attentions of
host fishes. As soon as the host fish tries to swallow the glochidia are ejected all around the fish mouth,
making it possible for the maximum of glochidia to fix with gills of the host fish. Similarly some
species uses to attract hosts by ejecting large attractive packages of glochidia (conglutinates) which
immediately release glochidia after biting by a host (Haag and Warren, 1999) (Fig. 1).
Figure 1: Life cycle of freshwater mussel
1.7 Functions of freshwater mussels
Some well-known and noteworthy activities are performed by the fresh water mussels in aquatic
environment (Vaughn and Hakenkamp, 2001; Strayer, 2008; Vaughn et al., 2008; Haag, 2012).
1.7.1 Regulating services: mussels as biofilters that purify water
Freshwater mussels, being one of the strong filter feeders, tend to filter the particulate
matters from the sediments belonging to water as well as interstitial localities (Vaughn et al., 2008).
Inspite of the established facts that prove them to be fed mainly via phytoplankton, still the recent and
3
advanced research studies on certain nutrients including stable isotopes as well as the fatty acids reveal
that they are specialized sort of Omnivores that alter their diet with alteration in habitat as well as the
accessibility to food (Christian et al., 2004; Vaughn et al., 2008; Newton et al., 2013). The studies
performed by Ismail et al., (2014) disclosed that certain species including Anodonta californiensis
and Corbicula fluminea are able to eliminate various products from the water including herbicides,
flame retardants, personal care materials and various drugs etc. They may also store these materials
in their tissue in the form of bio-deposits (Ismail, Müller, Morgan, & Luthy, 2014).
Figure 2: The variety of roles paid by freshwater mussels.
1.7.2 Supporting services: nutrient cycling and storage
These Freshwater mussels use to be nourished with a variety of nutrients, converting them
into soft tissues bio deposits as well as the shells inside their bodies followed by their slow dissolution
(Fig. 1) (Strayer, 2014). Accordingly whenever they experience any material having a huge biomass,
they pay an extra ordinary role by eliminating these soluble nutrients into the aqueous columns and
thus serving a great part in recycling process of the nutrients. (Vaughn & Hakenkamp, 2001). Such
nutrients are absorbed immediately by heterotrophic bacteria as well as the algae, resulting in the
tumbling up of aqueous food webs (Fig. 2) (Vaughn et al., 2008; Bril et al., 2014), These fresh water
mussels are found to execute alteration of algae populations in the water streams as well as the
restriction of nutritional substances, ultimately resulting in affecting quality of water (Atkinson et al.,
4
2013a). The mussel’s crowded play a huge share in nitrogen elimination from the aqueous ecosystem
by altering the denitrification process (Turek and Hoellein, 2015).
1.7.3 Supporting services: mussels as habitat and habitat modifiers
As a universal rule, the shells of fresh water mussels serve as an addition of physical structure
into the aquatic environment, ultimately producing biogenic habitat as in instance oyster reefs
(Gutierrez et al., 2003). The shells of fresh water mussels serve as a habitat for the various other
organisms and pay a significant share in biogeochemical cycling (Strayer and Malcom, 2007).
Aggregated Mussels usually aid lavish as well as diversified sorts of populations of micro
invertebrates as compared to those which lack mussels (Howard & Cuffey, 2006; Vaughn & Spooner,
2006; Aldridge et al., 2007). On the one hand the entire shell bodies serve as a habitats in soft natured
sediments while on the other hand the fissures in them guard communities of such habitats from being
flown or predated. The living mussels hold a variety of animal populations in their shells more
efficiently than the dead ones or stones (Vaughn et al., 2008; Bo´dis et al., 2014; Ilarri et al., 2015a,
b).
1.7.4 Supporting services: mussels support food webs
By providing the energies as well as nutritional substances via the bottom-up, mussels pay
their share positively in an eco-system’s food web. The fresh water mussels usually occur in the
aggregation inside the rivers which are termed as mussel beds, that are not only highly crowded with
up to 100 ind m2 but also contain multiple (10-20) species (Atkinson&Vaughn, 2015). Due to their
forced conversion to stable sediments with minute shear stresses, the beds of fresh water mussels are
uniformly disseminated in the form of patches patterns in the water streams (Haag, 2012). So within
the river stream these beds are usually separated by distant patches where either they are absent or
found slightly (Atkinson and Vaughn, 2015; Newton et al., 2011). The studies of Atkinson et al.,
suggested that the mussels elimination may provoke 40% of nitrogen especially in nutrients deficient
environment as they are proved to provide almost 19% of nitrogen in a specified trophic level of a
food web (Atkinson et al., 2014).
1.7.5 Supporting services: mussels as environmental monitors
Freshwater mussels have the potential to serve as important biomonitors of environmental

change, revealing past conditions and monitoring future change. As they are non-motile and perform
5
the process of filtration of water, they bioaccumulate particles, allowing measurements of stressor
levels in their soft tissue (Green et al., 1985; Rocha et al., 2015). Geochemical status of the shells of
fresh water mussels may enable us to now about the physical as well as the chemical circumstances
shown in the past on the temporal as well as spatial scales (Brown et al., 2005). The pollution related
incidents happened in passed days may be estimated by the study of trace elements incorporated into
these shells (Jamil et al., 1999; Brown et al., 2005) and future periods (Langlet et al., 2007).
Ecological circumstances from the ancient times may be found from the isotopes C13and O18 in fresh
water mussels (Blazejowski et al., 2012). Soft tissues of fresh water mussels may be used to find the
environmental conditions for shorter terms of time. In order to find the quality of any water, various
portions of fresh water mussels are used as sub-lethal indicators including its chemical components
present in its hemolymph, gills, mantle as well as the foot tissues which work on the mechanism of
immune reaction or the stress mediated techniques (Newton & Cope, 2007; Fritts et al., 2015;
Goodchild et al., 2015; Jasinska et al., 2015; Kolarevic et al., 2016). Mussels use to accumulate the
drugs contents more than other aqueous organisms like fishes (Du et al., 2014). Ecotoxicological
studies have revealed mussels to be a prominent indicator of various biological attributes (Cope et al.,
2008).
1.7.6 Provisioning and cultural services
The utilities of mussels include as ornaments, tools, food and utensils. U.S., archeological
studies reveal that the inhabitants of ancient America used to farm mussels for fulfilling their
nutritional needs almost before 10,000 years (Haag, 2012). They are used as a food substance in south-
eastern Asian states (Bolotov et al., 2014), but modern studies show their near to extinction position
in these states (Ziertitz et al., 2016, 2017).
1.7.7 Losses, restoration and conservation
Studies approved the fresh water mussels to be as vital contributors in aquatic eco-system
because of their uplifted conservation rate as revealed from Vaughn and Hakenkamp 2001 and
Gutiérrez et al., 2003. For the global preservation of mussel’s species and their hosts, development of
certain firm and direct strategies is mandatory. These species are considered as one of those groups of
organisms which are at high risk across the globe (Lydeard et al., 2004; Lopes-Lima et al., 2014).
North America is facing extinction of almost thirty from last one hundred years. While the 65%
among the existing 300 species are at high risk of extinction there (Haag & Williams, 2014). Similarly
6
the turning down of mussels is causing not merely the loss of species but it is bringing a huge downfall
in the biomass and frequency of certain other familiar species (Haag & Williams, 2014).
As far as the suggestions of Haag & Williams (2014) are considered, they reveal that theme
behind the strategies for preservation must include the safety of mussels or the betterment of aquatic
habitats instead of its reversals. Uplifting in the budget for such goals will also bring fruitful results.
In those areas where the extinction or decline of mussels species are observed must be introduced
again with these species along with providing of a sufficient stock of host fishes (Zettler and Jueg,
2007).
1.8 Description of Study area:
1.8.1 River Kabul:
The origin of Kabul River is present in basal areas of Unai Pass that is located in the mountains
of Paghman inside Afghanistan. Right after origin this river flows along the East, travelling via the
northern range of Koh-e-Sufaid. It reaches Kabul after flowing for 72 km from the site of its origin.
In its way River Kabul meets various other rivers. Immediately after Jalalabad, the River Kunar joins
it (Gress well and Huxley, 1965). The story of Kunar river starts with Chitral River which is originated
from Hindu Kush Mountains in Pakistan. Arandu is the site located in Chitral where the Chitral River
enters Afghanistan where a branch from Nuristan joins it. From this joining point it is known as Kunar
River. This Kunar river and the River Kabul are joined together right after Jalalabad. This extended
form of Kabul River gets entered into Pakistan at a point in Khyber Agency known as Shalman. The
River Kabul moves ahead via the Khyber Agency as well as Mohmand Agency throughout which it
is covered from the sides by Koh-i-Sufaid Mountains till it touches the Warsak Dam in Peshawar.
Just after the Dam it splits into three major twigs that are nominated as Shah Alam, Nagoman and
Adezai. Heavy metal, sometimes called as trace elements, plays a vital role in biological systems. Still
it is a matter of attention that they act as very poisonous when existed in elevated amounts (Ibok et
al., 1989). There are various aspects of modern world that are affecting the eco-system and its
associated communities and ultimately resulting in the disturbances of organisms residing in water.
All of the said branches serve to irrigate the fields of Peshawar, Charsadda and Nowshera Districts.
Finally all of these three branches fall in the River Indus at the point of Kund (Yousafzai et al., 2008
b). Thirty five (35) km ahead the Warsak Dam Shah Alam and Nagoman branches are flanked.
Similarly the Adezi branch in combined with River Swat. All of the described branched are combined
with each other 1km ahead at the point of Akberpura. At the same point of joining, the River Bara is
too flanked with them making the Main Kabul River. The main Kabul River is combined with Indus
7
River at the site of Khairabad that is located 90km ahead of Warsak Dam. The Adezai branch is r-
isolated from the main Kabul River at a point neighbor to Larmandi village. The sewages and
drainages of about 40 villages (having a population of 150,000 people) fall in it, making it the branch
with the highest volume among the three branches of River Kabul (Yousafzai et al., 2008 b). The
Nagoman branch separates out adjacent to Machini where it accepts waste materials from tanneries
and sewages of 27 villages located in the adjacent areas. There are various aspects of modern world
that are affecting the eco-system and its associated communities and ultimately resulting in the
disturbances of organisms residing in water. Shah Alam branch is detached of Kabul River at a point
located in the vicinity of Kander Landi at Daudzai. It collects the drainages from whole Peshawar
District (about 30 villages) through the Ganda Erab and Budni nulla (Yousafzai et al., 2008).
1.8.2 Human population:
Asadabad is regarded as the pioneer habitat on the River Kunar. Kabul, the capital city of
Afghanistan is the host of a major branch of Kunar River which is known as Kabul Branch. This is
the reason it was given the name of River Kabul. Heavy metal, sometimes called as trace elements,
plays a vital role in biological systems. Still it is a matter of attention that they act as very poisonous
when existed in elevated amounts (Ibok et al., 1989; Ajimaet al., 2015). Jalalabad, being a
neighboring site of Kabul and Kunar, is the last prominent place before the entrance of the river to the
territories of Pakistan. There are various aspects of modern world that are affecting the eco-system
and its associated communities and ultimately resulting in the disturbances of organisms residing in
water. Immediately after the Warsak Dam the River Kabul flows via the belt that contains maximum
density of population in Khyber Pakhtunkhwa and is regarded as one of the most packed rural areas
of Pakistan with respect to the population. The Peshawar district is located in close proximity with
Shah Alam branch, having about One Million populations. Similarly certain huge towns like
Nowshera and Charsadda lies near the river banks. The Peshawar city is near to the Shah Alam
Branch where population is approximately one million, while other two cities such as Nowshera and
Charsadda are also closed to bank of river Kabul. Several colonies of Afghan refugees are situated
near the main River and its various branches (IUCN, 1994).
1.8.3 Industries along River Kabul:
The reports of a survey conducted at Khyber Pakhtunkhwa reveals that there are a total of about
348 industries comprising of both small and large units. Among these 348 units, about 80 industrial
plants release their waste products either straightly or ultimately into the river Kabul. Among these 80
8
industries, 4 are sugar mills, 2 are distillation plants, 3 are edible oil and ghee plants, 5 are textile mills,
2 are woolen mills, 12 are tanneries, 3 are board and paper mills, 10 are chemical and pharmaceutical
industries, 4 are match industries, 10 are soap factories, 1 is petroleum refinery, 10 are photo
laboratories, 4 are varnish and paint plants and 11 are plastic and rubber factories (IUCN, 1994).
1.8.4: Industries at Aman Garh industrial zone:
There are fifteen (15) diversified sorts of small as well as large industries are existed in
Nowshera and the adjoining Aman Gar industrial zone. All of these industrial plants dispose off their
effluents into the Kabul River without having their proper treatments straightly or via small canals
and nullas. The major names in these plants are Adamjee chemical plant, Feroz sons laboratories and
its colony, Sarhad textile mills, go downs of Petrol, Adamjee boards and paper factory, Nowshera
engineering works, Nowshera DDT factory and tanneries adjecent to the railway bridge in Nowshera,
edible oils and Ghee mills (Yousafzai, 2004). Heavy metal, sometimes called as trace elements, plays
a vital role in biological systems. Still it is a matter of attention that they act as very poisonous when
existed in elevated amounts (Ibok et al., 1989; Ajima et al., 2015). These toxic wastes not only
contaminate the water in river but also make the sub-surface unsuitable for drinking and irrigation
purposes (IUCN, 1994; Khan et al., 1999). The people residing near the river banks of River Kabul
have complained regarding the water pollution in the River Kabul, which is revealed by various
signals including periodic killing of fishes, depletion of the entire population of fishes in certain areas
reported because of pollution mediated oxygen exhaustion in river waters and various health issues
produced in people of surrounding areas (A. Khan et al., 1999).
1.8.5 Disposal of sewages and industrial effluents to River Kabul.
Discharging the untreated wastes products from industrial plants has been a common practice
across the globe especially in underdeveloped countries just like Pakistan. The River Kabul along
with its branches is serving as a transportation tool for the untreated industrial wastes from industries,
sewages from various cities, towns and villages and drains from densely populated colonies of Kunar,
Jalalabad, Kabul, Chitral, Peshawar, Mardan, Charsadda districts and Khyber, Mohmand and
Malakand Agencies. The lower branch of the river flows mainly via the highly populated plain areas.
In these areas a huge number of sewage lines and drainage tracks fall in it. Similarly industrial
effluents of various industries enter into the river straightly or via canals and nullas. These industries
include sugar mills, paper plants, distilleries, edible oil and ghee industries, tanneries and textile mills,
9
adding their shares in the overall water pollution in River Kabul (IUCN, 1994). There are various
aspects of modern world that are affecting the eco-system and its associated communities and
ultimately resulting in the disturbances of organisms residing in water. Due to lack of legislation
regarding control of such activities in Pakistan has resulted in immense urbanization and
industrialization which ultimately created the environmental pollution level to be at an alarming point
within the country. Moreover, the untreated agricultural, industrial and public wastes has polluted the
majority of available water resources (Murtaza and Zia, 2012).
1.8.6 Water contamination:
Contaminated water is not only a risk for biotic eco system but also for the human beings.
Deteriorated water results in production of a number of illnesses in human beings including cholera,
hepatitis A, typhoid, skin and gastro-intestinal diseases which are acting as a major cause of several
millions deaths across the globe (McGhee and Steel, 1991). Reports reveal that water borne illnesses
comprises 30% cases of overall diseases and 40% of all deaths in Pakistan (Haider, 1981). In the same
way 40% of overall adult and 60% of all the infant deaths in Pakistan are because of water-borne
illnesses like typhoid, infective hepatitis and diarrhea etc. it is also found that one quarter of all the
under five years deaths are due to G.I.T problems produced because of contaminated water
(Yousafzai et al., 2010b).
1.8.7 Heavy metals:
Thirty five (35) metals listed in periodic table are considered to be concerned with public and
professional exposure. Among these 35, merely 23 are regarded as the heavy metals. Such metals are
originated in trace amounts usually from corrosion of natural rocks, volcanic explosions, release of
industrial waste products, public sewages etc. in order to maintain a healthy life the trace amounts of
these metals are vital in the body. However the concentration of these heavy metals is going to
increasing day by day because of mining activities, paints and dye manufacturing, electroplating,
battery making industries etc. The heavy metals are considered as toxic and dangerous because of
their two major attributes that include imperishability and permanent nature plus their affinity and
tendencies towards accumulation in the ecosystem (Zhuang et al., 2009). Unlike organic matters the
heavy metals does not destroy on its own. Similarly the heavy metals have the behavior of
accumulation in the form of sediments in sub-surface rocks along with other organic materials. Eighty
(80) elements among the total of one hundred (104) listed in the periodic table are regarded as metals.
10
17 in this table are listed as non-metals. The remaining 7 are categorized as metalloids. The metals
having densities to be or above 6 g/cm3 are called as heavy metals (Yousafzai, 2004).
1.8.8 Sources of heavy metals:
Heavy metals mediated aqueous pollution has been an alarming issue across the globe. The
well-known sources of heavy metals include industrial as well as mining practices, burning of
petroleum products, processing plants, effluents treatment protocols, sewages, earth quakes, sliding
of lands, tornadoes, environmental condensation and cyclones etc. (Nathaniel et al., 2000; Zhuang et
al., 2009; Siraj, 2016).
1.8.9 Metals in the Environment:
Assessment of human tissues reveal the presence of a variety of metals in small or larger
extents. It is not astonishing to know that the edible food materials contains one or more heavy metals
which is sign of their widespread existence in the environment. The fertile soils contain diversified
sorts of metals, sewages clumps, chemical fertilizers and various other products. Mining raw products,
industrial effluents, fuel burnings, smoke and dusts of fossils are the sources of heavy metals to the
heavy metals mediated environmental pollution. Water also puts its share by serving as the
transportation tool for these heavy metals and having pollution in its course. Heavy metals
concentration in the soil depends upon the nature of originating rocks, their extent of mineralization
and many more other aspects. By the level of heavy metals in the water we may predict the presence
of industrial zone in nearby areas and also the nature of the rocks and soil of that area. Moreover the
reticulated water usually puts its share of metals because of having plumbing and containers (Wang
et al., 2009).
1.8.10 Metals in the body of an organism:
The concentrations of metals in the plant as well as animal based foods are dependent on
various factors including ecological conditions, production mode and the way of processing etc. It is
even possible that the metallic contents of food may vary among the foods from an identical group
(Wang et al., 2009). The level of metals may be different in various segments of a specific kind of
food. Similarly inside the body of animals the allotment of metals is uneven as certain metals prefer
to be stored in one part and others in somewhere else inside the body. As in instance, the preferred
sites for the accumulation of poisonous metal Cadmium are kidneys (12 mg) and to some extent in
11
blood stream (0.76 mg). Moreover the Lead will be amassing mainly in blood (1.3 mg) and Kidneys
(0.12 mg). Similarly sites for the Chromium storage are muscles (2.4 mg) and Kidneys (0.019 mg) (
Somero, et al., 1977; Wang et al., 2009).
1.8. 11 Essential and non-essential metals:
Minute numbers of metals present inside the body are considered to be vital for the normal
functioning of the body. The dearth of any of these metals will lead to the emergence of certain
pathological manifestations. Majority of all other metals are regarded as functionless masses in the
body.
On the basis of the needed amount heavy metals are categorized into two groups including
Macro-Nutrients and Micro-Nutrients. Micro-Nutrients comprise of zinc, manganese, iron, copper,
cobalt, molybdenum, silicon, chromium, tin and nickel. While the Macro-Nutrients contain calcium,
sodium potassium and magnesium. The metals whose metabolic activity is still not identified are
called non -essential metals which are composed of mercury, gold, lead, bismuth , silver, boron,
titanium, antimony beryllium, gallium, lithium and various other metals apart from arsenic, cadmium,
barium, vanadium and strontium (Reilly, 2008).
1.8.12 Role of heavy metals in the body of an organism:
Essential metals usually act in three major ways inside the body including to be as a soluble
salts in the bones and teeth, aiding to maintain the body fluid and cells composition inside the body
and as a vital companion of several enzymes and active proteins. Macro-nutrients plays an important
role inside the body while the micro-nutrients usually aid enzymatic activities. A minute number of
proteins can carry on their catalytic function alone. Majority of them needs a non-protein companion
for their functioning which is called as prosthetic group. The separable prosthetic group is called as
co-enzyme which is usually a combination of organic molecules and trace elements or solely trace
elements. The sole trace element serving as a co-enzyme is called as activator. As in instance, the Iron
and Copper serves as activators of oxidative enzymes. Similarly Zinc and Manganese act as activators
of enzymes engaged with metabolic activities at cellular level (Reilly, 2008).Such enzymes involving
micro-nutrients as their activators are called as metalloenzymes. These metals are also the constituents
of various body substances including hormones and vitamins. The insulin production and storage
usually needs Zinc inside the pancreases. Similarly the Hemoglobin, being the vital carrier of Oxygen
in blood involves Iron. Cobalt is a constituent of Vitamin B12 (Reilly, 2008).
12
1.8.12.1 Cadmium:
Cadmium, being a heavy metal, has atomic weight of 112.4, density of 8.6g/cm3, atomic
number 48, melting point 321.0C˚, boiling point 767C˚. It is believed to be involved in renal and bone
impairments. It is found to a cancerous agent affecting kidney and bones (Fowler, 2009). It is utilized
in batteries, finishing of metals, electroplating, metallurgy, paints, mining and dyes factories. It is a
proved heavy metal attributed by the specific gravity values higher than water by 8.65 times (Lide,
1992). Because of not undergoing metabolic alterations, the heavy metals are accumulated in soft
tissues which ultimately leads to toxicities. Symptoms appeared after mild Cd poisoning in the human
bodies include nausea, abdominal discomfort, vomiting and respiratory depression. The symptoms of
sever subjection to Cd include baldness, deficiency of blood, inflammation of joints, mental issues,
growth deformities, migraine, osteoporosis, cardiovascular problems and emphysema etc. while its
consequences include pulmonary obstruction, kidney problems and bone weaknesses etc. Clinical
results with Cd blood level beyond 5 mcg/dL and urine creatinine level higher than 10 mcg/dL reveal
the occurrence of Cd poisoning (Dupler, 2001). Cadmium (Cd), being a non -essential and non-
corrosive element, is widely preferred to be utilized in fertilizers, cements, paints and dyes etc. (Jarrup,
2003). It is believed to be poisonous to embryo, kidney and other organs, teratogenic and cancerous
in human beings affecting the smokers at its most (Sunderman et al., 1991). Cadmium (Cd), after
absorption into the body via lungs or Gastro intestinal tract, competes with essential elements like Fe,
Cu, Ca, Zn etc. for bindings to the proteins, receptors and metalloenzymes antagonistically (Wright
and Frain, 1981). It acts as a stressor agent producing metabolic modification leading to starvation
like conditions.
Cadmium(Cd), being a toxic metal, prevents certain proteins including phosphofructokinase,
lactate dehydrogenase etc. slows down glycolysis, alter glycogen as well as lipid level and generate
reactive oxygen which usually acts as molecule showing signals for production of gene expressions
as well as apoptosis (Waisberg et al., 2003). Because of inability to be metabolized, the heavy metals
are accumulated in various parts of the body including kidneys, liver, lungs, placenta, bones and brain
etc., causing their severe toxicities. Cd damages oxidative DNA producing breakages of DNA
strands, prevention of cross-linking of DNA-protein and stoppage of repairment process of the DNA
(Nemmiche, et al., 2011)
1.8.12.2 Chromium:
Chromium, having weight of 52, melting point 1860C˚, atomic number 24, density 7.2
g/cm3, is a really lustrous, hard, brittle, corrosion resistant and abundant metal. Its occurrence is
13
different in different soils but one thing is clear that in all of them its concentration is nutritionally
enough for plants and animals. Soils, as it is an essential element to life (Reilly, 2008). It is found in
the form of ores. Its valencies range from 2-6. It is utilized in production of stainless steel, as a planting
base, as corrosion preventer, as a photography helper, as a caustic in textile production, in
anticorrosion paints and varnishes, in leather tanning, in inks and in glazes etc. it is occurred in drain
treatment plants and effluents of chromium industries (Klein, et al., 1974). It is a toxic element whose
action inside the bodies of humans and animals is well known. It causes various tissue problems in
men and animals (Yousafzai, 2004). The Cr is a contaminant of water and so is hazardous to aquatic
eco system (Förstner & Wittmann, 2012). The factors that may alter the course of Chromium
mediated toxicities include size of the body of organism, species type, stage of life, water pH, its
salinity, hardness as well as temperature (Nriagu & Nieboer, 1988).
Like various other heavy metals, the major supplier of chromium to the environment is
sewage slush which is reported to contain 8g of chromium per each Kg of the sludge (Yousafzai,
2004). After ingestion, the chromium is absorbed and reaches the systemic circulation, via which it is
distributed into various segments and organs of the body mainly in to liver where it shows its trivalent
chemistry. The chromium gets excreted out of the body usually via urinary tract. It is worth
mentioning that its highest concentration resides in hair where it is found as 0.2-2.0 mg/kg (Mertz,
1969). Chromium induces certain prominent alterations in the metabolites levels within the blood as
well as tissues. It may also produce hepatic glycogenolysis and cerebral hypergycemia (Ginter et al.,
1989). As a result of mild chromium poisoning uplifted level of cholesterol in the blood, kidneys,
liver, testis as well as ovaries (Gauglhofer, 1984). Inspite of higher level of chromium resistance in
fishes as compared to other water organisms still, they may experience sub-fetal toxicities after
subjection to 0.013 - 50 mg/l or above of chromium molecules (Farag, et al., 2006).
1.8.12.3 Copper:
Copper, being available in the form of tough, ductile, soft, conductive pure element, has an
atomic weight of 63.64, density of 8.96 g/cm3, atomic number of 29, Melting point 1083 C˚ and
oxidation states of +1 as well as +2. Being the second best conductor after silver, Copper is widely
utilized in the industries of electrical appliances as well as utensils. Being one of the softening attribute,
it has been using in the ornaments for centuries. Its ability to form alloys with several other metals like
cadmium, silver, zinc and tin etc. made people use it for the manufacturing of weapons, cooking
equipments and vessels. It usually makes two kinds of compounds including Cu (I) cuprous and Cu
(II) cupric. Being available in the form of colored ores, it is very much to be identified. In the form of
14
crystals, copper attributes reddish color (Yousafzai., 2004). Because of its brilliant conductance, it is
vital component of almost half of all the electrical utilities including cables etc. that demands huge
extent of electrical conductance. Copper may also be used in heating as well as planting etc. it has
certain valuable utilities in agriculture as well as in pharmaceuticals e.g. in pesticides and in the
antifungal sprays that contain Bordeaux mixture which is a fungicide used in treatment of various
kinds of diseases in grapes as well as potatoes. Being an essential element, it has been a vital
component of various enzymes including tyrosinase enzymes that is vital for melanin pigments
production, cytochrome oxidase, amine oxidase, urinase and super oxide dismutase etc. it is important
for the employment of iron in hemoglobin production (Yousafzai., 2004). Reports reveal that the
rivers flowing in the United States of America comprises of an average of 0.83 to 1.5 µg/l of copper.
Like other heavy metals, copper also exhibits certain systemic poisonous impacts. Copper used as a
component of insecticides or fungicides, makes its way to the bodies of animals. Its toxic effects are
identified in juvenile Salmon. After intake, copper is absorbed and reaches the systemic circulation
where the albumin carries it to liver. Copper is stored in the brain, liver, heart, muscles and kidneys,
exhibiting its maximum concentration in the bodies of humans and animals (Piscator, 1979;
Dollwet,and Sorenson, 1985)
Transportation of copper is done from liver to other tissues and blood by heavy protein called
ceruloplasmin which itself is formed in liver. Within the tissues and blood it is bound to protein which
may or may not be enzymes (Yousafzai, 2004). Studies reveal the accumulation of copper in the
freshwater mussel’s gills, kidney and mantle. Its excretion is done via bile through the hepatic
metabolism protocol (Croteau et al., 2004). Extensive hepatic copper storage comprises Wison’s
disease or hepatolenticular degeneration that is an innate metabolic issue. Its root cause may include
problems in liver excretion mechanism into the bile. Accidental intake of huge copper contents may
lead to G.I problems, hematologic issues e.g. hemolysis, hepatic impairment and kidney failure
(Yousafzai, 2004).
1.8.12.4 Iron:
Iron is a heavy metal having an atomic number of 26, atomic weight of 55.8, density
7.8g/cm3, average melting point of 536 0C and a boiling point of 2861 0C. Iron overload produce
different kinds of physiological and pathological conditions and alteration (Brissot, et al., 2012).
15
1.8.12.5 Mercury:
Mercury, being an element with atomic No. of 80, atomic mass 200.6, density 13.5 g/cm3,
melting point of -38.8C˚, boiling point of 356.7C˚, is extensively toxic, naturally occurred as organic
as well as inorganic form and widespread substance. Methyle Mercury, being its organic form is
prepared using microbial methylation and extremely poisonous and easily able to be accumulated.
The major fonts of Mercury mediated pollutions include chloride-alkaline industry, mining, utilities
of Mercury, mercuric atmospheric component produced by the environmental burns, fossil fuels
burns etc. Study of Cyprinus carpio gill cells showed a mercury-mediated genotoxicity (Weiner et
al., 2003; Arabi., 2004). Release of Mercury into the Minamata bay was observed in 1950 and 1960s
in Japan. In the aquatic environment, the Mercury is shifted to its methylated form under the action
of a bacteria that is easily absorbable which is immediately utilized by fishes ultimately resulting in
the cases of Mercury poisoning or Minamata disease. Mercury poisong and Minamata disease caused
107 and 800 deaths respectively by 1970. Inspite of the healthy looks of mothers, several cases of
Mercury mediated toxicities were observed occurring due to the eating of contaminating fishes by
their mothers, making them to show cerebral paralysis as well as mental problems (Hodgson, 2004).
Mainly the cancerous and mutagenic materials damage DNA inside the cells. Mercury, being a potent
DNA damaging substance, breaks the strands of DNA inside the cell. Main fonts of organic Mercury
include Fishes and sea foods. Hyper-exposure to mercury may result in disturbances in nervous
system, kidney, immune system and liver in human beings. Mercury, being considered as one of the
potent poisonous heavy metals, shows extensive toxicity, bioaccumulative attributes and other
hazardous impacts on life including genetic modification or mutagenesis (Al-Sabti & Metcalfe,
1995).
1.8.12.6 Manganese:
Manganese, being discovered in 1774, is grayish-pink in color, chemically active and

reactive, easily oxidizable, physically hard and brittle, has an atomic weight of 55, atomic mass of
54.9 g mol -1, boiling point 2061°C, melting point 1247°C and density 7.4 g/cm3. It burns in Oxygen
presence, reacts easily with water, dissolves quickly in diluted acids and difficult to melt. Upon
cutaneous absorption it produces permanent co-ordination collapse resulting in tremors. Chronic over
dosage, usually resulted from extensive inhalation of Manganese dust or fumes, may lead to
development of tumor in animals. Signs produces after its chronic ingestion include tranquility,
weakness, emotional issues, repeated cramps in legs and ultimate paralysis, spastic gait etc. High
16
grade respiratory issues are common among the labors subjected to its fumes and dusts. These are
under experiment carcinogenic agents (Nordberg, et al., 2014). Manganese, being a vital component
of bone anatomy, reproductive system and well-being of enzymatic actions. It is a micro-nutrient at
optimum concentration but at higher level it causes toxicities (Watts, 1990).
1.8.12.7 Nickel:
Nickel, being a tough silver- white, malleable, elastic, conductor, soluble in diluted acids and
air resistant metal has an atomic weight of 58.71, atomic no. 28, a melting point of 1453 C˚ and a
density of 8.9 g/cm3. It is found in combination with other elements including sulphur, antimony and
arsenic. It has high electrical and thermal conductivities and can be drown, rolled, forged and polished.
It contains Oxidation states of 0, +1, +2, +3 among with merely +2 is frequent. It’s primarily important
Compounds include nickel hydroxide, oxide, sulfate, subsulfide, and chloride. Nickel carbonyl
Ni(CO)4, being a liquid which is volatile with no color, is an important substance of it found in nature.
The occurrence of Nickel is in the form of four fundamental ores including, arsenide, silicate, sulphide
and laterite, (Galvin, 1996). Nickel, after absorption usually is accumulated in liver, lungs and kidney.
The excretion of them is done mainly through urinary system. Absorption of Nickel-carbonyl
is done from the lungs after which its rapid decomposition is done into nickel and carbon mono oxide.
Right oxidation, the Nickel is excreted via the renal route. Nickel and its compounds are sensitizing
substances causing dermatitis. It may also produce nasal mucosal inflammation. Subjection to nickel
carbonyl may result in the acute toxicities which is usually fetal and s usually characterized mainly by
impairment of lungs (Yousafzai, 2004). Slightly less than half of total nickel produced is utilized in
the production of steel. Steel having nickel in their structure are highly resistant to corrosion. It may
also be utilized in manufacturing of steel with heat resistant properties and cast iron. Such steels and
irons are used in production a variety of utensils and equipments. In the hydrogenation oils the nickel
is utilized as a catalyst. nickel carbonates may also be used as a component of electronic machines
including vacuum tubes as well as transistor cans etc. various anthropogenic actions including
electroplating, mining and steel plant activities usually leads to the Ni release to the atmosphere.
Solubility of nickel is 0.005 - 0.010 mg/l (Galvin, 1996). Nickel shows its solubility at pH below 6.5,
above which it mainly produce nickel hydroxide which is insoluble in nature (Dalls and Day, 1993).
Being a prominent part of tobacco, Ni contribute towards the major cause of lung cancer with its
carbonyl compound. As tobacco is sewed in several sites and lands irrigated with river Kabul so the
chances of leaking out of the nickel present in the water of river Kabul to the tobacco is obviously
prevailed (Yousafzai, 2004). . Inside the human body albumin is the carrier protein for nickel. Nickel
17
plasmin, alpha macro globulin containing nickel is a famous nickel substance in the human body
similarly ultra-filterable serum fraction also contains Nickel (Friberg and Nordberg, 1979; Norseth
and Piscutor,1979).
1.8.12.8 Lead
Lead, being a soft, bendable, malleable, silver bluish white colored metal with poor
conductance attributes, have atomic number 82, density of 11.3 g/cm3, atomic weight 207.19 and
melting point 327.5 C˚, boiling point 1725 C˚ and several oxidation states including +2 etc. Its +2
form is considered as the most stable one in which it prevails in water organisms. It is not found in
pure form instead occurs in combination with other metals and as well as salts. The entrance of lead
to the aquatic eco life via leaking off from soils, lead based dust falling, gasoline burnings, effluent
and drain released from various localities and industries (Holmes, 1996; DWAF, 1996). Reports of
international lead and zinc study Group (1976) shows that consumptions of lead done in 1974 was in
production of storage batteries (40%), formation of alkyl lead (12%), in pigments (12%), for the
sheathing of cables(9.2%), in alloys (10.8%) and in the development of semi-conductors (12%). Other
fields involving lead include welding, breaking of ships, production of plastics, printings, rubber
factories and vehicle industries. Among the 4.1 million tons of lead which was consuming across the
globe, about more than half was engaged in automobile factories (Friberg and Nordberg, 1979) .
Moreover it has been used in a variety of treatments and processing inside as well as outside the
industries. Being toxic metal, lead induces chronic health issues in both humans and animals via
accumulation in their body tissues. Poisonous effect of lead has been recorded in a variety of regions
internationally (Schuhamcher et al., 1990).
It is believed the Lead is deposited in the gills, bones, kidneys, livers and scale tissues of
various species of fishes (Yousafzai, 2004). The most usual symptom of lead toxicity is G.I colic. It
may also interfere with hemoglobin production or make shorter the erythrocytes life span both leading
to the anemia. The Lead impacts on nervous system are established facts, affecting both the central as
well as the peripheral components, leading to chronic encephalopathy, reduced nervous conduction,
lead colic, peripheral encephalopathy, benign and malignant tumors and renal effects etc.(Friberg and
Nordberg, 1979).
18
1.8.12.9 Zinc
Zinc, being an abundant crystalline, bluish- white lustrous, ductile and malleable metal, has
a density of 7.14 g/cm3, melting points 420 C˚, atomic number 30 and atomic weight 65.37. Zinc lies
along with various other elements including lead, cadmium and copper etc. in nature. Having an
oxidation number of 2, Zinc occurs sulfide ores including wurtzite and sphalerite. Emitting huge
extent of volatile portion during melting is its special action causing prominent ecological poisoning.
While crushing and mining of zinc, traces of it enters to environment (Kelly et al., 2012).
Main utilities of Zinc include formation of alloys that are non-corrosive in nature, brass
formation, galvanization of steel and production of iron etc. Undergoing oxidation on the surface,
Zinc shields the entrapped metal from environmental destruction. This property of zinc has been
utilized in a variety of fields including vehicle spare parts and home appliances etc. Anthropogenic
actions leads to enhanced proportion of environmental Zn that ultimately causes its hyper level in the
bodies of people utilizing this environments for food, recreation and other purposes. Hence the Zn
mediated aquatic pollution especially in fresh waters must be critically examined (Yousafzai, 2004).
Zn is generally a non-poisonous element which is vital portion of cells, needed by majority of
enzymes for their activities as a co-factor. It gets entered into the bodies of water based organisms
through their gills, body surface and G.I route during ingestion of polluted food. Its elimination is
done G.I canal (Krebs, 2000).
1.9 Heavy metals pollution in River Kabul:

The people residing near the banks of River Kabul have been complaining regarding
pollution in the River Kabul for previous few decades as they are constantly observing clear signals
of pollution arising due to one or the other reason mainly including intermittent killing of fishes within
the river. As a result of this pollution in the said river, people of the area are facing certain hazards
including skin diseases in humans, sufferings in live-stock and the reduction of crops production in
the lands irrigated through the water of the River Kabul. Excess of heavy metals beyond the minimum
toxic level, induced by various factors including expansion of populations, extensive urbanization,
wide-spreading of natural assets, uplifted coverage of irrigation and agricultural activities and the
absence of policies for environmental protections are causing numerous issues now a days (Yousazai,
2004). Heavy metals-mediated water pollution has become a global matter these days. Various factors
including excessive industrialization, extensive outflow of agricultural as well as commercial
chemicals into the natural water reservoirs have made toxic effects on all the aquatic organisms
(Yaucob and Gad, 2012). A variety of emancipations were recorded in the waters of River Kabul that
19
include industrial, originated from rivers and streams, produced from sewage and flown-off from
drains. These all factors were studied comparing them with one another. An average of 4,600 cusecs
of poisonous water from Mardan is entered via the Kalpana River into the River Kabul at Risalpur.
This polluted water contains sewage wastes as well discharges of sugar mills from Mardan showing
a huge amount of organic pollutants and heavy metals making these waters of even more poorer
quality as compared to that in Bara River categorized in class 3 standards. It is definitely diluted to a
huge extent upon addition to the River Kabul but still have certain hazardous effects on the standards
of water especially in the neighboring water. The leading sources of heavy metals in the waters of
River Kabul include Khazana Sugar Mills (industries) as well as the Gandaerab and Badni Nullas
(Rivers and streams) that carries sewage from Peshawar. The minute extent of water which is heavily
loaded with toxic chemicals from the sugar mills really alters the oxygen and ammonia contents of
water making it lethal for fishes especially when the mills are passing via the routine cleaning
procedures (Siraj, 2016). The industrial vicinities that discharge their toxic waste materials into the
different branches of River Kabul are eight in number. Besides these eight, there are a variety of small
commercial openings that are adding their share in the form of heavy metals and organic toxins from
various small industries into the River Kabul. Similarly the numerous drains that fall in River Kabul
are also a source of pollution of waters of the River Kabul. The entering of heavy metals and organic
toxins in the waters of River Kabul is putting hazardous impacts of the flora and fauna ( Siraj, 2016).
The Industrial stat present in the Hayatabad area of Peshawar district has various industries.
Almost all of them release their untreated wastes to the Bara and Burni Nulla that ultimately fall in
the River Kabul, delivering a huge amount of heavy metals and organic toxins in it. Similarly drains
and sewages from the well-populated area of Peshawar add their share in the waters of River Kabul
in the form of various toxic metals (Khan et al., 2011). The Paper, Ghee, Textile, and Tanneries mills
from the Charsadda get rid of their waste products by discharging them all in the various small canals
that finally enter in the River Kabul, making the waters of this river sub-standard. These all toxic
elements pollute the waters of River Kabul, which had been a foundation of fresh water for drinking,
fisheries as well as agricultural purposes. The industrial toxic wastes not only include ordinary
pollutants but also certain highly poisonous metals like lead, Cadmium, Chromium, Mercury and
uplifted contents of toxic organic solvents including organic halides (Ali et al., 2009).
1.10 Effects of Heavy Metals on human beings:
At lower concentrations heavy metals are vital for the enzymatic actions as well as several
other biological functions occurring in living organisms. But at higher concentrations their role is
20
turned into toxic effects. Besides the advantageous impacts there are certain hazardous effects that
may be appeared in the human beings after ingestion of fishes contaminated with heavy metals
(Alloway and Ayres, 1993; Mozaffarian et al., 2006). The toxicities produced due to heavy metal
excessive intake may lead to the impaired mental and central nervous system activities, sub-optimum
energy levels, altered blood composition, impairments in the lungs, kidneys, liver and various other
vital organs in the humans. The toxic effects prevail when certain important functions inside the body
are impaired like excretion, metabolism, storage and detoxification etc. Persistent disclosure of human
beings to the various types of heavy metals may leads to the sluggish progression of degeneration at
physical, muscular and neuronal level that may ultimately result in Alzheimer's disease, Parkinson's
disease, multiple sclerosis, muscular dystrophy etc. (Joseph et al., 2012).
Various cases of heavy metals mediated toxicities have been observed in both the fishes and
its users. Excessive amounts of essential metals cause sub-lethal impacts in some organisms and act
as fetal factors for other. As below the optimum level of these metals is also hazardous so they are
considered to carry double toxic threshold (Rainbow, 2007). Heavy metals contaminated water may
create certain serious health issues in humans because of their bioaccumulation or due to straightly
utilization of surface water. Heavy metal may provoke renal or connective tissue problems causing
kidney damage and cancer of lungs and various other organs. The recorded signs of heavy metal
mediated poisoning in humans include abdominal pain, nausea, breathing issues and vomiting.
Pulmonary obstructions, renal failure, bone weaknesses may also be resulted from heavy metals over
dosage. Symptoms of their over dosage include hair loss, anemia, learning deformities, joints pain,
growth issues, migraine, emphysema, osteoporosis and cardio vascular disorders diseases ( Dupler,
2001; Jarrup, 2003; Sharma and Agarwal, 2005; Mates et al., 2010; Jose et al., 2010).
1.11 Aims and objectives:
Heavy metals mediated aquatic pollution has been a leading ecological issue across the globe.
Industrial effluents, petroleum combustion, atmospheric condensation and drains serve as the sources
of heavy metal induced pollution. Natural processes including earth quacks, landslides and cyclones
also add their share in the form of heavy metals to the habitat. Absence of enough scientific
information, public education, social awareness is making it possible to add tons of heavy metals into
the environment via industrial and mining activities, fuel combustion and sewages throughout the
Pakistan. The huge extent of heavy metals is damaging the ecosystem resulting in the extinction of
fresh water life including fishes. Those aquatic organisms which manage to survive in spite of so
much heavy metals intake, not just experience retarded growth patterns but also serves as the carriers
21
of these toxic metals into the human bodies. The heavy metals are likely to induce serious health
issues. Actually the majority of heavy metals are destroyable and non-degradable one. So they use to
be accumulated in one or the other component of ecosystem creating toxicities. Adoption of straight
forward regulations in this regards is the great need of the era. Making public awareness via the mass
media will surely help reducing heavy metal induced pollutions. Proving their health hazards on
scientific basis will surely bring a revolution among the societies. Making industries treat their
effluents before discharging them out will be another fruitful step to protect the human beings and
animals from the poisonous effects of these heavy metals.
1. To conduct a gross examination of the freshwater mussel’s morphology, parasitic infestation,
morphometric measurements and the mucus production due to the impact of heavy metals.
2. To investigate the types and levels of toxic components, both physico-chemical parameters
and heavy metals in water and sediment of River Kabul.
3. To study bioaccumulation of selected heavy metals in fresh water mussels of River Kabul.
4. To evaluate histopathological and genotoxical effects due to heavy metals in fresh water
mussels of River Kabul.
22
CHAPTER 2
PHYSICAL AND CHEMICAL CHARACTERISTICS OF RIVER KABUL
Abstract
This research study was performed in the River Kabul which is now-a-days a victim of
untreated industrial waste products from a variety of industries and sewages from densely populated
nearby areas. These toxic industrial effluents and sewages from various cities, towns and villages
finally fall in the River Kabul. During this study four samples of waters were obtained from River
Kabul at various spots. These collections were collected in the following manner;
Sample A was collected from Michini Bridge, Sample B, C and D was collected from
polluted sites in River Kabul during low (winter) and high flow (summer) periods respectively. After
that all these samples were assessed to evaluate their physic-chemical characters as well as the heavy
metal concentrations. The results revealed that industries working in the near-by areas of River Kabul
are releasing their untreated waste products that contain excessive concentrations of TSS and
Mercury. The concentrations of TSS and Mercury were found beyond the allowed limits established
by National Environmental Quality Standards (NEQS) for effluents. However the Physico-chemical
parameters were found to be within the acceptable limits provided by NEQS. Similarly the heavy
metals concentrations were also observed to be lying within the permissible limits of NEQS. The
samples C and D that were obtained from polluted sites revealed an uplifted level of heavy metals and
higher tendencies of Physico-chemical parameters as compared to samples of water A and B. The
different samples of water obtained from a variety of sites showed a sequence of D > C > B > A in
this regards. It discloses the fact that sample D contained the highest levels of heavy metals and
physico-chemical parameters followed by the water samples C, B and A where the sample A
contained the minimum levels. The maximum physico-chemical and heavy metal parameters
observed in Sample D shows that this particular spot of River Kabul is polluted at its maximum level
by the industrial effluents and public drains. The spot from where sample D was mounted receives
the industrial wastes and drains from a variety of areas including Mardan, Nowshera and Aman Garh.
It means that the industries and localities in the above mentioned areas are discharging their untreated
effluents as well the drains into the River Kabul at its peak level as compared to the other sites. All the
materials and physico-chemical parameters for which these waters (Sample C and D) were tested
including TDS, TSS, EC, TA, Na, Cl, K and pH and heavy metals e.g. Zn, Ni, Cr, Cu, Cd, Pb, Fe,
Mn and Hg were found to be at higher levels as compared to the water collected from Michini Bridge.
23
2.1 Introduction
Water, being a vital factor for the survival of all sorts of lives, is a huge blessing of the creator.
Human being utilizes an average of 2 liters of water per day. Water covers approximately 80% of
water. Among the total of 1011 million Km 3 of total water present on earth crust, merely 3340 m3 of
water is able to be drunk, utilized in agricultural, domestic and industrial activities. The remaining
water is engaged in ocean as salt water, glaciers, polar ice caps and underground reservoirs. Rapid
industrialization and enhanced ignition activities have led to the uplifted water demands. Similarly
drains, effluents and a huge extent of artificial chemical contaminate a huge portion of this
considerable portion of this limited reservoirs of water. The development of water borne illnesses has
not only been a problem for merely humans but also for water life like fishes. So the maintenance of
quality and quantity of pure water availability is essential for the wellbeing of human beings (Dara,
1993).
2.2 Physico-chemical Parameters:
The River Kabul waters were tested for a variety of physico-chemical factors including pH,
total dissolved solid, total suspended solid, electrical conductance, total alkaline pattern, Na, K and Cl
etc. all of the mentioned parameters were found to be uplifted than the established standards of
national environmental quality (Yousafzai, 2004). Analysis of water of river Kabul reveals various
parameters including, total suspended solids, pH, and total dissolved solids to be at higher levels. The
effects of industrial wastes outflow on the quality of River Kabul water at Aman Garh, Nowshera was
assessed for a variety of chemical and biochemical factors including pH, suspended solids, electrical
conductivity, alkalinity, chlorides, sulfates, sodium, potassium etc. the findings revealed that the
localized contamination within half kilometer near the convergence has made water deteriorated.
Similarly the hyper concentration of total suspended solids was found in water of River Swat at
Mingora, Pakistan (Siraj, 2016).
The effects of industrial wastes outflow on the quality of River Kabul water at Aman Garh,
Nowshera was assessed for a variety of chemical and biochemical factors including pH, suspended
solids, electrical conductivity, alkalinity, chlorides, sulfates, sodium, potassium etc. showing the hyper
levels of the above parameters in water of river Kabul (Khan et al.,1999). The water in various rivers
including Kerala, Cauvery, Gomti, Krishna, Godavari, Mahanadi, Narmada, Tapti, Indus,
Brahamputra and Ganges was assessed to test total alkalinity, all of which revealed versatile findings
regarding values of total alkalinity. The river Kabul water has a hyper concentration of total
24
conductivity in the entire river as compared to levels of total dissolved solids, total suspended solids
and major ions (Siraj, 2016).
The testing of effluents entering river Kabul showed a huge extent of total dissolved solids.
The TDS resided within the National Environmental Quality Standards limits suggested for the public
and commercial discharges of Pakistan. Higher total alkalinity was observed in River Kabul. The
waters of Kabul water, assessed for TDS showed a hyper levels during both as well as low flow
scenarios (Yousafzai, 2004). The industrial mediated water contamination is leading issue. The
assessment of water for a variety of factors revealed hyper levels of pH, total suspended solids and
total dissolved solids. Total suspended solids (TSS) is composed of silt, clay, fine particles of organic
and inorganic matter, soluble organic compounds and microscopic organisms. In spite of higher levels
of these all parameters still, they comprised of the natural attributes of the river Kabul because of its
catchments and release properties. Occurrence of natural erosion, a major source of TSS, is found to
be frequent in hilly as well as volcanic areas. Because of its turbidity, which is prevailed due to hyper
TSS values during both high as well as low flow conditions, the river Kabul is described frequently
as a dirty river (Siraj, 2016).
Total suspended solid (TSS) values were found to be occur beyond the NEQS limits at all
the sampling sites. These results may be correlated to the extreme flood condition during high flow
because of snow melting, summer’s monsoon raining, extensive deforestation, weathering, soil
erosion, mining and other anthropogenic actions occurring near the banks of the river in Pakistan and
Afghanistan (Yousazai et al., 2010 a).
The River Kabul, being a receiver of wastes from the concerned industrial plants beside
Nowshera, was tested for various parameters including electrical conductivity, alkalinity, potassium,
chloride and pH. The values of mentioned parameters were found higher in the above waters as
compared to the below ones A hyper level of Total Alkalinity was found in River Swat water at
Mingawora district Swat. An analysis of river Kabul was done to check its alkalinity, chlorides,
potassium and sodium levels and found to be high during both high and low flow conditions. It was
due to extensive discharge of effluents and drains that rendered the chloride, potassium and sodium
levels in samples both during low and high flows (Yousazai et al., 2010a; Ali, 2012).
Agricultural, industrial and domestic waste, being the leading origins of chlorides, potassium
and sodium in the River Kabul are important environmental factors. In household drains, the chlorides
are obtained from kitchen and human waste products. Industries may also have a major share in
chlorides of the river. A hyper level of chlorides is an indication of industrial contaminants in water
organisms ( Khan. et al., 2003; Ramakar et al., 2005). A hyper alkalinity was found in the water of
Warsak Dam. A variety of physical, chemical and biological factors was assessed in the River Kabul
25
at Nowshera, showing less effects of industrial effluents on the water of Main River. The effects of
industrial wastes and drainages were assessed on the quality of waters of River Kabul. The efforts
were made as a pioneer step in KPK with respect to the industrial wastes mediated water pollution in
river Kabul. pH, electrical conductivity, total dissolved solids, total suspended solids and chloride etc
were observed to be hyper than recommended standards for industrial effluents (Yousafzai., 2004).
The effects of industrial wastes was done river Kabul waters was done. The variety of industrial
wastes entering the River Kabul showed a hyper level of pH, chloride, potassium, sodium and salinity
but they all resided within permissible limits. The wastes of industries resided at industrial estate
Kohat road Peshawar was assessed and it was revealed that a hyper level of TSS, TDS, potassium
and chloride and sodium in them. These are of huge importance as all of them are to be drowned into
the River Kabul (Jan et al., 2002).
2.3 Heavy metal parameters:
Because of heavy metal mediated poisoning, precise information are vital specifically from
natural and pure ecosystems (Janssen et al., 2000). Assessment of heavy metals such as Pb, Ni, and
Cu etc. in the water of River Kabul showed them to be below the acceptable limits. The effects of
industrial wastes was tested. A variety of industrial wastes showed hyper levels of heavy metals like
Cu, Zn, Fe, Cd, Pb and Ni as studied. Concentrations of majority of trace elements were found to be
beyond the limits for irrigation water (Khattak and Rehman, 1992). The water of the river Kabul tested
for a variety of heavy metals like Cd, Cr, Cu, Hg, Fe, Mn, Ni, Pb and Zn. The Kabul River receives
drains and wastes from Peshawar via Budni nalla and Ganda vinda and alters its environmental status,
showing a hyper level of Cd, Cu, and Fe. The wastes of the specified industries resided at small
Industrial Estate, Kohat Road, Peshawar was tested for heavy metals and found higher levels of Fe,
Mn and Crin it. Hyper values of such factors is important as at the end these wastes have to be fallen
into River Kabul. The heavy metals such as Cu, Fe, Pb, Cd, Mn and Zn levels in the water of El-
Rahawy drain and River Nile at El- Kanater El-Khyria were periodically assessed. Findings reveal
that heavy metals levels in water from El-Rahawy drain region were higher as compare to those
collected from River Nile (Jan et al., 2002; Jan et al., 2010; Authman et al., 2013; Mohammad et al.,
2013).
Heavy metals, being one of the main kind of poisonous contaminants usually present in
surface waters and highly poisonous to marine and fresh water aquatic life. Several investigation have
done for detection of heavy metals in water (Sabae and Abdel-Satar, 2001; Soualili et al., 2008). The
heavy metals were detected to be available in the water of Abu Za'baal Lakes in Egypt including Fe,
26
Zn? Mn, Pb, Cu and Cd. Studies revealed that extent of heavy metals is present in high concentration
usually in river water. The black Smiths’s active engagement in metal crap functions has resulted in
the introduction of heavy metals in the lake of Geriyo. Immense agricultural and irrigational actions
as well as the discharging and storage of public drains near the lake bank were the reasons behind that
all Ogun river water was analyzed for heavy metals levels including lead and cadmium and were
found to reside beyond the WHO limits (Fatima & Gad, 2005; Jonathan and Maina, 2009: Jaji et al.,
2007). It is found that waste water as well as the surface waters consist of complex chemicals
including heavy metals. Such statements were supported by a study performed on the municipal water
of Ivanja Reka area where heavy metals like Sr, Hg, As, Cu, Cr, Mn were found with hyper levels.
The wetland ecosystems that use to accept complex wastes involving huge extent of heavy metals are
of extreme importance for the ecological territories specifically for water life that have to be exposed
polluted waters as their environment. Distribution of heavy metals among the components of habitat
for recycling purposes via accumulation in the sediments and bodies might be the reason of their
demolished concentrations in the water and fish samples collected from the lower zone of sampling
collection. Metals may sediment in the bottom where they stay for several years. Such sediments are
believed to make the main heavy metal reservoirs in the water habitats (Johnson et al., 2005, Tipping
et al., 2006; Raychaudhuri et al., 2008; Olowu et al., 2010).
2.4 Materials and Methods
2.4.1 Study area description
For detail see page- 07

2.4.2 Sampling sites
The water samples were collected from different sites of river Kabul. The Kabul River enters
Pakistan through Shalman in Khyber Agency. It then flows through the Khyber and Mohmand
Agencies until it reaches Warsak dam constructed in 1960 without any fish ladder for upward
migration of inhabitant fish. Below the dam, it is divided into three main branches are known as Shah
Alam, Nagoman and Adezai and then joins the River Indus at Kund (Attock). River Swat also joins
River Kabul a few km below the dam (Siraj, 2016).
27
2.4.3 Sampling points
To evaluate the quality and quantity of physico-chemical and heavy metal parameters at
Michini Bridge and adjacent Amangarh industrial area of River Kabul, water from the following
points of the main River were collected (Fig.3).
Figure 3: Water sampling site 1(water sample A), site 2 (water sample B), site 3 (Water sample C)
and site 4 (water sample D) at River Kabul.
2.4.4. Water sample from Michini Bridge below Warsak dam
Water sample(A) was collected from reference site 3 (Warsak dam) water reservoir
constructed on River Kabul in 1960, which is about 60 km upstream of the polluted part of the River
and can be called safe in the sense of being far away from the dense human and industrial population.
This was considered as control sample.
28
2.4.5. Water samples from the main River
Water samples (A), (B), (C) and (D) were collected from four of the main River at, Michini
Bridge (site 1), Sardaryab (site 2), Aman Garh (site 3) and Peer Sabaq (site 4). Sites 2, 3 and 4 are at
the main river after city sewage and industrial effluent discharged into it. While in site 1 have no any
industry is present.
2.4.6. Collection of water samples
Water samples were collected from the sampling sites in 100ml or 500ml bottles already
cleaned by vim and distilled water. At the time of sampling these bottles were also washed with the
respective river water. The samples collected below the surface about 2-3 feet away from the river
banks in such a way that no bobbles were allowed. Conductivity, TDS and pH for the river water
were determined immediately on the spot and rest of the parameters were analyzed in the laboratory.
Sampling was conducted three times a year from January 2013 to December 2015. Water samples
from Main River were collected once in high flow season in the month of June and two times a year
in low flow season in the months of January and December to highlight the effect of water volume
on the quantity and quality of water pollution.
2.4.7. Preservation of water samples
Water samples for heavy metals estimations were collected in separate 1 liter plastic bottle
and were preserved with 5ml nitric acid (50%) per liter to prevent metal absorption on the inner
surface of container. Samples for physico-chemical parameters were collected in separate 1 liter
plastic bottle. The water samples were then stored at 4c° in the refrigerator before analysis for various
physico-chemical and heavy metal parameters.
2.4.8. Water analysis
To obtain information about the degree of contamination caused by industrial, domestic and
agricultural effluents with respect to concentration of chemical, physical pollutants and heavy metals,
samples were collected from three polluted sites (Akber pura, Aman garh and Nowshera)
downstream and from one spot of non-polluted site (Warsak dam) upstream of the River Kabul.
Which is about 35 Km upstream of the polluted site B of the river and can be called safe in the sense
of being far away from the dense human population and industrial activities. This was considered as
29
control sample. The physico-chemical parameters like pH, total suspended solid (TSS), total
dissolved solid (TDS), alkalinity, chloride, electrical conductivity, sodium (Na) and potassium (K)
were analyzed in the water samples from both polluted and non-polluted sites of River Kabul and the
heavy metals such as cadmium, chromium, copper, iron, mercury, manganese, nickel, lead, and zinc
were determined in the water samples from both polluted and control portions of River Kabul.
2.4.9. Physico-chemical parameters
2.4.9.1. pH
Model; 7020- pH meter.

pH values were determined on the spot in the quickest possible time. First the pH meter was
calibrated with the buffer 4 and 7. Then the pH values of the water samples from three spots of
polluted sites and one from non-polluted site of River Kabul were measured through pH meter.
2.4.9.2. Electrical conductivity
Model; AGB 1000.
The instrument was calibrated with distilled water. Then conductivity of the water samples
from polluted and non-polluted sites of River Kabul measured in microsiemenes per centimeter
through conductivity meter.
2.4.9.3. Total dissolved solid (TDS)
The instrument was calibrated with distilled water. Then total dissolved solid of the water
samples from polluted and non-polluted sites of River Kabul measured in milli gram per liter through
TDS meter.
2.4.9.4. Total suspended solid (TSS)
First of all filter paper was taken and dried in oven at 103 - 105 % for an hour. After heating
the filter paper kept in desiccator for moisture content absorption. The weight of filter paper noted and
then a known amount of unfiltered water from polluted and reference sites of River Kabul taken in
flasks and filtered. Filter paper was again kept in the oven at 103 - 105.The total suspended solid
calculated and reported as mg/l.
2.4.9.5. Chloride
The chloride was determined through PC multi direct spectrophotometer. Firstly filled a clean
vial (24 mm) with 10ml of water samples and closed the vial with cap tightly. Then placed the vial in
30
the sample chamber and pressed the zero key. Then removed the vial from the sample chamber and
added one chloride T1 tablet to the water sample and crushed through stirring rod and dissolved the
tablet. Then another chloride T2 tablet added to the same water sample and crushed tablet through
stirring rod and dissolved the tablet. Then closed the vial with the cap tightly and swirl the vial several
times until the tablet is dissolved. Then placed the vial in the sample chamber and pressed the key and
waited for a reaction period of 2 minutes. After the period completion, the reading starts automatically
and the result is shown in the display in mg/l chloride.
2.4.9.6. Total alkalinity
The total alkalinity was also determined through PC multi direct spectrophotometer. Firstly
filled a clean vial (24 mm) with 10 ml of water samples and closed the vial with cap tightly. Then
placed the vial in the sample chamber and pressed the zero key. Then removed the vial from the
sample chamber and added one Alka-M- Photometer tablet straight from the foil to the water sample
and crushed the tablet using a clean stirring rod and dissolved the tablet. Then closed the vial with the
cap tightly and swirl the vial gently several times until the tablet is dissolved. Then placed the vial in
the sample chamber and pressed the key and was waited for a reaction period of two minutes. After
the period completion, the reading starts automatically and the result is shown in the display in mg/l
CaCO3.
2.4.9.7. Sodium and Potassium
Atomic absorption spectrophotometer (Spectra-AA-700) was used for the determination of

sodium and potassium in the water samples. The same method of sample digestion and preparation
was followed as adopted for the heavy metals determination. Wave lengths set were 589.0 nm for
sodium and 766.5 for potassium in the presence of air acetylene flame.
2.5 Heavy metals parameters
Heavy metals like zinc, nickel, chromium, copper, cadmium, lead, manganese, iron and
mercury were determined in the water samples from both polluted and control sites of River Kabul.
Water sample (100 ml) in the volumetric flask was acidified with 5 ml nitric acid (55%) and kept on
evaporated on a hot plate to about 20 ml. 5ml additional nitric acid (55%) and perchloric acid (70%)
and a few drops of beads were added to prevent bumping. The mixture was then evaporated until a
brown fumes change into dense white fumes. The sample was removed from hot plate, cooled down
to room temperature and diluted to 100 ml volumetric flask. The solutions were then aspirated into
flame Atomic Absorption Spectrophotometer (Spectra-AA-700) for determination of Cd, Cr, Cu, Fe,
31
Hg, Mn, Ni, Pb and Zn under the following operating parameters. The flame used was air acetylene
(A-AC). Standard curve were prepared and the optical density obtained were calibrated against the
standard curves to know the contents of heavy metals in the water.
Table 1: Operating data of Atomic absorption spectrophotometer for determination of metals
Elements Wave length (nm) Flame Working range (µg/mL-1)

Cd 228.8 AA (R) 0.5-2
Cr 357.9 AA (R) 2-8
Cu 324.7 AA (L) 2-8
Fe 248.3 AA (L) 1-4
Hg 253.7 AA (L) 100-400
Mn 279.5 AA (L) 1-4
Ni 232.0 AA (L) 3-12
Pb 217.0 AA (L) 5-20
Zn 213.9 AA (L) 0.4-1.6
Abbreviations, AA: Air Acetylene, R: Fuel-rich, L: Fuel-lean

Cd-Cadmium, Cr-Chromium, Cu-Copper, Fe-Iron, Hg-Mercury, Mn- Manganese, Ni-Nickel, Pb-
Lead, Zn-Zinc.
2.6. Statistical Analysis
Statistical analysis was done by using ANOVA software for windows. Mean and standard
deviation values of the data were determined. The different sets of data were analyzed for statistical
differences by using student’s t – test (two-tailed). P value <0.05 was considered to show statistical
significance.
32
2.7 Results and Discussion
2.7.1 Water Analysis of River Kabul
In the present study water samples from polluted sites 2, 3 and 4 (Sardaryab, Aman Garh and
Peer Sabaq) and reference site 1, Michini bridge below Warsak dam of River Kabul were taken from
2013 to December 2015 and was studied for different Physico-chemical and heavy metal parameters.
Physico-chemical parameters include pH, total suspended solid, total dissolved solid, total alkalinity,
chloride, electrical conductivity, potassium and sodium and heavy metals parameters include
cadmium, chromium, copper, iron, manganese, nickel, lead and zinc.
2.7.1.1 Michini Bridge water sample from site 1 (Sample A= Reference site)
A. Physico-Chemical Parameters
Water samples from Michini Bridge below Warsak dam (reference site 1) were analyzed for
both Physico-chemical and heavy metal concentrations and showed less Physico-chemical and heavy
metals contents than polluted sites 2, 3 and 4 (Tables 2 - 17 and Figs. 4 - 20).
Water sample from this site of river Kabul had a pH ranged between 7.4 and 7.9 with mean
values of 7.6 ± 0.2, 7.6 ± 0.2 and 7.5 ± 0.1 during summer season and ranged between 7.4 and 7.9
with mean values of 7.5 ± 0.2, 7.7 ± 0.2 and 7.6 ± 0.1 during winter season respectively. Total
suspended solid (TSS) concentration was in the range of 587- 729 mg L-1 with mean values of 599.3
± 13.0 mg L-1, 706.6 ± 4.0 mg L-1 and 725.0 ± 4.0 mg L-1 for summer high flow and was in the range
of 381-729 with mean values of 405.0 ± 10.4 mg L-1, 385.0 ± 4.0 mg L-1 and 415.3 ± 0.3 mg L-1 for
winter season (low flow). Total dissolved solids (TDS) content was varied between 301-389 mg L-1
with mean values of 385.3 ± 3.5 mg L-1, 306.0 ± 5.0 mg L-1 and 345.3 ± 3.7 mg L-1 for summer and
content varied between 412-613 with mean values of 607.0 ± 4.5 mg L-1, 416.3 ± 4.0 mg L-1 and
610.0 ± 3.0 mg L-1 for winter. The electrical conductivity in water sample from site 1 was ranged
between 269-302 µs/cm with mean values of 285.0 ± 4.0 µs/cm, 297.3 ± 4.5 µs/cm, and 270.6 ± 2.0
µs/cm during summer season and ranged between 336-375 µs/cm with mean values of 339.0 ± 3.0
µs/cm, 371.6 ± 3.0 µs/cm and 372.0 ± 3.0 µs/cm during winter periods. The chloride ranged between
6-15 mg L-1 with mean values of 8.3 ± 2.5 mg L-1, 12.0 ± 3.0 mg L-1 and 11.6 ± 2.5 mg L-1 for summer
season and was ranged between 12-19 mg L-1 with mean values of 17.6 ± 1.5 mg L-1, 16.6 ± 1.5 mg
L-1 and 15.0 ± 3.0 mg L-1 for winter seasons.
The potassium (K) range was 2.3 -2.10 mg L-1 with mean values of 2.4 ± 0.2 mg L-1, 2.5 ±
0.2 mg L-1 and 2.5 ± 0.4 mg L-1 during summer and ranged between 1.3-1.9 mg L-1 with mean values
of 1.5 ± 0.1 mg L-1, 1.7 ± 0.2 mg L-1 and 1.5 ± 0.3 mg L-1 during winter. The sodium concentration
33
was ranged between 7-13 mg L-1 with mean values of 10.6 ± 2.0 mg L-1, 11.0 ± 2.6 mg L-1 and 9.3 ±
2.5 mg L-1 for summer and ranged between 9-19 mg L-1 with mean values of 17.0 ± 2.0 mg L-1, 14.6
± 3.2 mg L-1 and 10.0 ± 3.6 mg L-1 for winter seasons. Similarly water from this site had total alkalinity
in the range of 55-101 mg L-1 with mean values of 59.3 ± 6.6 mg L-1, 87.3 ± 5.6 mg L-1 and 97.0 ±
4.0 mg L-1 for summer and values ranged between 83-119 mg L-1 with mean values of 113.0 ± 4.5
mg L-1, 87.3 ± 4.5 mg L-1 and 90.0 ± 3.0 mg L-1 for high flow periods. The present results of higher
TDS, TSS, EC and TA contents in water sample from this site of River Kabul agree with the previous
findings (Khan et al., 1999; Akif et al., 2002; Siraj, 2016). On comparison with NEQS recommended
values for effluents, all physico-chemical parameters except total suspended solid (TSS) were within
recommended minimum limits.
Total suspended solid (TSS) had mean values of 745.0 ± 3.0 mg L-1, 706.6 ± 4.0 mg L-1 and
725.0 ± 4.0 mg L-1 for summer and had mean values of 607.0 ± 4.5 mg L-1, 416.3 ± 4.0 mg L-1 and
610.0 ± 3.0 mg L-1 for winter seasons respectively. These values were higher than NEQS
recommended limit of 150 mg L-1 for this parameter. This shows that TSS concentration was the
highest during high flow and lowest during low flow. The highest concentration of TSS during high
flow was associated to flooding and soil erosion in the river. Due to high turbidity of River Kabul
many people has described it as a ‘dirty river’. This could be correlated to greater level of TSS in the
river, which ranged from 10 to 800 mg L-1 during low flow and from 340 to 1,310 mg/l during high
flow conditions (Yousufzai, 2004). Similarly Siraj (2016) had also reported high TSS concentration
with a mean values of 418.8 ± 398.9 mg L-1 for low flow and 715.0 ± 138.0 mg L-1 for high flow
seasons from the same water resources of River Kabul that verifying the validity of our findings (Siraj,
2016). Comparing our study with the findings of other workers showed an increasing level of TSS in
water from this site in the last few years. Rest of the physico-chemical parameters like TDS, EC, TA,
Cl, Na, pH and K in water from this point were below the permissible limits proposed by NEQS. The
physicochemical parameters were found in the order of TSS > TDS > EC > TA > Cl > Na > pH > K
for summer and TDS > TSS > EC > TA > Cl > Na > pH > K for winter.
The reference site Michini Bridge below Warsak dam is safe and quiet fit for aquatic life. Because
this site is being far away from industrial activities and densely human population. The only parameter
which exceeds the NEQS limits is TSS value, which can be correlated to excessive flooding in the
River Kabul during summer (June, July and August). During these months snow melt occurs on the
tops of surrounding hills, both in Pakistan and Afghanistan and causes flooding in the River Kabul,
moreover moon soon rains also add to flooding. Other reason can be correlated to mining activities
and deforestation in the adjacent hills. As freshwater mussels have most of the above stated
adaptations for such environment, therefore TSS can no way be a limiting factor.
34
Table 2: Physico-chemical characteristics of water sample from Michini Bridge during winter
and summer during 2013-2015
Parameter S. 2013 W.2013 S.2014 W.2014 S.2015 W.2015
pH 7.6 ± 0.2 7.5 ± 0.2 7.6 ± 0.2 7.7 ± 0.2 7.5 ± 0.1 7.6 ± 0.1
TSS (mg/l) 599.3 ± 13.0 405.0 ± 10.4 706.6 ± 4.0 385.0 ± 4.0 725.0 ± 4.0 415.3 ± 0.3
TDS (mg/l) 385.3 ± 3.5 607.0 ± 4.5 306.0 ± 5.0 416.3 ± 4.0 345.3 ± 3.7 610.0 ± 3.0
EC (µs/cm) 285.0 ± 4.0 339.0 ± 3.0 297.3 ± 4.5 371.6 ± 3.0 270.6 ± 2.0 372.0 ± 3.0
Cl (mg/l) 8.3 ± 2.5 17.6 ± 1.5 12.0 ± 3.0 16.6 ± 1.5 11.6 ± 2.5 15.0 ± 3.0
K (mg/l) 2.4 ± 0.2 1.5 ± 0.1 2.5 ± 0.2 1.7 ± 0.2 2.5 ± 0.4 1.5 ± 0.3
Na (mg/l) 10.6 ± 2.0 17.0 ± 2.0 11.0 ± 2.6 14.6 ± 3.2 9.3 ± 2.5 10.0 ± 3.6
TA (mg/l) 59.3 ± 6.6 113.0±4.5 87.3 ± 5.6 87.3 ± 4.5 97.0 ± 4.0 90.0 ± 3.0
1, Mean± Standard deviation

2, Abbreviations: S, Summer, W, Winter, (Total suspended solid (TSS), Total dissolved solid (TDS),
Electrical conductivity (EC),) Chloride (Cl), Potassium (K), Sodium (Na), Total alkalinity (TA),
Dissolved oxygen (DO) , NEQS, National environmental quality standards, NA, Not available.
35
Table 3: National Environmental Quality Standards (NEQS) for municipal and liquid
industrial effluents of Pakistan
Parameters (mg/l) Existing Standard

pH 6-10
TSS 150
TDS 3500
Cl 1000
B. Heavy Metals Parameters

Water samples A from reference site 1 showed less concentration of heavy metals as compare
to polluted sites (Tables 2 - 17 and Figs. 4 - 20). From amongst the heavy metal parameters, water
sample A from Michini Bridge below Warsak Dam had cadmium in the range of 22-38 µg L-1 with
mean values of 33.3 ± 4.0 µg L-1, 36.0 ± 2.6 µg L-1and 23.6 ± 2.0 µg L-1 during summer and values
ranged between 21-25 µg L-1with mean values of 23.3 ± 2.0 µg L-1, 22.6 ± 1.5 µg L-1 and 24.0 ± 0.9
µg L-1 during winter seasons. The minimum concentration recorded for Chromium was 17 µg L-1 and
maximum concentration of 25 µg L-1 with mean values of 23.4 ± 1.3 µg L-1, 21.9 ± 1.1 µg L-1 and
19.0 ± 2.0 µg L-1 for summer and minimum concentration 12 µg L-1 and maximum 19 µg L-1 with
mean values of 16.6 ± 2.2 µg L-1, 14.1 ± 2.0 µg L-1 and 16.3 ± 3.0 µg L-1 for winter. Copper content
ranged between 32 µg L-1 and 47 µg L-1 with mean values of 35.3 ± 4.1 µg L-1, 43.6 ± 2.8 µg L-1 and
37.6 ± 4.1 µg L-1 for summer and content ranged between 18 µg L-1 and 24 µg L-1 with mean values
of 20.6 ± 2.5 µg L-1, 21.2 ± 1.6 µg L-1 and 23.3 ± 0.6 µg L-1 for winter periods. Iron concentration
ranged between 20 and 34 µg L-1 with mean values of 21.8 ± 2.3 µg L-1, 24.3 ± 1.5 µg L-1and 32.0 ±
2.0 µg L-1 for summer and concentration ranged between 12.8 and 20.9 µg L-1 with mean values of
14.2 ± 1.6 µg L-1, 22.6 ± 1.5 µg L-1 and 20.6 ± 0.5 µg L-1 for winter seasons. The present result found
higher concentration of Cd, Cr, Cu and Fe as compare to previous investigations (Khattak and
Rehman, 1992; Yousufzai, 2004; Siraj, 2016), who reported low level of these metals from the same
site of River Kabul. Similarly manganese at this point had minimum range of 24 µg L-1 and maximum
range of 37 µg L-1 with mean values of 27.0 ± 3.6 µg L-1, 30.9 ± 1.9 µg L-1 and 34.6 ± 2.0 µg L-1 for
summer and minimum range 18 µg L-1 and maximum 24 µg L-1 with mean values of 20.3 ± 2.3 µg
L-1, 20.6 ± 2.5 µg L-1 and 23.0 ± 1.0 µg L-1for winter seasons. The nickel had a concentration varying
between 59-73.2 µg L-1 with mean values of 62.0 ± 3.0 µg L-1, 71.7 ± 1.6 µg L-1 and 69.0 ± 2.0 µg L-
1
for high flow and concentration varying between 35-44 µg L-1 for low flow periods. This is in
agreement with the findings reported by Khan, et al., (1999). Similarly the minimum concentration
36
recorded for lead was 32.1 µg L-1 and maximum concentration of 55 µg L-1 with mean values of 33.3
± 1.1 µg L-1, 42.9 ± 0.7 µg L-1 and 48.6 ± 6.0 µg L-1 for high flow and minimum concentration 27 µg
L-1 and maximum 47 µg L-1 with mean values of 29.3 ± 2.0 µg L-1, 41.6 ± 3.5 µg L-1 and 48.3 ± 2.3
µg L-1 for low flow periods. Zinc content ranged between 98 µg L-1 and 115 µg L-1with mean values
of 102.0±4.5 µg L-1, 111.6±3.5 µg L-1 and 109.0±5.0 µg L-1 for summer and content ranged between
43 µg L-1 and 99 µg L-1 with mean values of 92.6±4.0 µg L-1, 95.6±3.0 µg L-1 and 45.6±3.0 µg L-1for
winter. Heavy metals in water sample A from this point was in order of Zn > Ni > Pb > Cu > Cd >
Mn > Hg > Fe > Cr for summer and Zn > Ni > Pb > Cd > Cu > Mn > Fe > Hg > Cr for winter seasons.
These results are in agreement with those observed by many investigators (Khan, et al., 1999:
Yousufzai, 2008; Ahmad. et. al., 2013), who have also studied higher content of metals in water from
other resources. Many investigations were previously carried out on the level of heavy metals in water
(Pan & Wang, 2012; Peric, et al., 2012; Siraj, 2016). In this study the average mean shows that levels
of all these parameters were lowest during winter and highest during summer seasons. Highest
concentration during summer could be correlated to increased river volume, atmospheric
condensation, earth quake, landslides, tornadoes and cyclones in river. Heavy metals except Hg
studied in water sample A from Michini Bridge were within permissible limits laid down by National
Environmental Quality Standards. All the heavy metals except Hg at this point were within
permissible limits (10µg/l) lay down by NEQS. The Hg mean values for low flow and high flow
periods were 17.3 ± 4.5 µg L-1 and 30.8 ± 11.5 µg L-1, respectively. Therefore Michini Bridge water
area is safe for aquatic life including freshwater mussels
37
Table 4: Heavy metals concentration (µg/L) of water sample from Michini Bridge during
winter and summer (2013-2015).
Heavy S. 2013 W.2013 S.2014 W.2014 S.2015 W.2015

Metals
Cd 33.3 ± 4.0 23.3 ± 2.0 36.0 ± 2.6 22.6 ± 1.5 23.6 ± 2.0 24.0 ± 0.9
Cr 23.4 ± 1.3 16.6 ± 2.2 21.9 ± 1.1 14.1 ± 2.0 19.0 ± 2.0 16.3 ± 3.0
Cu 35.3 ± 4.1 20.6 ± 2.5 43.6 ± 2.8 21.2 ± 1.6 37.6 ± 4.1 23.3 ± 0.6
Fe 21.8 ± 2.3 14.2 ± 1.6 24.3 ± 1.5 22.6 ± 1.5 32.0 ± 2.0 20.6 ± 0.5
Hg 18.5 ± 6.5 17.3 ± 4.5 30.8 ± 11.5 18.9 ± 6.5 30.3 ± 11.7 27.3 ± 11.2
Mn 27.0 ± 3.6 20.3 ± 2.3 30.9 ± 1.9 20.6 ± 2.5 34.6 ± 2.0 23.0 ± 1.0
Ni 62.0 ± 3.0 37.8 ± 2.4 71.7 ± 1.6 40.3 ± 2.0 69.0 ± 2.0 43.3 ± 1.1
Pb 33.3 ± 1.1 29.3 ± 2.0 42.9 ± 0.7 41.6 ± 3.5 48.6 ± 6.0 48.3 ± 2.3
Zn 102.0 ± 4.5 92.6 ± 4.0 111.6 ± 3.5 95.6 ± 3.0 109.0 ± 5.0 45.6 ± 3.0
1. Mean± Standard deviation

2. Abbreviations: S-Summer, W-Winter, Zn-zinc, Ni-nickel, Cr-chromium, Cu-copper, Pb- lead, Cd-
cadmium, Fe-iron, Mn-manganeez, Hg-mercury.
38
Parameters Concentration (mg/l) Concentration (µg/l)

Cd 0.1 100
Cr 1.0 1000
Cu 1.0 1000
Fe 8.0 8000
Hg 0.01 10
Mn 1.5 1500
Ni 1.0 1000
Pb 0.5 500
Zn 0.5 500
39
2.7.1.2. Water sample from Sardaryab site 2 receiving sewages (Water sample-B)
a. Physico-Chemical Parameters
Water samples B from Sardaryab (site 2) of River Kabul downstream to Michini Bridge and
upstream to confluence point was analyzed for different physico-chemical and heavy metal
parameters concentration. At this point sewages from Peshawar and Charsadda districts and effluents
from marbel industries of Mohmand agency are dumped into River Kabul (Tables 2 - 17 and Figs. 4
- 20).
From amongst the physico-chemical parameters, water samples from this site had pH in the
range of 7.4 and 7.9 with mean values of 7.5 ± 0.2, 7.7 ± 0.2 and 7.6 ± 0.2 during summer (high flow)
and values ranged between 74. And 7.9 with mean values of 7.5 ± 0.2, 7.7 ± 0.2 and 7.6 ± 0.2 during
winter (low flow) periods. Total suspended solid (TSS) concentration was in the range of 711-741
mg L-1 with mean value of 717.0 ± 6.0 mg L-1, 728.6 ± 5.0 mg L-1 and 737.6 ± 4.1 mg L-1 for summer
season and concentration ranged between 412-457 mg L-1 with mean values of 452.0 ± 5.5 mg L-1,
418.3 ± 5.6 mg L-1 and 415.3 ± 0.3 mg L-1 for winter season. The present result of higher TSS content
in water sample from this site agree with the findings of Siraj (2016), who had also reported high TSS
values in water sample from this site of River Kabul. Similarly in a past research, Khan et al (1999)
have studied the quality of water and the effects of industrial effluents on River Kabul at Aman Garh,
Nowshera and analyzed water from this site for various chemical and biochemical parameters. They
also have found high level of total suspended solid (TSS) from the same water resources. Total
dissolved solid at this point had minimum range of 321 mg L-1 and a maximum range of 398 mg L-1
with mean values of 393.6 ± 4.1 mg L-1, 326.0 ± 5.0 mg L-1 and 395.3 ± 3.7 mg L-1 for summer season
and minimum range of 521 mg L-1 and maximum range of 459 mg L-1 with mean values of 623.6 ±
7.3 mg L-1, 529.0 ± 9.1 mg L-1 and 752.3 ± 7.0 mg L-1 for winter season. The present result found low
level of pH, TSS and TDS in water samples from this site than reported by Yousufzai, (2004), who
had reported 8.5,1230 mg L-1 and 2893.5 mg L-1 values for pH, TSS and TDS respectively from the
same water of River Kabul.
The electrical conductivity in water samples from site 2 of main river was ranged between
279- 431 µs/cm with mean values of 285.3 ± 5.6 µs/cm, 343.0 ± 5.5 µs/cm and 424.3 ± 6.1 µs/cm
during high flow period and values ranged between 383 - 429 µs/cm with mean values of 388.0 ± 4.5
µs/cm, 421.6 ± 7.0 µs/cm and 391.3 ± 2.5µs/cm during low flow period. The chloride of all the
samples of water at this point was in the range of 9-19 mg L-1 with mean values of 12.3 ± 4.1 mg L-1,
16.3 ± 3.0 mg L-1 and 15.6 ± 4.1 mg L-1 for summer and ranged between 9 - 27 mg L-1 for winter
season. The potassium content ranged between 2.1 mg L-1 and 2.9 mg L-1 with mean values of 2.6 ±
40
0.2 mg L-1, 2.5 ± 0.4 mg L-1 and 2.8 ± 0.1 mg L-1 during high flow and content ranged between 1.3-
1.9 mg L-1 16.6 ± 2.5 mg L-1, 15.6 ± 3.2 mg L-1 and 15.0 ± 3.6 mg L-1 during low flow periods. In a
previous research Khan et al., (2011) had reported high levels of different parameters including
temperature, pH, conductivity, dissolved solid, suspended solid, alkalinity, chloride and nitrate in the
water of River Swat. The sodium concentration varied between 7- 19 mg L-1 with mean values of 9.0
± 2.6 mg L-1, 12.3 ± 6.1 mg L-1 and 16.3 ± 3.0 mg L-1 for summer season and concentration varied
between 12-19 mg L-1 with mean values of 16.6 ± 2.5 mg L-1, 15.6 ± 3.2 mg L-1 and 15.0 ± 3.6 mg
L-1 for winter season. Similarly water from this site had total alkalinity in the range of 91-113 mg L-1
with mean values of 109.0 ± 4.0 mg L-1, 95.3 ± 3.7 mg L-1 and 110.0 ± 3.6 mg L-1 for high flow and
values ranged between 98 - 123 mg L-1 with mean values of 114.6 ± 11.1 mg L-1, 102.3 ± 4.5 mg L-1
and 103.0 ± 4.0 mg L-1 for low flow seasons. These parameters in water sample from this site was in
sequence of TSS > TDS > EC > TA > Cl > Na > pH > K for summer and TDS > TSS > EC > TA >
Cl > Na > pH > K for winter seasons. In this study all physico-chemical parameters in water samples
from this point were below NEQS recommended values for effluents except TSS, which exceeds the
NEQS recommended limit of 150 mg/l for this parameter.
Total suspended solid (TSS) concentration was in the range of 711 - 741 mg L-1 with mean
value of 717.0 ± 6.0 mg L-1, 728.6 ± 5.0 mg L-1 and 737.6 ± 4.1 mg L-1 for summer season and
concentration ranged between 412 - 457 mg L-1 with mean values of 452.0 ± 5.5 mg L-1, 418.3 ± 5.6
mg L-1 and 415.3 ± 0.3 mg L-1 for winter season. All these values were higher than the value of 150
mg L-1 proposed by NEQS for this parameter. All parameters like pH, TDS, EC, Cl, Na, K and TA
were having increasing tendency on comparison with Michini Bridge below Warsak dam water
samples. The high level of TSS could be correlated to mining activities, deforestation, and natural
process of weathering and poor agricultural practice in adjoining hills of River Swat during low flow
season, which also joins River Kabul below Warsak dam. In a recent studies by (Siraj, 2016) and
Yousazai et al (2010a) had also reported high TSS values at this sampling site of the River Kabul
exceeded the NEQS value of 150 mg/l for this parameter. This can be correlated to high flooding
during high flow due to snow melt on the peaks of surrounding hills, both in Pakistan and Afghanistan
and monsoon rains during summer months, excessive deforestation, weathering, soil erosion, mining
and other anthropogenic activities along the banks of river. Similarly in past investigations by Khan.
et. al., (1999); Sabae and Abdel-Satar, 2001; Kane et al., 2012; and Siraj, 2016, have also analyzed
water of River Kabul and found the water sample from this site to be contained high level of total
suspended solid.
41
Table 6: Physico-chemical characteristics of water sample from Sardayab during winter and
summer (2013-2015).
pH 7.5 ± 0.2 7.5 ± 0.2 7.7 ± 0.2 7.7 ± 0.2 7.6 ± 0.2 7.6 ± 0.2
TSS (mg/l) 717.0 ± 6.0 452.0 ± 5.5 728.6 ± 5.0 418.3 ± 5.6 737.6 ± 4.1 415.3 ± 0.3
TDS (mg/l) 393.6 ± 4.1 623.6 ± 7.3 326.0 ± 5.0 529.0 ± 9.1 395.3 ± 3.7 752.3 ± 7.0
EC (µs/cm) 285.3 ± 5.6 388.0 ± 4.5 343.0 ± 5.5 421.6 ± 7.0 424.3 ± 6.1 391.3 ± 2.5
Cl (mg/l) 12.3 ± 4.1 15.0 ± 3.6 16.3 ± 3.0 13.0 ± 3.6 15.6 ± 4.1 22.3 ± 5.0
K (mg/l) 2.6 ± 0.2 1.6 ± 0.3 2.5 ± 0.4 1.7 ± 0.1 2.8 ± 0.1 1.7 ± 0.2
Na (mg/l) 9.0 ± 2.6 16.6 ± 2.5 12.3 ± 6.1 15.6 ± 3.2 16.3 ± 3.0 15.0 ± 3.6
TA (mg/l) 109.0 ± 4.0 114.6 ± 11.1 95.3 ± 3.7 102.3 ± 4.5 110.0 ± 3.6 103.0 ± 4.0
1, Mean± Standard deviation.

2, See table. 3 for abbreviations

pH 6-10
TSS 150
TDS 3500
Cl 1000
42
b. Heavy Metals Parameters
Heavy metals in water sample B from Sardaryab (site 2) showed higher concentration than
water sample A. Water sample B from this site had cadmium concentration ranged between 79-87
µg L-1 with mean values of 83.0 ± 4.0 µg L-1, 81.9 ± 1.0 µg L-1 and 85.6 ± 2.3 µg L-1 for high flow
(summer) and concentration ranged between 61 - 68 µg L-1 with mean values of 63.3 ± 2.5 µg L-1 ,
27.6 ± 0.5 µg L-1 and 65.6 ± 2.0 µg L-1 for low flow (winter) periods (Tables 2 - 17 and Figs. 4 - 20).
The minimum concentration recorded for chromium was 45 µg L-1 and maximum
concentration of 57 µg L-1 with mean values of 49.0 ± 4.0 µg L-1, 52.3 ± 1.5 µg L-1 and 54.6 ± 2.0 µg
L-1 for summer and minimum concentration 23 µg L-1 and maximum 31.1 µg L-1 with mean values
of 28.6 ± 2.0 µg L-1, 29.7 ± 1.5 µg L-1 and 24.3 ± 1.5 µg L-1 for winter season. The copper of all the
water samples at this site was in the range of 24-28.6 µg L-1 with mean values of 26.3 ± 2.0 µg L-1,
26.5 ± 2.3 µg L-1 and 27.9 ± 1.0 µg L-1 for high flow and was ranged between 27-37 µg L-1 with mean
values of 33.3 ± 1.1 µg L-1, 27.6 ± 0.5 µg L-1 and 35.3 ± 1.5 µg L-1 for low flow seasons. The iron
concentration ranged between 51 µg L-1 and 57 µg L-1 with mean values of 54.0 ± 3.0 µg L-1, 54.1 ±
1.0 µg L-1 and 54.9 ± 0.9 µg L-1 during summer and concentration ranged between 31 µg L-1 and 39
µg L-1 with mean values of 35.0 ± 3.6 µg L-1, 64.8 ± 2.5 µg L-1 and 32.3 ± 1.5 µg L-1 during winter
season. In this study values for Zn, Ni, Cr, Cu and Cd were higher and Fe, Mn and Pb and Hg were
lower as compare to values mentioned by Yousufzai, (2004). Similarly manganese at this point had
minimum concentration of 51 µg L-1 and maximum concentration of 58 µg L-1 with mean values of
54.0 ± 1.7 µg L-1, 55.7 ± 1.9 µg L-1 and 55.0 ± 3.6 µg L-1 during high flow and concentration varied
between 39.4-43 µg L-1 during low flow seasons. The nickle concentration was 70-78 µg L-1 with
mean values 75.3 ± 3.0 µg L-1, 72.6 ± 3.0 µg L-1 and 74.5 ± 3.1 µg L-1 during summer and
concentration was 61-68 µg L-1 during high winter seasons. In a past study Khattak & Rehman, (1992)
had investigated the water of the River Kabul for different heavy metals like Cd, Cr, Ca, Pb, Fe, Mn
and Zn. The analysis of these effluents showed high concentration of Cd, Cu, and Fe. Lead
concentration had varied a range from 164-189 µg L-1 with mean values of 181.6 ± 7.0 µg L-1, 172.6
± 3.0 µg L-1 and 166.6 ± 3.0 µg L-1 for high flow and concentration varied ranged 71-78 µg L-1 with
mean values of 75.3 ± 3.0 µg L-1, 72.9 ± 1.7 µg L-1 and 74.3 ± 2.8 µg L-1 for low flow seasons.
Similarly minimum concentration recorded for zinc was 233 µg L-1 and maximum concentration of
243 µg L-1 with mean values of 236.0 ± 4.3 µg L-1, 241.0 ± 3.5 µg L-1 and 235.9 ± 1.0 µg L-1 for
summer and minimum concentration 211 µg L-1 and maximum concentration 234 µg L-1 with mean
values of 215.0 ± 3.0 µg L-1, 232.4 ± 1.5 µg L-1 and 213.3 ± 2.5 µg L-1 for winter season. These results
are in agreement with the researches of Akif, et al., 2002 and Yousufzai, et. al., (2008). The sequence
of these parameters in water sample B was Zn > Pb > Cd > Ni > Mn > Fe > Cr > Hg > Cu and Zn >
43
Pb > Ni > Cd > Fe > Mn > Cu > Cr > Hg for winter season. Comparing our study with the findings
of above researchers showed that heavy metal and physico-chemical parameters concentrations are
the main pollutants for the River Kabul and levels of these parameters have increased in River Kabul
in the last few years and both heavy metal and physico-chemical parameters also showed increasing
tendency in water samples from this point as compare to those with Michini bridge below Warsak
dam. The increasing in level of heavy metal and physico-chemical parameters could be correlated
with effluents from factories, mills and sewages from Peshawar city. All the nine heavy metals like
Cd, Cu, Cr Fe, Hg, Mn, Ni, Pb and Zn studied in the water sample B from River Kabul showed
increasing tendency on comparison with water samples from reference site 1. All heavy metals except
Hg at this point were within permissible limits laid down by NEQS.
As again all physico-chemical and heavy metal parameters except TSS had values within the
permissible range proposed by NEQS, therefore water quality at this point seem fit for aquatic life.
However all the parameters were showing increasing tendency when compared to reference site
Michini Bridge below Warsak dam water samples. This water sample point was 35 km downstream
from the Warsak dam and 20 km upstream from the confluence point at Amangarh (Nowshera). On
downstream journey from Warsak dam, the river passes through a number of villages and the city of
Peshawar. Therefore city sewage and effluents from many factories and mills and other installations
in the vicinity of River Kabul tributaries also joins the river. Moreover dirty water of River Bara also
joins the river on the downstream. Therefore it is obvious that water quality of the river would be
deteriorated downstream.
44
Table 8: Heavy metals concentration (µg/L) of water sample from Sardaryab during winter
and summer (2013-2015).
Heavy S. 2013 W.2013 S.2014 W.2014 S.2015 W.2015

Metals
Cd 83.0 ± 4.0 63.3 ± 2.5 81.9 ± 1.0 27.6 ± 0.5 85.6 ± 2.3 65.6 ± 2.0
Cr 49.0 ± 4.0 28.6 ± 2.0 52.3 ± 1.5 29.7 ± 1.5 54.6 ± 2.0 24.3 ± 1.5
Cu 26.3 ± 2.0 33.3 ± 1.1 26.5 ± 2.3 27.6 ± 0.5 27.9 ± 1.0 35.3 ± 1.5
Fe 54.0 ± 3.0 35.0 ± 3.6 54.1 ± 1.0 64.8 ± 2.5 54.9 ± 0.9 32.3 ± 1.5
Hg 36.3 ± 15.0 18.5 ± 6.8 37.3 ± 12.5 31.3 ± 11.5 36.3 ± 15.0 18.5 ± 6.5
Mn 54.0 ± 1.7 41.6 ± 1.5 55.7 ± 1.9 40.1 ± 0.8 55.0 ± 3.6 41.6 ± 1.5
Ni 75.3 ± 3.0 63.3 ± 2.5 72.6 ± 3.0 63.0 ± 2.0 74.5 ± 3.1 65.6 ± 2.0
Pb 181.6 ± 7.0 75.3 ± 3.0 172.6 ± 3.0 72.9 ± 1.7 166.6 ± 3.0 74.3 ± 2.8
Zn 236.0 ± 4.3 215.0 ± 3.0 241.0 ± 3.5 232.4 ± 1.5 235.9 ± 1.0 213.3 ± 2.5
P < 0.05
1, Mean± Standard deviation. 2, See table 3 for abbreviations.

Cd 0.1 100
Cr 1.0 1000
Cu 1.0 1000
Fe 8.0 8000
Hg 0.01 10
Mn 1.5 1500
Ni 1.0 1000
Pb 0.5 500
Zn 0.5 500
45
2.7.1.3 Water sample C from River Kabul at Aman garh (site 3)
Water samples C from polluted site 3 downstream to the site B were taken and studied for
various physico-chemical and heavy metal parameters as already mentioned to examine the degree
of pollution in the River Kabul at this point. This was the site from where freshwater mussels’ samples
were collected. Comparing the data from Michini Bridge below Warsak dam (reference site) shows
that a considerable increase in pollution has occurred. Most of the parameters have increased showing
signs of increased localized pollution in the river, most probably caused by effluents (Tables 2 - 17
and Figs. 4 - 20).
The water sample C from this site of River Kabul had a pH ranged between 7.3 and 7.9 with
mean values of 7.5 ± 0.3, 7.7 ± 0.1 and 7.5 ± 0.1 during summer (high flow) and ranged between 7.4
and 7.8 with mean values of 7.5 ± 0.3, 7.6 ± 0.1 and 7.5 ± 0.2 during winter (low flow) seasons
respectively. Total suspended solid (TSS) concentration was in the range of 423-731 mg/l with mean
values of 429.6 ± 6.1 mg L-1, 722.6 ± 9.0 mg L-1 and 547.3 ± 6.0 mg L-1 for summer and values ranged
between 509-743 mg L-1 with mean values of 514.0 ± 6.2 mg L-1, 629.3 ± 5.6 mg L-1 and 737.6 ± 5.5
mg L-1 for winter season. Total dissolved solid (TDS) (of all water samples C was in the range of 423-
472 mg L-1 with mean values of 429.6 ± 6.1 mg L-1, 443.6 ± 16.0 mg L-1 and 463.6 ± 10.4 mg L-1 and
ranged between 542-743 mg L-1 with mean values of 593.6 ± 10.0 mg L-1, 556.0 ± 13.5 mg L-1 and
729.0 ± 15.7 mg L-1 for both high and low flow seasons respectively.) The electrical conductivity in
water sample from Warsak dam was ranged between 279 µs/cm and 565 µs/cm with mean values of
285.3 ± 5.6 µs/cm, 364.6 ± 6.5 µs/cm and 556.0 ± 8.1µs/cm during summer and values ranged
between 383-471 µs/cm with mean values of 388.0 ± 4.5 µs/cm, 464.6 ± 7.7 µs/cm and 445.6 ± 12.3
µs/cm during winter. The present results of higher pH, TSS, TDS contents in samples of water taken
from this site agree with the findings of Adeogun (2012). On the other hand, the present data for K,
total alkalinity, Na, Cl and electrical conductivity agree with those of Subramanian (2004) and Ali, et
al., (2012). In this study chloride concentration ranged between 8 - 22 mg L-1 with mean values of
11.3 ± 3.0 mg L-1, 15.0 ± 3.6 mg L-1 and 17.3 ± 4.0 mg L-1 for high flow and concentration varied
between 9-13 mg L-1 with mean values of 14.6 ± 3.5 mg L-1, 13.0 ± 4.0 mg L-1 and 17.6 ± 4.1 mg L-1
for low flow seasons. The potassium (K) range was 2.4-3 mg L-1 with mean values of 2.6 ± 0.2 mg L-
1
, 2.7 ± 0.3 mg L-1 and 2.6 ± 0.2 mg L-1 during for summer and concentration ranged between 1.4-2.7
mg L-1 with mean values of 1.7 ± 0.2 mg L-1, 1.8 ± 0.5 mg L-1 and 1.9 ± 0.6 mg L-1 during winter
seasons. The sodium ranged between 11 - 18 mg L-1 with mean values of 15.6 ± 4.1 mg L-1, 14.6 ±
46
2.5 mg L-1 and 15.6 ± 3.2 mg L-1 for high flow and ranged between 11 - 19 mg L-1 with mean values
of 15.0 ± 3.6 mg L-1, 17.3 ± 2.8 mg L-1 and 15.0 ± 2.0 mg L-1 for low flow seasons. Similarly water
sample from this site had total alkalinity in the range of 92 -123 mg L-1 with mean values of 112.3 ±
5.5 mg L-1, 100.0 ± 7.5 mg L-1 and 116.6 ± 6.0 mg L-1 for summer and values varied between 96-121
mg L-1 with mean values of 111.3 ± 8.6 mg L-1, 108.6 ± 11.1 mg L-1 and 117.6 ± 4.1 mg L-1 for high
flow periods. These parameters in the water sample from this site were in the sequence of TSS >
TDS > EC > TA > Na > Cl > pH > K for summer and TSS > TDS > EC > TA > Na > Cl > pH > K
for winter seasons respectively. This study found more values for TDS, TSS, EC, TA, Na, Cl, K and
pH as compare to the previous investigations (Akif et al., 2002). Previously Yousafzai (2004) and
Siraj (2016) have also reported high mean values for pH, TDS, EC, Cl, K, Na and TA from the same
water resource of River Kabul that verifying the validity of present study. Comparing our data with
the finding of other mentioned studies indicate that all studied physico-chemical parameters, when
compared with Michini Bridge (reference site) are great in concentration and showing an increasing
tendency in river at this point.
All the physico-chemical parameters were below the permissible limits proposed by NEQS
except TSS, which exceeds the NEQS recommended limit of 150 mg L-1 for this parameter. Total
suspended solid (TSS) concentration was in the range of 423 - 731 mg L-1 with mean values of 429.6
± 6.1 mg L-1, 722.6 ± 9.0 mg L-1 and 547.3 ± 6.0 mg L-1 for summer and values ranged between 509-
743 mg L-1 with mean values of 514.0 ± 6.2 mg L-1, 629.3 ± 5.6 mg L-1 and 737.6 ± 5.5 mg L-1 for
winter season. All these values were greater than the value of 150 mg L-1 proposed by NEQS for this
parameter. These values 429.6 ± 6.1 mg L-1, 722.6 ± 9.0 mg L-1 and 547.3 ± 6.0 mg L-1 and 514.0 ±
6.2 mg L-1, 629.3 ± 5.6 mg L-1 and 737.6 ± 5.5 mg L-1 for both lower and higher flows were higher
than the recorded values for the same periods 599.3 ± 13.0 mg L-1, 706.6 ± 4.0 mg L-1 and 725.0 ± 4.0
mg L-1 and 405.0 ± 10.4 mg L-1, 385.0 ± 4.0 mg L-1 and 415.3 ± 0.3 mg L-1 from Michini bridge below
Warsak dam. The TSS value was highest for high flow and lowest for low seasons. This could be
correlated to sewage, industrial effluents, flooding, soil erosion etc. in the river at this site. All the
physico-chemical parameters showed a considerable increase during low and high flow seasons in
relation to Michini Bridge (reference). Rest of the parameters were within the NEQS range. A
decrease in the pH level as compare to water sample A from Michini Bridge and water sample B
from the main river upstream was also observed showing deterioration of the river quality
downstream. Water sample C possessed all the parameters higher than the water sample A from
Warsak dam and water sample B upstream from the main river.
47
Table 10: Physico-chemical characteristics of water sample from Aman Garh during winter
and summer (2013-2015).
pH 7.5 ± 0.3 7.5 ± 0.3 7.7 ± 0.1 7.6 ± 0.1 7.5 ± 0.1 7.5 ± 0.2
TSS (mg/l) 429.6 ± 6.1 514.0 ± 6.2 722.6 ± 9.0 629.3 ± 5.6 547.3 ± 6.0 737.6 ± 5.5
TDS (mg/l) 429.6 ± 6.1 593.6 ± 10.0 443.6 ± 16.0 556.0 ± 13.5 463.6 ± 10.4 729.0 ± 15.7
EC (µs/cm) 285.3 ± 5.6 388.0 ± 4.5 364.6 ± 6.5 464.6 ± 7.7 556.0 ± 8.1 445.6 ± 12.3
Cl (mg/l) 11.3 ± 3.0 14.6 ± 3.5 15.0 ± 3.6 13.0 ± 4.0 17.3 ± 4.0 17.6 ± 4.1
K (mg/l) 2.6 ± 0.2 1.7 ± 0.2 2.7 ± 0.3 1.8 ± 0.5 2.6 ± 0.2 1.9 ± 0.6
Na (mg/l) 15.6 ± 4.1 15.0 ± 3.6 14.6 ± 2.5 17.3 ± 2.8 15.6 ± 3.2 15.0 ± 2.0
TA (mg/l) 112.3 ± 5.5 111.3 ± 8.6 100.0 ± 7.5 108.6 ± 11.1 116.6 ± 6.0 117.6 ± 4.1
P < 0.05
1. Mean± Standard deviation. 2. See table 3 for abbreviations.

pH 6-10
TSS 150
TDS 3500
Cl 1000
48
b. Heavy Metals Parameters (Aman Garh).
From amongst the heavy metals water sample C from site 3 had cadmium concentration ranged
between 83.8-89 µg L-1 with mean values of 87.6 ± 1.5 µg L-1 85.4 ± 1.4 µg L-1 and 86.9 ± 0.9 µg L-1
for summer and ranged between 65 - 76 µg L-1 with mean values of 72.3 ± 3.2 µg L-1, 67.0 ± 2.0 µg
L-1 and 70.7 ± 1.3 µg L-1 for winter. (Tables 2 - 17 and Figs. 4 - 20).
The minimum concentration recorded for chromium was 51 µg/l and maximum concentration of
62.7 µg L-1 with mean values of 55.3 ± 4.0 µg L-1, 59.0 ± 2.3 µg L-1 and 43.5 ± 2.0 µg L-1 for high
flow and minimum concentration was 29 µg L-1 and maximum concentration was 39.8 µg L-1 with
mean values of 37.9 ± 2.5 µg L-1, 33.0 ± 2.6 µg L-1 and 30.7 ± 1.5 µg L-1 for low flow periods. The
copper of all the water samples at this site was in the range of 24-34 µg L-1 with mean values of 26.6
± 2.5 µg L-1, 27.9 ± 1.0 µg L-1 and 31.9 ± 2.5 µg L-1 and ranged between 24.8-35.3 µg L-1 with mean
values of 34.1 ± 1.1 µg L-1, 26.2 ± 1.6 µg L-1 and 33.2 ± 1.1 µg L-1 for both high and low flow seasons.
Iron content ranged between 54 µg L-1 and 64 µg L-1 with mean values of 55.6 ± 2.0 µg L-1, 58.2 ±
1.0 µg L-1 and 59.0 ± 1.7 µg L-1 during summer and values ranged between 32 µg L-1 and 45 µg L-1
with mean values of 37.5 ± 11.2 µg L-1, 35.3 ± 3.5 µg L-1 and 37.3 ± 0.8 µg L-1 during winter season.
Similarly manganese at this point had minimum range of 53 µg L-1 and maximum range of 62.4 µg
L-1 with mean values of 60.8 ± 1.7 µg L-1, 59.0 ± 3.0 µg L-1 and 53.4 ± 0. µg L-1 during high flow and
minimum range of 41 µg L-1 and maximum range of 48 µg L-1 with mean values of 44.6 ± 1.5 µg L-
1
, 35.3 ± 3.5 µg L-1 and 47.0 ± 1.7 µg L-1 during low flow seasons. Nickel concentration was ranged
between 72-79.2 µg L-1 with mean values of 77.4 ± 1.6 µg L-1, 76.6 ± 1.5 µg L-1 and 73.8 ± 1.6 µg L-
1
during summer and ranged between 64-74 µg L-1 with mean values of 69.5 ± 1.2 µg L-1, 65.9 ± 2.7
µg L-1 and 71.8 ± 2.3 µg L-1 during winter seasons. The lead of all the water samples at this site was
in the range of 173 - 190 µg L-1 with mean values of 184.8±3.6 µg L-1, 173.8 ± 0.8 µg L-1 and 189.2
± 1.1 µg L-1 and values varied between 74 - 86 µg L-1 with mean values of 75.5±1.5 µg L-1, 77.7 ± 2.3
µg L-1 and 84.8 ± 1.2 µg L-1 for low and high flows seasons respectively. These results are in
agreement with previous findings as reported by (El-Ezaby, 1994; IUCN, 1994; Khan et al., 2011).
Zinc contents had a range varying between 232 - 248.7 µg L-1 with mean values of 235.0 ± 3.6 µg L-
1
, 247.5 ± 1.4 µg L-1 and 246.6 ± 1.5 µg L-1 during summer and ranged varied between 219 - 238 µg
L-1 with mean values of 219.9 ± 2.6 µg L-1, 235.6 ± 2.1 µg L-1 and 227.6 ± 1.2 µg L-1 during winter
season. (Mercury was in the range between 27-41 µg/l with mean values of 37.3 ± 12.5µg/l for low
flow and 43.0 ± 12.9 µg/l for high flow periods. All the heavy metals except Hg at this point were
within permissible limits laid down by NEQS and showed an increasing tendency at this point than
the Michini Bridge water. The heavy metal parameters in water sample C from this site were in the
order of Zn > Pb > Cd > Ni > Mn > Fe > Cr > Hg > Cu for summer and Zn > Pb > Cd > Ni > Mn >
49
Hg > Fe > Cr > Cu for winter seasons respectively. In a previous finding Ali et al (2012) have
investigated that throughout the world, pollution due to heavy metals in the aquatic environment is a
very growing and serious problem. Similarly in another finding Enrique et al (2007) and Authman,
et al., (2013) have found that increasing in the number and amount of commercial agricultural and
industrial chemicals that are dumped into the aquatic environment had various harmful effects on
aquatic organisms. The increased load of heavy metals in Kabul River has adverse effects on flora
and fauna.
All heavy metals like Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn studied in the water sample C
from River Kabul showed increasing tendency on comparison with water samples from Michini
Bridge below Warsak dam and water sample B. All heavy metals except Hg at this point were within
permissible limits laid down by NEQS. The Hg mean values for low flow and high flow periods were
of 37.3 ± 12.5µg/l and 43.0 ± 12.9 µg L-1 respectively. All the studied heavy metals when compared
with Michini Bridge below Warsak dam (reference site) are high in concentration and showing
increasing tendency due to dumping of industrial effluents in river at this point. Zinc from this site
showed great increase due to various zinc emitting industries present in the vicinity of River Kabul.
The heavy metals concentration at this point of the river were higher than the Michini bridge below
Warsak dam samples and main River upstream to the confluence point samples, indicated increasing
tendency due to dumping of industrial effluents into River Kabul (Tables 2 - 17 and Figs. 4 - 20).
50
Table 12: Heavy metals concentration (µg/L) of water sample from Aman Garh during winter
and summer (2013-2015).
Heavy S. 2013 W.2013 S.2014 W.2014 S.2015 W.2015

Metals
Cd 87.6 ± 1.5 72.3 ± 3.2 85.4 ± 1.4 67.0 ± 2.0 86.9 ± 0.9 70.7 ± 1.3
Cr 55.3 ± 4.0 37.9 ± 2.5 59.0 ± 2.3 33.0 ± 2.6 43.5 ± 2.0 30.7 ± 1.5
Cu 26.6 ± 2.5 34.1 ± 1.1 27.9 ± 1.0 26.2 ± 1.6 31.9 ± 2.5 33.2 ± 1.1
Fe 55.6 ± 2.0 37.5 ± 11.2 58.2 ± 1.0 35.3 ± 3.5 59.0 ± 1.7 37.3 ± 0.8
Hg 36.3 ± 15.0 37.3 ± 12.5 43.0 ± 12.9 39.7 ± 11.8 41.0 ± 14.8 37.3 ± 12.7
Mn 60.8 ± 1.7 44.6 ± 1.5 59.0 ± 3.0 35.3 ± 3.5 53.4 ± 0.5 47.0 ± 1.7
Ni 77.4 ± 1.6 69.5 ± 1.2 76.6 ± 1.5 65.9 ± 2.7 73.8 ± 1.6 71.8 ± 2.3
Pb 184.8 ± 3.6 75.5 ± 1.5 173.8 ± 0.8 77.7 ± 2.3 189.2 ± 1.1 84.8 ± 1.2
Zn 235.0 ± 3.6 219.9 ± 2.6 247.5 ± 1.4 235.6 ± 2.1 246.6 ± 1.5 227.6 ± 1.2
P < 0.051, Mean± Standard deviation. 2, See table 3 for abbreviations.

Cd 0.1 100
Cr 1.0 1000
Cu 1.0 1000
Fe 8.0 8000
Hg 0.01 10
Mn 1.5 1500
Ni 1.0 1000
Pb 0.5 500
Zn 0.5 500
51
2.7.1.4 Polluted River Kabul water from Peer Sabaq, site 4 receiving cities sewages (Water
sample D)
Water sample D from site 4 downstream to site 3 was taken and analyzed for various physico-
chemical and heavy metal parameters as already mentioned. This was the site from which freshwater
mussels samples were collected. At this point Kalpani nalla from Mardan district joins the River
Kabul on opposite side. The quantity of water down stream of this point is considerably deteriorated
(Siraj, 2016). All physico-chemical parameters were below the permissible limits proposed by NEQS
except TSS, which exceeds the NEQS recommended limit of 150 mg L-1 for this parameter. Samples
of Water from this site were analyzed for different physico-chemical and heavy metal parameters to
quantify the extent of pollution at this point (Tables 2 - 17 and Figs. 4 - 20).
Some physico-chemical parameters are components of industrial waste products, which are
discharged into the River Kabul along with other industrial effluents and caused aquatic pollution. All
physico-chemical parameters showed a considerable increase during low and high flow periods in
relation to Michini Bridge (reference site). The water sample D from this point had a pH in the range
of 7.4 - 7.9 with mean values of 7.5 ± 0.2, 7.6 ± 0.1 and 7.7 ± 0.1 during summer and had rang 7.4 -
7.9 with mean values of 7.5 ± 0.2, 7.6 ± 0.2 and 7.7 ± 0.2 during winter seasons. In a past study IUCN
(1994) have also reported higher concentration for pH in water of River Kabul above than the
recommended standard for industrial effluents. Total suspended solid (TSS) was in the range of 741
- 798 mg/l with mean values of 790.0 ± 7.5 mg L-1, 755.6 ± 15.5 mg L-1 and 745.0 ± 3.0 mg L-1 for
high flow and values ranged between 412 - 798 mg L-1 with mean values of 427.3 ± 14.5 mg L-1,
564.3 ± 11.7 mg L-1 and 547.0 ± 10.5 mg L-1 for low period. The TSS value was greatest for high flow
and lowest for low flow seasons. This could be correlated to sewage, industrial effluents and flooding
and soil erosion in the river. In another investigation Ali (1991) had also reported a high TSS level in
the water samples from River Swat. Similarly total dissolved solid (TDS) at this point had minimum
range of 531 mg L-1 and a maximum range of 682 mg L-1 with mean values of 608.3 ± 63.9 mg L-1,
540.0 ± 8.1 mg L-1 and 551.0 ± 10.1 mg L-1 during summer and minimum range 432 mg L-1 and a
maximum range 761 mg L-1 with mean values of 444.0 ± 12.5 mg L-1, 651.0 ± 17.6 mg L-1 and 754.0
± 9.6 mg L-1 during winter seasons. The electrical conductivity in water sample D was ranged between
354 - 782 µs/cm with mean values of 358.3 ± 4.0 µs/cm, 480.6 ± 114.1 µs/cm and 778.6 ± 4.1 µs/cm
during high flow period and values had ranged 433 - 789 µs/cm with mean values of 441.0 ± 9.1
µs/cm, 777.6 ± 10.2 µs/cm and 685.0 ± 5.5 µs/cm during low flow period. The chloride concentration
of all the samples at this point was in the range of 12 - 25 mg L-1 with mean values of 17.6 ± 5.5 mg
52
L-1, 19.0 ± 5.5 mg L-1 and 21.3 ± 2.5 mg L-1 for summer season and ranged between 15 mg L-1 and 21
mg L-1 with mean values of 17.0 ± 2.1 mg L-1, 17.3 ± 2.0 mg L-1 and 19.6 ± 1.5 mg L-1 for winter
season. The potassium concentration ranged between 2.3 mg L-1 and 3.3 mg L-1 with mean values of
2.5 ± 0.2 mg L-1, 2.8 ± 0.4 mg L-1 and 2.6 ± 0.2 mg L-1 during high flow and ranged between 1.9 mg
L-1 and 2.9 mg L-1 with mean values of 2.6 ± 0.3 mg L-1, 2.6 ± 0.3 mg L-1 and 2.3 ± 0.4 mg L-1 during
low flow periods. These values were greater than the findings of other workers from the same water
resources (Guidelines., 1996; Khan et al., 1999; Akif et al., 2002; Yousafzai et al., 2010 b).
The sodium concentration was ranged between 13 mg L-1 and 20 mg L-1 with mean values of
17.3 ± 2.0 mg L-1, 16.6 ± 3.2 mg L-1 and 17.6 ± 2.5 mg L-1 for high flow and ranged between 14 mg
L-1 and 23 mg L-1 with mean values of 17.3 ± 2.0 mg L-1, 17.6 ± 2.5 mg L-1 and 19.3 ± 3.5 mg L-1 for
low flow seasons. Similarly the water samples from this site had total alkalinity in the range of 106 -
127 mg L-1 with mean values of 109.6 ± 3.5 mg L-1, 120.0 ± 7.9 mg L-1 and 120.6 ± 7.7 mg L-1 for
summer and in a range of 109 - 123 mg L-1 with mean values of 117.6 ± 3.5 mg L-1, 115.6 ± 7.0 mg
L-1 and 117.6 ± 7.5 mg L-1 for winter periods. These parameters in water sample D from this site were
in the order of TSS > TDS > EC > TA > Cl > Na > pH > K for summer and EC > TDS > TSS > TA
> Na > Cl > pH > K winter seasons respectively. In a past finding Siraj (2016) and Yousafzai (2004)
have also reported greater mean values for pH, total dissolved solid, electrical conductivity, chloride,
potassium, sodium and total alkalinity from the same water resource of River Kabul verifying the
validity of our study. All physico-chemical parameters in water sample D had higher concentration
than the water samples A, B and C. Total suspended solid (TSS) was in the range of 741 - 798 mg/l
with mean values of 790.0 ± 7.5 mg L-1, 755.6 ± 15.5 mg L-1 and 745.0 ± 3.0 mg L-1 for high flow
and values ranged between 412 - 798 mg L-1 with mean values of 427.3 ± 14.5 mg L-1, 564.3 ± 11.7
mg L-1 and 547.0 ± 10.5 mg L-1 for low period were greater than the NEQS recommended limit of
150 mg L-1.
53
Table 14: Physico-chemical characteristics of water sample D from Peer Sabaq during winter
and summer season during 2013-2015.
pH 7.5 ± 0.2 7.5 ± 0.2 7.6 ± 0.1 7.6 ± 0.2 7.7 ± 0.1 7.7 ± 0.2
TSS (mg/l) 547.0 ± 10.5 427.3 ± 14.5 755.6 ± 15.5 564.3 ± 11.7 745.0 ± 3.0 790.0 ± 7.5
TDS (mg/l) 608.3 ± 63.9 444.0 ± 12.5 540.0 ± 8.1 651.0 ± 17.6 551.0 ± 10.1 754.0 ± 9.6
EC (µs/cm) 358.3 ± 4.0 441.0 ± 9.1 480.6 ± 114.1 777.6 ± 10.2 778.6 ± 4.1 685.0 ± 5.5
Cl (mg/l) 17.6 ± 5.5 17.0 ± 2.1 19.0 ± 5.5 17.3 ± 2.0 21.3 ± 2.5 19.6 ± 1.5
K (mg/l) 2.5 ± 0.2 2.6 ± 0.3 2.8 ± 0.4 2.6 ± 0.3 2.6 ± 0.2 2.3 ± 0.4
Na (mg/l) 17.3 ± 2.0 17.3 ± 2.0 16.6 ± 3.2 17.6 ± 2.5 17.6 ± 2.5 19.3 ± 3.5
TA (mg/l) 109.6 ± 3.5 117.6 ± 3.5 120.0 ± 7.9 115.6 ± 7.0 120.6 ± 7.7 117.6 ± 7.5
1, Mean± Standard deviation.

2, See table 3 for abbreviations.
54
b. Heavy Metals Parameters
All heavy metal parameters in water from this site 4 showed a considerable increasing
tendency during low and high flow seasons in relation to reference water samples from Michini
Bridge below Warsak dam (Tables 2 - 17 and Figs. 4 - 20).
Among the heavy metal parameters, water sample D from this site had cadmium in the range
of 89 - 98.9 µg L-1 with mean values of 96.4 ± 1.2 µg L-1, 90.6 ± 1.5 µg L-1 and 96.5 ± 1.6 µg L-1
during summer season and values ranged 72.8 - 83.7 µg L-1 with mean values of 76.9 ± 4.6 µg L-1,
89.8 ± 5.2 µg L-1 and 81.6 ± 2.9 µg L-1 during low flow seasons. The minimum concentration recorded
for chromium was 63.8 µg L-1 and maximum concentration of 67 µg L-1 with mean values of 64.2 ±
0.6 µg L-1, 65.4 ± 1.4 µg L-1 and 65.2 ± 1.0 µg L-1 for high flow and minimum concentration was 36.9
µg L-1 and maximum 48.9 µg L-1 with mean values of 44.6 ± 1.4 µg L-1, 44.8 ± 3.6 µg L-1 and 37.7 ±
0.9 µg L-1 for low flow periods. The findings of this research are accordance to those found by many
researchers (Akoto et al., 2008; Abida et al., 2009; Ajima et al., 2015), who have also studied high
levels of heavy metals in water from other resources. The copper content ranged between 29.5 µg L-
1
and 41 µg L-1 with mean values of 33.6 ± 1.5 µg L-1, 31.5 ± 2.2 µg L-1 and 38.2 ± 3.0 µg L-1 and
content ranged between 31 µg L-1 and 41 µg L-1 with mean values of 37.6 ± 1.5 µg L-1, 34.8 ± 3.9 µg
L-1 and 39.1 ± 2.2 µg L-1 for high flow and low flow periods respectively. The iron concentration
ranged varying between 54 - 71 µg L-1 with mean values of 55.9 ± 1.6 µg L-1, 61.3 ± 2.0 µg L-1 and
67.8 ± 3.0 µg L-1 for summer and concentration range varied between 41 - 51.8 µg L-1 with mean
values of 49.5 ± 1.2 µg L-1, 47.7 ± 0.6 µg L-1 and 42.4 ± 1.2 µg L-1 for summer seasons. Similarly
manganese at this point had minimum concentration of 63 µg L-1 and maximum content of 68 µg L-
1
with mean values of 64.5 ± 1.3 µg L-1, 59.7 ± 10.4 µg L-1 and 64.2 ± 0.6 µg L-1 for high flow and
minimum concentration 46.7 µg L-1 and maximum 51 µg L-1 with mean values of 49.5 ± 1.2 µg L-1,
49.0 ± 1.2 µg L-1 and 48.9 ± 2.1 µg L-1 for high flow seasons.
The nickle in all the samples from this site had a concentration varying between 78 - 83.9 µg
L-1 with mean values of 80.0 ± 1.9 µg L-1, 78.6 ± 4.2 µg L-1 and 80.9 ± 2.9 µg L-1 for summer season
and concentration varying between 73.2 - 81 µg L-1 with mean values of 75.2 ± 1.2 µg L-1, 75.9 ± 2.7
µg L-1 and 76.9 ± 3.9 µg L-1 for winter season. Lead had a range from 181 -205.8 µg L-1 with mean
values of 186.9 ± 1.7 µg L-1, 183.6 ± 2.5 µg L-1 and 203.4 ± 2.1 µg L-1 for high flow and ranged from
85 - 123 µg L-1 with mean values of 120.3 ± 3.0 µg L-1, 89.8 ± 5.2 µg L-1 and 93.1 ± 2.2 µg L-1 for low
flow seasons. Similarly minimum concentration recorded for zinc was 343 µg L-1 and maximum
concentration of 354 µg L-1 with mean value of 345.3 ± 2.5 µg L-1, 350.6 ± 2.9 µg L-1 and 350.3 ± 2.0
µg L-1 for summer and minimum concentration 323 µg L-1 and maximum concentration 342.8 µg L-
1
for winter season. The mercury content ranged between 34µg/l and 45µg/l with mean values of 29.3
55
± 12.4 µg/l for low flow and 41.0 ± 13.9 µg/l for high flow periods respectively. Heavy metal
parameters in water sample D from this site were in the order of (Zn > Pb > Cd > Ni > Cr > Mn > Fe
> Hg > Cu for summer and Zn > Pb > Cd > Ni > Mn > Fe > Cr > Cu > Hg for winter season
respectively. All heavy metals except Hg at this point were within permissible limits laid down by
NEQS. The Hg mean contents for low and high flow seasons were 37.3 ± 12.5 µg L-1 and 41.0 ± 14.8
µg L-1 respectively and show an increasing tendency at this point than Michini Bridge below Warsak
Dam water. This study found maximum level of examined metals from this point as compare to the
previous findings reported by IUCN (1994).
Heavy metals at this site showed higher concentration as compare to Michini Bridge and
upstream of water samples B, C and D and indicating increasing heavy metal pollution in downstream
water. Heavy metals except Hg determined at this point were within permissible limits laid down by
National Environmental Quality Standards (NEQS). But still alarming due to their bioaccumulation
capability especially during low flow season when water volume shrinks. Overall result showed that
industries in the vicinity are discharging effluents containing higher levels of TSS and Hg into the
River Kabul. Comparing heavy metal contents to Michini bridge water (reference site) again showed
a drastic increase in contents of physico-chemical parameters both during winter and summer seasons.
Similarly a further increase had also occurred in heavy metals concentration as compare to water
sample C. As this sample was collected from the portion of the river, where sewages from Nawshera
and Mardan cities also join the river a little upstream, most probably this city sewage may be further
increasing the heavy metal concentration and physico-chemical concentrations. The overall results
show that industries in the vicinity dumping effluents and sewages containing higher levels of TSS
and Hg into River Kabul especially at sites C and D. Thus both sewages and effluents causing both
organic and inorganic pollution in River Kabul. The TSS and Hg in upstream from Michini bridge
(water sample A) and downstream from main river (water sample B), from polluted site 3(water
sample C) and polluted site 4 (water sample D) exceeds the permissible limits proposed by NEQS
during both summer and winter seasons. Both physico- chemical and heavy metal parameters in
downstream river water samples also showed an increasing tendency when compared with upstream
samples showing both physico-chemical and heavy metal stress in downstream portion of River
Kabul. This extensive study confirmed that River Kabul has higher pollution downstream the Michini
Bridge and there is a localized pollution in the vicinity of Peshawar and Nowshera. Similar reports
regarding River Kabul pollution have also been reported in the previous studies (IUCN, 1994; Akif
et al., 2002; Yousafzai, 2004; Siraj, 2016).
This pollution plug might be preventing the freshwater mussels from their migration which
definitely play a vital role in reduction in whole mussel’s population, which are considered as clean
56
water lover and breed in clean water. Moreover this pollution will be lethal to eggs and juveniles as
compare to adults and there is the sure danger for loss of mussels.
Table 15: Heavy metals concentration of water sample D from Peer Sabaq during winter and
summer seasons (2013-2015)

Heavy S. 2013 W.2013 S.2014 W.2014 S.2015 W.2015
Metals
Cd 96.4 ± 1.2 76.9 ± 4.6 90.6 ± 1.5 89.8 ± 5.2 96.5 ± 1.6 81.6 ± 2.9
Cr 64.2 ± 0.6 44.6 ± 1.4 65.4 ± 1.4 44.8 ± 3.6 65.2 ± 1.0 37.7 ± 0.9
Cu 33.6 ± 1.5 37.6 ± 1.5 31.5 ± 2.2 34.8 ± 3.9 38.2 ± 3.0 39.1 ± 2.2
Fe 55.9 ± 1.6 49.5 ± 1.2 61.3 ± 2.0 47.7 ± 0.6 67.8 ± 3.0 42.4 ± 1.2
Hg 41.2 ± 14.8 32.3 ± 11.8 39.3 ± 13.5 29.3 ± 12.4 41.0 ± 13.9 31.3 ± 11.5
Mn 64.5 ± 1.3 49.5 ± 1.2 59.7 ± 10.4 49.0 ± 1.2 64.2 ± 0.6 48.9 ± 2.1
Ni 80.0 ± 1.9 75.2 ± 1.2 78.6 ± 4.2 75.9 ± 2.7 80.9 ± 2.9 76.9 ± 3.9
Pb 186.9 ± 1.7 120.3 ± 3.0 183.6 ± 2.5 89.8 ± 5.2 203.4 ± 2.1 93.1 ± 2.2
Zn 345.3 ± 2.5 328.3 ± 3.0 350.6 ± 2.9 341.9 ± 0.9 350.3 ± 2.0 231.3 ± 8.0
P < 0.05
1. Mean± Standard deviation.
2. See table for abbreviations.
57

Cd 0.1 100
Cr 1.0 1000
Cu 1.0 1000
Fe 8.0 8000
Hg 0.01 10
Mn 1.5 1500
Ni 1.0 1000
Pb 0.5 500
Zn 0.5 500
2.7.1.5 Conclusions and Remarks
The following tables 3.10 heavy metal parameters of different water samples collected from
Michini Bridge (water sample A), polluted River Kabul (water samples B, C and D) during winter
and summer.
Overall results show that industries in the vicinity are discharging effluents containing high
level of TSS and Hg. Level of these parameter was exceeding the permissible limits laid down by
NEQS for effluents but the remaining parameters were within permissible limits provided by NEQS.
The downstream water samples B, C and D from polluted sites 2, 3 and 4 showed an increasing
tendency of both physico-chemical and heavy metal parameters as when compared with water
samples A showing metal stress in downstream portions of river. During low flow period in winter
due to less snow melt at the top of hills, the river water volume reduces about three times, but the
sewage and effluent contents remain the same. During high flow in summer, snow melts at the hills
and results in flooding and hence increases the river volume, which dilutes the pollutants in the river
in these months, except TSS, which exceeded the NEQS limits even in high flow months. Among
heavy metals except Hg studied in the all water samples A, B, C and D were within permissible limits
laid down by NEQS. The high TSS and Hg content reduces light penetration in deeper strata of river
water and thus results in less growth of plankton and algae, which naturally will reduce food and
consequently reduce the muscles yield. Deforestation and mining activities on the hills resulted in soil
erosion and hence increases TSS concentration in river water. Thus different water samples were in
58
the trend of D > C > B > A. This shows that water sample D had highest physico-chemical and heavy
metal parameters followed by water samples C, B and A.
The highest parameters in water sample D may be attributed to effluents and sewages
discharging into River Kabul at this site. Water sample D has received effluents and sewages from
Mardan and Nowshera cities and also received effluents from upstream industries of Aman Ghar.
Therefore here at site D the pollution is high as compare to remaining studied sites of River Kabul.
All investigated metals including Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn, physico-chemical
parameters like TDS, TSS, EC, TA, Na, Cl, K and pH in water samples from polluted water showed
increasing tendency as compare with reference water from Michini bridge. The possible reasons for
this tremendous increase in individual metal and physico-chemical parameter level in water of River
Kabul could be correlated to city sewages and industrial effluents discharges, mining, agricultural and
other anthropogenic activities in the vicinity.
59
Table 17: Comparison of Heavy metals concentration (µg/L) in water of study sites collected
during the summer and winter seasons (2013 – 2015)
Site Season Concentration of Heavy Metals (µg/L)

Cd Cr Cu Fe Mn Ni Pb Zn
2013 S 33.3±4.0 35.3±4.1 23.4±1.3 21.8±2.3 27.0±3.6 62.0±3.0 33.3±1.1 102.0±4.5
2013 W 23.3±2.0 a 20.6±2.5 a 16.6±2.2 a 14.2±1.6 a 20.3±2.3 37.8±2.4a 29.3±2.0 a 92.6±4.0 a
2014 S 36.0±2.6 43.6±2.8 21.9±1.1 24.3±1.5 30.9±1.9 71.7±1.6 42.9±0.7 111.6±3.5
1 2014 W 22.6±1.5 a 21.2±1.6 a 14.1±2.0 a 22.6±1.5 a 20.6±2.5 a 40.3±2.0 a 41.6±3.5 95.6±3.0 a
2015 S 23.6±2.0 37.6±4.1 19.0±2.0 32.0±2.0 34.6±2.0 69.0±2.0 48.6±6.0 109.0±5.0
2015 W 24.0±0.9 23.3±0.6 a 16.3±3.0 20.6±0.5 a 23.0±1.0 a 43.3±1.1 a 48.3±2.3 45.6±3.0 a
2013 S 83.0±4.0 49.0±4.0 26.3±2.0 54.0±3.0 54.0±1.7 75.3±3.0 181.6±7.0 236.0±4.3
2013 W 63.3±2.5 b 28.6±2.0 b 33.3±1.1 b 35.0±3.6 b 41.6±1.5 b 63.3±2.5 b 75.3±3.0 b 215.0±3.0b
2014 S 81.9±1.0 52.3±1.5 26.5±2.3 54.1±1.0 55.7±1.9 72.6±3.0 172.6±3.0 241.0±3.5
2 2014 W 27.6±0.5 b 29.7±1.5 b 27.6±0.5 64.8±2.5 b 40.1±0.8 b 63.0±2.0 b 72.9±1.7 b 232.4±1.5 b
2015 S 85.6±2.3 54.6±2.0 27.9±1.0 54.9±0.9 55.0±3.6 74.5±3.1 166.6±3.0 235.9±1.0
2015 W 65.6±2.0 b 24.3±1.5 b 35.3±1.5 b 32.3±1.5 b 41.6±1.5 b 65.6±2.0 b 74.3±2.8 b 213.3±2.5 b
2013 S 87.6±1.5 55.3±4.0 26.6±2.5 55.6±2.0 60.8±1.7 77.4±1.6 184.8±3.6 235.0±3.6
2013 W 72.3±3.2 c 37.9±2.5 c 34.1±1.1 c 37.5±11.2 c 44.6±1.5 c 69.5±1.2 c 75.5±1.5 c 219.9±2.6c
2014 S 85.4±1.4 59.0±2.3 27.9±1.0 58.2±1.0 59.0±3.0 76.6±1.5 173.8±0.8 247.5±1.4
3 2014 W 67.0±2.0 c 33.0±2.6 c 26.2±1.6 35.3±3.5 c 35.3±3.5 c 65.9±2.7 c 77.7±2.3 c 235.6±2.1 c
2015 S 86.9±0.9 43.5±2.0 31.9±2.5 59.0±1.7 53.4±0.5 73.8±1.6 189.2±1.1 246.6±1.5
2015 W 70.7±1.3 c 30.7±1.5 c 33.2±1.1 37.3±0.8 c 47.0±1.7 c 71.8±2.3 84.8±1.2 c 227.6±1.2 c
2013 S 96.4±1.2 64.2±0.6 33.6±1.5 55.9±1.6 64.5±1.3 80.0±1.9 186.9±1.7 345.3±2.5
2013 W 76.9±4.6 d 44.6±1.4 d 37.6±1.5 d 49.5±1.2 d 49.5±1.2 d 75.2±1.2 d 120.3±3.0 d 328.3±3.0 d
2014 S 90.6±1.5 65.4±1.4 31.5±2.2 61.3±2.0 59.7±10.4 78.6±4.2 183.6±2.5 350.6±2.9
4 2014 W 89.8±5.2 44.8±3.6 d 34.8±3.9 47.7±0.6 d 49.0±1.2 d 75.9±2.7 89.8±5.2 d 341.9±0.9 d
2015 S 96.5±1.6 65.2±1.0 38.2±3.0 67.8±3.0 64.2±0.6 80.9±2.9 203.4±2.1 350.3±2.0
2015 W 81.6±2.9 d 37.7±0.9 d 39.1±2.2 42.4±1.2 d 48.9±2.1 d 76.9±3.9 93.1±2.2 d 231.3±8.0 d
a
P<0.05, bP<0.05,cP<0.05, dP<0.05
60
pH
2013 2014 2015
7.75
7.7
7.65
7.6
7.55
7.5
7.45
7.4
Summer Winter Summer Winter Summer Winter Summer Winter
Michini bridge Sardaryab Aman Garh Peer Sabaq
Figure 4: Comparison of pH water samples from four different sites of River Kabul during summer
and winter.
TSS mg L-1
2013 2014 2015
900
800
700
600
500
400
300
200
100
0
Figure 5: Comparison of TSS (Total Suspended Solids milli gram per liter) in water samples from four different
sites of River Kabul during summer and winter.
61
TDS mg L-1
2013 2014 2015
800
700
600
500
400
300
200
100
0
Figure 6: Comparison of TSS (Total Suspended Solids milli gram per liter) in water samples from four different
EC µs/cm
2013 2014 2015
900
800
700
600
500
400
300
200
100
0
Figure 7: Comparison of EC (Electric Conductivity) of water samples from four different sites of
River Kabul during summer and winter.
62
Cl mg L-1
2013 2014 2015
25
20
15
10
0
Figure 8: Comparison of Cl (Chlorine in milli gram per liter) in water samples from four different
K mg L-1
2013 2014 2015
2.5
1.5
0.5
0
Figure 9: Comparison of K (Potassium in milli gram per liter) in water samples from four different
63
Na mg L-1
2013 2014 2015
25
20
15
10
0
Figure 10: Comparison of Na (Sodium in milli gram per liter) in water samples from four different
TA mg L-1
2013 2014 2015
140
120
100
80
60
40
20
0
Figure 11: Comparison of TA (Total Alkalinityin) in water samples from four different sites of River
Kabul during summer and winter.
64
Figure 12: Comparison of Cd (Cadmium in micro gram per liter) in water samples from four different
Cr µg L-1
2013 2014 2015
70
60
50
40
30
20
10
0
Figure 13: Comparison of Cr (Chromium in micro gram per liter) in water samples from four
different sites of River Kabul during summer and winter.
65
Cu µg L-1
2013 2014 2015
50
45
40
35
30
25
20
15
10
5
0
Figure 14: Comparison of Cu (Copper in micro gram per liter) in water samples from four different
Fe µg L-1
2013 2014 2015
80
70
60
50
40
30
20
10
0
Figure 15: Comparison of Fe (Iron in micro gram per liter) in water samples from four different sites
of River Kabul during summer and winter.
66
Hg µg L-1
2013 2014 2015
50
45
40
35
30
25
20
15
10
5
0
Figure 16: Comparison of Hg (Mercury in micro gram per liter) in water samples from four different
Mn µg L-1
2013 2014 2015
70
60
50
40
30
20
10
0
Figure 17: Comparison of Mn (Menganese in micro gram per liter) in water samples from four different sites
of River Kabul during summer and winter.
67
Ni µg L-1
2013 2014 2015
90
80
70
60
50
40
30
20
10
0
Figure 18: Comparison of Ni (Nickle in micro gram per liter) in water samples from four different sites of River
Pb µg L-1
2013 2014 2015
250
200
150
100
50
0
Figure 19: Comparison of Pb (Lead in micro gram per liter) in water samples from four different
68
Zn µg L-1
2013 2014 2015
400
350
300
250
200
150
100
50
0
Figure 20: Comparison of Zn (Zinc in micro gram per liter) in water samples from four different sites of River
69
CHAPTER 3
HEAVY METALS CONCENTRATION IN SEDIMENTS OF RIVER

KABUL.
3.1. Introduction
In modern environmental research, pollution due to metals in the aquatic ecosystem have got
universal consideration due to their accumulation, persistence and environmental toxicity. Due to
expanding industrial and agricultural production and in addition to fast global growth of population
and severe household actions, great amount of toxic materials particularly heavy metals have
discharged into freshwater all over the world. In large cities the rivers have been also correlated with
problems of water qualities due to the practices of releasing of crud industrial and domestic wastes in
water which results to raise the level of metals in riverine water (Yuane et al., 2011; Srebotnjak et al.,
2012; Su et al., 2013; Islam et al., 2014; Shari et al., 2015).
Heavy metals could go through several variations in their speciation during transport,
adsorption, because of condensation, complexation as well as dissolution process. These variations
influence the bioavailability and manner of heavy metals. The fundamental and active portion of a
river basin is sediment, which have varieties of environments and habitats. Though, information about
total concentrations of metals, are insufficient for determination of ecological impacts of pollution due
to sediment that leads to specific attention of chemical composition of them ( Nwuche and Ugojie,
2010; Nouri et al., 2011; Mohiuddine et al., 2012; Shari et. al., 2015). In aquatic environment, the
relationship of metals with different geochemical phases of sediments strongly influenced the overall
behavior of heavy metals. Geo-chemical distribution as well as speciation of heavy metals in the
definite chemical portion of sediment also been used in guessing the bioavailability, motility and
prospective contamination (Kabala and Singh, 2001; Pueyo et al., 2003; Morillo. et al., 2004; Caeiro
et al., 2005). Therefore, it is essential to evaluate heavy metals distribution and concentrations of in
riverine ecosystem.
Heavy metals are ordinary components of ecosystem, but normally little amount. Level of
metal ions have been increased by human activities in many of these natural aquatic ecosystems.
Mostly, due to domestic and industrial wastes the load of metals in waters increased and finally
accumulated in sediments. River sediment which is a habitat for aquatic living organisms also serves
as a major source of nutrients for them. while living organisms require some essential metals in trace
amounts, excessive levels can be harmful to them (Ansari et al., 2004; De Vries et al., 2007) with the
70
exception of cadmium (Cd), mercury (Hg) and lead (Pb) which are lethal even in least concentrations
(Galas-Gorcher, 1991).
Riverine sediment reveal the past record of contamination of the river. The concentration of
heavy metals in sediments near about this areas can help investigators with indication of the manmade
activities on aquatic environment and consequently, help to assess the hazards related to
anthropogenic wastes discharge. Major ecological complications are produced due to increase of
heavy metals in sediments for indigenous populations, in addition to the water quality of the river. For
instance, many organisms use these sediment as a source of food, these organisms may be the food
of mussels and can be vulnerable to contaminated metals bioaccumulation in the body of mussels.
Now this bioaccumulation could be possibly warn the health of various species like birds, fish and
humans at the top of the food chain due to bio magnification (Jain, 2004; Shari et. al., 2015).
The most important sink of heavy metals and other contaminants in the aquatic system is the
river sediments, as they have the potential to associate with the sediments (Zahra et al., 2014; Diop et
al., 2015 Morina et al., 2015). Heavy metals are serious pollutants because of their non-degradability,
persistence, and toxicity in the ecosystem. In a water column the quality of sediment is an excellent
indicator of heavy metals contamination, where they tend to precipitate. High concentrations of these
heavy metals possibly will effect on sediments dwelling organisms, which may ultimately cause
health risks to human beings (Diop et al., 2015; Naji and Sohrabi, 2015).
Riverine and sedimentary contamination with different contaminants like heavy metals
presents a complex, ever-lasting environmental problem, predominantly in areas with high
anthropogenic pressure. Among the most common environmental contaminants are heavy metals
their presence in sediments and living organisms indicating the existence of anthropogenic or natural
source (Adaikpoh et al., 2005; Akoto et al., 2008; Esmaeilzadeh et al., 2016). Depending upon the
physicochemical condition sediments are the reservoir of nutrients and heavy metals. Hence,
sediments are not only considered as carriers of contaminants like heavy metals but it also serve as
probable alternate origin of pollutants in an aquatic ecosystem Thus, the exploration of sediment is
very significant to assessing the health of aquatic ecosystem (Malherbe et al., 2015; Kralj et al., 2016;
Aamir et al., 2016 b).
Unfortunately, erosion of local soil, overpopulation, intensive deforestation and inadequate
management of water use have extremely diminished the qualities of riverine water and sediment.
Considerably, great uncontrolled effluents discharges, as well as heavy metals from urban and
industrial sources, have contributed to increased pollution of the mainstream and rivers and have
accumulated in the sediments. When environmental conditions of the water changed then from the
sediments heavy metals are released into the water, thereby worsening the water quality (Yao et al.,
71
2007, Mishra, 2008). In the mainstream and rivers, a geochemical sediment analysis of heavy metals
is critical to evaluating the ecological hazards of heavy metals. The speciation and total concentration
determined the possible environmental risks of each heavy metal in sediments (Pagnanelli et al., 2004;
Clozel et al., 2006; Shari et al., 2015).
Heavy metals as well as other contaminants are present in considerable amount in sediments
(C. Ip et al., 2004) and have paid great consideration (Hu et al., 2013a; Zhang et al., 2015a; Zhang et
al., 2015b). Water quality can be effected by contamination due to heavy metal in sediments and
therefore the bioaccumulation and bio-assimilation in organisms, results in ever-lasting consequences
for human health and whole aquatic ecosystem. The depositional characteristics of surface sediments
and pollutants thorough understandings are essential for the evaluation aquatic contamination due to
heavy metals (Ip et al., 2007; Xu et al., 2015b; Xu et al., 2015c).
Sediments of aquatic system have been regarded as an important reservoir for trace metals
and contaminated sediments due to these metals may have a range of toxicological effects on benthic
fauna and associated species. Moreover, heavy metal elements cannot be constantly present in
sediments, as environmental conditions such as pH, salinity, redox potential and suspension changed,
can release some of the sediment-bound metals back to water columns (Roberets, 2012; Hiill. et al.,
2013). Thus, sediments have a basic function in deposition and transmission of the metals. It is useful
to do topographical analysis of metal content in the sediments to guess pollution status in the aquatic
environment and to contribute essential reports for the assessment of ecological health threats (Longe.
et al., 2006; Simpson and Batley, 2007; Li. et al., 2012).
Sandy sediments of polluted stations contain more metals contaminants than fine grain
sediments from non-polluted stations. In sediments at stations near landfills the leachable metals
concentration is 100 times higher than background values. In detrital sediments, there are two main
causes of heavy metals, i.e. anthropogenic (manmade) contaminations and natural inputs; the first one
include fossil fuel combustion (natural gas, petroleum and coal), and biomass, waste burning, along
with smelting and mining industries (Nriagu and Pacyna, 1988; Pan and Wang, 2012). The
comparative influences of manmade sources to worldwide discharges of heavy metals have been
significantly altered time to time. For chronological study of anthropogenic pollution the sediment in
aquatic environments has become an ecological record (Qiu et al., 2007; Krome et al., 2009). For the
assessment of the effects of natural and anthropogenic processes on depositional environments the
geochemical composition of ancient sediment cores are outstanding tools. To examine and update the
past records of pollutants discharges in these environments, the sediment cores from lake and river
have used in various current investigations (Audrye et al., 2004; Keldrman and Osman, 2007;
72
Morellie et al., 2012; Townsende and Seene, 2012; Azourye et al., 2013; Duaan et al., 2014; Xu et
al., 2014; Vallius, 2014; Choi et al., 2015).
However, the environmental proceedings and bioavailability of metals in sediments are not
only associated to their total concentrations, but also are determined to a large extent by their chemical
speciation. Thus, the research about the transportation and speciation of heavy metals in sediments
has become one of the most significant part of environmental study (Chakraborty et al., 2012; Yang
et al., 2013; Shari et al., 2015).
In aquatic ecosystem, the sinks and carriers of pollutants are the sediments. The great cause
of environmental contamination in rivers sediments and living organisms is heavy metal and its
quantity in both waters and living things designate the occurrence of anthropogenic or natural sources.
The chief human actions, industrialization and wastes from cities and the weathering of sediments are
the natural sources of metals which are dumped into aquatic environment. The metals could be both
accumulated in benthic organism or adsorbed onto sediments, occasionally to toxic stage.
Consequently the successive toxicity and bioavailability of metals is a main assessment field (Klavins
et al., 2000; Gonzalez et al., 2000).
During transport heavy metals could be scattered in two phases the aqueous and in the form
of small suspended sediments. By the process of precipitation or adsorption, the suspended load and
sediments of river have the essential function of buffering of concentrations heavy metal. Numerous
researches have investigated that in the hydrological systems the metals concentrations in basin and
suspended sediment could be best index of pollutants. The particle size and composition of the
sediments can affect the occurrence of trace metals in sediments (Vutukuru, 2005; Yousafzai and
Shakoori, 2008). The sediments of river play important role in transportation of heavy metals to the
oceans which is more than 97 %. Through joint actions of precipitation, hydrolysis and adsorption
merely a greater quantity of free metal ions get accumulated in the sediments while a minor quantity
dissolved in water. Though, for the overlying column of waters, the conversion of sediments takes
place from the main sink to sources of heavy metals when environmental conditions changed. In
sediments the content of heavy metals are frequently detected to give rudimentary information for the
assessment of ecological risks. In lakes and rivers, contaminants have polluted many of the sediments.
Due to bioaccumulation, some of the contaminants in the sediments are taken up by benthic fauna.
When larger organisms eat these contaminated organisms, these toxins go to the food chain (Jain and
Sharma, 2001; Gaur et al., 2005; Prica et al., 2008; Shari et. al., 2015). As sediments are very
important in the aquatic ecosystems, so the investigation about sediments is frequently comprised in
researches on environmental assessments (Adeakola. and Eletta 2007; Li et al., 2006; Shari et al.,
2015).
73
3.2 Materials and methods;
3.2.1. Study area description

3.2.2. Sampling sites
The samples of sediment were collected from four different sites of River Kabul. Through
Shalman in Khyber Agency the Kabul River enters Pakistan. It then flows through the Agencies of
Khyber and Mohmand until it reaches Warsak dam constructed in 1960. It is divided into three main
branches below the dam, branches are known as Shah Alam, Nagoman and Adezai and then joins
the River Indus at Kund (Attock). River Swat also joins River Kabul a few km below the dam. There
are about eighty one (81) industrial units, the heavy metals containing effluents of which are
discharged directly or indirectly in the River Kabul. Besides that sewages of Peshawar city, several
large towns, large number of villages and several Afghan refugees’ camps ultimately drain into River
Kabul. (IUCN, 1994; Khan et al., 1999).
3.2.3. Sampling points
To evaluate the heavy metal concentration in sediments at Michini Bridge, Sardaryab,

Amangarh (adjacent industrial area of River Kabul) and Peer Sabaq, samples of sediments from the
following points of the main River and Warsak dam were collected (Fig. 21).
74
Figure 21: Sedimets Sampling Sites; Michini Bridge, Sardaryab, Amangarh (adjacent industrial area
of River Kabul) and Peer Sabaq.
3.2.4. Sediment samples from Michini Bridge below Warsak dam
Sediment samples were collected from least polluted site 1 (below Warsak dam water
reservoir constructed on River Kabul in 1960, which is about sixty kilo meter upstream of the
contaminated part of the River and can be called safe in the sense of being far away from the dense
human population and industrial area. This were considered as reference samples.
3.2.5. Sediment samples from the main River
Sediment samples (B) (C) and (D) were collected from polluted part of the main River at
Sardaryab (site 2), Aman garh (site 3) and Peer Sabaq (site 4). Site 4 is at the main river after city
sewage discharged into it. Site 2 is at the main River after industrial effluent dumped into it and site 2
is at the main River after city sewages flows into it.
75
3.2.6. Collection of Sediments samples
To determine the total concentrations of selected heavy metals like (Cd, Cr, Cu, Fe, Hg, Mn,
Ni, Pb and Zn), the sediment samples were obtained from each collection site and season. By using
plastic spoons sediment samples were collected, transported to the laboratory and stored in
polyethylene bags. Then, Oven was used for drying out in until constant weight at 70 ± 5 0C and then
the dried samples were sieved so as to get the lowest portion (<62 lm). This portion has considered
the most identified to examine concentrations of heavy metal in samples of sediment (Amat Infanate
et al., 2002; Pekey, 2006). Sampling was conducted two times in summer and winter season in each
year from 2013 to December 2015.
3.2.7. Preparation of Sediment Samples.
20 g of sediment sample was dried at 105∘C. from each sample one gram was weighed
carefully and to each sample 5mL of concentrated nitric acid (Merck, 99.99%) was added. The
sample, then, was heated up to 80∘C until near dryness. The addition of acid and the process of heating
were repeated two more times. An amount of water was supplemented to the residual material. The
suspension was filtered (Whatman filterMerck, 0.45 𝜇m) and the filtrate was diluted to 50 mL by
deionized water which is a final volume.
3.2.8. Chemical analysis of sediments
To obtain information about the degree of contamination caused by industrial, domestic and
agricultural effluents with respect to concentration of heavy metals, samples were collected from three
polluted sites (Sardaryab, Aman garh and Peer Sabaq) downstream and from one spot of least
polluted site (Michini bridge below Warsak dam) upstream of the River Kabul, Which is a reference
site and about sixty kilo meter up-stream of the contaminated sites of the river and can be called nearly
safe in the sense of being far away from the dense human population and industrial activities.
Sediments samples from contaminated and reference portions of River Kabul and the heavy metals
such as manganese, copper, nickel, cadmium, iron, mercury, chromium, lead and zinc were observed
in the sediment samples from contaminated and reference portions of River Kabul.
76
3.2.9. Heavy metals parameters in sediment samples
The solutions were then aspirated into flame Atomic Absorption Spectrophotometer
(Spectra-AA-700) for determination of Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn under the following
operating parameters. The flame used was air acetylene (A-AC). Standard curve were prepared and
the optical density acquired were calibrated against the standard curves to know the contents of heavy
metals in the water.
Table 18: Operating data of Atomic absorption spectrophotometer for determination of metals
Elements Wave length (nm) Flame Working range (µg/mL-1)

Cd 228.8 AA (R) 0.5-2
Cr 357.9 AA (R) 2-8
Cu 324.7 AA (L) 2-8
Fe 248.3 AA (L) 1-4
Hg 253.7 AA (L) 100-400
Mn 279.5 AA (L) 1-4
Ni 232.0 AA (L) 3-12
Pb 217.0 AA (L) 5-20
Zn 213.9 AA (L) 0.4-1.6
Abbreviations, AA: air acetylene, R: Fuel-rich, L: Fuel-lean
3.2.10. Statistical Analysis
differences by using student’s t –test (two-tailed). P value <0.05 was considered to show statistical
significance.
77
3.3. Result and discussion
3.3.1. Chemical analysis of sediments samples
In the present study sediments samples from polluted sites (Sardaryab, Aman Garh, Peer
Sabaq) and reference site (Michini Bridge area below Warsak dam) of River Kabul were taken from
2013 to December 2015 on monthly basis and was studied for heavy metal parameters. Heavy metals
parameters including cadmium, chromium, copper, iron, mercury, manganese, nickel, lead and zinc.
3.3.1.1. Heavy Metals concentration in Sediment Sample A from site 1, Michini Bridge
(Sample A= Reference site)
Heavy metals in sediment sample A from Michini bridge (site 1), had cadmium concentration
ranged between 1.5 - 8 (mg kg-1 with mean) values of 3.0 ± 1.2 mg kg-1, 7.4 ± 0.5 mg kg-1 and 1.7 ±
0.2 mg kg-1 for high flow (summer) and concentration ranged between 3.2 - 5.4 mg kg-1 with mean
values of 4.0 ± 0.2 mg kg-1, 5.1 ± 0.2 mg kg-1 and 3.6 ± 0.4 mg kg-1 for low flow (winter) periods
(Tables 19 - 22 and Figs 22 - 30).
The minimum concentration recorded for chromium was 54 mg kg-1 and maximum
concentration of 83 mg kg-1 with mean values of 82.4 ± 0.9 mg kg-1, 69.9 ± 2.6 mg kg-1 and 56.5 ± 2.3
mg kg-1 for summer and minimum concentration 45.3 mg kg-1 and maximum 67.4 mg kg-1 with mean
values of 46.3 ± 1.2 mg kg-1, 56.4 ± 1.5 mg kg-1 and 66.5 ± 1.3 mg kg-1 for winter season. These results
are in accordance with previous reports revealing indiscriminate disposal of waste effluents as a major
source of increased pollution in certain rivers of the country (Yousafzai, et. al., 2008; Nergis. et. al.,
2013; Siraj, 2016). The copper of all the sediments samples at this site was in the range of 5 - 14.9 mg
kg-1 with mean values of 13.1 ± 2.1 mg kg-1, 6.2 ± 1.1 mg kg-1 and 12.2 ± 1.1 mg kg-1 for high flow
and was ranged between 9.4 - 13.9 mg kg-1 with mean values of 11.4 ± 1.8 mg kg-1, 12.3 ± 1.1 mg kg-
1
and 13.3 ± 0.5 mg kg-1 for low flow seasons. The iron concentration ranged between 180 mg kg-1
and 280 mg kg-1 with mean values of 244.0 ± 3.6 mg kg-1, 185.3 ± 4.7 mg kg-1 and 278.3 ± 2.0 mg
kg-1 during summer and concentration ranged between 122 mg kg-1 and 249 mg kg-1 with mean values
of 245.6 ± 4.1 mg kg-1, 124.0 ± 2.6 mg kg-1 and 224.0 ± 1.5 mg kg-1 during winter season. In this study
values for Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn, were higher as compare to values mentioned by
Demirak. et. al., (2006). The mercury concentration 0.01 mg kg-1 with mean values of 0.01 ± 0.005
mg kg-1 and 0.01 ± 0.006 mg kg-1 during summer and concentration ranged between 0.01 mg kg-1 and
0.02 mg kg-1 with mean value of 0.03 ± 0.008 mg kg-1 during winter season. Similarly manganese at
78
this point had minimum concentration of 66 mg kg-1 and maximum concentration of 73 mg kg-1 with
mean values of 70.8 ± 2.3 mg kg-1, 66.6 ± 0.5 mg kg-1 and 68.6 ± 2.0 mg kg-1 during high flow and
concentration varied between 42 - 70.3 mg kg-1 with mean values of 68.0 ± 1.9 mg kg-1, 62.0 ± 1.1
mg kg-1 and 42.6 ± 0.5 mg kg-1 during low flow seasons. The nickle concentration was ranged 46.4 -
78.5 mg kg-1 with mean values of 47.7 ± 1.3 mg kg-1, 76.9 ± 1.3 mg kg-1 and 68.7 ± 2.8 mg kg-1 during
summer and concentration was varied between 62.9 - 89 mg kg-1 with mean values of 62.1 ± 2.0 mg
kg-1, 62.7 ± 2.0 mg kg-1 and 75.9 ± 1.7 mg kg-1 during winter seasons. In a past study Morelli, et al.,
(2012) had investigated the sediments for different heavy metals like Cd, Cr, Pb, Ni and Zn. The
analysis of these effluents showed high concentration of Cd and Cu. Lead concentration had varied a
range from 20 - 41 mg kg-1 with mean values of 23.7 ± 1.7 mg kg-1, 38.3 ± 3.0 mg kg-1 and 21.3 ± 1.5
mg kg-1 for high flow and concentration varied ranged 26 - 37.1 mg kg-1 with mean values of 28.4 ±
2.1 mg kg-1, 36.1 ± 0.8 mg kg-1 and 32.9 ± 1.0 mg kg-1 for low flow seasons. Similarly minimum
concentration recorded for zinc was 53 mg kg-1 and maximum concentration of 98.4 mg kg-1 with
mean values of 78.7 ± 1.1 mg kg-1, 54.2 ± 1.5 mg kg-1 and 97.8 ± 0.7 mg kg-1 for summer and
minimum concentration 61 mg kg-1 and maximum concentration 83.9 mg kg-1 with mean values of
65.5 ± 0.6 mg kg-1, 62.7 ± 2.0 mg kg-1 and 83.2 ± 1 mg kg-1 for winter season. These results are in
agreement with previous researches revealing indiscriminate disposal of waste effluents as a major
source of increased pollution in certain rivers of the inside and outside of the country (Nergise. et al.,
2013; Paramasivame, 2015). The sequence of these parameters in sediments sample A was Fe > Zn
> Cr > Mn > Ni > Pb > Cu > Cd > Hg for summer season and Fe > Zn > Ni > Cr > Mn > Pb > Cu >
Cd > Hg for winter season. These findings of the present study were similar to the results of ( Zahra,
et al., 2014; Zhang, et al., 2014; Wang. et. al., 2015; Yang et. al., 2015) in different parts of the world.
Comparing our study with the findings of above researchers showed that heavy metal concentrations
are the main pollutants for the River Kabul and levels of these parameters have increased in River
Kabul in the last few years.
79
Table 19: Heavy metals concentration (mg/kg) of sediment sample A from Michini Bridge
during winter and summer (2013-2015).
Heavy S. 2013 W.2013 S.2014 W.2014 S.2015 W.2015

Metals
Cd 3.0±1.2 4.0±0.2 7.4±0.5 5.1±0.2 1.7±0.2 3.6±0.4
Cr 82.4±0.9 46.3±1.2 69.9±2.6 56.4±1.5 56.5±2.3 66.5±1.3
Cu 13.1±2.1 11.4±1.8 6.2±1.1 12.3±1.1 12.2±1.1 13.3±0.5
Fe 244.0±3.6 245.6±4.1 185.3±4.7 124.0±2.6 278.3±2.0 224.0±1.5
Hg 0.01±0.006 ND ND ND 0.01±0.005 0.03±0.008
Mn 70.8±2.3 68.0±1.9 66.6±0.5 62.0±1.1 68.6±2.0 42.6±0.5
Ni 47.7±1.3 62.1±2.0 76.9±1.3 62.7±2.0 68.7±2.8 75.9±1.7
Pb 23.7±1.7 28.4±2.1 38.3±3.0 36.1±0.8 21.3 ± 1.5 32.9 ± 1.0
Zn 78.7 ± 1.1 65.5 ± 0.6 54.2 ± 1.5 62.7 ± 2.0 97.8 ± 0.7 83.2 ± 1
1, Mean± Standard deviation

2, Abbreviations: ND-Not Detected, Cd-cadmium, Cr-chromium, Cu-copper, Fe-iron,
Hg-mercury, Mn-manganeez, Ni-nickel, Pb-lead, Zn-zinc.
80
3.3.1.2. Heavy Metals concentration in Sediment Sample B from Sardaryab, site 2
Heavy metals in sediments samples B from Sardaryab (site 2) showed higher concentration
than sediments sample A. sediments sample B from this site had cadmium concentration ranged
between 4.2 mg kg-1 and 7.8 mg kg-1 with mean values of 5.3 ± 0.4 mg kg-1, 6.4 ± 0.4 mg kg-1 and 4.2
± 0.5 mg kg-1 for high flow (summer) and concentration ranged between 4 mg kg-1 and 7.9 mg kg-1
with mean values of 7.0 ± 0.8 mg kg-1, 6.4 ± 0.4 mg kg-1 and 3.7 ± 0.2 mg kg-1 for low flow (winter)
periods (Tables 19 - 22 and Figs 22 - 30).
The minimum concentration recorded for chromium was 64 mg kg-1 and maximum
concentration of 103.8 mg kg-1 with mean values of 65.2 ± 1.1 mg kg-1, 102.2 ± 2.0 mg kg-1 and 87.7
± 0.1 mg kg-1 for summer and minimum concentration 76.3 mg kg-1 and maximum 90 mg kg-1 with
mean values of 89.2 ± 1.1 mg kg-1, 83.0 ± 0.9 mg kg-1 and 78.0 ± 1.5 mg kg-1 for winter season. The
copper of all the sediments samples at this site was in the range of 10 - 18 mg kg-1 with mean values
of 11.3 ± 1.1 mg kg-1, 12.7 ± 0.7 mg kg-1 and 17.4 ± 0.5 mg kg-1 for high flow and was ranged between
11.5 - 23.7 mg kg-1 with mean values of 23.0 ± 0.8 mg kg-1, 12.7 ± 1.9 mg kg-1 and 15.0 ± 1.7 mg kg-
1
for low flow seasons. The iron concentration ranged between 285 mg kg-1 and 478 mg kg-1 with
mean values of 286.2 ± 1.0 mg kg-1, 476.6 ± 1.5 mg kg-1 and 377.6 ± 1.5 mg kg-1 during summer and
concentration ranged between 197 mg kg-1 and 387 mg kg-1 with mean values of 274.0 ± 4.3 mg/kg,
197 ± 0.7 mg kg-1 and 383.3 ± 3.2 mg kg-1 winter season. The mercury concentration ranged between
0.02 mg kg-1 and 0.03 mg kg-1 with mean values of 0.03 ± 0.008 mg kg-1and 0.02 ± 0.004 mg kg-1
during summer and concentration ranged between 0.001 mg kg-1 and 0.01 mg kg-1 with mean values
of 0.01 ± 0.005 mg/kg and 0.00 ± 0.006 mg kg-1 winter season. In this study values for Zn, Ni, Cr, Cu
and Cd were higher and Fe, Mn and Pb and Hg were lower as compare to values mentioned by Wang
et al., (2004). Similarly manganese at this point had minimum concentration of 53.1 mg kg-1 and
maximum concentration of 89 mg kg-1 with mean values of 53.9 ± 0.8 mg kg-1, 88.0 ± 1.0 mg kg-1
and 74.6 ± 1.5 mg kg-1 during high flow and concentration varied between 46 - 99 mg kg-1 with mean
values of 46.5 ± 0.5 mg kg-1, 97.7 ± 2.1 mg kg-1and 89.8 ± 1.2 mg kg-1 during low flow seasons. The
nickle concentration was ranged 81 - 88 mg kg-1with mean values of 81.7 ± 0.9 mg kg-1, 87.5 ± 0.4
mg kg-1 and 86.0 ± 2.7 mg kg-1 during summer and concentration was varied between 72.4 - 83 mg
kg-1 with mean values of 72.9 ± 0.8 mg kg-1, 74.4 ± 0.5 mg kg-1 and 81.6 ± 1.8 mg kg-1 during winter
seasons. In a past research Sitasward (1984) observed the concentrations of copper and zinc in greater
quantity in bed sediments of the River Yamuna, India as associated to the findings of present research
in which the concentration of lead is almost same. Lead concentration had varied a range from 23.3 -
81
53 mg kg-1 with mean values of 46.1 ± 1.2 mg kg-1, 52.2 ± 0.6 mg kg-1 and 23.6 ± 0.3 mg kg-1 for high
flow and concentration varied ranged 24 - 57.1 mg kg-1 with mean values of 24.6 ± 0.5 mg kg-1, 55.5
± 1.5 mg kg-1 and 43.0 ± 1.7 mg kg-1 for low flow seasons. Similarly minimum concentration recorded
for zinc was 76.9 mg kg-1 and maximum concentration of 101.9 mg kg-1 with mean values of 91.0 ±
0.8 mg kg-1, 78.0 ± 0.9 mg kg-1 and 104.9 ± 61 mg kg-1 for summer and minimum concentration 73.2
mg kg-1 and maximum concentration 89.8 mg kg-1 with mean values of 73.5 ± 0.4 mg kg-1, 89.4 ± 2.2
mg kg-1 and 89.6 ± 1.3 mg kg-1 for winter season. The order of heavy metals parameters in the
sediments was Fe > Zn > Cr > Ni > Mn > Pb > Cu > Cd > Hg in summer and Fe > Zn > Cr > Mn >
Ni > Pb > Cu > Cd > Hg for winter season. These results are in agreement with the researches of
Wang et al., (2004) (H Wang et al., 2004) and Kralj et al., 2016 (Kralj et al., 2016) . These findings
of the present study were similar to the results of Zahra et al., 2014; Zhang et al., 2014 and Naji. et
al., 2015. The increasing in level of heavy metal parameters could be co-related with effluents from
factories, mills and sewages from Peshawar city. All the nine heavy metals like Cd, Cu, Cr Fe, Hg,
Mn, Ni, Pb and Zn studied in the sediments sample B from River Kabul showed increasing tendency
on comparison with sediments samples from reference site 1.
82
Table 20: Heavy metals concentration (mg/kg) of sediment sample B from Sardaryab, site 2,
in winter and summer seasons during 2013-2015.
Heavy S. 2013 W.2013 S.2014 W.2014 S.2015 W.2015

Metals
Cd 5.3 ± 0.4 7.0 ± 0.8 6.4 ± 0.4 6.4 ± 0.4 4.2 ± 0.5 3.7 ± 0.2
Cr 65.2 ± 1.1 89.2 ± 1.1 102.2 ± 2.0 83.0 ± 0.9 87.7 ± 0.1 78.0 ± 1.5
Cu 11.3 ± 1.1 23.0 ± 0.8 12.7 ± 0.7 12.7 ± 1.9 17.4 ± 0.5 15.0 ± 1.7
Fe 286.2 ± 1.0 274.0 ± 4.3 476.6 ± 1.5 197 ± 0.7 377.6 ± 1.5 383.3 ± 3.2
Hg 0.03 ± 0.008 0.01 ± 0.005 ND 0.01 ± 0.006 0.02 ± 0.004 ND
Mn 53.9 ± 0.8 46.5 ± 0.5 88.0 ± 1.0 97.7 ± 2.1 74.6 ± 1.5 89.8 ± 1.2
Ni 81.0 ± 0.9 72.9 ± 0.8 87.0 ± 0.4 74.4 ± 0.5 86.0 ± 2.7 81.6 ± 1.8
Pb 46.1 ± 1.2 24.6 ± 0.5 52.2 ± 0.6 55.5 ± 1.5 23.6 ± 0.3 43.0 ± 1.7
Zn 91.0 ± .8 73.5 ± 0.4 78.0 ± 0.9 89.4 ± 2.2 104.9 ± 61 89.6 ± 1.3
P < 0.05
83
3.3.1.3. Heavy Metals concentration in Sediment Sample C from Aman Garh, site 3.
Heavy metals in sediments samples C from Aman Garh (site 3) showed higher concentration than
sediments sample A and B. sediments sample C from this site had cadmium concentration ranged
between 3.5 mg kg-1 and 7.1 mg kg-1 with mean values of 3.7 ± 0.2 mg kg-1, 6.7 ± 0.4 mg kg-1 and 4.7
± 0.5 mg kg-1 for high flow (summer) and concentration ranged between 4.6 mg kg-1 and 8.6 mg kg-1
with mean values of 5.0 ± 0.5 mg kg-1, 7.9 ± 0.6 mg kg-1 and 7.1 ± 0.1 mg kg-1 for low flow (winter)
periods) (Tables 19 - 22 and Figs 22 - 30).
The minimum concentration recorded for chromium was 83.8 mg kg-1 and maximum
concentration of 104 mg kg-1 with mean values of 102.9 ± 1.7 mg kg-1, 85.7 ± 1.7 mg kg-1 and 85.6 ±
1.7 mg kg-1 for summer and minimum concentration 81 mg kg-1 and maximum 87 mg kg-1 with mean
values of 85.3 ± 2.2 mg kg-1, 86.0 ± 1.7 mg kg-1 and 82.1 ± 1.1 mg kg-1 for winter season. The copper
of all the sediments samples at this site was in the range of 13.9-21.8 mg kg-1 with mean values of
15.3 ± 1.2 mg kg-1, 14.9 ± 0.8 mg kg-1 and 20.9 ± 0.9 mg kg-1 for high flow and was ranged between
11 - 18 mg kg-1 with mean values of 12.0 ± 0.9 mg kg-1, 17.6 ± 0.4 mg kg-1 and 17.2 ± 0.4 mg kg-1 for
low flow seasons. The iron concentration ranged between 343 mg kg-1 and 589 mg kg-1 with mean
values of 344.2 ± 1.5 mg kg-1, 581.0 ± 7.0 mg kg-1 and 431.6 ± 5.5 mg kg-1 during summer and
concentration ranged between 272 mg kg-1 and 385 mg kg-1 with mean values of 276.0 ± 3.6 mg kg-
1
, 382.5 ± 2.2 mg kg-1 and 381.0 ± 1.6 mg kg-1 during winter season. These results are in agreement
with the researches of Akif et al., (2000) and Siraj, (2016). The mercury concentration ranged between
0.01 mg kg-1 and 0.03 mg kg-1 with mean values of 0.01 ± 0.005 mg kg-1 and 0.03 ± 0.007 mg kg-1
during summer and concentration ranged between 0.01 mg kg-1 and 0.02 mg kg-1 with mean values
of 0.02 ± 0.004 mg kg-1, 0.01 ± 0.006 mg kg-1 and 0.02 ± 0.004 mg kg-1 during winter season. Similarly
manganese at this point had minimum concentration of 80.1 mg kg-1 and maximum concentration of
94 mg kg-1 with mean values of 91.7 ± 1.9 mg kg-1, 82.6 ± 2.2 mg kg-1 and 84.7 ± 2.6 mg kg-1 during
high flow and concentration varied between 74.9 mg kg-1 and 92 mg kg-1 with mean values of 87.7 ±
1.4 mg kg-1, 77.2 ± 2.0 mg kg-1 and 89.3 ± 2.5 mg kg-1 during low flow seasons. The nickle
concentration was ranged 53.9-80 mg kg-1 with mean values of 54.6 ± 1.1 mg kg-1, 71.8 ± 1.4 mg kg-
1
and 80.4 ± 1.3 mg kg-1 during summer and concentration was varied between 78 mg kg-1 and 96 mg
kg-1 with mean values of 79.1 ± 1.1 mg kg-1, 85.6 ± 1.5 mg kg-1 and 94.2 ± 1.5 mg kg-1 during winter)
seasons. In a past study Aamir et al., 2016 also found the same results of heavy metals concentrations.
Lead concentration had varied a range from 32-67.4 mg/kg with mean values of 44.6 ± 2.2 mg kg-1,
64.1 ± 5.3 mg kg-1 and 33.0 ± 0.9 mg kg-1 for high flow and concentration varied ranged 38.8-57 mg
84
kg-1 with mean values of 39.5 ± 1.2 mg kg-1, 55.9 ± 1.0 mg kg-1 and 40.0 ± 0.9 mg kg-1 for low flow
seasons. Similarly minimum concentration recorded for zinc was 84 mg kg-1 and maximum
concentration of 116 mg kg-1 with mean values of 91.7 ± 1.6 mg kg-1, 85.5 ± 1.4 mg kg-1 and 113.3 ±
23 mg kg-1 for summer and minimum concentration 81 mg kg-1 and maximum concentration 88.9 mg
kg-1 with mean values of 82.9 ± 1.9 mg kg-1, 87.9 ± 0.9 mg kg-1 and 87.6 ± 1.5 mg kg-1 for winter
season. The order of heavy metals concentration in sediments of sample C from this was Fe > Zn >
Cr > Mn > Ni > Pb > Cu > Cd > Hg for summer season and Fe > Ni > Zn > Mn > Cr > Pb > Cu >
Cd > Hg for winter season. These results were in agreement with Zahra et al., (2014) and Naji and
Sohrabi (2015). The increasing in level of heavy metal parameters could be co-related with effluents
from factories, mills and sewages from Peshawar and some parts of Nawshera city. All heavy metals
like Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn studied in the water sample C from River Kabul showed
increasing tendency on comparison with water samples from Michini Bridge below Warsak dam and
sediments sample B.
85
Table 21: Heavy metals concentration (mg/kg) in sediment sample C from Aman Garh site 2,
in winter and summer seasons during 2013-2015.
Heavy S. 2013 W.2013 S.2014 W.2014 S.2015 W.2015

Metals
Cd 3.7 ± 0.2 5.0 ± 0.5 6.7 ± 0.4 7.9 ± 0.6 4.7 ± 0.5 7.1 ± 0.1
Cr 102.9 ± 1.7 85.3 ± 2.2 85.7 ± 1.7 86.0 ± 1.7 85.6 ± 1.7 82.0 ± 1.1
Cu 15.3 ± 1.2 12.0 ± 0.9 14.9 ± 0.8 17.6 ± 0.4 20.9 ± 0.9 17.2 ± 0.4
Fe 344.2 ± 1.5 276.0 ± 3.6 581.0 ± 7.0 382.5 ± 2.2 431.6 ± 5.5 381.0 ± 1.6
Hg 0.01 ± 0.005 0.02 ± 0.004 0.03 ± 0.007 0.01 ± 0.006 ND 0.02 ± 0.004
Mn 91.7 ± 1.9 87.7 ± 1.4 82.6 ± 2.2 77.2 ± 2.0 84.7 ± 2.6 89.3 ± 2.5
Ni 54.6 ± 1.1 79.1 ± 1.1 71.8 ± 1.4 85.6 ± 1.5 80.4 ± 1.3 94.2 ± 1.5
Pb 44.6 ± 2.2 39.5 ± 1.2 64.1 ± 5.3 55.9 ± 1.0 33.0 ± 0.9 40.0 ± 0.9
Zn 91.7 ± 1.6 82.9 ± 1.9 85.5 ± 1.4 87.9 ± 0.9 113.3 ± 23 87.6 ± 1.5
P < 0.05
86
3.3.1.4. Heavy Metals concentration in Sediment Sample D from Peer Sabaq, site 4.
Heavy metals in sediments sample D from Peer Sabaq (site 4) showed higher concentration
than sediments sample A, B and C. Sediments sample D from this site had cadmium concentration
ranged between 4.7 mg kg-1 and 9.1 mg kg-1 with mean values of 5.2 ± 0.6 mg kg-1, 8.4 ± 0.5 mg kg-
1
and 8.3 ± 0.4 mg kg-1 for high flow (summer) and concentration ranged between 5.4 mg kg-1 and 8
mg kg-1 with mean values of 6.4 ± 1.3 mg kg-1, 6.7 ± 0.7 mg kg-1 and 7.5 ± 0.5 mg kg-1 for low flow)
(winter) periods (Tables 19 - 22 and Figs 22 - 30).
The minimum concentration recorded for chromium was 68.5 mg kg-1 and maximum
concentration of 95.9 mg kg-1 with mean values of 96.6 ± 1.8 mg kg-1, 65.6 ± 5.0 mg kg-1 and 89.1 ±
0.8 mg kg-1 for summer and minimum concentration 71.1 mg kg-1 and maximum 110 mg kg-1 with
mean values of 108.6 ± 2.2 mg kg-1, 72.7 ± 1.4 mg kg-1 and 79.2 ± 1.7 mg kg-1 for winter season. The
copper of all the sediments samples at this site was in the range of 5-24.6 mg kg-1 with mean values
of 18.5 ± 1.4 mg kg-1, 5.5 ± 0.6 mg kg-1 and 22.5 ± 1.7 mg kg-1 for high flow and was ranged between
15.9-22 mg kg-1 with mean values of 16.9 ± 1.0 mg kg-1, 20.9 ± 1.1 mg kg-1 and 15.1 ± 1.7 mg kg-1
for low flow seasons. The iron concentration ranged between 434 mg kg-1 and 745 mg kg-1 with mean
values of 742.0 ± 3.6 mg kg-1, 592.3 ± 4.1 mg kg-1 and 439.6 ± 6.6 mg kg-1 during summer and
concentration ranged between 467 mg kg-1 and 801 mg kg-1 with mean values of 472.6 ± 4.9 mg kg-
1
, 658.0 ± 3.6 mg kg-1 and 794.3 ± 9.0 mg kg-1 during winter season. The mercury concentration
ranged between 0.03 mg kg-1 and 0.12 mg kg-1 with mean values of 0.03 ± 0.007 mg kg-1 and 0.12 ±
0.004 mg kg-1 during summer and concentration ranged between 0.01 mg kg-1 and 0.11 mg kg-1 with
mean values of 0.01 ± 0.004 mg kg-1, 0.02 ± 0.006 mg kg-1 and 0.11 ± 0.006 mg kg-1 during winter
season. In this study values for Zn, Ni, Cr, Cu and Cd were higher and Fe, Mn and Pb and Hg were
lower as compare to values mentioned by Yousufzai, (2004). Similarly manganese at this point had
minimum concentration of 77.2 mg kg-1 and maximum concentration of 92.8 mg kg-1 with mean
values of 90.2 ± 3.0 mg kg-1, 82.9 ± 0.9 mg kg-1 and 79.1 ± 1.6 mg kg-1 during high flow and
concentration varied between 78 mg kg-1 and 86 mg kg-1 with mean values of 84.6 ± 0.5 mg kg-1, 83.9
± 2.0 mg kg-1 and 79.4 ± 2.0 mg kg-1 during low flow seasons. The nickle concentration was ranged
86-99.3 mg kg-1 with mean values of 87.1 ± 1.0 mg kg-1, 93.0 ± 4.0 mg kg-1 and 97.4 ± 1.7 mg kg-1
during summer and concentration was varied between 89 mg kg-1 and 98.9 mg kg-1 with mean values
of 96.8 ± 2.5 mg kg-1, 90.3 ± 1.5 mg kg-1 and 95.6 ± 2.9 mg kg-1 during winter seasons. Lead
concentration had varied a range from 48.9-78.9 mg kg-1 with mean values of 50.7 ± 2.0 mg kg-1, 77.6
± 1.4 mg kg-1 and 55.5 ± 3.8 mg kg-1 for high flow and concentration varied ranged 42.1-58 mg kg-1
with mean values of 46.9 ± 1.9 mg kg-1, 43.4 ± 1.4 mg kg-1 and 55.9 ± 2.1 mg kg-1 for low flow
87
seasons. Similarly minimum concentration recorded for zinc was 75 mg kg-1 and maximum
concentration of 123.2 mg kg-1 with mean values of for 92.1 ± 1.6 mg kg-1, 78.5 ± 3.2 mg kg-1 and
122.4 ± 12 mg kg-1 summer and minimum concentration 85 mg kg-1 and maximum concentration
101.4 mg kg-1 with mean values of 86.8 ± 1.6 mg kg-1, 89.3 ± 2.0 mg kg-1 and 99.7 ± 1.5 mg kg-1 for
winter season. In sediments, the permissible limits of Ni, Cd, Pb, Cu, and Zn are 35, 0.8, 85, 36, and
140 mg/kg, respectively, as recommended by Dutch Target Limits (Tabinda et al., 2013). The
sequence of the concentration of heavy metals in sample D of sediments was Fe > Zn > Ni > Mn >
Cr > Pb > Cu > Cd > Hg in summer and Fe > Zn > Ni > Cr > Mn > Pb > Cu > Cd > Hg for winter
season. In sediments also the increase in metal content was found from the upstream to downstream
sites as was recorded in water, because of sewages effluents that release into the river. These findings
of the present study were similar to the results of Zhang, et al., 2014; Yang et. al., 2015; Xu. et. al.,
2015 in different parts of the world. Comparing our study with the findings of above researchers
showed that heavy metal concentrations are the main pollutants for the River Kabul and levels of
these parameters have increased in River Kabul in the last few years. All heavy metals parameters
like Cd, Cu, Cr, Fe, Hg, Mn, Ni, Pb and Zn were having increasing tendency on comparison with
Michini Bridge below Warsak dam sediment samples. The high level of heavy metals could be
correlated to mining activities, deforestation, natural process of weathering and poor agricultural
practices in adjoining hills of River Swat during low flow season, which also joins River Kabul below
Warsak dam. The increasing in level of heavy metal parameters could be co-related with effluents
from factories, mills and sewages from Peshawar, Charsada, Nawshera and Mardan city. All the nine
heavy metals like Cd, Cu, Cr Fe, Hg, Mn, Ni, Pb and Zn studied in the sediments sample D from
River Kabul showed increasing tendency on comparison with sediments samples A, B and C from
sites 1, 2 and 3 respectively.
88
Table 22: Heavy metals concentration (mg/kg) of sediment sample D from Peer Sabaq During
winter and summer (2013-2015).
Heavy S. 2013 W.2013 S.2014 W.2014 S.2015 W.2015

Metals
Cd 5.2 ± 0.6 6.4 ± 1.3 8.4 ± 0.5 6.7 ± 0.7 8.3 ± 0.4 7.5 ± 0.5
Cr 96.6 ± 1.8 108.6 ± 2.2 65.6 ± 5.0 72.7 ± 1.4 89.1 ± 0.8 79.2 ± 1.7
Cu 18.5 ± 1.4 16.9 ± 1.0 5.5 ± 0.6 20.9 ± 1.1 22.5 ± 1.7 15.1 ± 1.7
Fe 742.0 ± 3.6 472.6 ± 4.9 592.3 ± 4.1 658.0 ± 3.6 439.6 ± 6.6 794.3 ± 9.0
Hg 0.03 ± 0.007 0.01 ± 0.004 0.12 ± 0.004 0.02 ± 0.006 ND 0.11 ± 0.006
Mn 90.2 ± 3.0 84.6 ± 0.5 82.9 ± 0.9 83.9 ± 2.0 79.1 ± 1.6 79.4 ± 2.0
Ni 87.1 ± 1.0 96.8 ± 2.5 93.0 ± 4.0 90.3 ± 1.5 97.4 ± 1.7 95.6 ± 2.9
Pb 50.7 ± 2.0 46.9 ± 1.9 77.6 ± 1.4 43.4 ± 1.4 55.5 ± 3.8 55.9 ± 2.1
Zn 92.1 ± 1.6 86.8 ± 1.6 78.5 ± 3.2 89.3 ± 2.0 122.4 ± 12 99.7 ± 1.5
P < 0.05
89
Cd mg/kg
2013 2014 2015
9
8
7
6
5
4
3
2
1
0
Figure 22: Comparison of Cd (Cadmium in milli gram per kilogram) in sediment samples from four
different sites of River Kabul during summer and winter.
Cr mg/kg
2013 2014 2015
120
100
80
60
40
20
0
Figure 23: Comparison of Cr (Chromium in milli gram per kilogram) in sediment samples from four
different sites of River Kabul during summer and winter
90
Cu mg/kg
2013 2014 2015
25
20
15
10
0
Figure 24: Comparison of Cu (Copper in milli gram per kilogram) in sediment samples from four
Fe mg/kg
2013 2014 2015
900
800
700
600
500
400
300
200
100
0
Figure 25: Comparison of Fe (Iron in milli gram per kilogram) in sediment samples from four
91
Hg mg/kg
2013 2014 2015
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
Figure 26: Comparison of Hg (Mercury in milli gram per kilogram) in sediment samples from four
Mn mg/kg
2013 2014 2015
120
100
80
60
40
20
0
Figure 27: Comparison of Mn (Menganese in milli gram per kilogram) in sediment samples from
four different sites of River Kabul during summer and winter
92
Ni mg/kg
2013 2014 2015
120
100
80
60
40
20
0
Figure 28: Comparison of Ni (Nickle in milli gram per kilogram) in sediment samples from four
Pb mg/kg
2013 2014 2015
90
80
70
60
50
40
30
20
10
0
Figure 29: Comparison of Pb (Lead in milli gram per kilogram) in sediment samples from four
93
Zn mg/kg
2013 2014 2015
140
120
100
80
60
40
20
0
Michini bridge Sardaryab Aaman garh Peer Sabaq
Figure 30: Comparison of Zn (Zn in milli gram per kilogram) in sediment samples from four different sites of
River Kabul during summer and winter
94
CHAPTER 4
BIOACCUMULATION OF HEAVY METALS IN SOFT TISSUES OF

FRESHWATER MUSSELS.
4.1. Introduction
Accumulation of materials, which are not components of an organism body, is

termed bioaccumulation of metals (Siraj, 2016). The last decade has shown that bioaccumulation and
biomagnifications of chemicals in biota through the food chain or trophic webs may be a prior
condition for adverse effects to the species, individuals, ecosystems because environmental
xenobiotics concentrations are often too small to have a harmful effect (Nordberg et al., 2014) . Fig.
31 presents the effects of heavy metals bioaccumulation in a trophic chain.
Figure 31: Heavy metals bioaccumulation in a trophic chain
In the mid-1970s, the use of the mussel was initially recommended, to examine trends and
levels of chemical pollutants mainly heavy metals in aquatic environment (Goldberg, 1975).
Biomonitoring is a process which is established on the capability of mussels to accumulate chemical
pollutants in their bodies to an extent relative to the availability of these chemicals. The study of found
95
that the biomonitoring could be applied on majority of bivalent metals and all aquatic organisms
including fresh water mussels (Cossea, 1988). Due to the process of bioaccumulation covers a
duration long-lasting even months, the higher levels of pollutants found in mussel could be measured
more easily if the daily variations of water masses is excluded. For this purpose two contrasting plans
of action have been used. Many investigators work on cultivated or native inhabitants of natural
ecosystem which is passive bio-monitoring. Other trust on transfer mussel’s samples after a site of
reference to the studied area which is active bio-monitoring (Goldberg, 1975; Clisse, 1989; Fabrise et
al., 1994).
Heavy metals have received considerable attention among the myriad of various inorganic
and organic substances dumped into this aquatic environment due to their potential bioaccumulation
in different aquatic organisms. Many of these heavy metals are well recognized to be persistent, non
– biodegradable, extremely toxic with their deleterious health effects even at lower concentration
levels, aside their unfortunate ability to easily bio-accumulate in aquatic ecosystems species (Liu et
al., 2010; Tunca et al., 2013). Heavy metals are of great interest since they go in and store in the food
chain which is called biomagnification. Some of these are essential in low concentrations for the
development and growth of living organisms, but others like Cd, Cr, Hg and Pb are biologically very
toxic and non-essential to all living organisms. If essential metals are present in a concentration above
the permissible level, even they may become toxic (Reilly, 2008; Chellali and Guendouz, 2015).
Among aquatic biota, mussels are desirable organisms for biomonitoring purposes. Since these
organisms are in direct contact with polluted parts of water and sediments of their habitats and can
accumulate high levels of heavy metals in soft parts of their body (Fournier et al., 2001; Farries, and
Hassel, 2007; Tunca et al., 2013; Chellali and Guendouz, 2015).
Freshwater mussels are useful in monitoring temporal and spatial trends of a wide range of
persistent pollutants in aquatic environments due to many advantages e.g., a wide geographical
distribution, sedentary life style, long lifespan, tolerance to a wide range of contaminants, poor
metabolic capacity for pollutants, correlation of pollutant content between organism and habitat,
adequate tissue for analyses (Tanabe & Subramanian, 2006) (Tanabe and Subramanian, 2006). A
freshwater mussel has been used as a unique bioindicator (bioaccumulator) for ‘Freshwater Mussel
Watch’ research in the Taihu Lake of China (J. Yang, Wang, Zhu, Gong, & Yu, 2005) (Yang et al.,
2005) to assess the pollution caused by organotins (J. Yang, Harino, Liu, & Miyazaki, 2008) (Yang
et al., 2008), organo-chlorines (Bian, Liu, Gan, Li, & Yang, 2009) (Bian et al., 2009) and heavy
metals (Liu et al., 2010) (Liu et al., 2010). There have been numerous reports of heavy metals and
other elements in adult mussels ( Ravera et al., 2003a; 2003b; 2005; Usero et al., 2005) since the
Mussel Watch concept was proposed by Goldberg (1975). Adult Freshwater mussels have strong
96
capability of filtration of great volume of water then obtain food (e.g., detritus, zooplankton, bacteria,
and algae) principally by filter feeding). As a result, both diet and water can contribute to element
accumulation in tissues and the diet is considered as a major route (Christian and Smith, 2004;
Hedouin et al., 2007; Pernice et al., 2009).
The freshwater mussels can accumulate pollutants at levels that are significantly higher than
their abundance in the surrounding water, deprived of metabolizing them to any appreciable level. By
measuring the concentration of heavy metals in mussel’s soft tissues, they might be used as
bioindicator of heavy metals caused environmental pollution. However, the extrinsic and intrinsic
factors can affect the accumulation of heavy metals in tissues of mussels. The extrinsic factors
comprise, bivalve size and spawning season. Researchers have shown that the body size might change
the absorption of heavy metals. It is clear that body size can affect the rates of metal adsorption and
desorption. For instance, physiological changes are effected by body size, such as respiration, filtration
and pumping rate in the bivalve mussels, Mytilus edulis have been reported (Lobel et al., 1991; Adjei-
Boateng et al., 2010; Chellali and Guendouz, 2015).
Freshwater mussels are an inexpensive secondary indicator of water pollution compared to
water or sediment analysis (Phillips, 1976). In preference to passive samplers, the use of mussels has
been recommended for the research of food-chain effects (Brown et al., 2005). In a number of recent
researches, the usefulness of mussels for monitoring pollutants like heavy metals loads has been
revealed including those conducted by Perić et al. (2012) and Kibria et al. (2012), who used caged
mussels to examine the bioavailable heavy metal concentrations of different water samples.
Earlier studies have demonstrated the ability of fresh water mussels to accumulate pesticides
(Uno et al., 2001) and trace elements (Liu. et al., 2010). The Freshwater mussels have strong
capability of filtration of great volume of water for and have capability to concentrate and accumulate
contaminants in the body tissues at range greater than those present in the surrounding water.
Numerous researches had found that the differences of responses of mussels to an extensive range of
pollutants can be caused in part by spatial and seasonal patterns. Moreover, the bioaccumulation of
metal can be influenced by the interactions among chemical bioavailability, speciation and metal
concentration, physiological absorption, accumulation, growth and weight loss and environmental
food concentration and temperature factors (Sasikumare et al., 2006; Casas and Bacherb, 2006;
Pisanlli et al., 2009; Pan and Wange, 2012).
The criteria for being a good biomonitor do not solely depend on its accumulative index for
the Particular parameter. Previously Phillips, (1990) have recommended that an ideal biomonitor
should be sedentary, abundant in study area, tolerant to wide range of contaminants and accumulator
of contaminants. Freshwater mussels are useful in monitoring temporal and spatial trends of a wide
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range of persistent pollutants in aquatic environments due to many advantages e.g., a wide
geographical distribution, sedentary life style, long lifespan, tolerance to a wide range of
contaminants, poor metabolic capacity for pollutants, correlation of pollutant content between
organism and habitat, adequate tissue for analyses (Tanabe and Subramanian, 2006).
Bivalves such as mussels and clams accumulate chemical pollutants like heavy metals
particularly in the digestive diverticula. The cells of Digestive diverticula are not only involved in
digestion and absorption of food material, but are also involved in the bioaccumulation and
bioconcentration. Compared to sediments, mollusks like mussels show greater spatial sensitivity and
therefore, the mussels are used as the most reliable tool. Furthermore, fishes and mussels have been
recognized to store heavy and trace metals in the soft tissues. Meanwhile they are a significant part of
anthropological diet (Goldberge et al., 1978; Rainbow, 2006; Azarbad et al., 2010 and Ponnusamy et
al., 2014).
By eating mussels, metals enter into the human body, however they can also suffer from an
extensive range of ecological, physiological and metabolic factors. When greater amount of metals
may be present in water and sediments then they will be accumulated in soft tissues of mussels in
greater amount. Ayling (1974) demonstrated that by various ways the mussels uptake of heavy metals
like Cu, Cr, Zn and Cd within the body. These ways can vary with environmental as well as
physiological features (Bryan, 1973) and the sex and sexual status of an organism (Alexander and
Young, 1976).
Recently, many researches had paying attention about the assessment of heavy metal
bioaccumulation in the aquatic organisms including invertebrates like freshwater mussels (Liu et al.,
2010; Tuneca et al., 2013; Chellali and Guendouz, 2015).
4.2. Materials and Methods
4.2.1. Study area
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For detail see page 07
4.2.2. Sampling sites of freshwater mussels
Forty samples at different times were collected from highly polluted belt of the main river. In
first collection forty samples were from Michini Bridge (site 1) below Warsak dam free of industrial
effluents and present above Peshawar city. This site is the reference site of the study. Second collection
of forty samples was from Sardaryab (site 2) below Peshawar city and sewages from Peshawar city
enter into River Kabul before this point. Third collection was taken from region of almost three km
upstream the Bridge on Nowshera to Mardan Road to Amangarh industrialized area (site 3). It
receives discharges from Amangarh Industries. The fourth collection of forty samples was collected
about four kilo meter downstream the Bridge on Nowshera to Mardan Road (site 4). The sewages
from cities of Nowshera, Mardaan, Risaalpur and further neighboring townships join River Kabul at
this point. All samples collection from site 2, 3 and 4 on River Kabul were considered mussels
samples from polluted water (tested mussels sample) and were compared with first sample collected
from reference site Michini Bridge (site 1) which is 60 km up-stream the contaminated sites of the
River Kabul. This was the reference mussels’ samples collection (Fig. 32).
4.2.3. Collection of Freshwater mussels’ samples
The mussel samples were washed with water immediately at all collection sites, to remove
encrusted mussels samples and transported to the laboratory in ice boxes. Mussels were inspected on
arrival, and dead samples were discarded. Forty individuals were used for biometric characterization
and metal analyses. By using 0.01 mm vernier caliper, Shell width (W, maximum lateral axis) and
length (L, maximum measure along the anterior-posterior axis) of each mussel were measured. By
cutting the adductor muscle with a stainless steel scalpel, each mussel was opened and for removal of
water from shell each sample was placed with its ventral side on filter paper. Then, after dissection of
each organism, total wet weight (TWW) of each sample without internal water and soft flesh wet
weight (FWW) (up to 0.01) were then measured (Tables 24 – 27 and Figs. 35 - 36).
99
Figure 32: Freshwater mussels sampling sites 1, 2, 3 and 4 at River Kabul.
4.2.4. Storage of tissues of freshwater mussels
Freshwater mussels were collected from four sites of the River Kabul, Michini Bridge (site
1), Sardaryab (site 2), Amangarh (site 3) and Peer Sabaq (site 4). The collected mussels were dissected
and distilled water was used for washing the mussel’s tissues, then transferred to already marked and
sterilized polythene bags. For further analysis of heavy metals bioaccumulation in soft tissues, then
tissues of mussels were stored in the freezer (at -20 c°) in polythene bags.
4.2.5. Tissue digestion
For estimation of heavy metals, the digestion of tissues was take place in the Department of
Zoology, University of Peshawar. Distilled water was used for Tissues washing and in blotting papers
they were blotted and then transferred to 100 milli liter volumetric flasks, which were cleaned with
distilled water before tissues transfer into the flaks and for a few minutes in oven at 60C˚ were dried
100
the tissues. Then ( 50g ) of each tissue was moved in the volumetric flasks. Technique used by Siraj
(2016) was followed for tissues digestion according to which 1ml per chloric acid (70%) and 5 ml
nitric acid (55%) were poured to each flask then each flask was kept back for overnight. Second dose
of 4ml (70%) per chloric acid and 5 ml nitric acid (55%) were added to each flask on next day. For
digestion of tissues the flasks were then retained on hot plate at 200 to 250° C and admit to digest till
a clear and transparent solution was obtained. Tissues sample were cooled after digestion and with
distilled water diluted to 100 ml. Till the determination of heavy metals concentration tissues samples
were reserved in well clean bottles of glass.
4.2.6. Determination of heavy metals
By using (Spectra-AA-700), (Atomic Absorption Spectrophotometer) heavy metals was

determined in the Centralized Resource Laboratory (CRL) University of Peshawar. It was used for
determination of heavy metals like Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn concentrations in soft
tissue of freshwater mussels from contaminated and reference sites of River Kabul.
Table 23: The standards used to analyze the variable using atomic absorption
spectrophotometer (Spectra-AA-700).
Metals Wave length Flam Working range (µg/mL-1)

Cd 228.8 AA (R) 0.5-2
Cr 357.9 AA (R) 2-8
Cu 324.7 AA (L) 2-8
Fe 279.5 AA (L) 1-4
Mn 248.3 AA (L) 1-4
Ni 232.0 AA (L) 3-12
Pb 217.0 AA (L) 5-20
Zn 213.9 AA (L) 0.4-1.6
Abbreviations, AA: air acetylene, R: Fuel-rich, L: Fuel-lean

4.2.7. Statistical Analysis
Statistical analysis have been done by using SPSS (version 20.0) software for windows. Mean and
standard deviation values of the data determined. The different sets of data have been analyzed for
statistical differences by using Analysis of Variance (ANOVA) and student’s t–test (two-tailed). P
value < 0.05 was considered to show statistical significance
101
4.3. Result and discussion
In the present investigation heavy metals like Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn were
determined in soft tissues of freshwater mussels collected from site 1 (reference) and site 2, 3 and site
4 (polluted sites).
4.3.1. Bioaccumulation of heavy metals in soft tissues
Soft tissues of freshwater mussels from site 1 (reference site) and sites 2, 3 and 4 (polluted
sites) were taken out and processed for estimation of cadmium, copper, chromium, mercury, iron,
manganese, nickel, lead, and zinc. Soft tissues of freshwater mussels from site 2, 3 and 4 showed
greater concentration as when compared with those from site 1. (Tables 28 - 29 and Figs. 33 - 34).
The order of these metals in soft tissues of freshwater mussels was Fe > Zn > Mn > Pb > Cu
> Cr > Ni > Cd. It indicates that Fe existed the highest and Cd was the lowest accumulated metals.
Among heavy metals, Iron (Fe) had highest level in soft tissues of freshwater mussels from sites 2, 3
and 4 with mean values 101.0 ± 30.3 µg/g, 162.3 ± 90.2 µg/g and 136.2 ± 62.3 µg/g respectively
while lowest value of iron is present in reference site which is 76.3 ± 31.6 µg/g. Highest level of Fe
was found in soft tissues of mussels from three polluted sites and the least level was noticed in mussels
from reference site, accordingly where the lowest level of these metals were also found in sediment.
Predominantly, the present results are in agreement with earlier researches (Amin et al., 1996a, 1997;
Giarrtano et al., 2010).
Second highest concentration is of Zinc (Zn) in the soft tissues of freshwater mussels which is 46.1 ±
13.7 µg/g, 47.4 ± 13.2 µg/g and 50.4 ± 13.7 µg/g in sites 2, 3, and 4 respectively. While lowest
concentration of Zn is in site 1 (reference site) 41.7 ± 16.6. The concentration of Zn found here in this
study was high as compared to the results of Yousufzai, (2004) from the same River Kabul in tissue
of collected samples.
Manganese (Mn) have the concentration 38.2 ± 13.5 µg/g, 39.9 ± 13.7 µg/g and 35.1 ± 12.7 µg/g in
sites 2, 3 and 4 respectively while 45.3 ± 19 µg/g in site 1. Lead (Pb) have the concentration 26.5 ±
11.2 µg/g, 26.1 ± 7.3 µg/g and 31.9 ± 11.9 µg/g in sites 2, 3 and 4 respectively while 10.1 ± 10 µg/g
in site 1. Copper (Cu) have the concentration of 3.6 ± 0.6 µg/g, 3.2 ± 0.9 µg/g and 2.7 ± 1.3 µg/g in
sites 2, 3 and 4 respectively while 3.3 ± 1.0 µg/g in site 1.Our findings are in agreement with previous
research where Nano and ionic forms of copper were used in mussels soft tissues and significant
difference was studied in copper for comparison between exposed and controls mussel and it was
observed that the higher damage in soft tissues of ionic form of Copper exposed mussels (Gomes et
102
al., 2013). Moura et al., (2000) also suggested that Anodonta cygnea have the ability to detoxify
extreme levels of Copper. Therefore, it can show that the mussels samples in our research were still
at an intensive developmental stage and due to high metabolism essential metals are required that
resulted in a high capability to accumulate Cu, Fe, Mn and Zn in their soft tissues. In contrast to our
results, other researchers reported higher levels of heavy metals bioaccumulation in mussels including
species from both the Anodonta and Unio genus (Gundackar, 2000; Liu et al., 2010). Chromium (Cr)
have the concentration of 1.5 ± 0.7 µg/g, 1.7 ± 0.9 µg/g and 2.7 ± 1.3 µg/g in sites 2, 3 and 4
respectively while 1.1 ± 0.9 µg/g in site 1. Nickel (Ni) have the concentration of 11.5 ± 0.9 µg/g, 1.6
± 1.1 µg/g and 2.7 ± 4.7 µg/g in sites 2, 3 and 4 respectively while 2.3 ± 1.0 µg/g in site 1. Cadmium
(Cd) have the concentration of 1.2 ± 0.7 µg/g, 0.9 ± 0.8 µg/g and 1.0 ± 0.6 µg/g in sites 2, 3 and 4
respectively while 1.2 ± 0.9 µg/g in site 1.The concentration of heavy metals here recorded in this
study is high as compared to previous researches where low concentration of Cr was observed in
tissues of organisms which may cause deficiencies at minor level (Farag et al., 2006; Siraj, 2016).
4.3.2. Bioaccumulation of heavy metals in Gills of freshwater mussels
Gills of freshwater mussels from site 1 (reference site) and sites 2, 3 and 4 (polluted sites)
were taken out and processed for estimation of cadmium, copper, chromium, iron, mercury,
manganese, nickel, lead, and zinc. Gills of freshwater mussels from site 2, 3 and 4 showed greater
concentration as when compared with those from site 1. (Tables 28 - 29 and Figs 33 - 34).
The order of these metals in gills of freshwater mussels was Fe > Mn > Zn > Pb > Ni > Cu
> Cr > Cd > Hg. It indicates that Fe existed the highest and Cd was the lowest accumulated metals.
Among heavy metals, Iron (Fe) had highest level in gills of freshwater mussels from sites 2, 3 and 4
with mean values 97.8 ± 23.2 µg/g, 159.5 ± 84.1 µg/g and 156.8 ± 59.6 µg/g respectively while lowest
value of iron is present in reference site which is 56.3 ± 28.4 µg/g. Highest level of Fe was found in
gills of mussels from three polluted sites and the least level was noticed in mussels from reference
site, accordingly where the lowest level of these metals were also found in water and sediment.
Second highest concentration in gills, is of Manganese (Mn) which have the concentration 39.8 ±
17.9 µg/g, 45.2 ± 17.4 µg/g and 42.9 ± 15.2 µg/g in sites 2, 3 and 4 respectively while 35.8 ± 14.9
µg/g in site 1. Zinc (Zn) in the gills of freshwater mussels which is 31.8 ± 12.8 µg/g, 42.9 ± 12.9 µg/g
and 41.7 ± 18.1 µg/g in sites 2, 3, and 4 respectively. While lowest concentration of Zn is in site 1
(reference site) 23.6 ± 17.2. Lead (Pb) have the concentration 27.5 ± 9.2 µg/g, 32.8 ± 1.3 µg/g and
45.9 ± 14.7 µg/g in sites 2, 3 and 4 respectively while 21.1 ± 13.2 µg/g in site 1. Predominantly, the
present results are in agreement with earlier research ( Sohail et al., 2016).
103
Copper (Cu) have the concentration of 3.8 ± 1.9 µg/g, 4.2 ± 0.8 µg/g and 2.4 ± 1.7 µg/g in sites 2, 3
and 4 respectively while 2.3 ± 0.6 µg/g in site 1.Our results are in accordance with previous studies
where two different forms of copper were used in mussels and significant difference was studied in
copper for comparison between exposed and controls mussel and it was observed that the higher
damage in soft tissues of ionic form of Copper exposed mussels (Gomes et al., 2013). In contrast to
our findings, other researchers reported higher levels of heavy metals bioaccumulation in mussels
including species from both the Anodonta and Unio genus (Liu et al., 2010). Chromium (Cr) have
the concentration of 2.5 ± 0.5 µg/g, 2.7 ± 0.8 µg/g and 2.7 ± 0.9 µg/g in sites 2, 3 and 4 respectively
while 0.1 ± 0.8 µg/g in site 1. The concentration of chromium here recorded in this study is very low
as compared to previous researches where large concentration of Cr was observed in tissues of
organisms which may cause deficiencies at minor level (Farag et al., 2006; Siraj, 2016).
Nickel (Ni) have the concentration of 17.9 ± 8.4 µg/g, 23.7 ± 2.9 µg/g and 31.7 ± 12.7 µg/g
in sites 2, 3 and 4 respectively while 2.3 ± 1.0 µg/g in site 1. Cadmium (Cd) have the concentration
of 2.7 ± 0.9 µg/g, 0.5 ± 0.7 µg/g and 1.4 ± 0.7 µg/g in sites 2, 3 and 4 respectively while 0.4 ± 0.7
µg/g in site 1.
The present study indicated that as increasing the concentration of metals in the medium, the
bioaccumulation of heavy metals in the soft tissues and gills of the analyzed mussels also increased.
These results are in accordance to the conclusions of Giron et al., (2004) on oysters, Hook and Fisher
(2002) on copepods and Yousafzai et al., 2008 (Yousafzai et al., 2008 a) on fishes. Furthermore,
Hemelrad et al., (1990) found that due to high concentration of Cd in the soft tissues low mortality of
Anodonta cygnea was observed and no any difference was found from a control group. But contrast
it was found by Gil et al., (2006) that when in sediment no detectable levels of Cd were observed
even that higher concentrations of Cd were noticed in tissue of mussels. Our research results also
show agreement with those of other investigators that bivalves (mussels) accumulate metal in their
tissues very efficiently (Casas et al., 2006; Binelli, et al., 2010; Paurange et al., 2010; Sohail, et al.,
2016; Denil et al., 2017).
Our results showed that the order of heavy metals concentration and spatial differences in the
surrounding sediments is followed by the amount of metal accumulation in soft tissues which show
a powerful relationship between biological and environmental concentrations of heavy metals. It is
accordance to the results from studies on metal bioaccumulation including unionids, freshwater
mussel (Królake and Zdanowski, 2001; Liu et al., 2010) or Unio pictorum (Gundacker 2000). For
shorter time scales the soft tissues of mussel may be used to observe environmental conditions.
Chemical composition of hemolymph, foot, and/or mantle tissue of mussels may be taken as sub-
lethal biomarkers for monitoring the water quality. Recently, in lakes and rivers freshwater
104
representatives of the Unionidae mussels family is used in several researches for reflection of
surrounding contamination which is the main focus of these researches. There have been numerous
reports of heavy metals and other elements in different organs of adult mussels (Frittes et al., 2015;
Jasinsaka et al., 2015; Kolareviec et al., 2016; Denil et al., 2017), since the Mussel Watch concept
was proposed by Goldberg (1975).
In conclusion, the results indicate that the concentrations of heavy metals like Cd, Cr, Cu, Fe,
Hg, Mn, Ni, Pb and Zn in River Kabul were higher. These metals accumulated in soft tissues and gills
of freshwater mussels. The levels of these metals in the soft tissues and gills of freshwater mussels
depend on the metal availability. The results highlights that metals have the capability to accumulate
in tissues of freshwater mussels.
Table 24: Length of freshwater mussels expressed in terms of maximum, minimum, mean
length, and standard deviation collected from four different sites.
Sampling Total Maximum Minimum Mean Standard

Sites Number Length Length Length Deviation
(mm) (mm) (mm)
Michini
40 134 93 113.7 ±7.9
Bridge
Sardaryab 40 146 88 109.4 ±4.5
Aman Garh 40 124 73 105.2 ±9.1
Peer Sabaq 40 113 69 87.9 ±9.5
105
Table 25: Width of freshwater mussels expressed in terms of maximum, minimum, mean
width, and standard deviation collected from four different sites.
Sampling Total Maximum Minimum Mean width Standard

sites number width (mm) width (mm) deviation
Michini
40 102 66 87.4 ±6.3
Bridge
Sardaryab 40 102 61 92.3 ±5.9
Aman Garh 40 89 59 72.1 ±5.3
Peer Sabaq
40 83 56 74.2 ±9.3
Table 26: Weight (shell + soft tissues) of freshwater mussels expressed in terms of maximum,
minimum, mean weight, and standard deviation collected four from different sites.

Sites Number Weight Weight Weight Deviation
(gm) (gm) (gm)
Michini
40 384.0 77.3 239.6 ±14.9
Bridge
Sardaryab 40 301.1 98.4 213.8 ±8.4
Aman Garh
40 211.7 97.9 189.3 ±11.8
Peer Sabaq
40 198.2 77.8 145.9 ±9.7
106
Table 27: Weight (soft tissues) of freshwater mussels expressed in terms of maximum,
minimum, mean weight, and standard deviation collected from four different sites.

Sites Number weight weight weight Deviation
(gm) (gm) (gm)
Michini
40 68.4 28.1 56.3 ±5.6
Bridge
Sardaryab 40 91.3 22.8 63.2 ±7.2
Aman Garh 40 60.1 24.1 39.2 ±5.3
Peer Sabaq 40 46.0 22.6 34.9 ±5.9
Table 28: Heavy metals concentration (µg gm-1) in soft tissues of fresh water Mussel collected
from Four sites of River Kabul.
Site Cd Cr Cu Fe Hg Mn Ni Pb Zn
s
1 1.2±0.9 1.1 ±0.9 3.3±1.0 76.3±31.6 0.01±0.003 45.3±19. 2.3±1.0 10.1±10.7 41.7±16.6
5
2 1.2±0.7 1.5±0.7 3.6±0.6 101.0±30.3 0.12±0.007 38.2±13. 1.5±0.9 26.5±11.2** 46.1±13.7
5 *
3 0.9±0.8 1.7±0.9 3.2±0.9 162.3±90.2** 0.03±0.009 39.9±13. 1.6±1.1 26.1±7.3*** 47.4±13.2
* 7
4 1.0±0.6 1.8±0.9* 2.7±1.3 136.2±62.3** 0.14±0.001 35.1±12. 2.7±4.7 31.9±11.9** 50.4±13.7
5 7 *
Difference significant relative to site 1 at *P<0.03, **P<0.01, ***P<0.000
107
Table 29: Heavy metals concentration (µg gm-1) in gills of freshwater mussels collected from
four sites of River Kabul.
Sites Cd Cr Cu Fe Hg Mn Ni Pb Zn
1 0.4 ± 0.7 0.1 ± 0.8 2.3 ± 0.6 56.3 ± 28.4 ND 35.8 ± 14.9 13.0 ± 5.2 21.1 ± 13.2 23.6 ± 17.2
2 2.7 ± 0.9 2.5 ± 0.5 3.8 ± 1.9 97.8 ± 23.2 ND 39.8 ± 17.9 17.9 ± 8.4 27.5 ± 9.2 31.8 ± 12.8
3 0.5 ± 0.7 2.7 ± 0.8 4.2 ± 0.8 159.5 ± 84.1 ND 45.2 ± 17.4 23.7 ± 2.9 32.8 ± 1.3 42.9 ± 12.9
4 1.4 ± 0.7 2.7 ± 0.9 2.4 ± 1.7 156.8 ± 59.6 0.17 ± 0.009 42.9 ± 15.2 31.7 ± 12.7 45.9 ± 14.7 41.7 ± 18.1
Difference significant relative to site 1 at P<0.03, P<0.01, P<0.000

180
160
140
120
100
80
60
40
20
0
Cd Cr Cu Fe Hg Mn Ni Pb Zn
Figure 33: Heavy metals concentration (µg gm-1) in soft tissues of freshwater Mussels collected
from four sites of River Kabul.
108
180
160
140
120
100
80
60
40
20
0
Cd Cr Cu Fe Hg Mn Ni Pb Zn
Figure 34: Heavy metals concentration (µg gm-1) in gills of freshwater Mussels collected from four
sites of River Kabul.
109
Figure 35: Image showing the samples of freshwater mussels
Figure 36: Image showing the freshwater mussel while measuring the width.
110
CHAPTER 5
HISTOPATHOLOGICAL EFFECTS OF HEAVY METALS IN

FRESHWATER MUSSELS
5.1. Introduction
Histopathology is of the best way for evaluating long-term and short-term noxious effects for
field assessments. It is a powerful indicator of prior exposure to environmental pollutants like heavy
metals and the consequences of negative physiological and biochemical alterations in organisms. In
ecotoxicology researches for last two decades, the Unionidae mussel have arisen as a critical
organism for a discussion because of higher sensitivity to a diversity of other ecological stressors and
chemical exposures, in contrast of another organisms class (Marigómez et al., 2006; Stentiford et al.,
2009; Brusa et al., 2011; Sonawane, 2015; Chandurvelan, et al., 2016).
Histopathology, as a technique or tool of analysis, can provide an idea of the environmental
status. Similar to assay techniques, histopathologIcal alterations also provides biomarkers that are
responsive and sensitive since tissues and organs are being altered even at minor concentrations of
pollutants like heavy metals (Abdallah, 2004; Brooks et al., 2009; Chandurvelan, et al., 2016).
Histopathology together with other tools such as diseases, growth, and biochemical changes are
biomarkers used in assessing effects of both external aquatic environmental conditions and internal
conditions on aquatic organisms like freshwater mussels (Auffert, 1988; Stentiford et al., 2009).
Accumulation of heavy metals may cause histopathological changes in soft parts of freshwater
mussels. Synthesis of metallothionein is increased in organism during acute and chronic exposures to
heavy metals. High level of metals are present in metallothionein and extra metal ions pour out into
further partitions of cell and results in histopathological injury in different tissues of freshwater
mussels. In environmental monitoring programs on pollution effects the Pathology is now a standard
research area. (Mohamed & Gad, 2005; Marigómeze et al., 2006; Sonawane, 2015; Martinez-
Gomeze et. al., 2017). On the Basis of easily reproducible technique, histopathological studies yield
fundamental information on tissue lesions related to the general health of organisms, and evaluate the
host's susceptibility to parasitic infestation and infectious diseases. Most of these parameters may
serves as indicators of the effects of xenobiotic contamination in aquatic organisms (Aarab et al.,
2008; Stentiford et al., 2009; Chowdhury et al., 2016).
Numerous researches in laboratory have been administered on freshwater mussels in order
to recognize the role of pollutants (heavy metals Hg etc.) in the decline of the populations due to
production of pathological conditions (Valenti, Cherry, Neves, & Schmerfeld, 2005) (Valenti et al.,
111
2005; Casas & Bacher, 2006; Ingersoll et al., 2006). The mussel’s digestive gland is the principal
organ for regulation metabolism and homeostatic regulation of the internal medium and mechanisms
of defense by immune system, in addition to system of elimination of xenobiotic and detoxification
of heavy metals (Moore and Allene, 2002; Brooks et al., 2009; Martinez-Gomez et. al., 2017).
Exposure to pollutants like heavy metals results in serious alterations in different types of cell
in soft tissues of aquatic animals. The secretory or basophilic cells much less than digestive cells but,
due to exposure to heavy metals, an obvious rise in relative quantities of basophilic cells takes place
(Cajaraville et al., 1989; Marigo´mez et al., 1998; Stentiford et al., 2009). Studies on the histology
and histopathology have proved that it is a very beneficial tool in determining the pollutants like heavy
metals induced injury to whole animals. Toxic effect of copper and mercury has been extensively
studied. Histopathological studies on the crustacean gill have been carried out by various author. The
histopathological changes detected in the epithelium of digestive gland of the mussels as a reaction to
contaminants included lysosomal alteration, tubular atrophy, inflammation, granulocytomas, necrosis
and digestive cell’s vacuolation. Several researches have revealed the changes in the structure of
digestive gland epithelium in response to chemical contaminants (Koide et al., 1982; Allison and
Simpson, 1983; Moore et al., 1987; Auffert, 1988; Lowe and Clarke, 1989; Donderro et al., 2006;
Sonawane, 2015).
In South America disease called disseminated neoplasia has initially detected in bivalve
called Ostrea chilensis at Chiloé Island by Mix & Breese (1980). Then, this abnormality was observed
by Campalans et al., (1998) in M. chilensis from this vicinity again and Cremonte et al., (2011)
reported from the Channel of Beagle and southern Argentina. Disseminated neoplasia is growing
disorder observed in nearly fifteen bivalve species all over the world and results in destruction of these
bivalves’ species (Barber, 2004; Brooks et al., 2009; Carballal et al., 2011; Sonawane, 2015).
In bivalve oysters, Crassostrea virginica digestive diverticular epithelial atrophy have been
when exposed to wood preservative, chromatid copper arsenate (Weis et al., 1993). Samples
collection of American oysters, Crassotrea virginica from metal contaminated sites, the same
damaged cells in the digestive diverticula and gills was recorded (Gold-Bouchot, Sima-Alvarez,
Zapata-Perez, & Güemez-Ricalde, 1995) (Gold-Bouchot et al., 1995). In mussels along
Mediterranean Coast, Porte et al., (1998) and Nasci et al., (1999) recorded a severe, enlargement of
lysosome and heavy haemocytic infiltration and in the clam connective tissues which are transplanted
in an estuary highly metal contaminated located in industrialized and urban area of Tampa Bay. In
mussels, Mytilus edulis Granulocytomas and enlarged vacuolation of digestive tubules was observed
which were exposed to trace metal contamination (Wedderburn et al., 2000). Atrophy of the digestive
112
diverticula and fibrosis development in digestive diverticula in mussels exposed to prolonged sub
lethal heat stress (Braid et. al., 2005). Zorita. et al., 2006, found in mussels, Mytilus edulis, a significant
higher destabilized lysosomal membrane collected near a copper mine in Norway than the reference.
They also observed epithelial atrophy of digestive tubule from site which was copper contaminated.
In Norway Aarab et al., 2008, observed large lipid vacuoles and dilation of tubule in digestive
diverticula of mussel, Mytilus edulis, which were obtained from an aluminium contaminated site. In
their study, Al-Subiai et al., 2011, reported that the digestive gland, posterior adductor muscles, and
gills of Mytilus edulis, exposed to different concentrations (18 - 56 μg L-1) of Copper and observed
under Olympus microscope. The mussels showed various deformities, histological abnormalities and
pathological alterations ranging from loss of myocyte bundles, increased myocytes, necrosis, hypo
and hyperplasia and haemocyte infiltration.
The choice of histology as disease and contamination indicator, was established on earlier
researches which indicated powerful association between environmental contamination and the
histopathology of mantles, gills, digestive glands and gonads (Auffret, 1988; Lowe and Clarke, 1989;
Domouhtsidou and Dimitriadis, 2000; Soklova, 2004; Soklova et al., 2004; Soklova et al., 2005).
Many researches on histopathological alterations in the body of aquatic animals after
extensive exposure to heavy metals. Gills structural changes is usually realized responses to abnormal
ecological conditions and are exhibitive of chemical and physical stresses (Au,2004; Peric, et al.,
2012; Abdel-Moneim et al., 2012). The histopathology of gills is used as an environmental pollution
indicator. Around the world histopathological abnormalities in gills are used as indicators of extensive
heavy metals exposure have been previously used in various laboratories and field researches
(Gregory, et al., 2002; Zettler et al., 2007; Abdel-Moneim et al., 2012; Sonawane, 2015; Sohail et al.,
2016 ).
Several pathological abnormalities can be observed in the gills, kidneys and gonads as
exposed to sewages, industrial and agricultural contaminants. Pathological disorders in gills of fish
are related to specific classes of toxicants. Many researchers have investigated different pathological
alterations in various species after exposure to various contaminants. Histopathological lesions such
as degeneration and necrosis of hepatocytes as the elevation in transaminase activities may be
attributed to heavy metals in the liver of fish C. gariepinus (Moore and Allen, 2002; Siraj, 2016).
Histopathological effects of heavy metals were reported in some fish inhabiting Bardawil
lagoon. Heavy metals are the toxicants that induce histopathological abnormalities in different tissues
of animals and fish. Microscopic examination of hepatocytes and their nuclei of fish from
contaminated areas exhibit histopathological changes in comparison to control ones due to
contamination. Hepatocytes lose their normal boundaries. Histopathological changes in the liver and
113
pathological disorders such as secondary lamellar disorganization, rupture in lamellar epithelium and
epithelial lifting in the gills of different fish were observed after collection from polluted water. Several
histopathological lesions in fish liver after exposure to heavy metals were reported and supported by
many other studies deals with monitoring the fish health and environmental pollution in aquatic
ecosystem (Yacoub and Abdel Satar., 2003; Stentford et al., 2003; Metwally et al., 2010).
Hepatocellular alterations in fish hepatic tissue after exposure to heavy metals was seen. Most
previous studies have reported that exposure of fish to heavy metals is associated with structural
damage of gills epithelia. Most gills changes caused by pollutants in aquatic animals (mussels) like
epithelial lifting and inflammatory response of the tissue were seen in the gills (Metwally, et al., 2010:
Sonawane, 2015; Siraj, 2016).
The pathological lesions in different tissues of aquatic organisms confirmed that exposure of
these organisms to heavy metals may be resulted into histological and pathological disorders, as
reported already in previous findings by (Damek and Sawicka, 2003; Zhange et al., 2005; Martin et
al., 2006; Garmendia et al., 2011; Khan et al., 2015; Siraj, 2016).
114
5.2. Methods and Materials
5.2.1. Study area
5.2.2. Freshwater mussels sampling sites
5.2.3. Collection of freshwater mussel’s samples
5.2.4. Preservation of freshwater mussel’s tissues
For detail see page-99
5.2.5. Procedure (Tables 30 and 31)
5.2.6. Preparation of solutions for tissue processing
The following different solutions for tissues processing were prepared according the method
described by Prophet et al. 1992 (Prophet, Mills, Arrington, & Sobin, 1992).
5.2.7. Preparation of solution of fixation
10% neutrally buffered formalin (NBF) was used as a fixative and was prepared by mixing
10% formalin with PBS.
5.2.8. Preparation of PBS (Sodium phosphate dibasic dehydrate1.44 gm, Potassium chloride 0.2
gm, d.H2O 1000 ml, potassium phosphate monobasic 0.24 gm, pH 7.4)
5.2.9. Preparation of 10% NBF (37% formaldehyde 270.27 ml,PBS 1000 mL).
5.2.10. Preparation of different ethanol solutions
5.2.10.1. 50% ethanol solution (ethanol 250 ml,d.H2O 250 ml).

115
5.2.10.6. 95% ethanol solution (ethanol 950 ml, d.H2O50 mL).
5.2.11. Preparation of alcohol-xylene solution (Distilled alcohol 50 mL, 100% xylene 50 mL).
5.2.12. Preparation of xylene-paraffin solution (Paraffin 50 mL, 100% xylene 50 mL).
5.2.13. Preparation of different solutions for staining
5.2.14. Mayer’s albumin

Equal quantities of both fresh egg albumin and glycerol was mixed. Few drops of formaldehyde
37% were also added and then stored at 4oC.
5.2.15. Harris hematoxyl in stain (Hematoxylin powder 5.0 gm, mercuric oxid 2.5 gm, potassium
alum 100 gm, absolute ethanol 50 mL, glacial acetic acid 40 mL, d. H2O1000 mL).
5.2.16. Eosin stain
Working solution was prepared from eosin and phloxine-B stock solutions.
5.2.17. Eosin-Y stock solution (Eosin-Y powder 1gm, d.H2O 100mL, few drops of 37%
formaldehyde).
5.2.18. Phloxine-B stock solution (Phloxine-B 1g, d.H2O 100 ml, few drops of 37% formaldehyde).
5.2.19. Eosin-phloxine working solution (Eosin Y stock solution 100 ml, phloxine-B stock solution
10 ml,95% ethanol 780 mL, Glacial acetic acid 4 mL).
5.2.20. 1% acid-alcohol solution (Fuming HCl5ml, 70% ethanol 500 mL).
5.2.21. 1000mL ammonia solution (Ammonium hydroxide 2 ml, d. H2O 998 mL).
5.2.22. Tissue processing
The tissues were processed according to Siraj (2016).
5.2.23. Tissues fixation
Weighted tissues were kept in 10% NBF for 48 hours.
5.2.24. Tissues dehydration
Tissues were cut into a small pieces and kept in tissue cassettes. These cassettes were then
kept in a series of ethanol solutions (30%, 50%, 70%, 80%, 90%, 95%).To accelerate dehydration,
116
the tissues were kept on hot plate magnetic stirrer and adjusted the temperature between 55-57oC. The
tissues were dehydrated according to the following schedule.
5.2.24.1. 70% ethanol solution for 1 hour.
5.2.24.4. Two changes each of 100% ethanol solution for 1 hour.
5.2.25. Clearing of tissues
Tissues were transferred to clearing solution (50% Xylene, 50% ethanol) and kept at on hot
plate magnetic stirrer and adjusted the temperature between 45-47oC. The cleared tissues were finally
treated twice with 100% Xylene.
5.2.26. Paraffin infiltration of tissues
The tissues were infiltrated with melted paraffin wax. First tissues were incubated at 62oC
with combined solution of 50% xylene and molten paraffin wax for one hour and then kept twice for
I hour each in paraffin wax.
5.2.27. Embedding of tissues
Tissues were transferred to stainless steel (S.S). Melted paraffin was poured into moulds and
tissues were placed with heated forceps in it. Warm tissue cassettes were placed on the surface of
paraffin. The paraffin wax moulds were kept in freezer till solidified. After solidification the tissue
wax blocks were removed.
5.2.28. Sectioning of tissues
The tissues were sectioned through a rotary microtome. The sections were adjusted to 12μm
thickness. The section ribbon was removed from blade edge through forceps and the shiny side of the
sectioned tissue was kept in water bath containing clean water at a temperature of 10 degree below
the melting point of wax. Then the tissues were mounted on microscopic slides coated with Mayer’s
albumin and cooled at ambient temperature and placed in staining slide tray. The staining slide tray
was then placed in oven at 58oC for 10-15 minutes.
117
5.2.29. Staining of tissues
The tissues were stained with hemotoxylin and eosin in staining glass jars. Before staining
wax were removed and then rehydrated by xylene and then gradual ethanol solution and finally with
100 % ethanol. For hemotoxyline staining, tissue slides were kept in it for 5 min and then gently
washed with running tap water. The same tissue slides were then kept in eosin-phloxine solution for
8 min and washed gently with running tap water. Stained tissues were covered with canadabolsm and
then placed cover slips on it.
5.2.30. Observation of tissues under microscope
The dried stained tissues were observed under a compound microscope using all
magnification. Photomicrographs of tissues sections were obtained on a digital camera connected to
the microscope. Photomicrographs of low and higher resolutions were taken.
5.2.31. Statistical analysis
Statistical analysis was done by using ANOVA software for Windows. Percentage values of
pathological disorders were determined through the following formula:
No. of mussels in which pathological abnormalities were observed × 100

Total no. of mussels studied for pathological disorders
118
5. 3 Results and discussion
5.3.1 Gross and histopathological examination
After taking measurements, each specimen of freshwater mussels was examined for shell
deformities. Following the gross examination, each shell was opened carefully by inserting the tip of
scalpel between the shells at the ventral end, the scalpel was run along the length of shell till the
posterior adductor muscle was cut. The shell when in open position was examined for the abnormal
production of mucus and any other abnormality. Then the sex of the mussel was identified on the
basis of color of the gonads. In males the gonads were creamy white and in females were apricot in
color. The sexes identified on the basis of colorations were later confirmed by histological studies.
5.4. Identification and Measurements
The samples of freshwater mussels collected from the four sites that is, Michini bridge,
Sardaryab, Amangarh and Peer Sabaq were found to compose of single species, that is, Anodonta
cygnea. Length, width and weight of each sample from various sites are given in Tables 24 and 27.
The specimens of mussels collected from Michini Bridge were comparatively of larger size than the
specimens collected from the other three sites.
5.5. Gross pathology
The only significant gross change observed in the collected specimens was shell deformity.
The shell deformity was observed only in the specimens of Anodonta cygnea collected from one site,
i.e. Peer Sabaq. Of the 40 individuals of Anodonta cygnea examined from Aman Garh, only three
specimens showed the shell deformity. After the opening of shell, increased mucus production was
observed around the gills of almost all the specimens of mussels collected from 4 sites during the
present study.
5.6. Parasitic infestation
The specimens of were found to be infested by the parasites, i.e. Rickettsia like inclusions
(Figs. 55 and 58). Rickettsia were round in shape and were scattered in the epithelium of digestive
gland, gills and gonads. The specimens collected from all the four sites were found to be parasitized
to one extent or another. Table 33 shows the densities of parasites found in the specimens of mussels
digestive gland, gills and gonads at the four collection sites. In Michini Bridge 23 % in digestive gland,
2 % in gonads and 23% in gills were present. . In Sardaryab 37 % in digestive gland, 12 % in gonads
119
and 25 % in gills were present, in Aman Garh 78 % in digestive gland, 21 % in gonads and 51 %
in gills were present and In Peer Sabaq 74 % in digestive gland, 35 % in gonads and 38 % in gills
were present in total specimens were found to be infested by the parasites. The rickettsia like organism
(RLOs) are intra bacterial parasites, known to be common among mussels. Some RLOs have caused
severe disease and mortalities particular in cultured organisms (Comps and Tigs, 1999). The
population of freshwater mussels were heavily infected with RLOs at Peer Sabaq as compare to other
sites of studied area. The Peer Sabaq in addition to pollution by the industrial and domestic effluents
from cities of Nawshera and Sawabi (Yousufzai, 2010; Siraj, 2016). It explains the high RLOs
infection rate observed in the samples from Peer Sabaq as compared to other sites. Inflammatory
responses were also highly prevalent; within which haemocytic infiltration was the most frequent
alteration. This lesion is regarded as an important biomarker of inflammatory response in bivalves;
furthermore, some works link this alteration to severe lesions caused by parasitosis (Villalba et al.,
2001; Sheir and Handy, 2010).
5.7. Histopathology
The various pathological conditions observed in the digestive gland, gonads, intestine and gills of the
specimens of Anodonta cygnea collected during the present study are summarized in figures 38 – 46.
5.7.1. Digestive gland
5.7.1.1. Structure of normal digestive gland
The normal digestive gland of mussels consists of a series of diverticula connected with the
stomach by a sequence of branching ducts. The epithelium of the digestive diverticula is made of two
main cell types, digestive cells with vesicles and basophilic cells with epical granules. Depending
upon the feeding rhythm, the tubules show great variation in size from thick-walled tubules with
narrow lumen to wide thin-walled tubules. In healthy mussels, Lumina of diverticula appear tri-radiate
or quadri-radiate because of the thick epithelium of the diverticulum. The structure of normal
digestive tubule observed during the present study is described in Fig. 47.
5.7.1.2. Lesions observed in digestive gland
Histological lesions observed in the digestive gland of the mussels, Anodonta cygnea during
the present study included inflammation, granulocytoma, sloughing of digestive epithelium, hydropic
vacuolation, digestive tubule atrophy and necrosis (Figs. 38 to 46). As shown, the digestive tubules
120
necrosis was found in highest 65.5% of the samples of Anodonta cygnea at the four collection sites.
The second most frequent lesion observed in the digestive gland was inflammation (44.2% of the total
sample) followed by atrophy in the digestive gland (31.7% of the total sample).
The same histopathological alterations was also reported in the digestive gland epithelium of
the blue mussels Mytilus edulis as a response to contaminants included tubular atrophy, necrosis,
granulocytomas, inflammation and vacuolation of digestive cells ( Auffert, 1988; Lowe and Clarke,
1989; Aarab, et al., 2008; Khan et al., 2015).
5.7.1.3 Comparison of digestive gland lesions among the sites
Maximum types (six types) of digestive gland lesions were found in the specimens of
freshwater mussels collected from Michini Bridge, Sardaryab, Aman Garh and Peer Sabaq. The
necrosis of epithelium of tubules was found highest in 87% of the samples at Aman Garh (Figure 3).
The most common pathological condition in the specimens of freshwater mussels at Michini Bridge
was necrosis of digestive gland tubule epithelium (25% of the total sample) followed by the
inflammation (23% of the total samples) while lowest recorded lesion was granulocytoma which is
4% of total samples from this site. The most common pathological condition in the specimens of
freshwater mussels at Sardaryab was necrosis of epithelium of tubules (72.2% of the total sample)
followed by the sloughing of epithelium, 54.3% of the total samples from this site. The samples
collected from Aman Garh showed the highest necrosis of epithelium which was 87% and lowest
granulocytoma which was 21 of the total samples from this site and necrosis of digestive gland
epithelium (78.2%) highest and sloughing of epithelium (12%) of the total samples from this site.
Atrophy of digestive diverticular epithelium of oysters, Crassostrea virginica, exposed to wood
preservative, chromatid copper arsenate (Weis et al., 1993). The same report was recorded for
American oysters, Crassotrea virginica sampled from metal contaminated sites showed damage in
the digestive diverticula and gills of the mollusk (Gold-Bouchot, 1995). In this study, the observed
defensive reaction inflammation that occurs after pathogen invasion and/or cell injury is meant to
serve as a local defense reaction in host tissue, involving the recruitment of immune cells to injured
foci to isolate or eliminate the causes of cell damage, in addition to provoke damage itself, which have
been also studied by De Vico and Carella, (2012). Galimany et al., (2008 a, b, c) described similar
inflammatory responses (infiltration and diapedesis of haemo-cytes) and other pathological disorders
in blue mussels (Mytilus edulis) exposed to different contaminated environments. Our results are
consistent with previous observations that have found atrophic damage to the digestive epithelia and
inflammatory responses in the natural environment (Dyrynda et al., 2000; Wu et al., 2005; Garmendia
et al., 2011; Khan et al., 2015).
121
5.7.2. Gonads
5.7.2.1. Lesions observed in the gonads
Histological lesions observed in the gonads of freshwater mussels, Anodonta cygnea during
the present study were inflammation (haemocytic infiltration), necrosis, atresia, granulocytoma, and
lipofusin pigments in the connective tissues of the gonads. The most frequent lesion observed in the
gonads of freshwater mussels was Atresia in female gonads (51% of the total samples) followed by
inflammation (46.5% of the total samples) (Figs. 41 – 44).
5.7.2.2. Comparison of gonadal lesions among the sites
Maximum five types of lesions that is, inflammation, necrosis, granulocytoma, atresia and
lipofusin pigments were observed in the gonads of Anodonta cygnea at three sites Sardaryab, Aman
Garh and Peer Sabaq (Figure 41 - 44). Among the specimens collected from Michini Bridge the
common pathological condition was the granulocytoma of gonads (2% of the total samples) while
highest is inflammation in the gonads (43% of the total sample). Atresia was present in highest 75%
at Sardaryab and Aman Garh and was absent at Michini Bridge. Granulocytoma was observed in
connective tissues of the gonads of mussels collected from all the sites with highest 18% at Peer Sabaq
and lowest 2% at Michini Bridge respectively. At Peer Sabaq the specimens showed the highest
percentage of lipofusin pigments (74% of the total sample) followed by necrosis of gonads (59% of
the total sample). All these results are in agreement with researches which have reported the same
pathological disorders in the gonads of bivalve molluscs, (exposed to heavy metals) that is,
Mercenaria spp. (Hesselman et al., 1988), Mya arenaria (Barber, 1996), Mytilus galloprovincialis
(Figueras et al., 1991; Alonso et al., 2001), in the blue mussel Mytilus edulis (Elston et al., 1988), in
green-lipped mussel, Perna canaliculus, Chandurvelan (2013) and green mussels Perna viridis
(Khan, et al., 2015).
5.7.3. Gills
5.7.3.1. Lesions observed in the gills
Various lesions observed in the gills of mussels, Anodonta cygnea collected from the four
sites included swelling of gills, fusion of gill lamellae, inflammation and degeneration of cillia. The
122
most common gill lesion examined in the specimens was fusion of gill lamellae, that is, 87% of the
total sample showed fusion of gill lamellae (Fig. 45).
5.7.3.2. Comparison of gill lesions among the sites
Prevalence of different abnormalities observed in the gills of mussels is described in Figure

45. At Michini Bridge, the specimens of Anodonta cygnea showed all types of gill lesions with lowest
intensity. In the specimens at Michini Bridge the frontal cilia were degenerated was 26%; gills were
swollen was 5%, fusion of gills lamellae was 11% and filled with haemocytes was 9%. In specimens
of Sardaryab the highest found lesion was inflammation (76%) and degeneration of cilia 12%.
Inflammation and fusion of gills lamellae were lesions at Aman Garh with highest 43% and lowest
21% respectively. While pathological lesions in mussels specimens at Peer Sabaq was fusion of gills
lamellae with highest 87% and gills swelling lowest 12%. Sunila (1986) and Akaishi et al. (2007)
have reported the swelling and fusion of gill lamellae in the specimens of blue mussel, Mytilus edulis.
Most previous studies have reported that exposure to heavy metals is associated with structural
damage of gills epithelia. Most gills changes caused by pollutants like heavy metals in aquatic animals
(mussels) like epithelial lifting and inflammatory response of the tissue were seen in the gills
(Metwally et al., 2010: Sonawane, 2015).
5.7.4. Intestine
5.7.4.1. Lesions observed in the intestine
Lesions observed in the intestine of the mussel specimens were degeneration of intestinal
epithelial cillia and necrosis. Among the specimens of freshwater mussels examined during this study
the most common intestinal lesions observed was degeneration of epithelial cilia (79% of the total
sample from Peer Sabaq site) (Fig. 46).
5.7.4.2. Comparison of intestinal lesion among the sites
Lesions observed in intestine of the mussel, Anodonta cygnea are described in Figure 46.
Among the different sites, it was observed that the mussels collected from Peer Sabaq were affected
by two types of intestinal lesions i.e. degeneration of intestinal epithelium and necrosis with highest
79% and 53% respectively. In this study The specimens collected from Sardaryab and Michini Bridge
showed lowest percentage of two types of intestinal lesions each i.e. degeneration of intestinal
epithelium and necrosis, 29% and 14% respectively. The pathological lesions due to heavy metals in
123
the intestine of the blue mussels, the epithelial cells of the intestinal wall have been observed by Sunila
1986; 1988) (Sunila, 1986), (Sunila, 1988) , (Affret (1988) and Arabab et. al., (2008).
The present result indicates that the heavy metal contamination definitely affects the aquatic
life of the fresh water mussels. However, the high concentrations of the analyzed metals in the whole
body tissues investigated could be due to the storage role played by these tissues. Mussels
contaminated by heavy metals suffers from various pathological alterations, with consequent
inhibition of metabolic processes, hemolymph changes and decline in fertility and survival. Our result
further showed that heavy metals are toxic in nature and can be induced different histopathological
abnormalities in the aquatic organisms. The result also showed heavy metals pollution in the River
Kabul.
124
Table 30: Detailed process of Delafield's hematoxylin and eosin staining Deparaffinization
Xylene 5 min
Xylene 5 min
Xylene 5 min
Hydration
100% ethanol 1 min

100% ethanol 1 min
90% ethanol 2 min
70% ethanol 2 min
Distilled water 2-3 dips
Staining series
Primary stain 3-5 min

Distilled water 2-3 dips
70% ethanol 1 min
Counter stain 1 min
Dehydration
90% ethanol 2 min

100% ethanol 1 min
100% ethanol 1 min
Clearing
Xylene 1 min
Xylene 1 min
125
Xylene 1 min
Mounting
Canada balsam 1 week to dry
 Haematoxylin
 Eosin
126
Table 31: Detailed process of routine histological method
Dehydration
70% ethanol 30 min

90% ethanol 30 min
100% ethanol 30 min
100% ethanol 30 min
Clearing
Cedar wood oil 24 hrs or till clearing of tissue
Xylene 30 min
Xylene 30 min
 Infiltration
50% xylene  wax mix. 1- ½ hrs

100% wax 2-2 ½ hrs
Block making
100% wax 24 hrs or more

Sectioning
Microtome 6-7  thick (24 hrs to dry).
 The whole procedure of infiltration was carried out in the oven.
127
Table 3. Semi-quantitative scale for digestive gland a trophy according to Kim et al., (2004)
Score Description
0 Tubules with normal wall thickness, lumen nearly occluded, few tubules slightly atrophied.
1 wall thickness less than normal, but greater than one-half normal thickness, most tubule
showing atrophy, some tubule still normal
2 wall thickness averaging about one-half as thick as normal
3 wall thickness less than one-half of normal, most tubules wall significantly a trophied, some
walls extremely thin (fully atrophied)
4 wall extremely thin (100 % atrophied), nearly all tubules affected

5 lining of the tubules found in the lumen and only musculo-epithelial border present
Table 32: Occurrence of parasites at various sites in the freshwater mussels
Reckittsia like parasite Cestodes

Sampling sites Total number
Frequency
Machini bridge 40 9.0 0
Sardaryab 40 23.4 0
Amangrh
40 64.7 0
Peer sabaq
40 71.2 0
128
Table 33: Developmental stages (in percentage) of male gonads collected from various sites.
Sampling sites Immature Developing Ripe Partially Spent

spent
Michini Bridge 3 20 22 55 0
Sardaryab 12 36 23 17 12
Aman Garh 5 45 13 21 16
Peer Sabaq
8.1 27.2 23.6 33 8.1
Table 34: Developmental stages (in percentage) of freshwater mussel’s female gonads collected
from various sites. .
Sampling sites Immature Developing Ripe Partially Spent

Spent
Michini Bridge 7.0 11.6 45.7 23.4 12.3
Sardaryab 0.0 23.3 12.4 27.9 36.4
Aman Garh 5.2 26.5 44 9 15.3
Peer Sabaq
17.0 12.3 32.0 19.7 19.0
129
Table 35: Frequency (in percentage) of sexes in the freshwater mussels at various sites.
Sampling sites Total number Male Female Intermediate
Michini Bridge 40 37.0 63.3 0
Sardaryab 40 32.3 52.0 16.1
Aman Garh 40 30.0 67.0 03.3
Peer Sabaq
40 33.3 36.4 30.3
Table 36: Intensity of digestive tubules atrophy in mussels from various sites.
Sampling Total scoring of atrophy in digestive tubules

sites number 0 1 2 3 4 5
Michini
40 34.0 17.2 12.0 18.0 16.0 2.8
Bridge
Sardaryab 40 11.0 32.0 22.4 19.3 30.0 15.3
Aman
40 6.9 12.1 20.4 21.6 19.0 20.0
Garh
Peer
Sabaq 40 2.7 19.0 13.0 18.0 21.6 25.7
130
Table 37: Intensity of atretic oocytes in female gonads of mussels from various sites.
Sampling sites Total number scoring of atresia in female gonads

1 2 3 4
Michini
40 1 9 1 0
Bridge
Sardaryab 40 5 2 3 1
Aman Garh 40 5 3 4 1
Peer Sabaq 40 2 4 4 1
131
90
80
P
70
E
R 60
C
E 50
N DG
T 40
GN
A
30 GL
G
E 20
S
10
0
Mchini Bridge Sardaryab Amangarh Peersabaq
SAMPLING SITES
Figure 37: Parasitic infestation in digestive gland (DG), Gonads (GN) and Gills (GL) of freshwater
mussels from four different sites.
132
30
P
E 25
R
C 20
E
N 15
T
A 10
G
E 5
0
INF NEC HDV SLE GRN AT
LESIONS
INF: Inflammation, NEC: Necrosis, HDV: Hydropic, SLE: Sloughing of epithelium, GRN:
Granulocytoma, AT: Atrophy
from Michini Bridge
80
P 70
E
R 60
C 50
E
N 40
T
30
A
G 20
E
10
S
0
LESIONS
Figure 39: Histopathological lesions (percentages) in digestive gland of freshwater mussels collected from
Sardaryab
133
100
P 90
E 80
R 70
C
60
E
50
N
T 40
A 30
G 20
E 10
S 0
LESIONS
Fig. 40. Histopathological lesions (percentages) in digestive gland of freshwater mussels collected
from Aman Garh
90
P 80
E
70
R
C 60
E 50
N
40
T
A 30
G 20
E
S 10
0
LESIONS
from Peer Sabaq
134
35
P 30
E
R 25
C
E 20
N
T 15
A
G 10
E
S 5
0
INF LFP GRN
LESIONS
INF: Inflammation, LFP: Liposusin pigments, GRN: Granulocytoma
Michini bridge
45
40
P
E 35
R
30
C
E 25
N
T 20
A 15
G
E 10
S 5
0
INF LFP GRN
LESIONS

Figure 41: Histopathological lesions (percentages) in gonads of freshwater mussels collected from Sardaryab
135
60
P
50
E
R
C 40
E
N 30
T
A 20
G
E
10
S
0
INF LFP GRN
LESIONS
Aman Garh
80
P 70
E
60
R
C 50
E 40
N 30
T
20
A
G 10
E 0
S INF LFP GRN
LESIONS

Figure 43: Histopathological lesions (percentages) in gonads of freshwater mussels collected from Peer Sabaq
136
100
90
P
E 80
R 70
C
60
E INF
N 50
DC
T 40
A FG
G 30
GS
E 20
S
10
0
SAMPLING SITES
INF: Inflammation, DC: Degeneration of cilia, FG: Fusion of gills lamellae, GS: Gills swelling
Figure 44: Histopathological lesions (percentages) in Gills of freshwater mussels collected from four
different sites.
90
P 80
E
70
R
C 60
E 50
N
40 DGC
T
A 30 NC
G 20
E
10
S
0
SAMPLING SITES
DGC: Degeneration of cilia, NC: Necrosis
Figure 45: Histopathological lesions (percentages) in intestine of freshwater mussels collected from
four different sites
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Figure 46: Image showing Normal epithelium of digestive tubules
Figure 47: Image showing Atrophy of digestive tubular epithelium.
138
Figure 48: Image showing lumen of digestive tubule
Figure 49: Image showing necrosis of digestive epithelium
139
Figure 50: Image showing granulocytoma in digestive gland
Figure 51: Image showing hydropic vaculation
140
Figure 52: Image showing degeneration of digestive epithelium
Figure 53: Image showing lipofusin pigment and necrotic epithelium
141
Figure 54: Image showing Rickettsia like parasite in digestive tubules
142
Figure 55: Image showing normal eggs in gonads
Figure 56: Image showing atretic eggs in gonads
143
Figure 57: Image showing Rickettsia like parasites and necrosis in gonads
Figure 58: Image showing abnormal elongated eggs in gonads
144
Figure 59: Image showing granulocytoma in gonads
145
Figure 60: Image showing inflammation in gonads
Figure 61: Image showing lipofusin pigments in gonads
Figure 62: Image showing hemocytic infiltration in gonads
146
147
Figure 65: Image showing normal cilia in gills
Figure 66: Image showing fusion of cilia in gills
148
Figure 67: Image showing haemocytic infiltration in gills
Figure 68: Image showing degeneration of cilia in gills
149
Figure 69: Image showing degeneration of cilia in intestine
Figure 70: Image showing degeneration of epithelium in intestine
150
Figure 71: Image showing normal cilia in intestine
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CHAPTER 6
GENOTOXICAL EFFECTS OF HEAVY METALS IN FRESHWATER

MUSSELS.
6.1. Introduction
Genotoxicity is a lethal process, which affects the genetic material of cell (Smith, 1996).
Heavy metals are genotoxicants that can damage DNA of a living cell (Igwilo et al. 2006; Binelli et
al., 2007; 2010). These genotoxicants have investigated that they are related to alterations in
chromosomes since they produce tight covalent bonds with the DNA, which resulted into formation
of DNA adducts that prevent replication of DNA and resulted into DNA damage (Hartwell et al.
2000; Klobucar et al., 2008; Parolini et al., 2011). The comet technique is a sensitive, rapid and
reliable method that can be used for the determination of DNA lesion in prokaryotic and eukaryotic
cell’s nucleus (Bajpaye et al., 2005; Binelli et al., 2007; 2010). Among the various techniques so far
used to assess the genotoxicity of environmental pollutants, the comet assay is rapid and sensitive
technique that can be used for estimation of DNAs lesions in both multiplying and non-multiplying
cells. For the determination of DNA damage due to complex environmental contamination in aquatic
organisms, the DNA alkaline unwinding assay was also used (Batel et al., 1999; Gorbi et al., 2008;
Sarkaar et al., 2008; Oliveaira et al., 2010). In the beginning Micro Nuclear technique was developed
with mammalian species, which is commonly used today in fishes and other water living animals
comprising mussels, transplanted and wild animals. Most of the researches with the Micro Nucleus
assay had been used in different species of bivalves (Bolognesi and Hayaeshi, 2011; Siraj, 2016).
Researchers have found DNA fragmentation through comet assay before more severe
abnormalities in both terrestrial and aquatic organisms. For detecting DNA damage, the comet
technique is an authentic method that could be conducted at cellular level. In aquatic organism the
DNA damage due to environmental contaminants, is examined through this rapid technique. It is
generally applied technique for bio-monitoring of contaminant in aquatic organisms associated with
genotoxicity (Lee and Steinaert, 2003; Jhaa, 2008; Gorbi et al., 2008; Kolarevic et al., 2016).
Comet assay is a successful method that can be used for determination of DNA damage in
both field and laboratory investigations with different aquatic animals, both from fresh and marine
water. Therefore, sentinel organisms for genotoxic studies in aquatic environment (Landolt and
Kocan, 1983; Belpaeme et al., 1998; Pandey et al., 2006). The comet assay is a new technique that is
used in molecular epidemiology. It gives good results about genotoxicity which is produced as a result
of diseases, therapy in clinical trials occupational or environmental exposure to toxic chemicals for
152
limited time and in an inexpensive method. Comet assay has wide description and development.
Various investigations have imposed concentration on mammalian cells for comet assay but a number
have focused on marine and fresh water mussels and fish for determination of damage of DNA in
their hemocytes, blood cells, gill, liver and gut cells (Fauste et al., 2004; Klobucar et al., 2008;
Kolarevic et al., 2016; Siraj, 2016).
Comet assay technique is helping in the analysis of mutagenesis and genotoxicity in the fish
red blood cell collected from polluted environment (The advancement in most mutagenesis test
systems in plants, yeast, bacteria and animals comprising freshwater mussels are due to recently
increasing concern about the genotoxicity in water and land. It has been investigated that by the comet
assay, breaks of DNA strand was measured, which is acted as a biomarker of genotoxicity in aquatic
species like freshwater mussels (Mitchelmore and Chipman, 1998; Andrade et al., 2004; Binelli et
al., 2007). The researchers have focused on increasing genotoxins like heavy metals in the aquatic
environment and also busy in improvement of sensitive biomarkers for the discovery of genotoxic
effects of toxins in aquatic organisms. Environmental pollutants like heavy metals are genotoxic and
can be observed by a wide-ranging in vivo and in vitro biomarker techniques, but among these assays
it is very popular because of its sensitivity for detecting less amount of DNA lesions (Gedik et al.,
1992; Hayashei et al., 1998; Gorbi et al., 2008). It is essential to investigate the damage of DNA levels
could be determined by this technique is unlimited to the impacts of external ecological contaminants
merely but DNA damage due to oxidation also has key role in DNA damage and is pertaining to the
secondary impacts of various contaminants like heavy metals. Several heavy metals among them
when metabolized then can attack on DNA and leading to severe DNA damage (Livingstone, 2001,
Gabbianelli et al., 2003, Mamaca et al., 2005; Binelli et al., 2007; Kolarevic et al., 2016).
Among the most toxic environmental contaminants, the heavy metals can affecting various
organisms genetically. Heavy metals in high concentrations resulted into DNA damage in mussels.
The DNA damage due to heavy metals was detected in hepatopancreas. The percentage of DNA
damage is increased by the increasing the duration of exposure to heavy metals. Consequently the
maximum percentage of DNA damage was detected in organism after exposure to heavy metals like
zinc, cadmium, and lead (Machella et al., 2006; Yingmei et al., 2006; Gorbi et al., 2008). These are
dangerous to living things both plants and animals because they are toxic and carcinogenic in nature
and can induce different abnormalities in living organisms. In previous finding it has been investigated
that heavy metals can be bind with nucleic acid through attaching to the DNA sites, which are resulted
into mutations, adducts and many complexes. Metal actions were also investigated to impact DNA
replication (Chang et al., 1996; Klobucar et al., 2008; Bolognesi et al., 2011). Recent investigations
have reported that heavy metals are carcinogenic and free radicals and reactive oxygen species are
153
produced as a result of oxidative mechanism, which are attributed to DNA damage in water as well
as in land animals. Therefore heavy metals toxicity and carcinogenicity are other threats to animals in
aquatic environment ecosystem and the scientific community are worried about this issue. Different
abnormalities such as plasma membranes damage, resulting in binding to phospholipids and proteins,
inhibition activities of sodium and potassium dependent ATP enzymes, lipid peroxidation, enzyme
inhibition, prevention of transmembrane amino acid transport and DNA lesions are attributed to
heavy metal cytotoxicity in animals (Stohs and Bagchi, 1995; Chang et al., 1996; Bal and Kasprzak,
2002). Heavy metals can enter into the cell and disturbing metabolism of the cells but some time get
entry into the nucleus. There is formed ionic and coordinated bonds between heavy metals and DNA,
but cannot produce all the disorders seen in cellular chromatin materials. Therefor both direct and
indirect impacts of metals on nuclear chromatin must be essential to know about the DNA damage
(Kasprzak., 1995; Klobucar et al., 2008; Siraj, 2016).
Previously it is discussed that the DNA in nucleus is directly affected by metals and
nanoparticles and rarely during cell division which results in damage of DNA. Frequency of DNA
damage in RBCs of Balkan loaches was determined on the basis of tail length, moment and intensity
(% DNA). The findings indicate that Balkan loaches from the Sava River contain smaller degree of
DNA damage cells as compare to those from reference site and therefore this technique is very precise
for evaluation of genotoxicity. The result showed that the red blood cells showed low frequency of
DNA damage at clean Kupa site, intermediate degree in the site Sava 2 and greater degree in the
relatively polluted site Sava 1 (Nevenka et al., 2008; Karlsson, 2010; Gomes, et al., 2013). Recently
it has been investigated genotoxic disease syndrome in the fish. The reduction of DNA integrity in
red blood cells of aquatic animals affected with polluted water in their habitats (Gedik et al. 1992;
Kopjar et al., 2008; Gorbi et al., 2008; Kolarevic et al., 2016).
It is very easy to find out degree of DNA damage cells in different tissues and organs of fish
because no knowledge about metaphases and chromosome numbers are needed. DNA strand
breakage due to environmental hazards is one of the initial alarm (Belpaeme et al., 1998; Binelli et
al., 2007; Klobucar et al., 2008). Some variations like alkali attachment of DNA, strand breaks and
other alterations in the cells of aquatic animals are induced as a result of interaction of genotoxic
metals with DNA, owing elimination of damage nucleotides by enzyme may assist to greater level of
breaks in strand of DNA. All of the comet factors determined in the red blood cell of Balkan loach
highlights the occurrence of greater frequency of DNA damage in red blood cells netted from the
Sava River at the polluted Ivanja Reka and the result also showed aquatic environmental genotoxicity.
Genotoxic and carcinogenic effects of heavy metals like Ar and Cu have been determined. Both of
them are attributed to higher frequency of genotoxicity in aquatic organisms (Reiferschiede and
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Grummete, 2000; Gabbiaenelli, 2003; Nevenka et al., 2008). DNA repair activity, Genetic
susceptibility, metabolic activity, antioxidant concentrations, the number of alkali-labile sites and
heavy metals are different factors through, which DNA damage variability can be explained in the
aquatic organisms (Mitchelmore and Chipman., 1998; Akcha et al., 2003; Buschini et al. 2004).
Variation of DNA damage was estimated between the male and female sex of mussels.
However studies proposed notorious data on the involvement of sex in regulation of DNA damage.
In past few decades ago it is investigated that heavy metals like Fe, Cu, Cd, Hg, Ni, Pb and Ar have
ability to generate reactive radicals, which are resulting into cellular and DNA damage in living cells
of animals (Phillips, 1995; Devaux et al., 1998; Gorbi et al., 2008). Red blood and white blood cells
were examined to assess the degree of DNA damage due to heavy metals in aquatic organisms. The
cells showed greater degree of DNA damage in organisms sampled from polluted water as compare
to those from non-polluted water (Satish et al., 2008; Parolini et al., 2011; Kolarevic et al., 2016). It
had investigated that toxic chemicals like heavy metals had the capability to binded with DNA strand
and resulted into greater degree of DNA damage (De Flora et al., 1991; Bhaskaran et al., 1999), gene
mutations (Maccubbin & Ersing, 1991) (Maccubin et al., 1991) and syndromes of genetic disease
(Kurelec, 1993) in the aquatic organisms. The blood of different species was processed for
determination of genotoxicity. The heavy metals like Cd and Hg are two most toxic heavy metals,
which toxicity and genotoxicity for different aquatic organism have been investigated (Ayllon and
Garcia - Vazquez, 2000; Risso - de Faveney et al., 2001; Parolini et al., 2011).
The mussels are generally used as sentinel organisms for screening of pollution and potential
environmental hazards (Andral et al., 2004; Viarengo et al., 2007). Moreover, for assessing
genotoxicity, mussels are commonly employed (Meresch and Beauvais, 1997; Black and Belin,
1998; De Lafontain et al., 2000; Pavlica. et al., 2001; Klobucare et al., 2003; Rochere et al., 2006;
Binellie et al., 2007, 2010; Gomes et al., 2013). Numerous earlier researches also described that the
fifty percent or greater genotoxicity in cells of water animals i.e mussels were associated with heavy
metals in polluted sites or various reagents exposed in laboratory (Frenzilli et al., 2001, 2004; Regoli
et al., 2005; Machella et al., 2006; Gorbi et al., 2008). Moreover, haemolymph collected from mussels
for genotoxicity easily supply a single cell suspension; it can be easily collected and its usage permits
repeated sampling tissue of individuals (Štambuk et al., 2008). Though, the number of genotoxicity
researches focused on marine bivalves is far greater than those dealing with freshwater species. In
native environment there are not many research work measuring DNA damage (De Lafontaine et al.,
2000; Rochere et al., 2006; Binellie et al., 2007), and fresh water mussels in cages (Mersch and
Beauvais, 1997; Black and Beline, 1998; Pavlica et al., 2001; Klobucˇar et al., 2003; Martínez-Gómez
et al., 2017; Vaughn, 2017).
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6.2. Methods and Materials
6.2.1. Study area
6.2.2. Mussels sampling sites
6.2.3. Collection of the freshwater mussels samples
6.2.4. Collection of haemolymph from freshwater samples
A mussel was placed in clean plastic tray; by insertion of blade of a scalpel the shells of
mussel was opened about 2 cm gently, to allow the paper to run off all the water present in the shell
of mussel. By using a hypodermic syringe, at least 1 ml of haemolymph was obtained from the
posterior adductor muscle. Centrifugation of the haemolymph samples were for 10 min at 2000 rpm
was done and standard alkaline comet procedure was used for detection of DNA damage.
6.2.5. Comet assay
Comet assay was conducted according to the method described by Singh et al (1988) with
minor modification. The comet assay technique is suitable for determination of genotoxicity in the
aquatic animals due to its sensitivity (Kim and Hyun, 2006).
6.2.5.1. Preparation different solutions for comet assay
6.2.5.2. Lysing solution (, Nacl 146.1 gm, Trizma base 1.2 gm, EDTA 37.2 gm and, NaOH8 gm,
d.H2O 890 mL, pH 10.0 by using concentrated Na OH or HCl,).
6.2.5.3. Final lysing solution (10 % DMSO, 1 % Trition, lysing solution).
6.2.5.4. Phosphate buffer saline (PBS) (PBS 1 packet, 1000 mL d.H2O, pH 7.4).
6.2.5.5. Preparation of stock solutions
a. NaOH (200 g/500 mL d.H2O).

b. EDTA (14.89 g/200 mL d.H2O, pH 10).
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6.2.5.6. Electrophoresis buffer
For Electrophoresis Buffer Per liter, 30 mL NaOH and 5.0 mL EDTA were added, q.s. to
1000 mL, and were mixed well. The total volume was depended on our gel box capacity. The pH of
the Buffer was measured to be 13.
6.2.5.7. Neutralization buffer (0.4 M Tris 48.5 gm d.H2O 1000 mL, pH 7.5).
6.2.5.8. Staining solution (Acridine Orange, 20 μg/mL was used)
6.2.5.9. Stock solution (20mg / 20 mL, stored at room temperature and protected from the light).
6.2.5.10. Working solution (Stock solution0.4 mL, d.H2O19.6 mL=20 μg/mL).
6.2.5.11. Preparation of 1% and 0.5 % LMPA and 1% NMA
a.1% LMPA (500 mg / 50ml PBS)

b.1 % NMA (500 mg / 50 ml Mili Q H2O)
Both the low melting point agarose (LAMP) and normal melting agarose (NMA)
were boiled in microwave oven and kept in refrigerator until needed.
6.2.5.12. Preparation of base slides
For preparation of base slides, the NMA agarose was again melted briefly in
microwave. After boiling the NMA was poured in caplin jar and conventional slides were kept in the
caplin jar upto two-third the frosted area for a minute and then removed from the jar and placed in
tray for drying. The undersides of the slides were wiped with the help of another slide to remove the
agarose and were stored at room temperature until needed. The slides were labeled.
6.2.5.13. Layering of cells and LMPA on base slides
To the coated slide, 75 μL of LMPA mixed with 10 μL of blood and suspension of

grinded tissues. A cover slip was placed on it and the slide was kept on ice pack for 5 to 10 minutes.
The cover slip was then removed from the slide and added 85 μL LMPA to the slide. The cover slip
was again kept on this layer and the slide was returned to the ice pack for 5 to 10 minutes.
6.2.5.14. Placing of slides in final lysing solution (Total volume 25ml, Tritone 250µl, lysing solution
22.75ml, DMSO 2.5ml).
The cover slip was gently removed from the third LMPA layer and the slide was kept in the
glass tray and poured gently lysing solution in glass tray .The glass tray containing the slides was kept
in the refrigerator for 2 hours or for overnight at 4ºC.
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6.2.5.15. Electrophoresis of slides (Total volume 250ml, NaOH 7.5ml, EDTA 1.25ml, pH 13).
After 2 hours or overnight at ~4ºC, we carefully removed the slides from the lysing
solution and placed the slides closely side by side on the horizontal gel box at one end. Then the
horizontal gel box was gently filled with electrophoresis buffer (pH > 13) and allowed the slides to
remain in the electrophoresis buffer for 20 minutes to unwind the DNA. After 20 minutes the gel box
was kept in refrigerator and power supply was set at 24 Volts and 300 mill amperes current for 30
minutes time and turned on.
6.2.5.16. Neutralization of slides
After the completion of electrophoresis, the slides were removed from the gel box and
washed with neutralization buffer drop by drop. The slides were let sit for about 5 minutes and again
washed with neutralization buffer drop by drop and the process was repeated two more times.
6.2.5.17. Drying of slides
The slides were drained and kept for 20 minutes in cold 100% ethanol for dehydration. The
slides were dried in open air for drying.
6.2.5.18. Rehydration and staining of slides
Chilled distilled water was used for rehydration of slides. The slides were kept for about 30
minutes in it and then stained with 70μL DNA specific fluorescent dye acridine orange (20 μg / ml)
and kept for 5 minutes. To remove the excess of dye, the slides were dipped again in chilled distilled
water. Then the slides were covered with cover slips.
6.2.5.19. Scoring of slides and Visualization of DNA damage
From every slide 100 cells were randomly selected and images were taken at 400x by using
fluorescent microscope (Nikon Eclipse 80 i) equipped with an excitation filter of 450 - 490 nm. Comet
tail lengths (consisting of nuclear region and tail) were scored visually into 5 Comet classes.
6.2.5.20. Comet classes
Comet class 0 (no damage, hence no tail),

Comet class 1 (tail up to 1.5 times the diameter of the comet nucleus),
Comet class 2 (tail 1.5–2.0 times the diameter of the comet nucleus),
Comet class 3 (tail 2.0–2.5 times the diameter of the comet nucleus) and
Comet class 4 (maximally damaged with total DNA in its tail).
158
A final overall total comet score for all 100 cells was obtained by summing up the number of
cells in each class times the class number, giving a rating between 0 (completely undamaged) and
400 (maximum damaged) (Collins, 2004) i.e.
TCS = 0(n) + 1(n) + 2(n) + 3(n) + 4(n), Where (n) indicate the number of cells in each class. One slide
reader performed the overall scoring.
6.2.5.21. Statistical analysis
differences by using student’s t – test (two-tailed); a P value < 0.05 was considered to show statistical
significance.
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6.3. Results and discussion
The present investigation has determined genotoxicological effects of heavy metals like Cd,
Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn, in haemocytes of freshwater mussels collected from site 2, 3 and
4 site (polluted) of River Kabul and were compared with mussels samples collected from site 1
(reference site) to estimate degree of DNA damage like total comet score (TCS) and comet classes
caused by heavy metals through comet assay.
6.3.1. TCS and comet classes in Hemolymph
Haemocytes of the freshwater mussels from Michini Bridge below Warsak dam, Site1
(reference site) and polluted sites 2, 3 and 4 (Sardaryab, Amangarh and Peer sabaq) were collected
and processed for the determination of DNA damage in cells. The haemocytes of the freshwater
mussels from polluted water revealed greater level of DNA damage in cells as compare with those
from reference site, where lesser DNA damage were detected (Table 39 and Fig. 73).
Haemocytes of freshwater mussels from polluted sites 2, 3 and 4 of River Kabul revealed
lower frequency of comet class 0 with mean values of 41.6 ± 13.5 cells, 28.3 ± 8.1 cells and 19.3 ±
6.7 cells and indicated greater level with mean value of 56.1 ± 15.8 cells from control site 1 (Michini
Bridge), This highlights that comet class 0 was maximum in reference site and minimum in last
downstream site which is well polluted. In this finding degree of comet class 0 was lower than those
reported by (Pandrangi et al., 1995; Tice., 1995; Villarini et al., 1998; Wilson et al 1998; Siraj 2016).
The result shows that comet class 0 was lowest in haemocytes from polluted site and highest from
reference site. This could be related to fewer metals in this samples. Comparing our result with the
findings of above mentioned studies highlights that heavy metals are toxic in nature and can induce
genotoxicity in various tissues of terrestrial and aquatic animals.
The detected greater mean values for comet class 1 in haemocytes of freshwater mussels from
polluted sites 2, 3 and 4, were 21.2 ± 4.4 cells, 18.7 ± 4.1 cells and 17.3 ± 3.7 cells, whereas 20.1 ±
5.8 cells from reference site1. This indicates that site 2 Sardaryab showed high degree of comet class
1 and site 4 low degree of comet class 1. This greater degree of comet class 1 in haemocytes of mussels
could be the cause of greater concentration of heavy metals in this site. As the results showed that
reference site also have the class 1 cells in greater number, it means that here in this site also greater
amount of the heavy metals are present, which results in DNA damage. These findings are agree with
results of those reported by (Belpaeme et al., 1996; Mitchelmore and Chipman, 1998; Kim et al 2000;
Gorbi et al., 2008). Comparing the present result with the findings of previous workers revealed that
160
heavy metals are genotoxic in nature, which may be resulted into higher frequency of DNA damage
cells in hemolyph of the freshwater mussels.
Haemocytes of freshwater mussels from polluted sites 2, 3 and 4 contained highest degree of
comet class 2 with mean values of 14.9 ± 4.7 cells, 16.3 ± 4.2 cells and 16.6 ± 5.5 cells, while reference
site 1 contained lowest frequency with mean value of 11.7 ± 4.5 cells. These results are agreed with
the findings of (Chandra and Khuda, 2004), who had also reported greater degree of comet class 2 in
gills of Oreochromis mossambicus after exposure to cadmium chloride and azadirachtin.
Haemocytes of freashwater mussels from sites 2, 3 and 4 revealed greater degree of comet
class 3 with mean values of 11.6 ± 4.0 cells, 16.2 ± 3.0 cells and 21.5 ± 5.2 cells whereas site 1
showed less degree of class 3 with mean value of 8.1 ± 4.1 cells. The present result of higher comet
class 3 in haemocytes of freshwater mussels agree with the findings of Bertin and Averbeck (2006).
On the other hand, the present data agree with those of Cotelle and erard (1999) and Valko et al (2005)
for blood DNA damage in blood of fish. Heavy metals are associated with DNA damage in humans
and other animals. Hence accumulation of Heavy metals can induce DNA damage cells in aquatic
animals like freshwater mussels. The present investigation found more degree of damage DNA
haemocytes cells in hemolymph of examined mussels from polluted water as compare to reference
site of Michini Bridge below Warsak dam. This could be correlated to higher concentration of heavy
metals in body of studied mussels from polluted sites of River Kabul. This result also confirmed the
heavy metals pollution in River Kabul.
Cr has been significantly correlated with the DNA strand damage in the mussels sampled
from the wild (Rank et al., 2005). Several earlier researchers described the considerably greater levels
of DNA damage in aquatic organism collected from heavy metal contaminated water (Steinert et al.,
1998; Frenzilli et al., 2001; Nacci et al., 2002; Gorbi et al., 2008). These findings are in agreement
with our results where the DNA damage detected by the Comet assay seem to be correlated with the
comparative heavy metals concentrations in the surrounding environment. Al-Subiai et al., (2011)
found the genotoxic effects of Cu on the DNA strand breaks of freshwater mussels.
Haemocytes from polluted sites showed maximum degree of comet class 4 with mean values
of 10.6 ± 4.2 cells, 19.5 ± 3.9 cells and 25.7 ± 8.3 cells and showed minimum mean value of 4.1 ± 3.5
cells from reference site (Michini Bridge). This trend shows greater degree of comet class 4 in site 4
and smaller in site 1.These results are agreed with the findings of previous investigations reported by
(Bresler et al., 2001; Gorbi et al., 2008). By comparing the present result with the findings of above
mentioned studies revealed that heavy metals tend to be concentrated in soft tissues of mussels and
induce greater degree of comet class 4 in hemocytes of freshwater mussels. The result also showed
161
heavy metals pollution in the studied areas. The haemolymph of mussels from polluted portions of
River Kabul showed maximum frequency of comet class 4 as compare to reference portion.
Haemolymph of freshwater mussels from polluted sites 2, 3 and 4 had highest frequency of
total comet score (TCS) with mean values of 128.3 ± 34.3 cells, 178.1 ± 24.3 cells and 217.9 ± 28.3
cells and had lowest mean value of 84.1 ± 37.8 cells from reference site. This result found more TCS
than those reported by (Ayllon and Garcia, 2000; Bresler et al., 2001; Risso et al., 2001).
In this research we examined variants in the extent of DNA damage in the haemocytes of the
haemolymph collected from freshwater mussel and the relation between the detected changes in
response and the chemical and physical features of the sampling sites. Genotoxicity due to heavy
metals were examined on haemocytes because of their role in processes of the digestion and transport
of nutrients, and removal of toxic heavy metals from the body, due to which haemocytes are
constantly exposed to contaminated water (Makala and Oikari, 1990; Soares-da-Silva et al., 2002).
Results of the present and previous studies demonstrated that heavy metals are genotoxic in nature
and can induce genotoxicity in aquatic and terrestrial animals.
Table 38: Genotoxicity results in the hemocytes of freshwater mussels.
Sites Class 0 Class 1 Class 2 Class 3 Class 4 TCS
1 56.1 ± 15.8 20.1 ± 5.8 11.7 ± 4.5 8.1 ± 4.1 4.1 ± 3.5 84.1 ± 37.8
2 41.6 ± 13.5 21.2 ± 4.4 14.9 ± 4.7 11.6 ± 4.0 10.6 ± 4.2 128.3±34.3*
3 28.3 ± 8.1 18.7 ± 4.1 16.3 ± 4.2 16.2 ± 3.0 19.5 ± 3.9 178.1±24.3*
4 19.3 ± 6.7 17.3 ± 3.7 16.6 ± 5.5 21.5 ± 5.2 25.7 ± 8.3 217.9±28.3*
*Difference significant relative to site 1 at P ≤ 0.0001
162
Figure 72: Images showing different comet classes that are induced as a result of heavy metals
163
Conclusions and Recommendations
Conclusions
1. The industries in the vicinity of River Kabul discharge their effluents containing high levels
of TSS and Hg into the River Kabul. Thus raising the levels of these parameters beyond the
recommended levels of National Environmental Quality Standards.
2. Some of the parameters like TSS and Hg still remain higher in downstream water showing
sublethal contents of contaminants in the water of River Kabul.
3. The accumulated heavy metals like Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn in soft tissues of
freshwater mussels.
4. The data generated in these studies confirm the presence of sub lethal concentrations of
pollutants in the River Kabul that freshwater mussel’s population is surviving under stressful
conditions, which is apparent from the genotoxical and pathological disorders and heavy
metals load in the bodies of inhabitant population.
5. The decline in the freshwater mussel’s population in River Kabul can not only attributed to
detrimental effect on adult freshwater mussels health but also on the eggs and juveniles.
6. The damage to the freshwater mussels health is indicated by genotoxical and

histopathological abnormalities in samples collected from polluted portions of River Kabul.
164
Recommendations
1. A general biomonitoring programing needed to be established where the hydrological and

geomorphological characteristics, the chemical and physical water quality and the river
vegetation are taken into consideration as these will affects aquatic system.
2. Strict environmental laws should be implemented and public awareness should be created.
3. The processing of industrial effluents, before their disposal should be regulated by strict
vigilance and effective legislation.
4. The industrial effluents and sewages should be detoxified before discharging into River
Kabul.
5. The general biomonitoring programme is needed to be established to check the level of heavy
metals and physico-chemical parameters in the River water on routine basis.
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7.1. References
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FRESHWATER MUSSEL (ANODONTA CYGNEA) AS A

BIOINDICATOR FOR MONITORING OF POLLUTION STATUS IN

MUHAMMAD IFTIKHAR KHAN

A MANUSCRIPT PRESENTED TO THE DEPARTMENT OF ZOOLOGY, UNIVERSITY OF

MUHAMMAD IFTIKHAR KHAN

Table 1: Operating data of Atomic absorption spectrophotometer for determination of metals............ 32

MUHAMMAD IFTIKHAR KHAN

AAC: Air acetylene LMPA: Low melting point agarose

Key Words: River Kabul, Physico-chemical parameters, Heavy metals, Bioaccumulation,

1.1: General Introduction

1.3 Geographic Range

1.5 Habitat and Ecology

Figure 1: Life cycle of freshwater mussel

1.7 Functions of freshwater mussels

1.7.1 Regulating services: mussels as biofilters that purify water

Figure 2: The variety of roles paid by freshwater mussels.

1.7.2 Supporting services: nutrient cycling and storage

1.7.3 Supporting services: mussels as habitat and habitat modifiers

1.7.4 Supporting services: mussels support food webs

1.7.5 Supporting services: mussels as environmental monitors

Freshwater mussels have the potential to serve as important biomonitors of environmental

1.7.6 Provisioning and cultural services

1.7.7 Losses, restoration and conservation

1.8 Description of Study area:

1.8.1 River Kabul:

1.8.2 Human population:

1.8.3 Industries along River Kabul:

1.8.4: Industries at Aman Garh industrial zone:

1.8.5 Disposal of sewages and industrial effluents to River Kabul.

1.8.6 Water contamination:

1.8.7 Heavy metals:

1.8.8 Sources of heavy metals:

1.8.9 Metals in the Environment:

1.8.10 Metals in the body of an organism:

1.8. 11 Essential and non-essential metals:

1.8.12 Role of heavy metals in the body of an organism:

Manganese, being discovered in 1774, is grayish-pink in color, chemically active and

1.9 Heavy metals pollution in River Kabul:

1.10 Effects of Heavy Metals on human beings:

1.11 Aims and objectives:

1. To conduct a gross examination of the freshwater mussel’s morphology, parasitic infestation,

and heavy metals in water and sediment of River Kabul.

mussels of River Kabul.

PHYSICAL AND CHEMICAL CHARACTERISTICS OF RIVER KABUL

2.2 Physico-chemical Parameters:

2.3 Heavy metal parameters:

2.4 Materials and Methods

2.4.1 Study area description

For detail see page- 07

and site 4 (water sample D) at River Kabul.

2.4.4. Water sample from Michini Bridge below Warsak dam

2.4.6. Collection of water samples

2.4.7. Preservation of water samples

2.4.8. Water analysis

Model; 7020- pH meter.

2.4.9.2. Electrical conductivity

Model; AGB 1000.

2.4.9.3. Total dissolved solid (TDS)

2.4.9.4. Total suspended solid (TSS)

2.4.9.6. Total alkalinity

2.4.9.7. Sodium and Potassium

Atomic absorption spectrophotometer (Spectra-AA-700) was used for the determination of