unionidae) from the chashma lake-river indus pakistan

I

Thesis

For partial fulfillment of Ph.D. degree in Zoology

Title:

Bioaccumulation of heavy metals and genotoxicity in freshwater mussels (Bivalvia:

Unionidae) from the Chashma Lake-River Indus Pakistan

Submitted by

Muhammad Sohail

(Roll NO. Zp11-08)

Supervisor:

Prof. Dr. Muhammad Naeem Khan

Department of Zoology, University of the Punjab

Lahore, Pakistan

Co-Supervisor:

Prof. Dr. Naureen Aziz Qureshi

Government College Women University Faisalabad, Pakistan

Department of Zoology, University of the Punjab

Lahore, Pakistan

II

Declaration

I, Mr. Muhammad Sohail Roll No. ZP11-08 student of PhD in the subject of Zoology,

Session 2011-2016 hereby declare that the material printed in this thesis titled

“Bioaccumulation of heavy metals and genotoxicity in freshwater mussels (Bivalvia:

Unionidae) from the Chashma Lake-River Indus Pakistan” is my own work that has been

submitted as thesis or would be published in Pakistan or internationally.

Dated: 17-08-2016

Muhammad Sohail

Roll No. ZP11-08

Session 2011-2016

III

In the name of almighty "ALLAH" who

Is

RAHMAN

And

RAHEEM

Oh Lord,

Make me

An Instrument of Your Peace

Where, there is Hatred

Let me Show Love

http://www.google.com.pk/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjY_uLOx4TOAhXJzRoKHTpnAjsQjRwIBw&url=http://islamic-stuff-corner.blogspot.com/2012/12/beautiful-bismillah-calligraphy-arabic.html&psig=AFQjCNHwTUhgfZrEhT54XjHx10nwd79hiw&ust=1469190593211359

IV

Where, there is Injury, Pardon

Where, there is Doubt, Faith

Where, there is Despair, Hope

Where, there is Darkness, Light

And where, there is Sadness, Enjoy

Dedicated To

HOLY PROPHET MUHAMMAD (PBUH)

The Greatest Social Reformer

To,

My beloved

MOTHER

And my affectionate

FATHER

Mr. & Mrs. CH. Sher Muhammad

Those who live in my Mind, in my heart,

throughout the whole span of my life,

and are Nearest, Dearest and Deepest,

to me.

V

Acknowledgments

All acclamations and appreciation are for ALMIGHTY ALLAH, the Omnipotent, the

omnipresent, the Merciful, the Beneficent, who presented me in a Muslim community and

also bestowed and blessed me with such a lucid intelligence as I could endeavor my services

towards this manuscript and who gave me an opportunity to add a drop in the wide ocean of

knowledge. All respect to Holy Prophet (P.B.U.H) whose sayings enlightened my brain and

who showed us the purpose and meaning of life.

I deem it a rare privilege and source of pleasure in expressing my profound and

cordial gratitude to my respected, learned and reverend supervisor Prof. Dr. Muhammad

Naeem Khan Dean faculty of life Sciences, Department of Zoology, University of the Punjab,

Lahore, Pakistan. His encouragement, support, guidance, valuable suggestion, boundless

forbearance, indefatigable help with anything, anywhere, anytime, consummate advice and

thought-provoking instructions in piloting this research venture and keen supervision made this

work possible to reach its present effective culmination. I offer my profound thanks to Prof.

Dr. Javed Iqbal Qazi, Chairman Department of Zoology University of the Punjab, Lahore for

providing all possible facilities during academic and research session and for his technical help

whenever needed.

My greatest thanks go to my respected Co-supervisors, Prof. Dr. Naureen Aziz

Qureshi Vice Chancellor, Government College Women University, Faisalabad who are always

a source of inspiration and continuous encouragement especially during hard and desperate

phases of this research. Special thanks for them would always be due. I would like to express

my thanks to Dr. Abdul Shakoor Chaudhry School of Agriculture, Food and Rural

Development Newcastle University UK, for their cooperation, and guidance during the period

VI

of my research work at this prestigious institution. I deem it my utmost pleasure to avail an

opportunity to express my heartiest gratitude and deep sense of obligation to all my friends

especially Khurram Shahzad, Shahid Sherzada, Munir Ahmad, Dr. Khalil Khan, Dr.

Naveed Ahmad, Dr. Khalid Javed Iqbal and Arshad Mahmood, because they remained a

source of guidance and encouragement during my research work. I am very thankful to my

friend Mr. Shoaib Hussain Satti who helped me in collection of samples from the Chashma

Lake River Indus Pakistan and provides me statistical help. I am also very grateful to Dr.

Farhat Jabeen, Dr. Noor Khan, Dr Muhammad Hafeez-ur-Rehman and Dr Fayyaz

Rasool for their time to time guidance and support during the research work of my PhD.

My acknowledgement is incomplete without thanking to my family for their

everlasting love and support. I especially thank to my dear wife Khadija for her contributions

and courage and love for my two kids Muhammad Ayan and Muhammad Arib. Many

thanks to my brothers Muhammad Shoaib, Muhammad Zohaib, Muhammad Shehroz, My

Grandfather Nazir Ahmed (late) and my grandmother who is always a source of

inspiration and continuous encouragement for me. I deem it my utmost pleasure to avail an

opportunity to express my hearties gratitude and deep sense of obligation to my father and

mother in law Mr. & Mrs. Muhammad Jamil, my brothers in law Muhammad Asif,

Muhammad Atif and my sweet sister Ambreen. They always acted as a light house for me in

the dark oceans of life path. No words can really express the feelings that I have for my small

family. The name of these people will always be in front of my eyes, as I will look on the cover

of my thesis, even though my name may be printed on it. Their prayers, sacrifices,

encouragement, moral support and confidence make me able to score this glory of life.

VII

Special thanks to Higher Education Commission Pakistan for their funding under

International Research Support Initiative Program (IRSIP) to support my PhD Research work

at School of Agriculture, Food and Rural Development, Newcastle University, UK.

(May Almighty ALLAH bless them all)

Ameen

MUHAMMAD SOHAIL

VIII

Table of contents

DEDICATION IV

ACKNOWLEDGEMENTS V

TABLE OF CONTENTS VIII

LIST OF TABLES IX

LIST OF FIGURES XI

S. No. CHAPTERS PAGE No.

1 SUMMARY 1

2 INTRODUCTION 4

3 AIMS AND OBJECTIVES 12

4 REVIEW OF LITERATURE 13

5 MATERIALS AND METHODS 31

6 RESULTS 44

7 DISCUSSIONS 114

8 CONCLUSION AND RECOMMENDATIONS 124

9 REFERENCES 127

10 APPENDIX

Publications

IX

List of tables

S. NO. Title PAGE

1

Bioaccumulation of metals in foot, gills and mantle of freshwater mussels

(Anodonta anatina) exposed to different doses of Lead (Pb), Copper (Cu)

and Chromium (Cr). 45

2

Mineral composition of different portions of freshwater mussels (Anodonta

anatina) collected from the control treatment. 49

3


anatina) exposed to different doses of Lead (Pb). 51

4


anatina) exposed to different doses of Copper (Cu). 55

5

Mineral composition of different portions of Freshwater Mussels

(Anodonta anatina) exposed to different doses of Chromium (Cr). 59

6


anatina) exposed to different doses of combined treatment (Pb + Cu + Cr). 63

7

Chemical composition of different portions of freshwater mussel

(Anodonta anatina) collected from the control treatment. 76

8 Proximate composition of various portions of freshwater mussels

(Anodonta anatina) exposed to different doses of Lead (Pb). 78


(Anodonta anatina) exposed to different doses of Copper (Cu). 82


(Anodonta anatina) exposed different doses of Chromium (Cr).

85

11

Proximate composition of various portions of freshwater mussels

(Anodonta anatina) exposed to different doses of combination of metals

(Pb + Cu + Cr).

88

12 Concentration of metals and minerals in water samples from the Chashma

lake River Indus Pakistan. 98

X

13 Concentration (mg/kg) of metals and minerals in various tissues of

freshwater mussels from the Chashma lake River Indus Pakistan 100

14

Pearson correlation coefficient (r) among metals in tissues of freshwater

mussels (Anodonta anatina) from the Chashma Lake River Indus Pakistan

102

15

Mean value (±S.D) of Carbon and Nitrogen in tissues of freshwater

Mussels (Anodonta anatina) from the Chashma Lake River Indus Pakistan 103

16

Proximate composition (%) of foot, gills and mantle in freshwater mussels

(Anodonta anatina) from the Chashma Lake River Indus Pakistan 104

17

Pearson correlation coefficient (r) among nutrients in tissues of freshwater

mussels (Anodonta anatina) form the Chashma Lake River Indus Pakistan 106

18

Effect of different doses of Pb, Cu, Cr individually and in combination on

mean % tail DNA. 109

19

Effect of different doses of Pb, Cu, Cr individually and in combination on

mean comet tail length. 110

20

Effect of different concentration of Pb, Cu, Cr individually and in

combination of metals on mean Olive Tail Moment (OTM). 111

XI

List of figures

S. NO. Title PAGE

1 Study area viewing the location of sampling spot 32

2 Anatomy of freshwater mussel 34

3 Flow chart of material and methods 42

4 Variations in metals bioaccumulation among various portion of freshwater

mussels (Anodonta anatina) exposed to different doses of Lead (Pb), Copper

(Cu) and Chromium (Cr).

46

5 Variations in mineral element composition among various portions of

freshwater mussels (Anodonta anatina) exposed to different doses of Lead

(Pb).

52


freshwater mussels (Anodonta anatina) exposed to different doses of Copper

(Cu).

56


freshwater mussels (Anodonta anatina) exposed to different doses of

Chromium (Cr).

60

8 Variations in mineral composition among various portions of freshwater

mussels (Anodonta anatina) exposed to different doses of combined

treatment (Pb + Cu + Cr).

64

9 Fluctuation of Potassium (K) levels in foot of freshwater mussels (Anodonta

anatina) exposed for the different doses of various metals.

65

10 Fluctuation of Calcium (Ca) levels in foot of freshwater mussels (Anodonta

anatina) exposed for the different doses of various metals

65

11 Fluctuation of Sodium (Na) levels in foot of freshwater mussels (Anodonta


66

12 Fluctuation of Phosphorus (P) levels in foot of freshwater mussels (Anodonta


66

13 Fluctuation of Zinc (Zn) levels in foot of freshwater mussels (Anodonta


67

14 Fluctuation of Manganese (Mn) levels in foot of freshwater mussels

(Anodonta anatina) exposed for the different doses of various metals.

67

XII

15 Fluctuation of Phosphorus (P) levels in gills of freshwater mussels


68

16 Fluctuation of Sodium (Na) levels in gills of freshwater mussels (Anodonta


68

17 Fluctuation of Potassium (K) levels in gills of freshwater mussels (Anodonta


69

18 Fluctuation of Calcium (Ca) levels in gills of freshwater mussels (Anodonta


69

19 Fluctuation of Zinc (Zn) levels in gills of freshwater mussels (Anodonta


70

20 Fluctuation of Manganese (Mn) levels in gills of freshwater mussels


70

21 Fluctuation of Phosphorus (P) levels in mantle of freshwater mussels


71

22 Fluctuation of Sodium (Na) levels in mantle of freshwater mussels


71

23 Fluctuation of Potassium (K) levels in mantle of freshwater mussels


72

24 Fluctuation of Calcium (Ca) levels in mantle of freshwater mussels


72

25 Fluctuation of Zinc (Zn) levels in mantle of freshwater mussels (Anodonta


73

26 Fluctuation of Manganese (Mn) levels in mantle of freshwater mussels


73

XIII

27 Variation in proximate composition among various portions of freshwater

mussels (Anodonta anatina) exposed to different doses of Lead (Pb).

79

28 Variations in proximate composition among various portions of freshwater

mussels (Anodonta anatina) exposed to different doses of Copper (Cu).

82

29 Variations in proximate composition among various portions of freshwater

mussels (Anodonta anatina) exposed to different doses of Chromium (Cr).

85

30 Variations in proximate composition among various portion of freshwater

mussels (Anodonta anatina) exposed to different doses of combined metal


88

31 Fluctuation of Carbohydrate contents in foot of freshwater mussels


89

32 Fluctuation of Moisture contents in foot of freshwater mussels (Anodonta


89

33 Fluctuation of Ash contents in foot of freshwater mussels (Anodonta


90

34 Fluctuation of Fat contents in foot of freshwater mussels (Anodonta anatina)

exposed for the different doses of various metals.

90

35 Fluctuation of Protein contents in foot of freshwater mussels (Anodonta


91

36 Fluctuation of Carbohydrate contents in gills of freshwater mussels


91

37 Fluctuation of Moisture contents in gills of freshwater mussels (Anodonta


92

38 Fluctuation of Ash contents in gills of freshwater mussels (Anodonta


92

XIV

39 Fluctuation of Fat contents in gills of freshwater mussels (Anodonta anatina)


93

40 Fluctuation of Protein contents in gills of freshwater mussels (Anodonta


93

41 Fluctuation of Carbohydrate contents in mantle of freshwater mussels


94

42 Fluctuation of Moisture contents in mantle of freshwater mussels (Anodonta


94

43 Fluctuation of Ash contents in mantle of freshwater mussels (Anodonta


95

44 Fluctuation of Fat contents in mantle of freshwater mussels (Anodonta


95

45 Fluctuation of Protein contents in mantle of freshwater mussels (Anodonta


96

46 Fluctuations of metals and minerals concentration in various tissues of

freshwater mussels from the Chashma lake River Indus Pakistan.

100

47 Fluctuation in proximate composition (%) of foot, gills and mantle in

freshwater mussels (Anodonta anatina) from the Chashma Lake River Indus

Pakistan.

104

48 The image of comets by fluorescent microscope from the gills of freshwater

mussels (Anodonta anatina) collected from the control treatment.

106

49 The images of comets by fluorescent microscope from the gills of freshwater

mussels (Anodonta anatina) collected from the treated tanks.

107

50 DNA damage in gills of freshwater mussel (Anodonta anatina) expressed

in the form of mean tail DNA exposed for different doses of heavy metals.

111

XV

51 DNA damage in gills of freshwater mussel (Anodonta anatina) expressed in

the form of mean tail length exposed for different doses of heavy metals.

111

52 DNA damage in gills of freshwater mussel (Anodonta anatina) expressed

in the form of mean olive tail moment (OTM) exposed for different doses

of heavy metals.

112

1

1. Summary

Bioaccumulation of heavy metals and analysis of mineral element alongside

proximate composition and genotoxicity were carried out in this study. Freshwater mussels

(Anodonta anatina) were directly harvested from the Chashma Lake River Indus Pakistan

and also exposed to various doses of Pb, Cu and Cr singly and in combination (Pb + Cu + Cr)

in laboratory conditions. In laboratory exposure, the concentrations of all the studied heavy

metals in soft tissues of the mussels increased as the metal doses were increased from 0 to

360 µg/L of water. The highest concentration of Cu was observed in the gills of mussels at

the highest dose (360 µg/L) whereas the lowest concentration was observed in the Cr-

exposed mussels at the lower dose (120 µg/L). Amongst mineral elements, Ca was found to

be the most abundant element in all tissues. The maximum Ca (156906 ± 736 mg/kg) was

observed in the gills. The abundance order of the other mineral elements was P > Mn > Na >

K > Zn. Proximate analysis showed that the protein (15.45 ± 1.13%), fat (0.97 ± 0.10%) and

moisture (77.78 ± 1.20%) contents were significantly higher in the foot, whereas the

carbohydrate (15.15 ± 1.30%) and ash (10.55 ± 1.11%) contents were higher in mantle and

gills, respectively. It was found that the low dose exposure of Pb and Cu and the high dose

exposure of Cr caused higher protein content in the foot. It appeared that freshwater mussels

(Anodonta anatina) are an essential tool for biomonitoring studies. However specific

evaluation of mussel tissues was more effective than using the whole animal in these studies

In a wild experiment, Proximate composition and elemental analysis were also carried

out in edible (Foot, Mantle) and non-edible portion (gills) of freshwater mussels (Anodonta

anatina) harvested from various site of Chashma Lake, River Indus Pakistan. The nutritional

components were varied among the studied portion and muscular foot found to be the best

2

part for consumption. Protein and fat contents were significantly higher in foot

(15.90±0.88%, 1.19±0.26%) as compared to mantle (10.78±2.24%, 0.27±0.09%) and gills

(6.44±1.22%, 0.53±0.15%) respectively. For the macro minerals mantle had high

concentration of Ca (46838±984 mg/kg), Na (2706±343 mg/kg), P (6921±1063 mg/kg) and

Mn (7207±1046 mg/kg) as compared to foot. Heavy metals (Cd, Cu, Cr) concentration in

edible portions were lower than the permissible limit by WHO whereas the concentration of

Pb was slightly higher than the recommended value that might be the risk for the consumers.

Being filter feeder gills accumulated the high concentration of all the metals and found to be

the key portion for biomonitoring studies. Freshwater mussels of Chashma lake Indus River

are the rich source of protein and all the other micro and macro minerals therefore could be

used as an excellent source of food. Aquatic invertebrates are playing an important role in

assessment of the water contaminants and also serve as a major component of food chain.

Freshwater mussels are considered to be the good bioindicator species of aquatic

environment and widely used to determine the metals load.

The genotoxic effect of different levels of heavy metals was investigated on gill cells of

freshwater mussels (Anodonta anatina), a sentinel species in aquatic environment.

Freshwater mussels were exposed to none (0 µg L -1), low (120 µg L-1), medium (240 µg L-1)

and high (360 µg L-1) levels of Lead (Pb), Chromium (Cr) and Copper (Cu) singly and in

combinations (Pb +Cr +Cu) for 15 days in laboratory conditions. The gills of mussels were

used to determine the level of DNA damage by comet assay, a rapid and sensitive method to

evaluate the genotoxic effects of chemicals. The tail DNA %, comet tail length and olive tail

moment (OTM) were the parameter selected to detect damaging of DNA. Low doses (120 µg

L-1) of each metal induced significantly higher level of DNA strands breaks as compared to

3

medium dose (240 µg L-1) and very low level of DNA damaged was observed at high dose

(360 µg L-1). Copper and lead showed significantly higher value of % of tail DNA

(56.74±1.81, 47.36±1.23) and comet tail length (41.30±0.758, 49.15±1.90), respectively as

compared to chromium and combined treatment (Pb + Cu + Cr). The lowest level of DNA

damage for all the parameters were observed in combined treatment.. It is concluded that the

Cu and Pb induced more DNA damage as compared to Cr and combined treatments (Pb + Cu

+ Cr). Moreover, our results showed that the low dose treatment of metals have more

genotoxic effect as compared to the medium and high doses. Genotoxic effect of metals on

freshwater mussels is very important to assess the aquatic health and could be suggested as

biomarker; furthermore these findings might be helpful for future bio-monitoring studies.

4

2. Introduction

Freshwater bodies are continually polluted by various point and non-point sources which

may put deleterious effects on the aquatic life. The metal pollution is a serious threat which

reduces the biodiversity by altering the water conditions which were essential for survival of

aquatic biota. Animals with filter feeding habits are directly exposed to such contaminants

and could be more threatened due to extensive bioaccumulation of these metals. Indus River

is huge water bodies flowing across the Pakistan; started from Jammu Kashmir directed

along the entire length of Punjab Province and merge to Arabian Sea near Karachi Sindh

Province. Various sites has been investigated for scientific research in many previous studies

but the Chashma Lake is heavily used for the inland fisheries and is economically important

reservoir for irrigation and generation of electricity. This lake has been reported for its

richness in fauna and flora but only few studies have been conducted on invertebrates

including freshwater mussels.

Freshwater ecosystems such as lakes and rivers are very essential resources because they

contribute in biodiversity, regulation of climate, managing floods and to meet the demands of

drinking water (Ra et al., 2011; Hansen, 2012). Chashma Lake is the main site on Indus

River for the inland fisheries and irrigation purposes. Due to intense change in climate, water

extraction and agricultural runoff; Indus River is at great risk (Jabeen and Chaudhry, 2010).

Unfortunately, lakes are continually polluted due to increasing anthropogenic activities such

as urbanization; industrialization, agricultural expansion, and manipulation of mineral

resources which cause a severe damage in freshwater ecosystem especially in less develop

countries (Thevenon et al., 2013; Zan et al., 2012). Among the other harms, the risks of toxic

metals in freshwater bodies cause adverse effect to the health of ecosystem (UNEP, 2011).

5

Heavy metals enter into the lakes from variety of sources such as due to breakdown of rocks,

soil erosion, aerial dust, and the release of anthropogenic activities and ultimately deposited

in their sediments (Karbassi et al., 2008; Malik et al., 2010). So, the profile of these metals in

freshwater ecosystem may provide the evidence for human influence, and safety of

freshwater ecosystem (Ip et al., 2007; Ma et al., 2013). Most of the heavy metals first settled

in bottom of water bodies in sediments and then resuspended to deteriorate the aquatic

environment (Malferrari et al., 2009). Due to increase in urbanization the huge quantity of oil

and coal were used in running vehicles and to generate the electric power so the reasonable

amount of Pb, Zn, Cu, Ni, Cd, Cr are discharged into the freshwater by road runoff and

atmospheric deposition.

The pollution due to heavy metal-induction is a major concern for the maintenance of aquatic

ecosystem functions. The heavy metals can cause loss of diversity and disability of the food

webs when they are accumulated at different trophic level which results in degradation of

aquatic ecosystem. Heavy metals occur in water naturally, but anthropogenic activities

related to various industries, mining, agricultural practices and household waste also

contribute to an increase of metals in the aquatic environment. This may directly affect the

aquatic life by accumulating heavy metals in different tissues (Usero et al., 2005).

Bioaccumulation and dispersal of these metals in waters greatly increase the health risk

because most of the water reservoirs are used by humans. The heavy metals are

predominantly important because they can be dangerous to a wide range of aquatic species,

are determined, non-biodegradable, and can be toxic above a certain edge (Rainbow, 2007).

Moreover, these effects may considerably reduce the survival capacity of the organism by

increasing vulnerability to diseases and damage (Montaudouin et al., 2010). Some metals are

6

biologically required (e.g., Cu, Zn, Na, K, P, Mn) although may prove toxic at high level and

could be harmful for the aquatic ecosystem due to their perseverance (Turkmen et al., 2008).

A substantial amount of metals has been found in aquatic environment, causing numerous

biological effects from ecological degradations to molecular malfunctions that depending on

the level of concentration and exposure time (Van der Oost et al., 2003; Jebali et al., 2013.)

cadmium, lead, arsenic, chromium and mercury are known to the most dangerous compounds

even at very lower concentrations (Bagal-Kestwal et al., 2008; Patrick, 2006). Most of the

trace metal-related diseases in animals are usually caused by the lead (Pb), cadmium (Cd),

mercury (Hg), arsenic (As) and chromium (Cr) (Patrick, 2006; Flora et al., 2008). Mostly the

small quantities of these metals are important in different biological processes of cells such

as transportation and cell signaling (Valko et al., 2005). If these metal ions are deviated from

their pathways and active in wrong side then these interact with essential protein and leads to

the disturbance in natural metabolic processes (Sevcikova et al., 2011). The adverse effect of

these metals includes enzyme inhibition, oxidative stress and impairment in metabolism

(Valko et al., 2005; Flora et al., 2008). There are several essential mineral elements such as

Na, K, Zn, P, Mn etc which are important for making body mass and several other essential

biological processes in living organisms. Bioaccumulation of heavy metals could interfere

with theses mineral elements and lead to serious biological dysfunctioning. No pervious

study has been reported that showed such influence of heavy metal bioaccumulation on

mineral elements.

Lead (Pb) is one of more common and persistent environmental pollutants. It has no valuable

function for the living organism and said to be the non-essential metal (Johannesson, 2002),

it can also accumulates in the tissues and it interferes with the other bio elements such as Ca,

7

Zn and Cu which cause a variety of serious disorders (Berrahal et al., 2011). It is estimated

that about 2.5 million tons of Pb produced from industries every year (Osfor et al., 2010). In

metabolic processes it plays an important role (Needleman, 2004) but on the other hand also

affects the variety of physiological processes and brings toxicity in tissues (Patrick, 2006).

Trace amount of lead prove fatal to aquatic organisms (Angelo et al., 2007; Bollhofer, 2012;

Krause-Nehring et al., 2012). Pb has also one of the most dangerous pollutants that can easily

accumulate in sediments and the tissue of aquatic animals (Sharma and Dubey, 2005).

Due to the toxicity and non-degradability heavy metals are believed as serious pollutants in

the aquatic environment. Some of these metals (Hg, Cr, Cd, Ni, Cu, Pb and Zn) may convert

into persistent metallic compounds with greater toxicity (Hyun et al., 2006; Maanan, 2007).

Moreover, these metals can accumulate in aquatic organisms like mussels and amplify in the

food chain and thus have threat to human health (Zhou et al., 2008). Some heavy metals may

change into metallic compound and prove toxic for the aquatic organism (Hyun et al., 2006;

Maanan, 2007). The metallic lead (Pb) is transformed in soil by different ways like erosion of

rocks and oxidation that increases the availability and the solubility in the aquatic

environment, thus a danger to aquatic biota.

The concentration of copper in unpolluted water is less than 5 parts per billion (ppb)

(Soegianto et al., 1999) and it may reach almost about 3 parts per million (ppm) in highly

polluted environment (Parry and Pipe, 2004). The constantly increasing concentration of

copper in aquatic environment is a serious danger to the aquatic organisms. Though, copper

is vital for the proper functioning of living organisms. For example, it may be a cofactor for

the enzyme action, but high concentration may prove toxic to an organism when exposed

chronically to an aquatic environment (Gaetke and Chow, 2003).

8

The accumulation of metals in aquatic animals not only danger for the animal but also

contaminate the aquatic food chain. Many previous studies showed that the metals

accumulate in the soft tissue of animal (Brown and Depledge, 1998) which put a long term

effect on aquatic ecosystem (Kucuksezgin et al., 2010). Bioaccumulation in specific tissues

of aquatic animal like bivalves provides indication to assess the contamination in aquatic

environment (Kavun and Podgurskaya, 2009). Bioaccumulation of these heavy metals in

aquatic organism would cause biomagnifications in food chain, which may harmful to the

humans. (Fergusson, 1990; Zhou et al., 2008).

The health of aquatic ecosystem has been monitored through the chemical analysis of

pollutant level in soft tissues of bivalves (Goldberg, 1986; Cantillo, 1998). As being sessile,

mussels are ecologically very important as they can bioaccumulate a broad range of varying

type of contaminants from aquatic ecosystems (Kimbrough et al., 2008). Many species of

animal and plant have been suggested as “biomonitors” of chemical pollutants in aquatic

environment (Akhiat et al., 2000; Bodin et al., 2013; Hunt and Slone, 2010; Saavedra et al.,

2004), but the bivalve molluscs are particularly suitable for monitoring purposes (Apeti et al.,

2012; Kimbrough et al., 2008; Nakata et al., 2012; Sericano et al., 2014).

Mussels can accumulate high degree of heavy metals in concentrations than levels in the

surrounding aquatic environment (Casas and Bacher, 2006; Casas et al., 2008; Schintu et al.,

2008) making them outstanding tracers to assess water quality (Apeti et al., 2009, 2010;

Catsiki and Florou, 2006; Chase et al., 2001; Rainbow and Phillips, 1993). Many

invertebrates that found in aquatic ecosystem are sensitive to contaminants and they could

accumulate heavy metals form their surrounding water (Reinecke et al., 2003). Freshwater

mussels are good choice for assessing aquatic pollutants due to their filter feeding activity

9

and continuous exposure of tissues to ambient water. Analysis of soft tissues and body fluid

of aquatic animals are investigated in various biomonitoring studies for assessing the

environmental health.

Bivalve mussels are biologically very important for biomonitoring studies due to more

survival rate, feasible to maintain in the laboratory; and able to accumulate greater

concentrations of a wide range of pollutants (Boening, 1999; Kimbrough et al., 2008; Sarkar

et al., 2008; Zhou et al., 2008). In previous biomonitoring studies it was noted that toxicity

can be assessed by using molluscs (Goldberg, 1986; Cantillo, 1998), even when the pollutant

concentrations in water are very low or close to their detection limits (Viarengo and Canesi,

1991; Blackmore and Wang, 2003). Freshwater mussels are bottom dweller species in

aquatic system and also prevalent in lotic waters of world and have distinctive activities like

filtration of water and excretion of nutrient which put positive influence on freshwater

ecosystem (Vaughn and Hakenkamp, 2001; Vaughn et al., 2008) as they filter plenty of

water for intake of food (Strayer, 2008).

A freshwater mussel is the key species both in relations of their ability to accumulate

toxicants in their soft tissues, and through the effects of this exposure on key biological

processes (Rainbow and Phillips, 1993). These bivalves are the bioindicator species which

represent a process for monitoring environmental health, (Rainbow, 2002). The sessile nature

and annual availability of bivalves like mussels along their ability to bear variations in

temperature, oxygen levels and salinity make them favorable as a bioindicator (Goldberg,

1986). Besides that, many laboratory and experimental studies have revealed that mussels

accumulate trace metals in proportion to the availability of heavy metals in the surroundings

(Boening, 1999). This ability has been used in the Mussel Watch programme (Goldberg,

10

1986). Mussels have also been used to evaluate the biological effects of pollutants,

(Chandurvelan et al., 2012).

In order to assess toxicological effects and exposure of contamination, there are variety of

behavioural, physiological and other biomarkers are available in addition to chemical

analysis of heavy metals in soft tissues (De caprio, 1997; Shaw et al., 2011; Schettino et al.,

2012). Genotoxicity means the damaging in heredity material like chromosome or DNA

damaging due to exposure of different chemical and physical agents (Newman, 2009).

Damaging in DNA could cause gene mutation which results in many genetic disease and

severe disturbance in metabolic processes, and this effect may take a long time to appear

(Hagenaars et al., 2008; Villela et al., 2006).

It is essential to locate the early warning response at molecular level to protect the aquatic

ecosystem from sever damages (Kalpaxis et al., 2004). DNA strand breakage is considered to

be one of the primary signals of environmental deterioration (Binelli et al., 2007; Klobucar et

al., 2008) and this technique has become well known to evaluate the genotoxic effect of

contamination on organismic and population level (Depledge, 1998). Comet assay is a quick

method to assess the genotoxicity in aquatic organism due to environmental pollutants (Jha,

2008). This is said to be one of the commonly used method for contaminant biomonitoring in

aquatic animals related to genotoxicity (Chen et al., 2007; Picado et al., 2007). Bivalves also

having ability to show a variety of molecular, cellular, histological and physiological

response for the environmental pollutants and one of that response is the breakage in their

DNA strand (Pavlica et al., 2001; Binelli et al., 2007; Bolognesi et al., 2004; Rocher et al.,

2006; Coffinet et al., 2008; Klobucar et al., 2008). The burden of heavy metals in the tissue

of mussel can correlate to biological dysfunctioning (Chandurvelan et al., 2012). Mussels are

11

widely used as bouncer animals for the screening of contamination and potential threat to

environmental (Andral et al., 2004; Viarengo et al., 2007). Furthermore, freshwater mussels

are usually manipulated for determining genotoxicity (Mersch and Beauvais, 1997; Black

and Belin, 1998; De Lafontaine et al., 2000; Pavlica et al., 2001; Klobucar et al., 2003;

Rocher et al., 2006; Binelli et al., 2007, 2010; Parolini et al., 2011).

Freshwater mussels are also a good source of food along with many vital nutrients in perfect

balance. In Pakistan, this imperilled fauna still not be utilized as a food, due to this pressure

is continuously increasing of fish that leads to scarcity of protein rich diet. Freshwater

mussels can be used as an alternate source of food to cope with the future challenges of

proteinaceous diet in less developed countries. Bivalves, especially mussels, are also used as

human food in various parts of the world, and due to high nutritive values their demand is

increasing internationally (Orban et al., 2002; Fuentes et al., 2009; Karnjanapratum et al.,

2013; Pogoda et al., 2013). Mussels are a rich source of dietary components such as protein,

minerals, fatty acids and carbohydrates (Fernandez-Reiriz et al., 1996; Orban et al., 2002;

Grienke et al., 2014). Espana et al. (2007) and Fuentes et al. (2009) reported that mussels

were a cheap source of protein and many essential vitamins and minerals. To investigate the

nutritional and commercial value of mussels, analysis of their chemical composition could be

a good indicator as reported by Orban et al. (2006). However, the chemical composition of

mussels may be affected if they are exposed to metal polluted waters causing metal induced

changes in their body functions. Nevertheless, potential interactions between metal

concentrations and chemical composition of mussels have not been reported in the past.

12

3. Aims and objectives

The objective of this study was to estimate the bioaccumulation of trace metals in foot, gills

and mantle of freshwater mussels in order to evaluate the capacity of these tissues to store

various metals and their impact on body compositions. Moreover, the effect of different

doses of various metals on mineral elements, proximate composition and nutritional values of

freshwater mussels was also investigated in laboratory condition. Chashma Lake

continuously receiving many industrial effluent, pesticides and sewage from the neighboring

communities that may deteriorate the water quality and cause decline in diversity of aquatic

species. The investigation of the metal load, mineral elements and food components in foot,

mantle and gills was also carried out in freshwater mussels directly from the Chashma Lake

River Indus Pakistan. It is important to investigate the aquatic health by using this animal as

bioindicator and this study was also spot the top indicator part of freshwater mussels for

biomonitoring in aquatic environment. Furthermore, the effects of various metals on gills of

freshwater mussels were also evaluated with reference to their DNA strand breaks by comet

assay.

13

4. Review of literature

4.1. Bioaccumulation of heavy metals

Bivalves are excellent bioindicator and good choice to assess aquatic health. The heavy

metals induction affects the mussels by various ways. These are not only accumulated in

tissues of mussels but also make them unfit for consumption which could leads to the

contamination of aquatic food chain (Boening, 1999). Among the other aquatic animal,

bivalves are the suitable bioindicator to assess the contamination in aquatic environment.

They are having ability to accumulate variety of metals in their soft tissues. The gills of

mussels are the main target site which can accumulate high level of metals than other tissues

(Hussein and Azza, 2013). Due to sedentary life style, mussels can accumulate high degree

of heavy metals from the immediate aquatic environment than the other aquatic species. The

traces of various heavy metals in mussel tissues showed the degree of contaminants in

aquatic environment (Irnidayanti, 2015). So, bivalve mussels are good candidate to assess the

water quality due to the significant accumulation of various heavy metals in their body

tissues. These features suggested that the bivalves are valuable tool to monitor the impact of

trace metals in assessment of the environmental risk (Giltrap et al., 2013). Gills and the

mantle are main visceral and sensitive parts for the heavy metals accumulation. Higher level

of metal load was observed in these tissue exposed under polluted water, furthermore it is

also stated that in polluted conditions, the metal uptake by freshwater mussels are more

frequent (Lukashev, 2010).

The soft tissues of bivalves are the excellent target sites for accumulation of heavy metals.

The accumulation of various heavy metals such as cadmium, zinc, lead and nickel have been

http://www.scopus.com/authid/detail.url?authorId=24166311300&eid=2-s2.0-84883113990


14

accumulated in the kidneys and other soft tissues of the mussels, when continuously contact

with contaminated areas (Podgurskaya et al., 2004). Jha et al. (2005) also reported the

bioaccumulation of various chemical in freshwater mussels and results showed that the gut

accumulating the high amount of these chemicals, followed by other tissues such as mantle,

gills and foot.

The mussels form the industrial area showed significant concentration of various toxic

metals. Anthropogenic activities contributed to contaminate the water which ultimately

affects the aquatic life. The bivalve mussels from contaminated area contained toxins in their

soft tissues which make them unfit for consumption (Sasikumar et al., 2006). Human related

activities continuously added the large amount of cadmium and zinc in aquatic environment

which could accumulate in the soft tissues of mussels. The accumulation level of such heavy

metals was higher than the permissible limit set by various healthy organizations for human

consumption (Unlu et al., 2008). Naimo, (1995) reported that the freshwater mussels can

accumulate certain trace metals and the quantity was significantly higher than in dissolved

water. It was also suggested that the gills, mantle and kidney of adult bivalve mussels are the

most vulnerable site for metal accumulation. In another study, bivalve mussels were exposed

to combination of Cd, Cu, and Hg, and their effects was measured by using cytological and

other biological markers. The gills of bivalves were found to be the first target sites for

contaminants and significant structural and functional changes were observed induced by

metals (Varotto et al., 2013).

The reduction in diversity and the population density of freshwater mussel was reported in

one of the previous study and it was concluded the chronic and the constant exposure to the

trace metals is the main reason behind that. Freshwater mussels are bottom dwellers and filter

15

feeders so that they have direct contact with the suspended particles and other contaminants

that dissolved in water. Several general, cytogenetic, physiological and cellular defects were

observed in mussels due to these agents. This study also revealed that the mussels are very

important bioindicator to determine the environmental health status (Dailianis, 2011). The

water bodies are continuously loaded with the pollutants due to anthropogenic activities.

These pollutants not only a source of genetic and metabolic defects but also cause mortality.

Bivalve mussels are bouncer species and widely used to determine the amount of

contaminants in aquatic environments (Thomas et al., 2011).

Bioaccumulation of various heavy metals in bivalve mussels and their effect on vital

chemical process were reported in several previous studies. The long term exposure of

different heavy metals such as Cd, Pb, Mn, Zn and Fe in tissues of bivalves were studies and

stated that the accumulation of metals is correlated with the exposure time, longer the time

higher the accumulation. Furthermore, it was observed that the bioaccumulation of metals

was varied among tissues such as gills, digestive gland and muscles of bivalves. The rate of

bioaccumulation depends on ability of specific tissues to uptake metals (Jebali et al., 2014).

The higher level of metals bioaccumulation was found in those mussels which were collected

from the polluted site as compared to those from unpolluted water (Belabed et al., 2013). The

rate of bioaccumulation is affected due to fluctuations in water chemistry. Kumar et al.

(2015) reported the accumulation of heavy metals in mussels form the high and low salinity

and more accumulation of metals was observed in animals collected from the water with low

salinity so it was concluded that the bivalves from the freshwater lakes have more metals

accumulation in their soft tissues as compared to the marine environment.


16

Human activities such as urbanization, agricultural practices and economic development put

extra load on aquatic ecosystem in the form different toxic pollutants. These anthropic related

pollutants contain many genotoxic agents and the results of study confirmed that mussels are

highly affected by aquatic contaminants (Klobucar et al., 2008). Heavy metals accumulation

was investigated in bivalve mussels from the coast of southern Italy. The level of water

quality and health risk of consumers was assessed by analyzing the soft tissues of mussels.

Significant level of heavy metals bioaccumulation were found in mussels tissue but the

values were below the permissible limit except the chromium, moreover study suggested that

there is no risk for consumers but the metals accumulation should be monitored to ensure the

consumers health (Spada et al., 2013). The bioaccumulation of metals was linked with the

seasons and greater values of metals observed in summer-spring. The study also stated that

the accumulation of metals varies species to species (Maanan, 2008). Many heavy metals

especially the nanoparticles which are widely used in personal care products ends in aquatic

environment and untimely accumulated from in aquatic food chain. Zinc nanoparticles are

lethal for biological system and causing DNA fragmentation and other metabolic defects in

bivalves (Falfushynska et al., 2015). Anthropic related activities added contamination in

aquatic environment all the time which includes many heavy metals such as arsenic (As),

cadmium (Cd), copper (Cu), lead (Pb), nickel (Ni), and zinc (Zn) and this is increasing day

by day. The accumulation of such heavy metals in gills and other soft tissue of mussels are

prove to be toxic and caused many biological stress responses (Chandurvelan et al., 2015).

Gillis et al., 2014 also reported the effect of urban derived pollutants such as municipal waste

and roads runoff on the gills of freshwater mussels. It was suggested that the

bioaccumulation of Pb and Cr leads to many physiological and sub-cellular stresses which

17

could reduce the growth rate of bivalves. In another previous study the seasonal

bioaccumulation of heavy metals were studied in bivalves from the Tersakan River, Turkey.

The higher level of bioaccumulation of heavy metals was found in winter and autumn as

compared to summer and spring. All the studied metals were in acceptable limit of human

consumption except the Pb; the higher value in gills was observed the designated limit (Genc

et al., 2015). In another study it was showed the bioaccumulation of Cu, Cr and Pb in soft

tissues of bivalves found in small natural water reservoir which receive the discharge from

the neighboring river. The greater value of trace metals was observed in viscera of mussels

while the order of metals toxicity was Pb > Cu > Cr. This was also suggested that the

bivalves are the best candidate for the bioindication of heavy metal pollution in small water

reservoir (Shue et al., 2014). Topi et al. (2012) investigated the heavy metals concentration

in water, sediments and mussels from the lagoon area of Albania. The level of Cu and Pb was

above the normal standard reflecting the human activities such as agriculture practices and

building construction, while high level of chromium concentration marked the climate

change and weather related processes. The significant and incremental effect of heavy metals

(Cu, Cr, and Pb) was observed in gills of freshwater mussels which indicate the availability

of metals in aquatic environment due to urban input. Chronic and combined exposure of

heavy metals not only retards the growth of freshwater mussels but also contribute in

declining in mussels populations (Gillis, 2012). Fatoki et al. (2012) reported the

bioaccumulation of metals in black mussels from the harbor of South Africa by ICPMS. The

higher value of Cu and Zn was found in soft tissues while in all the other studied metals, the

average amount of heavy metals was observed. The average concentration of metals was still

found higher than the permissible level indicated by FAO and other international parameters.

18

Anthropogenic activities such as waste water and agricultural runoff enhance the availability

of various toxic metals. Mussel samples showed higher concentration of heavy metals as

compared to sediments samples that make them a good bioindicator of aquatic pollution.

Significant correlation is found between the Cu and Pb concentration in sediments and

mussels as well and no carcinogenic effect was observed due to the heavy metals (Khairy et

al., 2011). The availability of various metals was observed in particulate, dissolved and biota

from the estuary of Belgium. It was stated that the bioaccumulation was observed to be more

significant in the dissolved phase as compared to particulate phase. Furthermore studies

reveals that the metals load in mussel’s tissues were higher than the acceptable limit of

international standard regarding estuary (Shilla et al., 2008).

Maanan, (2007) investigated the pollution costal water of Morocco by using the bivalve

mussels as an indicator species. The increased level of concentrations of these metals was

observed in area where there are more anthropic-related activities as compared to the clean

station. Moreover studies suggested that the bivalve’s mussels are suitable indicators to

investigate the metal pollution level in aquatic environment. The concentrations of trace

metals in soft tissue as well as in separate organs (mantle, gills, muscle and digestive gland)

of wild bivalves from coastline area were studied. Significant bioaccumulation was

confirmed concerning heavy metals which is subjected to extended agricultural activity and

intense industrialization arising at the adjacent area and a river outflow (Sakellari et al.,

2013). Heavy metal pollution adversely affects the shellfish life in water that induced

changes in trophic level. Heavy metals that are widely distributed in immediate aquatic

environment can cause accumulation in soft tissues of bivalves. Bivalve mussels serve as

bioindicator species in water impacted by trace metals, and give proposals for carry out

19

biomonitoring assays (Boening, 1999). Lead accumulation in the soft tissue of bivalves

mussels, were studied and the result illustrate that higher concentration in the soft tissue of

mussels which was depending on exposure time and the concentration of target metal lead in

the immediate aquatic environment (Rahnama et al., 2011). In the comparative study of

heavy metals regarding their accumulation in soft tissues of bivalve mussels, results showed

the higher concentration of Cu in gills, foot and sediments, the concentration of Pb was

intermediate from all the reference site while cadmium found to be lowered in tissues and

sediment (Pourang et al., 2010). The bivalves are the key species to accumulate the huge

burden of trace metals in their soft tissues. The bioaccumulation pattern was studied in

different tissue of mussels like in gills it was Zn > Fe > Cu > Cd, and in digestive gland was

Fe > Zn > Cu > Cd (Giarratano and Amin, 2010). The input of heavy metals in water

increasing continuously so more concentration of heavy metals was observed in tissues of

mussels as compared to previous studies (Mubiana et al., 2005).

Freshwater mussels are the part of human food in different regions of the world so it is

essential to investigate the potential risk of toxic metals which are accumulated in them.

Water bodies receive variety of toxin all the time from the different sources which untimely

contaminate the aquatic food chain and at the end that toxic might be the part of human food

via mussel consumption. Furthermore, as contamination by metal pollutants continue to

increase in some parts of the world, mainly in developing countries, it is also important to

limit the level of pollution in the aquatic environment, particularly in regions where

aquaculture is anticipated and where the local population eats large amounts of mussels

(Stankovic et al., 2012). Bivalve mussels are exposed to different contaminated sites to

examine the water quality of coastal area by measuring heavy metals accumulation. High

20

concentration of iron and copper was accumulated in digestive gland and highest level of

zinc was found in gills of mussels (Giarratano et al., 2010). In another previous study the

bivalve clams were evaluated from contaminated and non-contaminated sites to assess the

heavy metal load in their soft tissues. Significantly higher level of metals accumulations were

observed in contaminated site as compared to reference site. Moreover, it is stated that the

soft tissues of bivalve clams provide useful biomonitoring of heavy metals in aquatic

environment (Tarique et al., 2012). Bellas et al. (2014) examined the pollution level in

coastal area Sweden by using caged bivalve mussels. High level of accumulation of

chemicals are was observed in soft tissues of mussels which was collected from the area

receiving untreated industrial effluents as compared to reference site. There are different

biological and molecular defects were also observed in mussels from contaminated sites.

Furthermore it is also stated that mussels are the key animals to assess the aquatic load of

chemicals and toxicants. Bioaccumulation of chemicals and biomarker response was

observed in mussels collected from the harbor which manipulated by dredging activity and

high level of toxicants were found in soft tissue of mussels from the beginning of dredging

activity due to more availability of chemicals to organism which are previously trapped in

sediments. Furthermore, it is also mentioned that the dredging activity in any harbor area

increase the concentration of pollutants which effect the biota in aquatic environment

(Martins et al., 2012).

Many other similar studies in the past were also reported the same results related to

bioaccumulation of various heavy metals. The high level of metal concentration was

observed in gut and mantle of mussels which were collected from site that linked with

anthropogenic activity as compared to the non-contaminated site. Moreover it was also





21

studied that the high concentration of these metals was accumulated in gut as compared to

mantle (Trinchella et al., 2013). In another study the bioaccumulation of heavy metals was

investigated in gill of bivalve mussels. It was concluded that gills had more accumulation of

these metals due to direct contact with ambient water and showed immediate biomarker

response for aquatic toxicant as compared to other tissues (Giarratano et al., 2014).

Freshwater mussels are also used as a food throughout the world so there is need to evaluate

the proximate composition to other essential dietary minerals. It is also important to know the

influence of heavy metal on chemical composition and mineral elements for ensuring the best

quality food for the consumers. The effect of heavy metals on the tissues glycogen of

freshwater mussels was investigated by Rajalekshmi and Mohandas, (1993).

4.2. Proximate Composition

The variation of glycogen level in gills and hepatopancrease of freshwater were observed

when exposed for the various doses of Cu, Cd and Hg. The glycogen level was found to be

depleted in gills and hepatopancrease when exposed for the various doses of Cu and Cd.

Furthermore, it was concluded that the heavy metals exposure in freshwater mussel enhance

the breakdown of glycogen. Karnjanapratum et al. (2013) reported the proximate

composition and dietary values of bivalve mussels from the Coast of Andaman Sea. The

edible portion foot and mantle are rich source of all the micro and macro minerals and

therefore suggested as a best food for the health of consumers. Proximate and mineral

element composition was also investigated in bivalve from the coast of Marmara Sea Turkey.

It was revealed that the bivalves are the common and cheap food available throughout the

world. The entire mineral elements were in perfect balance except the Pb and Zn which need

to be monitor in the future to ensure the consumer health (Colakoglu et al., 2011). The



22

mussels from the Marmara Sea were also investigated by Ozden et al. (2010) for the micro

and macro minerals. The higher concentration of Ca, Mg, P, Na and K and values were near

to 1 mg/ 100 g whereas the concentration of Zn and Fe was lower than 1 mg/ 100 g. The

seasonal fluctuations in proximate composition were also analyzed and higher contents of

protein and lipids were found in summer while the ash and moisture content was higher in

winter. For the micro minerals, the higher contents of K, Na and Ca were found in summer.

All the toxic metals such as Pb, Cu and Cd was found to lowered the than international

permissible limit. The chemical composition and fatty acid analysis of freshwater mussels

were carried out in another previous study. It was suggested that the freshwater mussel

species are source of good fatty acid and also contained protein, carbohydrates and other

mineral with perfect balance. The study concluded that the studied species of freshwater

mussels are good choice for consumers (Ersoy and Sereflisan, 2010).

Laxmilatha, (2009) reported the high meat contents in marine clams and are rich source of

protein and other minerals with low fat contents. It was suggested that the marine bivalves

are preferred for consumption as compared to other shellfish. The effect of seasonal variation

on meat weight and proximate composition were reported in one of the previous study. The

significant increase in protein and carbohydrate contents were observed in summer and

autumn as compared to winter and spring. Furthermore, significant weight loss was observed

in winter condition as compared to summer and it was concluded that the cultured mussels

would preferably marketed from May to December (Okumuş and Stirling, 1998). Orban et al.

(2002) reported the season fluctuations in meat condition and proximate composition of

bivalve mussels cultured in Italy. The low meat contents and decreased in glycogen reserve

and lipid were observed in winter as compared to summer. Furthermore, it was suggested that

23

the bivalve mussels were source of good fats and other minerals. The dietary and commercial

importance of bivalve clams from the Adriatic Sea was reported by Orban et al. (2006). The

fluctuations were observed in nutritional quality parameter such as protein, lipid, stored

glycogen and nitrogen percentage throughout the year and contrary to one of the previous

study, the values were highest in winter. Further more study reveled that the bivalves

contained all the essential nutrients and are good choice for consumer’s health. Fuentes et al.

(2009) investigated the proximate composition of mussels from the various sites in Spain.

Variations were observed in proximate composition in mussels from the different sites. It was

observed that the small size mussels have the highest meat yield. All the collected samples

showed low concentration of copper and manganese whereas the value of Na, K, P, Mg and

Ca was found to be higher. For the dietary components, Espana et al. (2007) studied the

mineral and trace element concentration in bivalve mussels from the state of Chile. Bivalve

mussels showed the higher concentration of the entire essential mineral except the K. He

observed that the 100g serving of these mussels will provide all the essential daily dietary

components such as Zn, Mg and P etc. Furthermore, it was also reported that the

consumption of these mussel also fulfill the daily Cd intake. Chi et al. (2012) suggested that

the mussels are excellent source of food by analyzing their nutritional composition. They

reported that the mussels contained higher contents of protein, carbohydrates and good fat.

The essential and non-essential amino acids were present in perfect combination as

recommended by FAO/WHO. Bongiorno et al. (2015) reported the seasonal changes in

nutritional quality of bivalve mussels from the North Adriatic Sea. The significant variations

were observed in protein, lipids and ash contents according to the changing conditions. All

the collected samples showed lower concentration of Mn, Ni, Cr, Cu, Zn and Na whereas

24

higher contents of macro minerals in the late spring, which related to the phase immediately

before spawning. Temporal and spatial variation in proximate composition in mantle and

digestive glands of mussel was investigated by Irisarri et al. (2015). The proximate

composition was varied according to the seasonal fluctuations and peaks values were found

in spring blooms. The protein contents were initially depleted in the digestive gland during

autumn whereas the mantle maintained the peak value till the summer. The lipids contents

were highest in mantle during winter and eventually decreased during spring season.

Furthermore, it was reported that the turbidity has no effect on the proximate composition

throughout the longer time scale.

4.3. Genotoxicity

Accumulation of heavy metals also induces genotoxicity in term of DNA strand breakage

and other molecular stresses. Single cell gel electrophoresis (comet assay) was used to detect

the DNA damaging in bivalves mussel as this could be a useful technique for estimation of

genotoxic contaminants in aquatic environment. Goswami et al. (2014) stated that the heavy

metals such as copper and cadmium exposure induced many changes at sub-cellular level

including biochemical’s, physiological and genotoxic effects at sub-lethal concentration. D’

Agata et al. (2014) reported the higher level of DNA strand breakage in mussel tissues from

contaminated water as compared to the non-contaminated water. Another previous study

investigates the seasonal effects of genotoxicant in freshwater mussels by using comet assay

from different contaminated sites. They showed that there were variety of factor such as

metals, temperature and dissolve oxygen which induce the DNA damaging remarkably in

summer as compared to winter. Furthermore it was also stated that the freshwater mussels are

sensitive animals to investigate the aquatic pollution (Kolarevic et al., 2013). Klobucar et al.


25

(2008) also reported the seasonal genotoxic response for various aquatic contaminants by the

mussels. The highest value of olive tail moment (OTM) was obtained in summer months, so

results specify that freshwater mussels could be a useful tool for active biomonitoring of

aquatic environments and highlights the significance of seasonal genotoxic monitoring.

Aquatic environment are continuously polluted due to anthropogenic activity and that’s why

it’s the special concern for researcher, public and environmental regulators to sort out

different techniques. The comet assay is valuable indicator of genotoxic damage in aquatic

animals which are essential to assess the load of heavy metals in aquatic environment. Dallas

et al. (2013) reported the significantly higher level of DNA damaging in area with more

concentration of lead and chromium. Copper is essential metals for organism but excessive

amount may cause genotoxic effects. Bivalves are exposed to realistic amount of copper in

laboratory which causes the significant DNA damage and other molecular stress. The gills

found to be sensitive parts because of continuous exposure to the ambient water (Al-Subiai et

al., 2011). Comet assay is very sensitive technique to assess the DNA damage in animals.

Freshwater mussels from the polluted environment is collected and analyzed by using the

technique of comet assay. The gill and haemocytes cells are used for this study and the high

degree of DNA damage was observed in highly contaminated environment (Halldorsson et

al., 2004).

Many chemicals including insecticides, herbicides and fertilizers enter into the aquatic bodies

in the form of agricultural runoff. Among all the chemicals the effect of copper showed high

genotoxic response in the form of DNA stands breakage. It was also demonstrated that the

freshwater mussels are the sentinel organism in aquatic ecosystem to assess the adverse

effects of chemicals in water (Conners and Black, 2004). Aquatic pollution is an alarming



26

issue for scientist now a days and bivalves were widely used in biomonitoring purposes due

to their wide geographical distribution. The mussels are considered to be the bouncer species

to assess the aquatic health. Among many biomarker responses the comet assay is widely

used technique in biomonitoring studies. Genotoxic response such as DNA damaging is

helpful to create a clear picture of aquatic contamination (Bolognesi and Cirillo, 2014).

Copper Nano-particles shows significant genotoxic effects in bivalve mussels but the ionic

form have more adverse effects than Nano-particles. Furthermore studies revealed that the

DNA strand breakage using comet assay is important biomarker to determine the genotoxic

effect in aquatic organism (Gomes et al., 2013). Among the genotoxicant, chromium in

polluted water showed the reasonable DNA damaging in the gills of bivalve (Rank et al.,

2005). Comet assay was used on gill cells of bivalves to monitor the DNA damaging due to

genotoxic waste water including trace metals. The high value of DNA tail moment was

observed in samples from the polluted water (Rank, 1999).

The bioaccumulation of heavy metals in soft tissues of bivalve mussels is linked to

availability of metals in aquatic environment because of the filter feeding habits of

freshwater mussels. Aquatic environment which receives agricultural, ports, domestic and

industrial input without any treatment are likely to be studied for genotoxic effects. The

samples of mussels was collected and analyzed; the high value of DNA damaging was

observed. The same sites were again chosen for studies after six year when most of the

industries were closed and addition of pollutants was discontinues. The genotoxic effects

would be reduced after six years due to low level of pollution, so studies indicates that the

pollutants specially the heavy metals are actually the genotoxic agents (Rada et al., 2012).



27

The urban waste water which are continuously added to water bodies cause genotoxic impact

in aquatic organism, freshwater mussels were used to assess the urban load on aquatic bodies.

The polluted and non-polluted waters were compared and higher value of DNA damaging

was observed from the contaminated sites (Kolarevic et al., 2011). Pisanelli et al. (2009)

investigated the genotoxic effects of copper in bivalve mussels by using the technique of

comet assay. The increased level of DNA damaging was observed in site that connected to

the anthropogenic activities. Moreover study also revealed that the significant correlation was

found in between the DNA strand breakage with the levels of heavy metals in aquatic

environment.

The health status of mussels was studies in polluted and contaminated water bodies by using

different biological technique. DNA strand breakage is more sensitive technique to indicate

the level of contamination in aquatic environment. Overall poor health and significantly high

level of DNA damaging was observed in contaminated water and low frequency of DNA

damaging with good health of mussels was observed in cleaned water. So study revealed that

the health of mussels and DNA damaging was correlated with the level of contamination in

aquatic environment (Brooks et al., 2009). Another previous study reported the genotoxicant

accumulation and different biological responses in mussels collected from the various

polluted sites. The main focus of this study was on the heavy metals which are the potential

toxicant evaluated in the soft tissues of mussels. The high level of metals accumulation and

DNA strand breakage were observed in polluted sites are which directly received the

industrial effluents (Rocher et al., 2006).

Many chemicals and agriculture run off ultimately ends up in water and effect the aquatic

biota. The chemicals have adverse effect on early life of freshwater mussels and copper cause


28

severe DNA strand breakage among the other studied metals. Moreover, it is also concluded

that the freshwater mussels are the sensitive animals for aquatic pollutants and DNA strand

breakage is valuable tool for screening of contamination in water (Conners and Black, 2004).

The gills and haemocytes of bivalves were widely used in biomonitoring studied. In one of

previous study it was showed that the gills are more sensitive than because the higher values

of DNA damaging was observed in gills and compared to haemolymph (Akcha et al., 2004).

Taban et al. (2008) studied the genotoxic effect of some chemicals on gills, haemocytes and

the sperm cells of mussels. Results showed that the sperms cells of mussels are more

sensitive than gill cells and the haemocytes. Another study was conducted to show the

relationship between DNA strand breakage and exposure of aquatic organism to

environmental pollutants. The higher value of accumulation of metals and high frequency of

DNA stand breakage was observed in contaminated water. Furthermore it is also stated that

copper have more notable value in soft tissue among the heavy metals (Steinert et al., 1998).

Gacic et al. (2014) found that lower dose of heavy metal induce more DNA damaging as

compared to higher doses. Freshwater mussels were exposed for 3 days at different

concentration of these anticancer chemicals to check the genotoxic response in bivalves. The

significant level of DNA damaging was observed at lowest concentration of chemical.

Cadmium being non-essential metals could cause harmful effects on aquatic organisms.

Cytogenotoxic effect of cadmium was observed on gill cell of bivalve mussels under

laboratory conditions. The significant level of DNA damage was found at sub-chronic

exposure and it was also revealed that cytogenetic would be the useful biomarker to monitor

the environmental contaminations (Chandurvelan et al., 2013).


29

Heavy metals enter into the aquatic environment by different anthropic related activities like

mining, agricultural practices and industrialization. The genotoxic effect of cadmium was

investigated in laboratory and results showed that the significant bioaccumulation of mercury

in digestive gland and it also caused several cellular and molecular stresses in bivalves

(Pytharopoulou et al., 2013). The significant increase in DNA strand breaking was observed

in gill and along with cytogenetic defects due to induction of cadmium (Vincent-Hubert et

al., 2011). Nickel showed the same result in another previous study and higher level of DNA

damaging and other physiological and cytotoxic effects was observed in bivalves (Millward

et al., 2012).

Siu et al. (2008) reported the significant strong correlation between organic contaminants and

level of DNA strand breaks in bivalves which were exposed to that coastal water which

received industrial waste water and domestic discharge. The high level of bioaccumulation of

metals was observed in mussel tissues from the area after dredging activity. The significantly

strong correlation was found between the pollutants in sediments and the mussel tissues.

Moreover study also revealed that the high frequency of DNA strand damaging was also

observed in bivalve mussel during experimental period (Bellas et al., 2007). In one of the

previous study, bivalves were exposed against different heavy metals and bimolecular test

was made to evaluate the potential as a bioindicator. Results showed the high level of

genotoxicity in mussels for aquatic contamination. Furthermore, it was also suggested that

the mussels are the sentinel bioindicator species in aquatic pollution (Villela et al., 2006).

Freshwater mussel was selected to test as “biomonitor” animal in aquatic environment. They

were exposed to different polluted sites to evaluate the pollution related disorders in aquatic

organism. Haemocytes of mussels was targeted in this studies and strong significant






30

correlation was found in between DNA strand damaging with the increasing pollution in

experimental site. Furthermore it was stated that the comet assay is the sensitive techniques

to monitor the aquatic genotoxicity (Klobucar et al., 2003). In another study, freshwater

mussels were exposed to different concentration of pentachlorophenol and the level of DNA

damage in haemocytes of mussels by comet assay. The higher level of DNA damage was

observed at medium concentration of pentachlorophenol. It was also confirmed that the

technique of comet assay on mussel haemocytes is the valuable tool to investigate the

potential genotoxicity of aquatic contaminants (Pavlica et al., 2001).

Bolognesi et al. (1999) studied genotoxicity in bivalve mussels that were exposed to different

concentration of Cu, Cd and Hg in laboratory. Hg caused high frequency of DNA damaging

and Cd show least genotoxic effects on mussel whereas moderate level of DNA damaging

was observed in Cu-exposed mussels. Furthermore this study also revealed that the trace

metals are the persistent contaminants in water which induce genotoxicity in aquatic

organisms. The susceptibility of DNA damage in soft tissues freshwater mussel due to

induction of heavy metals was reported by Black et al. (1996). The effect of lead (Pb) was

assessed on the sensitivity of a freshwater mussel to DNA damage following in laboratory

and field experiments. The significant level of DNA strand breakage was observed in foot

tissue from mussels exposed in the laboratory to the lowest concentration of Pb. In contrary

to these studies, Pruski and Dixon, (2002) observed no genotoxic effect of cadmium in gills

of mussel both in chronic and acute condition. Comet assay (single cell gel electrophoresis)

is a sensitive technique to investigate the impact of genotoxicological pollutants in aquatic

environment (Frenzilli et al., 2009; Mitchelmore et al., 1998; Nacci et al., 1992).




31

5. Materials and methods

5.1 Sampling sites:

There were two sampling sites for the present study; one was the Chashma lake River Indus

Pakistan, selected for the wild experiment. Another site was university fish research farms,

from where the animals were collected for laboratory experiments.

5.2 Chashma Lake:

Chashma Lake is located at River Indus, 25 Km southwest from Mianwali City of Pakistan.

This site of River Indus is heavily use for the inland fisheries and is economically important

reservoir for irrigation and generation of electricity. The samples of water and freshwater

mussels were collected from the various sites of Chashma Lake (30°16′45′′ N, 66°57′23′′ E)

(Figure 1). Samples were collected by trained fisherman or by hand picking where the water

was shallow. After that the shell was cleaned from epiphytes and inside was rinsed with

deionized water to remove sand and other particle from the shell body. The mussels was left

to dry on blotting paper about two hours before dissection. The samples of freshwater

mussels were analysed for bioaccumulation of heavy metals, mineral elements analysis and

proximate composition while the water samples were tested for metals concentration.

32

Figure 1. Study area viewing the location of sampling spot (Google map)

5.3 University fish research farm:

For the laboratory experimentation, freshwater mussels (Anodonta anatina) of average size

(64.5 ± 2 g) were collected from an unpolluted freshwater pond. The temperature of pond

water at the time of collection was approximately 20.5 ± 1.5 °C. Mussels were directly

carried to the fish hatchery at Manawa fisheries centre in cool plastic bags and placed in large

rectangular cemented tanks with filtered pond water. Animals were fed with the green algae

harvested from the same fish pond and acclimatization period of mussels was 10 days to

minimize stress before used for experimentation. Mussels were kept at 16.8 ± 1.2 °C and

33

water was changed daily during acclimatized period when no mortality was occurred. These

animals were exposed later on for various doses of metals to investigate the bioaccumulation,

mineral elements analysis and proximate composition.

5.4 Experimental design

Freshwater mussels collected from the university fish research farm, were exposed to 0 µg/L,

120 µg/L, 240 µg/L and 360 µg/L of lead (Pb), copper (Cu) and chromium (Cr) in glass tanks

(24ʺ× 18ʺ× 24ʺ) (5 animal per tank) with three tanks as replicates per treatment for 15 days.

Primary stock solutions of Pb, Cu and Cr were prepared in distilled water by using Pb (NO3),

Cr (NO3). 9H2O and CuSO4. 5H2O, respectively and further diluted to achieve the required

concentration for exposure. The water was changed after every 5 days with the renewal of

each chemical. The mussels were sacrificed at the end of experiment and gills, foot and

mantle were collected for estimation heavy metals, mineral analysis and proximate

composition (Figure 2).

5.5 Isolation of soft tissues

Mussel’s shell was open with the help of scalpel and small steel rod, animal was washed

properly in laboratory to remove the unwanted particles. Gills, foot and mantle was excised

carefully with the help of sharp scissor and washed well with double distilled water. Tissue

samples were then put into small plastic bottles and kept in a refrigerator at -4 ˚C. All the

samples were weighed and then put into freeze dyer for 7 days and after that dried samples

were ground, weighed and placed in desiccators until digestion.

34

Figure 2. Anatomy of freshwater mussel (Google image)

5.6 Preparation and digestion of water samples

The water samples were collected in a plastic bottle and then filtered along with acid

stabilization. For the estimation of metals, about 100 ml water samples were put into the

volumetric flask and evaporated to about 20 ml on hot plate in a fume hood. The remaining

water samples were then left for cooling and after that added 5ml concentrated nitric acid

(HNO3) and 10 ml perchoric acid (HCLO4). The mixture was again placed on the hot plate

and evaporated until the brown fumes changed into the clear opaque white fumes of HCLO4.

The samples were then allowed to cool and after that diluted to 100 ml with dH2O and put

into refrigerator until analysis.

35

5.7 Preparation and digestion of tissue samples

A weight of 0.2 g of freeze dried sample was carried in a digestion flask and 9 mL

concentrated nitric acid (HNO3) was added with 1 mL perchloric acid (HClO4). The digestion

flasks were left in fume hood for overnight. Each sample was heated on hot plate using

fuming hood at 100˚ C till evaporated to near dryness. The samples were cooled and diluted

to 10 mL with distilled water and centrifuge at 2000 rpm for 10 minutes. The supernatants

were transferred to separate volumetric plastic vials and stored in refrigerator until analysis.

5.8 Metals and mineral elements analysis

The concentrations of Potassium (K), Phosphorus (P), Sodium (Na), Calcium (Ca),

Manganese (Mn) and Zinc (Zn), Lead (Pb), Copper (Cu) and Chromium (Cr) were

determined by inductively coupled plasma optical emission spectroscopy (ICP-OES Vista-

Mpx simultaneous) according to the method of AOAC, (2000). The calibration of machine

was achieved over related concentration with certified standard from the Sigma-Aldrich, UK.

The metal concentrations in foot, gills and mantle of freshwater mussels were shown as

mg/kg dry weight while the unit was mg/L in case of metal concentration in water. All the

facilities and laboratory equipments were used in school of Agriculture, Food and Rural

development, Newcastle University, United Kingdom.

5.9 Proximate composition analysis

Freeze dried samples from foot, gills and mantle portion were analysed for the moisture,

proteins, fat, and ash contents according to AOAC, (1997).

36

5.9.1 Determination of moisture

Weight of 5g of sample were carried out in container and placed in drying oven at 105˚ C for

24 hrs. The container was removed carefully and allowed to cool in desiccators. After that

the sample was weighed with great care that not to exposed with the atmosphere. Total

moisture was calculated by following formula.

Moisture contents (%) = weight of wet sample-weight of sample after drying/weight of wet

sample*100

5.9.2 Determination of crude protein

Determination of crude protein was carried out by Elementar Vario Macro Cube. For carbon

nitrogen analysis a weight of around 1g samples was taken into a small tin foil cup. After

that, this was wrapped and compressed in the form a pellet to remove the air and this was

done by using a tool provided by elementar. The analysis was carried out in carbon nitrogen

mode involves combustion, post combustion and reduction tube in the furnace of the

analyser. The combustion tube was at 960˚ and the prepared sample was put into it by a

carousel and ball valve. The sample burning was carried out by using oxygen and the gas was

carried off in helium through both the post combustion (900˚) and reduction tube (830˚) to

the detector housed within the analyser. The analysis was carried out singly for each element

by obtaining the percentage figure. The specific standard was run before each analysis to

make sure the analyser was functioning properly. The same standards were also run midway

throughout the analysis to ensure the accuracy of analyser. Furthermore, there was a daily

factor figure which was worked out after each run to check the perfection of analyser. The

protein contents were obtained by multiplying the value of nitrogen with 6.25.

37

5.9.3 Determination of Ash contents

A weight of around 3g sample was taken into already weighed crucibles and then placed it

into muffle furnace and heat at 500˚ C. After 12 hours the crucibles were removed from the

furnace and placed it into desiccators to let it cool. The crucibles were again weighed

carefully with the ash and the ash contents were calculated by using following formula.

Ash contents (%) = weight of crucible with sample-weight of crucible with ash/weight of

sample* 100

5.9.4 Determination of crude lipids

Crude lipids were determined by using the Soxhelt extraction apparatus. Flasks and cellulose

thimbles were weighed and numbered accordingly prior to start the procedure. A weight of

3g of sample was taken in a thimble and closes its mouth with cotton to prevent the sample

coming out. Thimbles were placed carefully in extraction tubes of Soxhelt apparatus and

connected the flask containing petroleum ether at 2/3 of total volume of extractor. The

condenser tube was attached with continues circulation of water. Placed the apparatus on

heating sources and allows the petroleum ether to boil (40-60˚ C) and adjusted the heat to

obtain 8-10 refluxes per hour. After 6 hours of extraction the unit was turned off, removed

the flasks and put into oven for evaporate of petroleum ether. The flasks were placed into

desiccators to cool down and then weighed again. Crude lipids were calculated by following

formula.

Crude lipid contents = weight of flask with lipids-weight of clean dry flask/weight of

sample*100

38

5.9.5 Determination of carbohydrates

The value of carbohydrates was determined with by difference method by following formula.

Carbohydrates contents (%) = [100-(Crude proteins+ Crude lipids + Moisture + Ash

contents)]

5.10 Preparation of reagents for determination of genotoxicity by Comet

assay:

5.10.1 Phosphate buffered saline (PBS)

One tablet of PBS was dissolved in 100 ml of distilled water by adjusting the pH 7.4 and then

the solution was stored at room temperature.

5.10.2 Stock of lysing solution

Constituents over 1 Litre

2.5 M sodium chloride = 146.1 g

100 mM Ethylene diamine tetra acetic acid = 37.2 g

10 mM trisma base = 1.2 g

All these constituents were added in 700ml distilled water stirred the mixture thoroughly for

45 minutes. 8 g sodium hydroxide (NaOH) was added and put on the hot plate for about 20

minutes. The pH of mixture was adjusted to 10.0 with the help of Hydrochloric acid (to make

acidic) or Sodium hydroxide (to make alkaline).

39

5.10.3 Finalizing lysing solution

89 ml Lysing solution was mixed with 1ml fresh triton x and 10 ml DMSO to make 100 ml.

This final Lysing solution was stored in refrigerator for 30 minutes prior to use.

5.10.4 Gel preparation

5.10.5 Normal melting agarose (NMA)

500 mg NMA was mixed in 50 ml distilled water then put on magnetic stirrer and heat up to

boiling level and kept in refrigerator.

5.10.6 Low melting point agarose (LMPA)

250 mg LMPA was added to 50ml PBS then put on hot plate up to boiling level along with

magnetic stirrer and stored in refrigerator.

5.10.7 Electrophoresis buffer

5.10.8 Stock solution

10N Sodium hydroxide (NaOH) (200 g NaOH + 500 ml dH2O) was mixed with the 200 mM

EDTA (14.89 g EDTA + 200 ml dH2O) by adjusting pH 10. Stored at room temperature

maximum for two weeks.

5.10.9 Finalizing buffer

The buffer was made freshly prior to every electrophoresis run, by adding 30 ml sodium

hydroxide along with 5 ml Ethylene diamine tetra acetic acid to make volume up to 1000 ml

with distilled water.

40

5.10.10 Neutralizing buffer

48.5 g Tris base was added in 800 ml dH2O then stirrer well on hot plate and make volume

up to 1000 ml. The pH was adjusted at 7.5 and stored at room temperature.

5.10.11 Staining solution.

10 mg Ethidium Bromide was dissolved in 50 ml distilled water and stored at room

temperature.

5.11 Procedure of comet assay

The protocol of comet assay was used according to Singh et al. (1988).

5.11.1 Preparation of base slide

The first layer on frosted microscopic slides were applied by using 1% normal melting point

(NMP) agarose. Slides were then placed on clean surface and allow to air dried for overnight.

The second supportive layer was formed by using 80 ul of 1% NMP agarose to place gently

on the top of the previously 1% NMP layer and to spread over the slide using a cover slip.

After that the slides were placed on ice box for five minutes to allow the agarose to fix and

cover slips were removed afterward.

5.11.2 Cell Isolation

Small piece of gills was ground up in 1ml cold Hanks balanced salt solution (HBSS) and

minced into very fine pieces. About 30 ul of PBS containing cells was mixed with 70 ul of

1% low melting point (37 ˚C) agarose using the vortex mixer and pipette onto the supportive

layer of 1% NMP agarose and covered with a cover slip. The Slides were put into freshly

made cold lysis buffer solution (2.5 M sodium chloride, 100 mM Ethylene diamine tetra

41

acetic acid, 10 mM Tris amino methane, 1.5% Triton X-100, pH = 10) for 2 hours after

removing cover slips.

5.11.3 Gel electrophoresis

In the next step alkaline electrophoresis buffer solution was prepared by using 300 mM

sodium hydroxide and 1mM Ethylene diamine tetra acetic acid at pH 13 and poured into

electrophoresis chamber. All the slides were placed in chamber and proceeded

electrophoresis at 25 Volts with 300 milli Ampere current for 20 minutes to achieve DNA

unwinding. Neutralizing buffer was prepared freshly by using 0.4 M Tris with pH 7.5 and

placed there slides for 15min.

5.11.4 Staining and scoring

20 uL Ethidium bromide (2 ug/ml) was used for staining per slide and then examined with

fluorescence microscope at magnification power of x400. The scoring of microscopic images

was carried out by using computer software Comet IV. Three slides were prepared from each

sample and the images of 20 cells were scored from each slide. Olive tail moment (product of

tail length and the fraction of total DNA in the tail), comet tail length and amount of DNA in

comet tail were the parameters assessed for DNA damaging.

5.12 Statistical analysis

The data were examined by using the two-way analysis of variance (ANOVA) and described

as mean ± standard deviation (S.D). The means were compared by using the Tukey’s

pairwise test to assess the difference between the control and various doses of the studied

metals. The relationship among minerals and bivalve tissues were investigated by Pearson

correlation coefficient (r). Statistical significance was declared if p < 0.05. All the statistical

42

analysis was carried out by using the Minitab 17 software. Graph and Table were drawn with

the help of Microsoft excel 2007.

43

Laboratory Experiment Wild Experiment

Mussels collected from fish

pond

Mussels collected from

Chashma Lake

Exposed for the various metals

in controlled conditions

Isolation of soft tissues

Foot Mantle Gills

Transported to the laboratory

Analysis of bioaccumulation of

heavy metals, mineral element

and proximate composition

Analysis of DNA strands breaks

by Comet assay

Figure 3. Flow chart of methodology

44

6. Results

In laboratory experiment, mussels were exposed to four doses T0 (0 µg/L), low T1 (120

µg/L), medium T2 (240 µg/L) and high T3 (360 µg/L) of Pb, Cu, Cr and combined metal

treatment (Pb + Cu + Cr). Since T0 (0 µg/L) contained no metals, it was considered as a

control. The comparison was carried out among the various doses of different metals and

with the control conditions. The mean values of temperature, pH and dissolve oxygen (DO)

of aquarium water were 17.6 ± 1.3 ˚C, 7.15 ± 0.15 and 6.32 ± 0.420 mg/L respectively. No

mortality was observed throughout the experimental period.

6.1 Bioaccumulation of metals

The accumulation of heavy metals in foot, gills and mantle of the studied was observed to be

increased for all metal in all tissues as the dose was increased. Among the studied portions

gills stored highest concentration of metals in all cases as compared to foot and mantle

(Table 1). The overall accumulation of copper (Cu) was found to be significantly higher as

compared lead (Pb) and chromium (Cr). The gills had the maximum mean value of Cu

(149.57 ± 5.34 mg/kg), Pb (30.00 ± 3.45 mg/kg) and Cr (22.192 ± 1.228 mg/kg) at the

highest dose (360 µg/L) (p < 0.05). The maximum concentration of Cu, Pb and Cr in foot

was 35.271 ± 0.0720 mg/kg, 17.149 ± 1.521 mg/kg and 4.539 ± 0.520 mg/kg, respectively.

Although no significant difference was found in foot portion for different doses of Cu; the

values were T1 (29.210 ± 0.514 mg/kg), T2 (32.888 ± 0.173 mg/kg) and T3 (35.271 ± 0.0720

mg/kg), these were significantly higher than the control T0 (10.584 ± 0.963 mg/kg).

45

Table 1. Bioaccumulation of metals in foot, gills and mantle of freshwater mussels (Anodonta anatina) exposed to different doses

of Lead (Pb), Copper (Cu) and Chromium (Cr).

Metals Tissue Concentration (mg/kg)

T0 (0µg/L) T1 (120µg/L) T2 (240µg/L) T3 (360µg/L)

Lead (Pb) Foot 3.959±0.757ef 6.955±1.394def 11.208±1.450bcd 17.149±1.521a

Gills 4.523±1.057ef 10.95±2.99bcd 16.06±3.25bc 30.00±3.45a

Mantle 3.23±1.99f 4.386±1.718ef 5.71±2.01def 10.30±2.50cde

Copper (Cu) Foot 10.584±0.963h 29.210±0.514g 32.888±0.173g 35.271±0.0720g

Gills 15.50±5.64h 123.75±6.87c 136.53±6.16b 149.57±5.34a

Mantle 6.983±1.722h 76.33±5.00f 93.90±4.15e 107.87±5.35d

Chromium (Cr) Foot 0.852±0.068d 2.111±0.583cd 2.889±0.235cd 4.539±0.520c

Gills 1.357±1.013d 4.377±0.933c 13.72±2.72b 22.192±1.228a

Mantle 0.837±0.296d 1.307±0.500d 1.681±0.516cd 3.324±0.302cd

Data are mean ± standard deviation

Values in the same rows with different superscripts indicates significant difference (p<0.05).

46

Cr showed the least accumulation as compared to Pb and Cu where; the lowest concentration

was found in mantle at various doses [T0 (0.837 ± 0.296 mg/kg), T1 (1.307 ± 0.500 mg/kg),

T2 (1.681 ± 0.516 mg/kg) and T3 (3.324 ± 0.302 mg/kg)] (Table I). In Pb-exposed mussels,

the highest concentration in foot (17.149±1.521 mg/kg) was found at higher dose (360µg/L),

whereas the concentrations at medium (240µg/L) and low dose (120µg/L) was 11.208±1.450

mg/kg and 6.955±1.394 mg/kg, respectively. The mantle had highest concentration

(10.30±2.50 mg/kg) at high dose (360µg/L), than medium (240 µg/L) and low dose (120

µg/L) 5.71±2.01 mg/kg and 4.386±1.718 mg/kg, respectively. There were no significant

differences was found in mantle between medium and low doses at Pb and Cr-exposure (p >

0.05) (Figure 4).The order of tissues in the case of Pb and Cr accumulation was gills > foot >

mantle, whereas in Cu-exposed mussels mantle had higher concentration than foot in most

cases (Table 1). Bioaccumulation of Cr also showed the same trend, where the concentration

increased as the dose was increased. The highest accumulation in foot portion (4.539±0.520

mg/kg) was observed at high dose (360 µg/L). Although, no significant difference was found

among other doses (p > 0.05), but values of Cr were comparatively higher than the Control (0

µg/L) (p < 0.05) (Figure 4).

47

Figure 4: Variations in metals bioaccumulation among various portion of freshwater mussels

(Anodonta anatina) exposed to different doses of Lead (Pb), Copper (Cu) and Chromium

(Cr). Graph draw by log base 10.

0.1

1

10

100

1000

Foot Gills Mantle Foot Gills Mantle Foot Gills Mantle Foot Gills Mantle

0µg/L 120µg/L 240µg/L 360µg/L

Pb

Cu

Cr

mg

/k

g ef ef f

def

bcd

ef

bcdbc

def

a

a

cdeh

h

h

g

cf

g

be

g

ad

d

d

d

cd

c

d

cd

b

cd

c

a

cd

48

6.2 Mineral analysis

Mean mineral element compositions of various tissues of freshwater mussels from the control

tank are shown in Table 2. Some variations were observed in gills, foot and mantle for

different mineral elements. The concentration of Na was higher in mantle (1879.9 ± 50.6

mg/kg) than in foot (1680.6 ± 2.39 mg/kg) and gills (1577 ± 57.3 mg/kg), whereas the value

of Ca was found to be greater in gills (156,906 ± 736 mg/kg) than mantle (45,207 ± 241

mg/kg) and foot (3052 ± 29.7 mg/kg) (p < 0.05). The same trend was also observed for P, Mn

and Zn, for which gills showed higher values than foot and mantle; the exception was K,

which was higher in foot (675±30.6 mg/kg) than gills (655±36.3 mg/kg) and mantle

(527.3±32.3 mg/kg) (Table 2). The concentration of P in foot, gills and mantle were

3468.7±56.1 mg/kg, 44076±1607 mg/kg and 14457±517 mg/kg respectively. Mn also

showed variation among foot, gills and mantle; the values were 192.45±1.81 mg/kg,

20106±752 mg/kg and 6270±227 mg/kg respectively. Amongst all the studied mineral

elements, Zn showed the lowest concentrations (gills: 282.2 ± 49.5 mg/kg; mantle: 128.7 ±

17.3 mg/kg, and foot: 66.16 ± 5.94 mg/kg).

49

Table 2. Mineral composition of different portions of freshwater mussels (Anodonta anatina) collected from the control

treatment.

Minerals Concentration(mg/kg)

Foot Gills Mantle

Sodium (Na)

1680.6±2.39c

1577±57.3d

1879.9±50.6d

Potassium (K)

675±30.6d

655±36.3d

527.3±32.3e

Calcium (Ca)

3052±29.7b

156906±736a

45207±241a

Zinc (Zn)

66.16±5.94f

282.2±49.5d

128.7±17.3e

Manganese (Mn)

192.45±1.81e

20106±752c

6270±227c

Phosphorus (P)

3468.7±56.1a

44076±1607b

14457±517b

Data are mean ± standard deviation

Values in the same columns with different superscripts indicates significant difference (p<0.05).

50

Mineral elements were also analysed in tissues of metal-exposed mussels. Amongst the three

studied portions foot, gills and mantle of freshwater mussels, various fluctuations were

observed for different doses of heavy metals. The metal wise description of mineral elements

exposed for the various does of metals are given below.

6.2.1 Pb-exposed mussels

The fluctuations among mineral elements were observed different tissues of freshwater

mussels when exposed for different doses of Pb (Table 3 and figure 5). The value of K from

the control treatment in foot, gills and mantle was 675±30.6 mg/kg, 655±36.3 mg/kg and

527.3±32.3 mg/kg respectively (Table 2). In foot of mussels, there were no significant

difference was found in K between low dose and control treatment and values at low dose

were 636.4±32.3 mg/kg, while it was significantly decreased at medium and high doses,

values were 543±30.5 mg/kg and 555.2±30.4 mg/kg, respectively (Table 3). Almost the same

results were noted in gills, the observed values were found to be lower than control such as at

low dose: 389.2±29.3 mg/kg, at medium dose: 427.1±31.3 mg/kg and at high dose: 479±33.7

mg/kg. In case of mantle, the concentration of K was increased (615.4±34.2 mg/kg) at low

dose than control level and then it was decreased at the medium and high doses 466.7±30.9

mg/kg and 466.1±31.9 mg/kg respectively. The concentration of Ca in gills of mussels was

significantly higher (p < 0.05) in Pb-exposed mussels at all doses, namely: at 120 µg/L:

185616 ± 590 mg/kg; at 240 µg/L: 183854 ± 96 mg/kg; and at 360 µg/L: 185098 ± 795

mg/kg as compared to control. Whereas in the foot region, lower values were observed than

control at low and medium dose, while it was higher (4637.5±16.4 mg/kg) at the highest

dose.

51

Table 3. Mineral composition of different portions of freshwater mussels (Anodonta anatina) exposed to different doses of Lead

(Pb).

Dose Tissues Concentration (mg/kg)

Na K Zn Mn P Ca

120µg/L

Foot 1271.5±29.1d 636.4±32.3a 80.74±3.19c 108.71±1.17e 3361.6±76.9a 2145.1±9.11g

Gills 1774.4±57.6c 389.2±29.3d 347.9±46.8a 22555±831ab 50258±1703a 185616±590a

Mantle 2359.3±52.1a 615.4±34.2ab 116.08±9.82c 3712.9±147.9d 9280±130.3c 34692±162e

240µg/L

Foot 1189±30.6d 543±30.5bc 72.95±3.34c 122.61±1.28e 3340.4±85.6a 2414.4±20.8g

Gills 1275.5±77d 427.1±31.3d 338.3±46.6a 21772±802b 49841±1789a 183854±96b

Mantle 1640.9±49.4c 466.7±30.9cd 114.09±10.22c 4844±182d 11986±445c 41148±6d

360µg/L

Foot 1760.8±42.3c 555.2±30.4abc 81.64±4.04c 347.78±3.55e 3617.8±112.5a 4637.5±16.4f

Gills 1954.8±48.8b 479±33.7cd 329.4±47.7a 23385±854a 50446±1884a 185098±795a

Mantle 1219.5±61.9d 466.1±31.9cd 202.2±23b 8481±263c 20764±336b 43596±204c Data are mean ± standard deviation


52

Mantle showed lower concentration of Ca as compared to control level and the observed

values were 34692±162 mg/kg, 41148±6 mg/kg and 43596±204 mg/kg at low, medium and

high doses respectively. The Na concentration in foot region was found to be decreased than

control level when exposed for the low (1271.5±29.1 mg/kg) and medium dose (1189±30.6

mg/kg), whereas increased at high dose (1760.8±42.3 mg/kg). In case of gills, it was

significantly increased when exposed for high dose, whereas there were no significant

difference was found at low and medium doses. The decreasing trend was observed in mantle

region for Na concentration as the dose was increased and values at low, medium and dose

were 2359.3±52.1 mg/kg, 1640.9±49.4 mg/kg and 1219.5±61.9 mg/kg respectively. The

level of Zn in foot portion was increased than control at all the doses, and maximum

concentration (81.64±4.04 mg/kg) was found at the highest dose. In gills of freshwater

mussels, no significant difference was found in concentration of Zn among the various doses

but still it was higher than the control level. But in case of mantle, the concentration was

lower than the control at low and medium doses except at the high dose where it was

202.2±23 mg/kg. The values of Mn in foot region were 108.71±1.17 mg/kg, 122.61±1.28

mg/kg and 347.78±3.55 mg/kg at low medium and high doses, respectively (Figure 5). Gills

contained almost the same quantity of Mn as in the control treatment; hence no significant

difference was found. The mantle had lower mean value of Mn than control at low and

medium doses while found higher at the high dose. For the P concentration, no significant

difference was found in foot, between the control levels and at all the doses, but in case of

gills, the concentration was found to be higher at all the doses. The concentration of P in

mantle was greatly decreased at the low dose while at medium dose it was near to control

level and at higher dose found significantly increased than control level (Figure 5).

53

Figure.5. Variations in mineral element composition among various portions of freshwater

mussels (Anodonta anatina) exposed to different doses of Lead (Pb). Graph drawn by log base 10

1

10

100

1000

10000

100000

1000000

Foot Gills Mantle Foot Gills Mantle Foot Gills Mantle

120µg/L 240µg/L 360µg/L

Na

K

Zn

Mn

P

Ca

d

d

a

c

d

a

c

d

a

b

c ad d

c c bd

ad

ab bc d cd abc cd cd

g

a

e

g

b

d

f

a

c

c

a

c

c

a

cc

a

b

e

ab

d

e

b

d

e

a

c

mg/k

g

54

6.2.2 Cu-exposed mussels

The concentrations of Na and K in foot, gills and mantle were significantly lower (p < 0.05)

than control at all the doses of Cu-exposed mussels (Table 4, Figure 6). No significant

difference (p > 0.05) was found in Zn, Mn and P between control and the Cu-exposed

mussels to all the doses. Foot portion showed low concentration of K at the all doses, and

least amount (300.5±24) was observed at high dose. In case of gills, the concentration of K

was greatly decreased (287.8±25.6 mg/kg) at low dose exposure, while at medium and high

doses the concentration were 411.7±30.8 mg/kg, 419.6±30.8 mg/kg, respectively (Figure 6).

Mantle had the lower concentration of K as compared to control level at all the doses. The Ca

level in foot was elevated as the dose was increased and maximum value (9009.8±57.9

mg/kg) was observed at high dose. Gills contained higher concentration of Ca than control

level, when exposed for the various doses. The same trend was observed in mantle where Cu

exposures significantly enhance the concentration of Ca and the values were 112218±311

mg/kg, 116548±179 mg/kg and 197496±393 mg/kg at low, medium and high doses

respectively. The level of Na was decreased as the dose was increased and lowest

concentration (720.55±2.68 mg/kg) was observed at highest dose. The similar results were

observed in gills, where values of Na were significantly decreased in high dose exposure,

while no significant difference was found at low and medium doses.

55

Table 4. Mineral composition of different portions of freshwater mussels (Anodonta anatina) exposed to different doses of Copper

(Cu).


Na K Zn Mn P Ca

120µg/L

Foot 1215.7±30.1c 479.4±28.1a 76.28±3.54e 231.87±3.53f 3288.3±102.9f 3153.8±12.5i

Gills 1323.5±63c 411.7±30.8ab 349.6±51a 23315±838a 50522±1665a 187483±445b

Mantle 1092.4±44.5d 406.9±29.7ab 222.6±26.2c 11836±425d 27006±1013d 112218±311e

240µg/L

Foot 782.5±1.14e 382.1±25.9b 65.19±3.83e 354.05±3.5f 4140.7±59.6f 4832±22.1h

Gills 1743.2±56.3b 419.6±30.8ab 325.4±44.8ab 20280±760b 45136±1582b 165763±885c

Mantle 1270.8±49c 284.6±23.7c 231.1±30.6bc 14863±531c 32163±1016c 116548±179d

360µg/L

Foot 720.55±2.68e 300.5±24c 83.86±6.09de 701.88±11.45f 5154±174f 9009.8±57.9g

Gills 987.66±2.61d 287.8±25.6c 343.5±56.3a 24549±903a 52611±1998a 197496±559a

Mantle 2977.7±47.4a 276.5±24.5c 172.7±22.1cd 9630±360e 22554±811e 197496±393f Data are mean ± standard deviation


56

Contrary to other tissues, the concentration of Na in mantle portion was found to be lower at

the low and medium doses and the values were 1092.4±44.5 mg/kg and 1270.8±49 mg/kg

respectively, while it was significantly higher (2977.7±47.4 mg/kg) than control at the high

dose treatment. At the high dose, the concentration of Zn in foot region was increased

significantly as compared to the control level, while at low and medium doses no significant

difference was found. In gills of freshwater mussels, the concentrations of Zn at low, medium

and high doses were 349.6±51 mg/kg, 325.4±44.8 mg/kg and 343.5±56.3 mg/kg,

respectively. The values of Zn in mantle were 222.6±26.2 mg/kg, 231.1±30.6 mg/kg and

172.7±22.1 at low medium and high doses respectively when compared with the control level

(128.7±17.3 mg/kg). For the Mn concentration in foot of freshwater mussels, the values were

greatly exceeded (701.88±11.45 mg/kg) at the high dose than the control level (192.45±1.81

mg/kg) (Figure 6) Gills were showed the highest concentration of Mn at low and high doses,

while at the medium dose treatment no significant difference was found with control level. In

case of mantle tissue, the significantly higher concentration of Mn was observed than control

treatment and the values at low, medium and high doses were 11836±425 mg/kg, 14863±531

mg/kg and 9630±360 mg/kg respectively. The fluctuations were also observed in P

concentration for dose related metal exposure. The concentration for the control treatment

was 3468.7±56.1 mg/kg, 44076±1607 mg/kg and 14457±517 mg/kg in foot, gills and mantle

portions of freshwater mussels (Table 2). In foot, no significant difference was found

between P at low dose and control level, while at medium and high doses concentration was

significantly increased. For gills, the concentration of the P was higher than control at low

and high doses, whereas no significant difference was found at medium dose treatment.

57

Whereas in mantle region, the maximum value of P (32163±1016 mg/kg) was observed at

the medium dose (Table 4).

Figure 6. Variations in mineral element composition among various portions of freshwater

mussels (Anodonta anatina) exposed to different doses of Copper (Cu). Graph drawn by log base 10

1

10

100

1000

10000

100000

1000000


120µg/L 240µg/L 360µg/L

Na

K

Zn

Mn

P

Ca

e

b

c

e

a

c

e

a

d

cbc

cc c c

ac

ab

b cbc b c c

ac

a

fg

b

c

g

a

d

f

a

e

c

a

b

c

a

b

c

a

bcf

cd

f

a

d

f

b

e

mg

/kg

58

6.2.3 Cr-exposed mussels

The effects of Cr exposure were also observed on the mineral elements among the soft

tissues of freshwater mussels (Table 5, Figure 7). No significant difference of K was found in

foot region at low and medium doses and the mean values were 496.8±28.9 mg/kg and

497.9±29.6 mg/kg, respectively (p > 0.05); while the values was significantly higher

(587.9±28.7) at high dose treatment (p< 0.05). In gills, the concentrations of K were

394.8±30.1 mg/kg, 382.9±29.4 mg/kg and 390.1±29.5 mg/kg at low, medium and high doses

respectively, and it was significantly lower than the control value (655±36.3 mg/kg). In

control treatment, the concentration of K in mantle region was 527.3±32.3 mg/kg. This was

decreased when exposed for the low and medium dose, while increased at the high dose

treatment. The value of Ca in foot of freshwater mussels from the control treatment was

3052±29.7 mg/kg, the concentration was greatly decreased as compared to control when

exposed for various doses, and the lowest value (1760±22.4 mg/kg) was observed at medium

dose. In case of gill portion, the concentration of Ca from the control tank was 156906±736

mg/kg, and the concentration found to be increased at medium and high doses, while no

significant difference was found at the low dose (Table 5). The sample of mantle from the

control treatment contained 45207±241 mg/kg of Ca, while the value was greatly increased

67489±284 mg/kg and 64606±83.9 mg/kg at low and medium doses, whereas at high dose it

was 38909±155 mg/kg.

59

Table 5. Mineral composition of different portions of Freshwater Mussels (Anodonta anatina) exposed to different doses of Chromium (Cr).


Na K Zn Mn P Ca

120µg/L

Foot 1276.7±28.3c 496.8±28.9b 72.06±4.16c 132.58±1.25f 3365.7±127e 2073.7±24.8fg

Gills 2044±56.8bc 394.8±30.1c 326.2±46.4a 19148±721c 40837±1553b 143815±497b

Mantle 1938±1039c 453.9±32.4bc 174.3±21.4b 8823±324d 18747±1958c 67489±284c

240µg/L

Foot 1635.4±42.1c 497.9±29.6b 61.26±3.81c 99.973±0.744f 4122.3±159.7e 1760±22.4g

Gills 1480.3±49.6c 382.9±29.4c 348.3±55.2a 23993±868a 52603±1852a 193304±493a

Mantle 1909±55.2c 408.7±29.4c 177.1±21.9b 8516±345d 19331±562c 64606±83.9d

360µg/L

Foot 3442.8±47.9a 587.9±28.7a 68.03±4.26c 210.88±2.4f 1086.9±28.5e 2759.1±11.4f

Gills 1752.8±54.1c 390.1±29.5c 324±54.1a 21636±800b 52804±1938a 192965±422a

Mantle 2982.9±21.9ab 636.8±34.2a 129.76±14.81bc 4802±179e 11976±405d 38909±155e Data are mean ± standard deviation


60

The instability in concentration of Na was also observed in Cr exposed mussels, the values in

foot were 1276.7±28.3 mg/kg, 1635.4±42.1 mg/kg and 3442.8±47.9 mg/kg at low, medium

and high doses, respectively. The control level of Na in gills was 1577±57.3 mg/kg and it

was significantly increased when exposed for the low dose treatment, while there were no

significant difference was found at medium and high doses (Figure 7). The concentration of

Na in mantle portion from the control treatment was 1879.9±50.6 mg/kg. The significant

increase in Na concentration (2982.9±21.9 mg/kg) was observed at the higher dose, while at

low and medium dose it was near to the control level. The concentration of Zn in foot region

from the control tank was 66.16±5.94 mg/kg, there were no significant difference was found

in Cr-exposure and the values were 72.06±4.16 mg/kg, 61.26±3.81 mg/kg and 68.03±4.26

mg/kg at low, medium and high doses respectively. At control level the concentration of Zn

in gills was 282.2±49.5 mg/kg that showed considerable increase in treated groups (Figure

7). The highest concentration (348.3±55.2 mg/kg) was observed in medium dose treatment,

while the concentrations at low and high doses were 326.2±46.4 mg/kg and 324±54.1 mg/kg,

respectively. The concentration of Zn in mantle region was increased at low and medium

doses as compared to the control treatment, whereas it was decreased at the highest dose. The

control value of P in foot was 3468.7±56.1 mg/ kg, the concentration at low dose

(3365.7±127 mg/kg) was near to control while slight increase (4122.3±159.7 mg/kg) was

observed at medium dose treatment but surprisingly the concentration was greatly decreased

(1086.9±28.5) at the high dose. In gills, the value of P from the control tank was 44076±1607

mg/kg, and it was greatly increased when exposed for the medium and high doses, while no

significant difference was found at low dose exposure. The concentration of P in mantle from

the control treatment was 14457±517 mg/kg and wide fluctuations were observed when

61

exposed for the different doses. The values were 18747±1958 mg/kg, 19331±562 mg/kg and

11976±405 mg/kg for low, medium and high doses respectively. For Mn concentration in

foot portion, values were 132.58±1.25 mg/kg, 99.973±0.744 mg/kg and 210.88±2.4 mg/kg

for low, medium and high doses as compared with the control level (192.45±1.81 mg/kg).

Gills showed slight variation for Mn concentration at various doses, observed concentration

at medium dose was 23993±868 mg/kg, which was higher than the control value (20106±752

mg/kg), while at all the other doses no significant difference was found. Investigation of Mn

in mantle of mussels showed greater concentration at low (8823±324 mg/kg) and medium

doses (8516±345 mg/kg) as compared to control (6270±227 mg/kg), while it was decreased

at the high dose exposure (4802±179 mg/kg) (Figure 7).

Figure 7. Variations in mineral element composition among various portions of freshwater

mussels (Anodonta anatina) exposed to different doses of Chromium (Cr). Graph drawn by log base 10

1

10

100

1000

10000

100000

1000000


120µg/L 240µg/L 360µg/L

Na

K

Zn

Mn

P

Ca

f

ad

f

bc

f

a

e

c c de

bc

ed

a

a ab ab b abc c c c

i

be

h

cd

g

a

f

e

ac

e

abbc

de

a

cdf

a

d

f

bc

f

a

e

mg/k

g

62

6.2.4 Combined exposure of metals (Pb + Cu + Cr)

The results of mineral element analysis from the combined treatment of metals were shown

here (Table 6, Figure 8). The concentration of K in foot region was found to be lower than

control value (675±30.6 mg/kg) in all cases and the least concentration (385.6±25.2 mg/kg)

was found at the highest dose. Gills showed the significantly lower concentration of K at

low, medium and high doses 485.5±32.6 mg/kg, 443.1±31.5 mg/kg and 389.7±30.8 mg/kg

respectively as compared with the control level (1577±57.3 mg/kg). K in mantle at low dose

exposure (589.9±33.9 mg/kg), showed no significant difference with control treatment

(527.3±32.3), while it was found to be lower at medium and high doses 468.6±32.9 mg/kg

and 459.8±32.3 mg/kg respectively (Table 6). The concentrations of Ca in foot region of

mussels at low, medium and high doses were 4635.3±22.6 mg/kg, 2995.2±23.1 mg/kg, and

5812.8±41.3 mg/kg, respectively and these found to be significantly higher than except

medium dose, whose concentration was about to control level (3052±29.7 mg/kg). Gills

showed higher concentration of Ca for all doses and the values were 193676±650 mg/kg,

178998±985 mg/kg and 186552±489 mg/kg at low, medium and high doses respectively.

63

Table 6. Mineral composition of different portions of freshwater mussels (Anodonta anatina) exposed to different doses of combined



Na K Zn Mn P Ca

120µg/L

Foot 907.29±3.81d 525.4±28.7ab 74.08±5.03c 460.8±5.49e 3708.3±76.5e 4635.3±22.6g

Gills 1527.2±51.5c 485.5±32.6b 333.6±55.4a 22709±853a 52120±1770a 193676±650a

Mantle 2131.6±42.5a 589.9±33.9a 149.4±17.7bc 6706±242d 15545±549d 52271±92f

240µg/L

Foot 965.27±6.98d 497.9±28.1b 77.52±5.91bc 220.23±2.23e 3201.8±49.4e 2995.2±23.1h

Gills 891.4±38.8d 443.1±31.5bc 318.8±50.3a 21018±763b 48057±1822b 178998±985c

Mantle 2076.5±22.3a 468.6±32.9bc 174±23.6b 9252±345c 22726±797c 79352±381d

360µg/L

Foot 716.86±1.97e 385.6±25.2c 70.69±6.14c 425.21±5.86e 4229.8±82.1e 5812.8±41.3g

Gills 1743.9±45.5b 389.7±30.8c 347.9±60.7a 22961±835a 50466±1794ab 186552±489b

Mantle 1493.5±48.1c 459.8±32.3bc 160.3±23.5bc 9047±341c 20853±799c 77507±463e Data are mean ± standard deviation


64

The concentration of Ca in mantle region was found to be higher than the control level and

the amount for low, medium and high doses were 52271±92 mg/kg, 79352±381 mg/kg and

77507±463 mg/kg respectively (Figure 8). The values of Na in foot were 907.29±3.81

mg/kg, 965.27±6.98 mg/kg and 716.86±1.97 mg/kg at low, medium and high doses

respectively, and this was significantly lower than the control level. Gills contained

significantly decreased concentration of Na (891.4±38.8 mg/kg) at medium dose as

compared to control treatment (1577±57.3 mg/kg), while no significant difference was found

at the low dose exposure (1527.2±51.5 mg/kg). In case of mantle, no significant difference

was found in Na concentration, between control level and the low and medium dose, while it

was decreased at the high dose. Foot showed slight fluctuations in Zn concentration at all the

doses but no significant difference was found than control. The similar results were observed

in gills, where no significant difference was found in concentration of Zn between all doses

(low dose: 333.6±55.4 mg/kg, medium dose: 318.8±50.3 mg/kg, high dose: 347.9±60.7

mg/kg) and control level (282.2±49.5 mg/kg) but still it values was slight higher than the

control. While in case of mantle, the values of Zn were 149.4±17.7 mg/kg for low dose,

174±23.6 mg/kg for medium dose and 160.3±23.5 mg/kg for high dose treatment comparing

with the control level (128.7±17.3 mg/kg). Foot portion of mussels had higher concentration

of P (4229.8±82.1 mg/kg) as compared to control (3468.7±56.1 mg/kg), while at the low and

medium doses the value were 3708.3±76.5 mg/kg and 3201.8±49.4 mg/kg respectively,

which was close to the control treatment. The concentration of P in gills was found to be

higher at all the doses as compared to control. In case of mantle, P showed no significant

difference with control level at low dose exposure and the value was 15545±549 mg/kg,

whereas it was significantly higher at medium and high doses 22726±797 mg/kg and

65

20853±799 mg/kg respectively. Foot region of mussels had the highest concentration of Mn

at low (460.8±5.49mg/kg) and medium (425.21±5.86 mg/kg) dose than the control

(192.45±1.81), whereas no significant difference was found at medium dose (220.23±2.23

mg/kg). In case of gills, Mn showed no significant difference in-between control and treated

groups. The concentration of Mn in mantle at low dose was 6706±242 mg/kg, which showed

no significant difference with the control level (6270±227 mg/kg), while significantly higher

values were observed at medium and high doses 9252±345 mg/kg and 9047±341 mg/kg

respectively (Figure 8).

Figure 8. Variations in mineral composition among various portions of freshwater mussels

(Anodonta anatina) exposed to different doses of combined treatment (Pb + Cu + Cr). Graph draw by log base 10.

1

10

100

1000

10000

100000

1000000


120µg/L 240µg/L 360µg/L

Na

K

Zn

Mn

P

Ca

mg/k

g

dc

a

d d

a

e

b c

ab ba b bc bc c bc bc

c

a

bcc

ab

c

a

bc

e

a

d

e

b

c

e

a

c

e

a

d

e

bc

e

ab

mg/k

g

dc

a

d d

a

e

b c

ab ba b bc bc c bc bc

c

a

bcc

ab

c

a

bc

e

a

d

e

b

c

e

a

c

e

a

d

bc

e

ab

c

g

a

f

h

c

d

g

b

e

66

The overall fluctuations in mineral elements among various tissues of freshwater mussels due

to metal exposure are shown individually from figure 9-26.

Figure 9. Fluctuation of Potassium (K) levels in foot of freshwater mussels (Anodonta


Figure 10. Fluctuation of Calcium (Ca) levels in foot of freshwater mussels (Anodonta


67

Figure 11. Fluctuation of Sodium (Na) levels in foot of freshwater mussels (Anodonta


Figure 12. Fluctuation of Phosphorus (P) levels in foot of freshwater mussels (Anodonta


68

Figure 13. Fluctuation of Zinc (Zn) levels in foot of freshwater mussels (Anodonta anatina)


Figure 14. Fluctuation of Manganese (Mn) levels in foot of freshwater mussels (Anodonta


69

Figure 15. Fluctuation of Phosphorus (P) levels in gills of freshwater mussels (Anodonta


Figure 16. Fluctuation of Sodium (Na) levels in gills of freshwater mussels (Anodonta


70

Figure 17. Fluctuation of Potassium (K) levels in gills of freshwater mussels (Anodonta


Figure 18. Fluctuation of Calcium (Ca) levels in gills of freshwater mussels (Anodonta


71

Figure 19. Fluctuation of Zinc (Zn) levels in gills of freshwater mussels (Anodonta anatina)


Figure 20. Fluctuation of Manganese (Mn) levels in gills of freshwater mussels (Anodonta


72

Figure 21. Fluctuation of Phosphorus (P) levels in mantle of freshwater mussels (Anodonta


Figure 22. Fluctuation of Sodium (Na) levels in mantle of freshwater mussels (Anodonta


73

Figure 23. Fluctuation of Potassium (K) levels in mantle of freshwater mussels (Anodonta


Figure 24. Fluctuation of Calcium (Ca) levels in mantle of freshwater mussels (Anodonta


74

Figure 25. Fluctuation of Zinc (Zn) levels in mantle of freshwater mussels (Anodonta


Figure 26. Fluctuation of Manganese (Mn) levels in mantle of freshwater mussels (Anodonta


75

6.3 Proximate composition

The mean values of proximate composition of foot, gills and mantle of freshwater mussels

from the control tank are shown in Table 7. The moisture, protein and fat contents were

highest in foot as compared to mantle and gills (p < 0.05), but on the contrary carbohydrates

were higher in the mantle, and ash in gills (p < 0.05) than other parts. The range of water

content was 76.32 - 79.45%, 71.49 - 74.28% and 72.85 - 76.25% for foot, gills and mantle

respectively. Based on the wet weight, protein contents were 13.88 - 16.93% in foot, 6.05 -

7.41% in gills, and 5.90-6.475% in mantle. The fat contents in foot, gills and mantle were

0.91 - 1.15%, 0.56 - 0.89% and 0.28 - 0.62% respectively. The values of carbohydrates in

foot, gills and mantle were 3.85 - 6.46%, 6.04 - 12.30%, 13.80 - 17.20% respectively. The

ash contents in foot, gills and mantle were 0.44 - 0.73%, 9.34 - 11.82% and 3.05 - 3.63%

respectively (Table 7). Proximate composition was also analysed in mussels exposed to

different doses of metals. The protein content was significantly increased (p < 0.05) in foot

portion of the Pb (22.39 ± 1.44%) and Cr (21.46 ± 0.97%) exposed mussels at the low dose

(120 µg/L) and then it was decreased at the medium dose (240 µg/L) of Pb (20.23 ± 1.75%)

and Cr (17.67 ± 1.23%). The protein content was lowest (10.54 ± 1.14%) at the high dose

(360 µg/L) of Pb (p < 0.05). There were no significant differences (p > 0.05) in any other

nutrients amongst tissues of Pb and Cr-exposed mussels (Table 8, 10). In Cu-exposed

mussels again the protein content was significantly higher in foot (24.70 ± 0.61%) at the high

dose (p < 0.05) while the values at the medium and low doses were 19.28 ± 1.32% and 10.95

± 0.98% respectively (Table 9). Nutrients other than protein in Cu-exposed mussels did not

vary significantly with respect to the control level (Table 7, 9)

76

Table 7. Chemical composition of different portions of freshwater mussel (Anodonta anatina) collected from the control

treatment.

Tissues Compositions (%)

Moisture Ash Proteins Fats Carbohydrates

Foot 77.78±1.208A 0.57±0.1078c 15.45±1.132a 0.97±0.1037a 5.23±1.114c

(2.62±0.390)C (71.39±1904)A (4.47±0.421)A (24.32±5.75)B

Gills 72.73±1.285B 10.55±1.113a 6.53±0.575b 0.72±0.1427b 9.47±2.66b

(45.33±0.651)A (28.10±1.498)B (3.07±0.539)B (42.03±15.99)A

Mantle 74.74±1.237B 3.40±0.224b 6.24±0.229b 0.47±0.1725c 15.15±1.304a

(13.12±0.543)B (24.11±1.360)C (1.83±0.706)C (58.72±7.32)A Data are mean ± standard deviation

Values in the same columns with different superscripts indicates significant differences (p<0.05).

In parenthesis the values are shown in dry matter (DM)

77

6.3.1 Pb-exposed mussels

The results of chemical composition of foot, gills and mantle at various doses (low: 120µg/l,

medium: 240µg/l and high: 360µg/l) of Pb-exposed mussels are shown in Table 8 and Figure 27.

Effect of metals on chemical composition of mussels was not fully understood but we found

some significant variations. The moisture contents in foot from control level were 77.78±1.208

% and found to be higher (80.03±1.283 %) at highest dose but lower at medium (73.89±2.37 %)

and low dose treatment (72.32±1.978 %). In case of mantle, the higher concentration

(78.05±1.925%) was found at the highest dose than control, while no significant difference was

found for the other doses. In gills, no significant difference was found between control and all

the other doses. For the ash contents in foot, 0.57±0.1078% was observed from the control level

and no significant difference was found with low and high doses, while the increased level

(0.70±0.1545 %) was found at medium dose treatment. Similar results was observed in case of

gills where the ash contents were higher at medium dose treatment than control level, while at

low and high doses it was near to control. Mantle tissues showed no significant differences for

ash contents between control and all the doses. Protein contents of foot were varied when

exposed to various doses. At control level, the values of were 15.45±1.132 % and it was

increased when exposed for lower (22.39±1.444 %) and medium (20.23±1.754) doses, white it

found to be lowered (10.54±1.144 %) at higher dose treatment (Table 8).

78

Table 8. Proximate composition of various portions of freshwater mussels (Anodonta anatina) exposed to different doses of Lead

(Pb).

Dose Tissues Compositions (%)


120µg/L Foot 72.32±1.978cd 0.63±0.0903e 22.39±1.444a 1.19±0.241b 3.46±0.329e

Gills 74.11±1.088bcd 12.37±0.741b 6.69±0.481de 0.68±0.1578cd 6.16±2.119de

Mantle 75.84±2.88bc 4.08±0.289d 5.83±0.814e 0.14±0.0667e 14.11±2.67a

240µg/L Foot 73.89±2.37cd 0.70±0.1545e 20.23±1.754b 1.76±0.516a 3.43±0.496e

Gills 71.33±1.760d 16.27±0.667a 7.70±0.586de 1.04±0.2103bc 3.67±1.205e

Mantle 72.86±2.107cd 5.79±0.444c 8.40±0.687d 0.33±0.1101de 12.63±1.755ab

360µg/L Foot 80.03±1.283a 0.57±0.0819e 10.54±1.144c 0.83±0.257bc 8.02±2.073cd

Gills 75.15±1.745bcd 11.70±0.964b 6.32±0.667de 0.58±0.1533cde 6.25±2.27de

Mantle 78.05±1.925ab 3.97±0.1965d 7.46±0.742de 0.28±0.1083de 10.24±1.164bc Data are mean ± standard deviation Values in the same columns with different superscripts indicates significant difference (p<0.05).

79

In gills and mantle tissues, protein contents were not fluctuated in dose exposure and values

were similar to control treatment. The value of fat in foot region at control level was

0.97±0.1037 while the slight changes were found at low, medium and high doses

1.19±0.241%, 1.76±0.516 % and 0.83±0.257% respectively. The quantity of fat in gill were

0.68±0.1578 %, 1.04±0.2103 % and 0.58±0.1533 % at low, medium and high doses

respectively comparing with control level (0.72±0.1427 %). Mantle had 0.47±0.1725 % fat

and the values at low, medium and high doses were 0.14±0.0667 %, 0.33±0.1101 % and

0.28±0.1083 % respectively. There were no significant difference was found in foot region

for carbohydrates between control and all the doses. Carbohydrates in case of gills were

9.47±2.66 % in control treatment, while at low medium and high doses treatment the values

were 6.16±2.119 %, 3.67±1.205 % and 6.25±2.27 % respectively. Mantle showed no

significant difference for carbohydrates at low and medium doses as compared to control

level while value was lower than control at highest dose treatment (Figure 27).

80

Figure 27. Variation in proximate composition among various portions of freshwater

mussels (Anodonta anatina) exposed to different doses of Lead (Pb). Graph drawn by log base 10

cd bcd bc cd d cd a bcd ab

e

b

d

e

a

c

e

b

d

a

de e

b

de dc

de de

b

cd

e

a

bc

de

bccde

de

e

de

a

ee

abcd de bc

0.1

1

10

100


120µg/L 240µg/L 360µg/L


%

81

6.3.2 Cu-exposed mussels

Mussels were exposed for the copper treatment at various doses and proximate composition

was investigated in foot, gills and mantle (Table 9 and Figure 28). Moisture contents in foot

region were decreased as compared to control, as the dose was increased while the Ash

contents were only higher at higher dose whereas at low and medium doses it was near to

control level. In gills, no significant difference was found in moisture contents at all the

doses, while in case of mantle it was higher than control at highest dose treatment. Ash

contents in gills were higher than at all the doses and maximum value was found at medium

dose. Similar results were observed in case of mantle, where the concentration of ash was

higher at all the doses. Protein contents in foot were increased as the dose was increased and

the highest concentration was found at high dose treatment. Gills contained 9.31±0.505 %,

9.36±0.511 % and 7.86±0.379 % protein in low, medium and high dose treatment while the

values at control level was 6.53±0.575 %. The same trend was observed in mantle region

where slight higher value of protein was observed at low and medium doses and in high dose

it found near to control. The concentration of fat in foot was found to be highest at high dose

treatment, while in case of gills, no significant difference was found with control for all the

doses (Table 9).

82

Table 9. Proximate composition of various portions of freshwater mussels (Anodonta anatina) exposed to different doses of

Copper (Cu).


Moisture Ash Protein Fats Carbohydrates

120µg/L Foot 79.80±1.751a 0.47±0.0756e 10.95±0.984c 0.73±0.1322b 8.048±1.532bc

Gills 72.02±1.994bc 13.17±0.738c 9.31±0.505d 0.85±0.1256b 4.64±2.153cd

Mantle 71.88±1.561bc 5.09±0.484d 9.37±0.804d 0.34±0.1120c 13.33±0.830a

240µg/L Foot 73.41±1.748bc 0.61±0.0743e 19.28±1.316b 0.98±0.2075b 5.72±2.81cd

Gills 71.01±0.927c 16.29±0.690a 9.36±0.511d 0.86±0.1682b 2.47±0.674d

Mantle 78.92±1.950a 5.70±0.399d 9.29±0.729d 0.34±0.0822c 5.75±2.30cd

360µg/L Foot 70.58±0.837c 0.84±0.1004e 24.70±0.614a 1.39±0.1648a 2.49±0.958d

Gills 71.97±1.133bc 14.36±0.801b 7.86±0.379d 0.69±0.229b 5.13±1.679cd

Mantle 74.67±2.65b 5.13±0.547d 8.68±0.454d 0.21±0.0913c 11.31±3.11ab Data are mean ± standard deviation


83

Mantle also showed no significant difference for fat al low and medium doses while at high

dose the value was slight lower than control level. For carbohydrates, the value in foot region

was increased than control at low dose treatment and decreased value was observed than at

high dose treatment, while at medium dose no significant difference was found. In case of

foot region lower values of carbohydrates were observed at all the dose than control, whereas

mantle had no significant difference at low and high dose than control while at medium dose

the concentration was found to be lowered than control level (Figure 28).

Figure 28. Variations in proximate composition among various portions of freshwater

mussels (Anodonta anatina) exposed to different doses of Copper (Cu). Graph drawn by log base 10

a bc bc bc c a c bc b

e

c

d

e

a

d

e

b

d

cd d

b

d d

a

d d

b b

c

b b

c

a

b

c

bccd

a

cd

d

cd

d

cd

ab

0.1

1

10

100


120µg/L 240µg/L 360µg/L


%

84

6.3.3 Cr-exposed mussels

Cr exposure also induced changes in dietary components in various portions of freshwater

mussels (Table 10 and Figure 29). Moisture contents in foot region was lower than control

level at low dose while no significant difference was found at medium and high doses. In

gills, there were no significant difference was found between control and all the doses for

metals. In case of mantle, again no significant difference was found between control and low,

and medium dose but at high dose the value was slightly higher than control. The

concentration of ash in foot, gills and mantle were similar to control level and no significant

difference was found. The concentration of protein in foot region was 21.46 ± 0.969 %, 17.67

± 1.228 % and 20.43 ± 1.325 % at low, medium and high doses respectively as compared to

control (15.45±1.132 %). In case of gills, the protein contents were slightly higher at low

dose treatment than control while at medium and high doses treatment the protein was near to

control treatment. The similar trend was observed in mantle tissues, where the concentration

was higher at low dose while at medium and lower dose no significant difference was found

with control level (Table 10 and 7).

85

Table 10. Proximate composition of various portions of freshwater mussels (Anodonta anatina) exposed different doses of

Chromium (Cr).



120µg/L Foot 71.97±0.595bc 0.63±0.1515e 21.46±0.969a 1.22±0.334a 4.72±1.907d

Gills 74.18±1.996bc 11.56±0.981b 10.54±1.077c 0.57±0.0951bc 3.15±1.099d

Mantle 70.76±3.42c 5.03±0.367c 10.46±0.792c 0.41±0.1685c 13.34±4.15a

240µg/L Foot 76.07±2.35ab 0.63±0.1426e 17.67±1.228b 1.03±0.336ab 4.59±2.89d

Gills 74.21±2.180bc 11.83±0.506b 6.45±0.672e 0.65±0.2146bc 6.86±2.28bcd

Mantle 75.35±3.21abc 3.81±0.246d 8.48±0.471d 0.36±0.1074c 11.99±3.49ab

360µg/L Foot 74.56±1.718abc 0.66±0.2145e 20.43±1.325a 1.41±0.370a 2.95±1.762d

Gills 73.56±1.649bc 13.49±0.786a 6.85±0.660de 0.68±0.276bc 5.43±1.583cd

Mantle 79.21±2.152a 3.50±0.396d 6.52±0.696e 0.29±0.0544c 10.47±2.54abc Data are mean ± standard deviation


86

The concentration of fat in foot region was higher than the control at low 1.22 ± 0.334 % and

high doses 1.41 ± 0.370 % while at medium dose 1.03 ± 0.336 % the value was near to

control (0.97 ± 0.1037 %). The values of fat in gills were showed no significant difference

with control while in case of mantle it was lower at high dose than the control. Carbohydrate

contents in foot region was found to be lowered at high dose as compared to control level

while at medium and low dose treatment, no significant difference was found with control. In

gills the value of carbohydrate lower than control at all the doses while in case of mantle, no

significant difference was found and the values were near to control level at all the doses.

Figure 29. Variations in proximate composition among various portions of freshwater

mussels (Anodonta anatina) exposed to different doses of Chromium (Cr). Graph drawn by log base 10

bc bc c ab bc abc abc bc a

e

b

c

e

b

d

e

a

d

a

c cb

ed

a

de e

a

bc c

abbc

c

abc

c

dd

a

d bcdab

dcd

abc

0.1

1

10

100


120µg/L 240µg/L 360µg/L


%

87

6.3.4 Combined metals treatment (Pb + Cu + Cr)

The variations in proximate composition were also studied in different portions of freshwater

mussels exposed for combined treatment of metals (Table 11 and Figure 30). A slight

decreased value of moisture contents were observed for all the doses than the control level in

foot region while the moisture contents in gills showed no significant difference between

control and metal-exposed mussels. In case of mantle, the moisture contents were slightly

higher at the low dose exposure while at medium and high dose treatment, there were no

significant difference was found with control treatment. The ash contents in foot region were

increased as the dose was increased and the lowest concentration was found at low dose

treatment which was near to control level. The ash contents in gills were 15.48±1.177 %,

13.13±0.796 % and 10.70±1.064 % at low, medium and high doses respectively as compared

to control level (10.55±1.113 %). In case of mantle, No significant difference was found in

ash contents at all the dose than control. Protein contents in foot region were 18.29±1.269 %,

21.05±2.151 % and 21.74±0.659 at low, medium and high dose respectively as compared to

control level (15.45±1.132 %). In gills, the protein contents were decreased as the dose was

increased and lowest concentration (7.96±0.959 %) was observed at high dose treatment

which was close to the control level (6.53±0.575) while in case of low and medium doses the

values were 11.12±1.222 % and 9.22±0.633 % respectively (Table 11). The protein contents

in mantle at low and medium doses showed no significant differences with the control level

while at the high dose treatment the protein contents were higher than control.

88

Table 11. Proximate composition of various portions of freshwater mussels (Anodonta anatina) exposed to different doses of

combination of metals (Pb + Cu + Cr).

Doe Tissues Compositions (%)


120µg/L Foot 72.49±1.914bc 0.65±0.0709f 18.29±1.269b 1.18±0.227a 7.39±2.092bcd

Gills 70.16±1.891c 15.48±1.177a 11.12±1.222c 0.70±0.1504b 2.55±1.784d

Mantle 79.75±1.028a 3.35±0.229e 6.20±0.364f 0.22±0.1153c 10.48±1.193ab

240µg/L Foot 71.61±0.931bc 0.72±0.1442f 21.05±2.151ab 1.23±0.274a 5.39±2.222bcd

Gills 72.35±2.078bc 13.13±0.796b 9.22±0.633cde 0.68±0.1136b 4.62±2.63cd

Mantle 74.04±3.13bc 4.12±0.1231e 7.08±0.679ef 0.27±0.0810c 14.49±2.99a

360µg/L Foot 71.82±1.544bc 0.96±0.0588f 21.74±0.659a 1.35±0.310a 4.13±1.686cd

Gills 74.17±2.47bc 10.70±1.064c 7.96±0.959def 0.71±0.1624b 6.46±4.22bcd

Mantle 75.07±2.61b 5.93±1.637d 10.53±2.53cd 0.39±0.1154bc 8.08±2.25bc Data are mean ± standard deviation


89

Foot contained higher fat contents in metal exposed mussels and the values were 1.18±0.227

%, 1.23±0.274 % 1.35±0.310 % at low, medium and high doses respectively as compared to

control treatment (0.97±0.1037 %). While in case of gills, no significant difference was

found in fat contents and the values at all the doses were close to control level. Mantle

showed low fat contents at low and medium doses as compared to control whereas at high

dose treatment no significant difference was found with control level. For carbohydrates

contents in foot region, the values at all the doses were near to the control level so no

significant difference was observed whereas in gill the fluctuations were observed and the

carbohydrates contents at low medium and high doses were 2.55±1.784 %, 4.62±2.63 % and

6.46±4.22 % respectively than control value (9.47±2.66 %). In case of mantle, the lower

value of carbohydrate was observed at lower and high dose treatment while at medium dose

it was close to the control level (Figure 30).

Figure 30. Variations in proximate composition among various portion of freshwater

mussels (Anodonta anatina) exposed to different doses of combined metal treatment (Pb +

Cu + Cr). Graph draw by log base 10.

0.1

1

10

100


120µg/L 240µg/L 360µg/L


%

bc c a bc bc bc bc bc b

f

a

e

f

b

e

f

c

d

b

c

f

ab

cdeef

a

def

cd

a

b

c

a

b

c

a

b

bc

bcd

d

ab

bcd cd

a

cd

bcd bc

90

The overall fluctuations in proximate composition of various tissues due to exposure of

metals are individually shown from figure 31-45.

Figure 31. Fluctuation of Carbohydrate contents in foot of freshwater mussels (Anodonta


Figure 32. Fluctuation of Moisture contents in foot of freshwater mussels (Anodonta


Treatment

Dose

Pb+Cu+CrPbCuCr

T3T2T1T3T2T1T3T2T1T3T2T1

12

10

8

6

4

2

0

%

4.13123

5.38907

7.38884

8.01534

3.427453.46384

2.49186

5.72382

8.04756

2.94838

4.59464.71934

Treatment

Dose

Pb+Cu+CrPbCuCr


82

80

78

76

74

72

70

%

71.821671.6115

72.4944

80.0392

73.8865

72.324

70.5811

73.4059

79.8009

74.5594

76.0729

71.9669

91

Figure 33. Fluctuation of Ash contents in foot of freshwater mussels (Anodonta anatina)


Figure 34. Fluctuation of Fat contents in foot of freshwater mussels (Anodonta anatina)


Treatment

Dose

Pb+Cu+CrPbCuCr


1 .1

1 .0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

%0.960438

0.723354

0.654296

0.573443

0.697601

0.634681

0.83542

0.611334

0.471662

0.6591090.6319640.629641

Treatment

Dose

Pb+Cu+CrPbCuCr


2.5

2.0

1 .5

1 .0

0.5

%

1.35189

1.225641.17567

0.830739

1.75685

1.18491

1.39129

0.983774

0.725627

1.40542

1.03341

1.22257

92

Figure 35. Fluctuation of Protein contents in foot of freshwater mussels (Anodonta anatina)


Figure 36. Fluctuation of Carbohydrate contents in gills of freshwater mussels (Anodonta


Treatment

Dose

Pb+Cu+CrPbCuCr


26

24

22

20

18

16

14

12

10

%21.7348

21.0504

18.2867

10.5412

20.2316

22.3926

24.7003

19.2752

10.9542

20.4277

17.6672

21.4615

Treatment

Dose

Pb+Cu+CrPbCuCr


14

12

10

8

6

4

2

0

%

6.46362

4.61848

2.54612

6.2489

3.66592

6.15809

5.12959

2.46895

4.63688

5.43083

6.85971

3.15326

93

Figure 37. Fluctuation of Moisture contents in gills of freshwater mussels (Anodonta


Figure 38. Fluctuation of Ash contents in gills of freshwater mussels (Anodonta anatina)


Treatment

Dose

Pb+Cu+CrPbCuCr


77

76

75

74

73

72

71

70

69

68

%

74.1684

72.3506

70.1625

75.146

71.3281

74.108

71.9648

71.0143

72.0264

73.5546

74.210974.176

Treatment

Dose

Pb+Cu+CrPbCuCr


18

17

16

15

14

13

12

1 1

10

9

%

10.6962

13.126

15.4788

11.7034

16.271

12.3693

14.3573

16.2932

13.1713.4852

11.829111.5623

94

Figure 39. Fluctuation of Fat contents in gills of freshwater mussels (Anodonta anatina)


Figure 40. Fluctuation of Protein contents in gills of freshwater mussels (Anodonta anatina)


Treatment

Dose

Pb+Cu+CrPbCuCr


1 .4

1 .2

1 .0

0.8

0.6

0.4

0.2

%

0.7075610.6845440.69596

0.578871

1.0372

0.6768010.692742

0.8626570.854683

0.6810780.651304

0.56565

Treatment

Dose

Pb+Cu+CrPbCuCr


13

12

1 1

10

9

8

7

6

5

%

7.96423

9.22038

11.1166

6.3228

7.69778

6.6878

7.85559

9.360829.31204

6.84826

6.44899

10.5429

95

Figure 41. Fluctuation of Carbohydrate contents in mantle of freshwater mussels (Anodonta


Figure 42. Fluctuation of Moisture contents in mantle of freshwater mussels (Anodonta


Treatment

Dose

Pb+Cu+CrPbCuCr


20

15

10

5

0

%

8.07626

14.487

10.483610.2348

12.6299

14.109

11.3117

5.75084

13.325

10.4706

11.9941

13.34

Treatment

Dose

Pb+Cu+CrPbCuCr


82.5

80.0

77.5

75.0

72.5

70.0

67.5

65.0

%

75.0685

74.0446

79.7446

78.0484

72.856

75.8446

74.6709

78.9196

71.8763

79.2093

75.3543

70.7577

96

Figure 43. Fluctuation of Ash contents in mantle of freshwater mussels (Anodonta anatina)


Figure 44. Fluctuation of Fat contents in mantle of freshwater mussels (Anodonta anatina)


Treatment

Dose

Pb+Cu+CrPbCuCr


8

7

6

5

4

3

%

5.93336

4.12397

3.35097

3.96901

5.78849

4.07567

5.12571

5.69924

5.09113

3.50425

3.80769

5.03054

Treatment

Dose

Pb+Cu+CrPbCuCr


0.6

0.5

0.4

0.3

0.2

0.1

0.0

%

0.387953

0.267897

0.222181

0.285503

0.32795

0.144467

0.214736

0.3405230.341464

0.291682

0.360636

0.409834

97

Figure 45. Fluctuation of Protein contents in mantle of freshwater mussels (Anodonta


Treatment

Dose

Pb+Cu+CrPbCuCr


14

13

12

1 1

10

9

8

7

6

5

%

10.534

7.07653

6.19869

7.46233

8.3977

5.82626

8.67696

9.289779.36606

6.52418

8.48333

10.4619

98

6.4 Wild experiment results

In wild experiment, the study site was Chashma Lake River Indus Pakistan. The samples of

water and soil were collected along with the freshwater mussels. The concentration of

various metals in water is shown in Table 12. The concentration of Ca was higher than all the

other studied minerals.

Table 12. Concentration of metals and minerals in water samples from the Chashma lake

River Indus Pakistan.

Metals Water (mg/L)

Lead (Pb)

0.31 ± 0.029

Copper (Cu)

0.33 ± 0.03

Chromium (Cr)

0.071 ± 0.11

Cadmium (Cd)

0.042 ± 0.007

Manganese (Mn)

0.23 ± 0.008

Zinc (Zn)

0.61 ± 0.04

Calcium (Ca)

114 ± 15

Potassium (K)

9.5 ± 0.79

Sodium (Na)

1.84 ± 0.24

Phosphorus (P) 0.44 ± 0.06

Data Means ± standard deviation

99

The concentration of Pb, Cu, Cr, Cd, Mn, Zn, Ca, K, Na, P in the soft tissues of freshwater

mussels Anodonta anatina harvested directly from the Chashma lake River Indus Pakistan

are summarized in Table 13. The means concentration of all the minerals was varied among

the tissues. Amongst three studied portions, gills had the highest concentration for all the

minerals followed by mantle and foot. The concentration of Ca was significantly higher in

gills (175346-179864 mg/kg) as compared to mantle (45117-47850 mg/kg) and foot (1823.5-

2407.4 mg/kg) (p < 0.05). No significant difference was found among the soft tissues for Pb

and the values in foot, gills and mantle were 1.645-6.500 mg/kg, 1.612-8.467 mg/kg and

2.240-10.104 mg/kg respectively. The same trend was observed for Cu where there was no

significant difference was observed among the studied portions but gills had slightly higher

value than mantle and foot (p > 0.05). The concentration of Cu in foot, gills and mantle were

10.10-21.54 mg/kg, 13.42-55.39 mg/kg and 21.17-54.67 mg/kg respectively. Gills and

mantle had higher value of Cr (0.967-3.728 mg/kg, 0.518-6.873 mg/kg), Cd (1.089-2.270

mg/kg, 1.055-3.175 mg/kg), Zn (155.2-300.1 mg/kg, 152.1-326.4 mg/kg) and Na (1541-3077

mg/kg, 2277-3136 mg/kg) respectively as compared with foot (Cr 0.7007-1.0511 mg/kg, Cd

0.3646-0.8903 mg/kg, Zn 70.83-106.69 mg/kg, Na 953.4-1454.5 mg/kg). The value of K had

again maximum in gills (740.3-963.5 mg/kg) followed by foot (530.0-722.3 mg/kg) and

mantle (366.3-489.1 mg/kg). The concentration of Mn and P was significantly higher in gills

(22179-25430 mg/kg, 45404-49080 mg/kg) with respect to mantle (5559-9146 mg/kg, 5399-

8402 mg/kg) and foot (18.7-86.8 mg/kg, 2198-3381 mg/kg) respectively. The graphical

representation of all the metals and minerals in foot, gills and mantle of freshwater mussels

are shown in Figure 46.

100

Table 13. Concentration (mg/kg) of metals and minerals in various tissues of freshwater

mussels from the Chashma lake River Indus Pakistan

Metals Concentration (mg/kg)

Foot Gills Mantle

Lead (Pb)

3.781±1.555a

4.037±2.348a

4.831±2.661a

Copper (Cu)

15.60±4.61a

29.06± 16.01a

30.07±13.70a

Chromium (Cr)

0.8278±0.1447b

1.977±0.951ab

2.659±2.459a

Cadmium (Cd)

0.6821±0.2216b

1.503±0.396a

2.011±0.768a

Manganese (Mn)

47.2±30.3c

23519±1050a

7207±1046b

Zinc (Zn)

84.85±11.59b

222.4±51.6a

212.4±56.2a

Calcium (Ca)

2157.6±235.4c

177698±1799a

46838±984b

Potassium (K)

610.2±66.9b

849.1±71.5a

420.6±38.2c

Sodium (Na)

1147.5±225.7b

2261±575a

2706±343a

Phosphorus (P)

2711±487c

47097±1278a

6921±1063b


Values in same rows with different superscripts are significantly different (p<0.05).

101

Figure 46. Fluctuations of metals and minerals concentration in various tissues of freshwater

mussels from the Chashma lake River Indus Pakistan. Graph drawn by log base 10

In bioaccumulation of different trace metals (Pb, Cu, Cr, Cd and Mn) gills showed the higher

concentrations of these metals as compared to foot and mantle. The strongly positive

correlation was observed among most of the studied elements (Table 14) whereas some

negative correlation were also observed such as Pb showed the negative correlation with the

Cu, Cd, Zn, Ca, K and P. In case of K, it showed the negative correlation with the Cu, Cr, Cd

and Na as well.

0.1

1

10

100

1000

10000

100000

1000000

Pb Cu Cr Cd Mn Zn Ca K Na P

mg

/kg

Foot

Gills

Mantle

aa a

a a

b

aba

ba

a

c

ab

b

a a

c

a

b

ba

c

baa c

a

b

102

Table 14. Pearson correlation coefficient (r) among metals in tissues of freshwater mussels

(Anodonta anatina) from the Chashma Lake River Indus Pakistan

Metals Pb Cu Cr Cd Mn Zn Ca K Na

Cu -0.032

P-Value 0.876

Cr 0.084 0.305

P-Value 0.676 0.123

Cd -0.012 0.068 0.005

P-Value 0.952 0.735 0.979

Mn 0.027 0.388 0.227 0.28

P-Value 0.893 0.046 0.254 0.157

Zn -0.093 0.412 0.004 0.489 0.634

P-Value 0.644 0.033 0.531 0.01 0.000

Ca -0.004 0.316 0.174 0.282 0.995 0.611

P-Value 0.644 0.108 0.385 0.154 0.000 0.001

K -0.054 -0.068 -0.152 -0.264 0.685 0.000 0.726

P-Value 0.789 0.735 0.449 0.183 0.000 1.000 0.000

Na 0.131 0.263 0.157 0.87 0.42 0.548 0.409 -0.127

P-Value 0.515 0.185 0.434 0.000 0.029 0.003 0.034 0.528

P -0.051 0.255 0.111 0.164 0.972 0.520 0.986 0.815 0.28

P-Value 0.799 0.198 0.582 0.413 0.000 0.005 0.000 0.000 0.157

103

Proximate composition of foot, mantle and gills of freshwater mussels (Anodonta anatina)

collected directly from the Chashma Lake River Indus Pakistan are shown in Table 16. The

% of carbon, nitrogen and their ratios were investigated and result revealed in Table 15.

There were no significant difference was found in foot, gills and mantle for the carbon and

nitrogen percentage whereas the values were slightly higher in foot and mantle as compared

to gills.

Table 15. Mean value (±S.D) of Carbon and Nitrogen in tissues of freshwater Mussels

(Anodonta anatina) from the Chashma Lake River Indus Pakistan.

Soft tissues Composition %

Carbon Nitrogen C/N Ratio

Foot

37.24±8.74a 9.31±3.01a 4.146±0.60a

Gills

32.12±5.12a 6.806±1.51a 4.787±0.51a

Mantle 38.86±4.08a 8.79±2.49a 4.63±0.98a Data Means ± standard deviation

Means (±S.D) in columns with different superscripts are significantly different (p<0.05).

Amongst all the studied parts, moisture contents found to be lowered in mantle (70.85-81.37)

as compared to gills and foot. The moisture contents in foot and gills were 74.340-80.190 %

and 74.380-82.393 % respectively. Protein contents were significantly higher in foot region

(14.694-17.629 %) followed by mantle (6.39-12.56%) and gills (4.76-7.82%) (Table 16). In

case of carbohydrates the higher concentration was observed in mantle region (4.362 -

12.480 %) and lowest was found in foot portion (1.848 - 6.181 %), while in gills it was 4.515

104

- 9.327 %. Percentage of ash was significantly higher in gills (6.13-11.18%) as compared to

mantle (4.67-5.79%) and foot (0.37-0.58%). The values of carbohydrate content were highest

in mantle (4.36-12.48%) than gills (4.51-9.33%) and foot (1.85-6.18%). In view of fat

contents, it was found that the foot had the highest content (0.89-1.69%) when compared

with gills (0.28-0.74%) and mantle (0.15-0.44%) (Table 16). The graphical representation of

proximate composition in foot gills and mantle of freshwater mussels are shown in Figure 47.

Table 16. Proximate composition (%) of foot, gills and mantle in freshwater mussels

(Anodonta anatina) from the Chashma Lake River Indus Pakistan.

Soft tissues Compositions (%)

Moisture Protein Carbohydrate Lipids Ash

Foot

78.22±1.70ab 15.90±0.88a 4.23±1.45b 1.19±0.26a 0.46±0.05c

Gills

78.96±2.86a 6.44±1.22c 6.55±1.69ab 0.53±0.15b 7.51±1.88a

Mantle

75.12±3.97b 10.78±2.24b 8.54±3.11a 0.27±0.09c 5.29±0.39b


Values in same columns with different superscripts are significantly different (p<0.05)

105

Figure 47. Fluctuation in proximate composition (%) of foot, gills and mantle in freshwater

mussels (Anodonta anatina) from the Chashma Lake River Indus Pakistan

Graph drawn by log base 10

Table 17 showed the correlation coefficient of various nutrients in soft tissues of freshwater

mussels. There were strong positive correlation was found between fat, moisture and protein

and between ash and carbohydrates whereas the negative correlation was found between

carbohydrates and other studied nutrients (Proteins, fats and moisture).

0.1

1

10

100

Moisture Protein Carbohydrate Lipids Ash

Foot

Gills

Mantle

%

ab ba

a

c

b

bab

a

a

b

cc

ab

106

Table 17. Pearson correlation coefficient (r) among nutrients in tissues of freshwater mussels

(Anodonta anatina) form the Chashma Lake River Indus Pakistan

Nutrients Moisture Protein Fat Carbohydrates

Protein -0.231

P-value 0.22

Fats 0.154 0.661

P-value 0.417 0.000

Carbohydrates -0.669 -0.396 -0.551

P-value 0.000 0.030 0.002

Ash -0.183 -0.823 -0.690 0.429

P-value 0.334 0.000 0.000 0.018

107

6.5 Genotoxicity by comet assay

Metal-induced genotoxicity was observed in gills of freshwater mussels (Anodonta anatina)

exposed for the various doses of heavy metals in laboratory conditions. The gills of mussels

being sensitive portion were chosen for this experiment due to direct exposure with ambient

water. The significant differences were observed between control and the metals-exposed

mussels. The control level was the same for all metals so had same value as mentioned in all

Tables. The value of tail DNA percentage, tail length and olive tail moment was decreased as

the dose was increased. The lowest values of theses parameters were observed at highest

dose treatment but still the value was higher than the control (p<0.05) except the chromium

and combined treatments where it was returned to the control level (p>0.05). The comet

image of gill cells of freshwater mussels collected from the control tank showed no damages

in DNA (Figure. 48) whereas significant damaging was observed in all treatment groups of

various heavy metals as shown in the Figure 49.

Figure 48. The image of comets by fluorescent microscope from the gills of freshwater

mussels (Anodonta anatina) collected from the control treatment.

108

Figure 49. The images of comets by fluorescent microscope from the gills of freshwater

mussels (Anodonta anatina) collected from the treated tanks.

109

For % of tail DNA highest values was observed in Cu-exposed mussels (56.74±8.11 %) as

compared to Pb (47.36±5.50 %), Cr (43.0±5.85 %) and combined treatment (25.94±5.21 %)

at low dose (120 µg / L). The least value of tail DNA was noted at high doses (360 µg / L)

such as for Cu (20.89±2.49 %), Pb (10.88+3.27 %), Cr (7.94±3.18 %) and combined

treatment (8.16±0.985 %). There were no significant differences between the Pb and Cr at

low dose treatment (120 µg / L) whereas both were significantly different (p<0.05) from Cu

and combined treatment (Pb + Cu + Cr) at the same doses. The percentage of DNA for

combined treatment at medium dose (240 µg / L) was significantly lower (19.60 ± 1.27) from

all the other treatments at the same doses (p<0.05) (Table 18, Figure 50).

Table 18. Effect of different doses of Pb, Cu, Cr individually and in combination on mean %

tail DNA.

Dose 0µg/L 120µg/L 240µg/L 360µg/L

Metals

Pb 1.34±1.28g 47.36±5.50b 26.89±4.35d 10.88+3.27f

Cu 1.34±1.28g 56.74±8.11a 30.03±1.91cd 20.89±2.49e

Cr 1.34±1.28g 43.05±5.85b 33.86±7.65c 7.94±3.18f

Pb + Cu + Cr 1.34±1.28g 25.94±5.21d 19.60±5.68e 8.16±4.40f

Any means (±S.D) in row and column with different superscripts are significantly different (p<0.05) and means

with the same superscript are not significantly different (p>0.05)

110

In case of comet tail length significantly higher value was observed in Pb-exposed mussels

(49.15±8.50) followed by Cu (41.30±3.39), Cr (26.6±3.51) and combined treatment

(18.20±1.13) at low dose. Trend for the same parameter in all metals at low dose was Pb >

Cu > Cr > Pb + Cu + Cr (Table II). The values of comet tail length at the medium dose of Pb,

Cu, Cr and Pb + Cu + Cr were 21.95 ± 3.99, 18.05 ± 2.61, 17.40 ± 5.17 and 8.750 ± 2.57

respectively. As usual the value of comet tail length was also decreasing with increasing the

concentration of metals. The lowest value of tail length was found at higher dose (360 µg / L)

of all metals exposure such as Cu (16.05±3.28), Pb (9.00±3.03), Cr (8.30±2.43) and

combined treatment (7.20±2.38) (Table 19, Figure 51).

Table 19.Effect of different doses of Pb, Cu, Cr individually and in combination on mean

comet tail length.


Metals

Pb 3.45±1.05g 49.15±8.50a 21.95±3.99d 9.00±3.03f

Cu 3.45±1.05g 41.30±3.39b 18.05±2.61de 16.05±3.28e

Cr 3.45±1.05g 26.65±3.51c 17.40±5.17e 8.30±2.43f

Pb + Cu + Cr 3.45±1.05g 18.20±5.07de 8.750±2.57f 7.20±2.38fg



111

The value of olive tail moment (OTM) was also varied among the different doses of metals.

Cu and Pb exposure showed significantly higher value of OTM (13.48±1.99, 12.91±2.02

respectively) at the low doses (P<0.05) and again the Cr and combined metal exposure

showed lower values (6.94±1.71, 3.89±0.94 respectively) at the same dose. The value of

OTM was significantly higher (7.75±0.75) in Cu treatment at the medium dose as compared

to other metals exposure at the same dose. There were no significant difference was found in

between Pb and Cr at medium dose and Cr and combined treatment at the high dose (p>0.05)

(Table 20, Figure 52). Chromium induced low DNA damage than Cu and Pb but higher than

the combined treatment at all the doses for all the parameters (Table 18, 19, 20).

Table 20. Effect of different concentration of Pb, Cu, Cr individually and in combination of

metals on mean Olive Tail Moment (OTM).


Metals

Pb 0.17±0.14h 12.91±2.02a 3.87±0.85cd 1.63±0.43fg

Cu 0.17±0.14h 13.48±1.99a 7.75±0.75b 3.38±0.47de

Cr 0.17±0.14h 6.94±1.71b 4.85±1.84c 1.30±0.50gh

Pb + Cu + Cr 0.17±0.14h 3.89±0.94cd 2.49±0.82ef 1.16±0.60gh



112

Figure 50. DNA damage in gills of freshwater mussel (Anodonta anatina) expressed in the

form of mean tail DNA exposed for different doses of heavy metals. Graph drawn by log base 10


form of mean tail length exposed for different doses of heavy metals. Graph drawn by log base 10

1

10

100

Pb Cu Cr Pb + Cu + Cr

0µg/L

120µg/L

240µg/L

360µg/LTai

l D

NA

(%

)

gg g g

b ab

ddcd c

e

f

e

f

f

1

10

100


0µg/L

120µg/L

240µg/L

360µg/LTai

l le

ngth

(µ

m)

gg g g

ab

c

ded de e

ff

e

f

fg

113


form of mean olive tail moment (OTM) exposed for different doses of heavy metals. Graph drawn by log base 10

0.1

1

10

100


0µg/L

120µg/L

240µg/L

360µg/L

OT

M

hh h h

a a

b

cdcd

bc

effg

de

gh

gh

114

7. Discussions

In this study, bioaccumulation of metals and analysis of mineral element alongside proximate

composition were carried out in laboratory exposed and wild sampled freshwater mussels. It

is recognised that the bivalve mussels can accumulate high levels of metal in their soft tissue.

Thus bivalves are good candidates to investigate water pollutants because of their sedentary

life style and the greater degree to accumulate the toxicants in their soft tissues. In the present

study Cu was prevalent among the studied metals and gills were found to be the highest

accumulating tissue. Usero et al. (2005) noted that bivalves were the desirable species for the

biomonitoring of environmental pollutants in aquatic system. In various other studies

different tissues of freshwater were used to indicate the metal loads in the surrounding

environment. According to Wadige et al. (2014) the bioaccumulation of Pb varied amongst

tissues of bivalves and the highest concentrations were observed in labial palps and gills as

compared to other tissues. In our results, gills contained higher concentration of all metals

than foot and mantle.

This is in agreement with the findings by Vincent-Hubert et al. (2011) who demonstrated that

the gills were the most sensitive organs for accumulating chemicals because they are directly

exposed to water pollutants as their immediate environment. Jones and Walker, (1979) also

reported that the highest levels of bioaccumulation in freshwater mussels were observed in

gills and the lowest values were observed in their foot. In another study (Yap et al., 2006) it

was concluded that the gills were the greater accumulator for zinc, lead, and cadmium than

the other visceral parts, and they were good bioindicators of aquatic pollution. Sasikumar et

al. (2006) investigated trace metal contaminants in green mussels collected from various sites

of southwest coast of India. Their results showed a significant input of cadmium, chromium

115

and lead from industrial sites. Yap et al. (2004) studied the heavy metal accumulation in

green lipped mussel (Perna viridis) collected from the coast of Peninsular Malaysia and

reported the concentrations of different heavy metals. The values ranged from 0.68 mg/g to

1.25 mg/g for cadmium, 7.76 mg/g to 20.1 mg/g for copper, 2.51 mg/g to 8.76 mg/g for lead,

and 75.1 mg/g to 129 mg/g for zinc.

In this study higher level of Cu accumulation was observed than the other studied metals.

Our results resemble a previous study where high level of copper was reported in D.

Trunculus as compared to other metals (Baraj et al., 2003). In our findings the accumulation

of metals increased with increasing doses; similar results were reported by Guidi et al. (2010)

who used freshwater mussels in biomonitoring studies, and stated that metal bioaccumulation

in tissues increased with elevated levels of metals in sediments. Maanan, (2007) investigated

the heavy metal concentration in Mytilus galloprovincialis harvested from the Safi coastal

water in Morocco. High levels of metals were observed in mussel tissues collected from area

near industrial coastal settlements, which was due to heavy concentration of metals in water.

The variations were observed among bivalve tissues for metals accumulation; the same

concept was reported by another study on freshwater mussels where it was stated that the

bioaccumulation of metals was tissue specific (Labrot et al., 1999). Patel and Anthony,

(1991) also reported a very high concentration of Cd in gills of Anadara granosa and

concluded that the gills could uptake most metals. Yap et al. (2003) reported that the Pb and

Zn were dominantly found in gills of M. edulis. Another study concluded that gills and

mantle had the highest levels of various metals such as Pb, Cu, Zn, Fe and Mn, whereas the

lowest value was observed in the foot samples of adductor muscles (Tessier et al., 1984). Our

findings also agreed with a previous study where significant bioaccumulation of Pb in mussel

116

tissues was reported at the highest dose, and least accumulation was observed at the lowest

dose (Rahnama et al., 2010). Furthermore, it was stated that the initial concentration of Pb

had a direct influence on its bioaccumulation in the soft tissue of the mussels.

There were several studies was conducted on fish fauna for metals, minerals and nutrients

analysis on the same site but the bivalves fauna would not be exploited so this was the first

study on freshwater mussels of Chashma Lake Indus River Pakistan. Biologically important

minerals (Ca, K, P, Mn, Na, and Cu) were abundantly observed in edible parts (Foot, Mantle)

of freshwater mussels from the Chashma lake Indus River Pakistan that might be the nutritive

source for the consumers. The concentration of Pb in edible parts of freshwater mussels from

Indus river was higher than the permissible value of WHO so it might be cause health

hazards to consumers whereas the accumulation of other metals were below the permissible

level. Another aim of our study was to use the freshwater mussels as a pollution indicator

species and results indicates the gills are best part for biomonitoring study as it had higher

accumulation of heavy metals when compared with foot and mantle.

Jabeen and Chaudhry, (2010) investigated the heavy metals in different tissues of cyprinus

carpio from the Indus River Pakistan and compared the value of Mn and Cr in fish organs

with the WHO and Federal environmental agencies standard and found that the concentration

were higher than recommended. Mytilus galloprovincialis harvested from Marmara sea

turkey contained Na (2146.764-4530.101 mg/kg) and K (1809.336-2930.825 mg/kg) in their

soft tissues (Ozden et al. 2010). Jebali et al. (2014) reported the comparative

bioaccumulation of trace metals in various tissues of bivalve pinna nobilis and stated that the

digestive gland stored maximum metals followed by gills and muscles and freshwater

mussels are sensitive animal for biomonitoring studies. Espana et al. (2007) compared

117

different minerals and trace elements in molluscs harvested from the Strait of Megellan

(Chile) and reported the high concentration of Na, Ca, Zn, Mn and Cd in Perunytitus

purpuratus. Elements composition of mytilus galloprovincialis collected from the different

coast of Spain and the authors reported the significant difference in metals concentrations

among the three sites. All the samples showed the high concentrations of Na, K, Ca and P

whereas the concentration of Cu and Mn was low (Fuentes et al., 2009).

Usero et al. (2005) reported the high concentration of Cr, Cu, Pb, Zn in D. trunculus as

compared to C. gallina collected from the Atlantic coast of southern Spain. Chemical

Composition of Chamelea gallina harvested from the various sites of Southern Coast of the

Marmara Sea has been studied and reported the 67 % moisture, 10.12% protein, 2.57% lipids

and 1.66% ash. They also reported the perfect concentration Cu, Cr, Mn and Cd tissues in

bivalves at most of the sites whereas the concentration of Pb and Zn was higher than the

critical concentration at some sites (Fatma et al., 2011). Our findings proposed that

freshwater mussels from the Chashma Lake Indus River Pakistan were the rich source of

nutrients like proteins carbohydrates and fats. The concentration of all the accumulated

metals was below the dangerous level except the Lead (Pb) which was slightly higher than

the recommended value by WHO. Gills (Non-edible part) had stored maximum concentrated

of all the metals that prove that it was the best parts to monitor the aquatic pollution by using

freshwater mussels as a key species.

In mineral elements composition Ca was found to be higher in gills of bivalves, but our

results are different from a previous study where the order of mineral elements was Na > K >

Ca > Mg (Karnjanapratum et al., 2013). Conversely, in our results the pattern of mineral

elements composition was similar to that found in other studies on bivalves (Espana et al.,

118

2007). Fuentes et al. (2009) reported a higher concentration of Ca in mussels as compared to

other mineral elements. Our results agree with studies which showed that the mussels were

good sources of Ca, Mg and P (King et al., 1990; Karakoltsidis et al., 1995). Amongst

different tissues, gills accumulated the most minerals when compared with mantle and foot as

reported by Karnjanapratum et al. (2013). Mineral elements play a very important role in

homeostasis so these are essential for maintaining various biological processes such as

osmotic balancing, membrane potentials and muscular contractions in animals.

In this study values of mineral elements were also investigated in bivalves exposed to

different doses of Pb, Cu, Cr and combined treatment (Pb + Cu+ Cr).There were no

significant differences in mineral elements except for Ca that appeared higher in metal-

exposed mussels than control. The burden of heavy metals in the tissue of mussels can

correlate to biological dysfunctioning (Chandurvelan et al., 2012). Heavy metals including

Cu showed adverse effects on Ca homeostasis in gills of mussels and also resulted in the

deterioration of enzyme action (Viarengo et al., 1996). Exposure to copper produces a variety

of serious defects in cell functioning by altering the Ca singling which can inhibit

biochemical reactions (Burlando et al., 2004). Lead accumulates in tissues and interferes

with other bio elements such as Ca, Zn and Cu, causing a variety of pathophysiological

effects (Berrahal et al., 2011).

Lower values of Na and K were observed in tissues of controlled as compared to the exposed

mussels but very few published data were found on this subject. Fluctuation in mineral

elements were shown when the mussels were exposed to different doses of heavy metals but

almost no effort was reported to find such associations between the exposure of organisms to

heavy metals and mineral composition of their tissues. Rahnama et al. (2010) reported the

119

effect of Pb accumulation on the filtration rate of zebra mussels harvested from the Anzali

wetland in the Caspian Sea and showed that the filtration rate was reduced by 40% at 455 µg/

L concentration of Pb. Amongst proximate composition; protein contents were highest in the

foot region of Anodonta anatina. Similar results were reported in a previous study where

different portions of bivalves were studied for proximate composition (Karnjanapratum et al.,

2013).

In our study the highest values of carbohydrates were found in mantle as compared to gills

and foot which is contrary to a previous study where higher value of carbohydrates was

found in foot than mantle (Karnjanapratum et al., 2013). The ash contents were higher in

gills as compared to mantle and foot, because of the higher accumulation of metals in gills of

bivalves. Ersoy and Sereflisan, (2010) reported that the minced whole freshwater mussels,

Potamida littoralis and Unio terminalis contained 11.9-12.0% protein, 1.6-1.7% ash, 1.1-

2.6% fat and 80.4-81.7% moisture. Striped Venus clam, C. gallina, harvested from the

Adriatic Sea contained 8.55-10.75% protein, 0.73-1.59% lipids and 2.25-4.96%

carbohydrates (Orban et al., 2006).

Karnjanapratum et al. (2013) also reported that the Asian hard clam, M. lusoria, collected

from the coast of the Andaman Sea contained 9.09-12.75% protein, 0.32-7.89%

carbohydrate, 1.58-6.58% fat and 1.23-2.58% ash. Protein was found to be highest in foot as

compared to mantle and viscera. In one of the previous study siphon and mantle of Pacific

clam, Panopea abrupt, were evaluated for proximate composition and observed value of

protein and moisture was 14.32-15.29% and 74.82-80.37% respectively (Oliveira et al.,

2011). The results of present study indicated that protein is a major constituent in Freshwater

mussels (Anodonta anatina) harvested from the Chashma Lake River Indus, Pakistan. Beiras

120

et al. (2003) investigated the accumulation of various metals and reported the high value of

Cu and Hg in mussels from the Ria of Pontevedra, Spain. The level of contamination was

investigated in mussels harvested from the various sites of Irish Sea.

The elevated level of Pb, Cd, Se and Ag was found in water and tissue of mussels. The

decreased in mussels population in various sites was associated with the increased level of

pollution (Widdows et al., 2002). In our results, fluctuations in term of minerals and

proximate composition were also observed when exposed for the combination of metals (Pb

+ Cu +Cr) and this is in agreement with the previous study, where the toxicological effect of

copper and cadmium on mussels was evaluated singly and in combination of metals (Cu +

Cd). The significant changes were in metabolism of mussels due to metal exposure. In

combined exposure of metal, the level of various amino acids was increased whereas the

level of carbohydrates was decreased. Cu-exposed mussels also showed disturbance at

various levels of metabolism, and the results were similar to combined exposure of metals

(Wu and Wang, 2010).

In case of genotoxicity, Cu and Pb both induced high damaging of DNA in gill cells as

compared to Cr and combined exposure of metals. Pb showed slightly higher value for the

tail length than copper at low dose as difference in means was 7.85±1.92 (n=20) but in case

of % of tail DNA Cu shows more value than Pb and mean difference was 9.38±2.01 (n=20)

at the same dose. For the olive tail moment at the same dose the little difference was

observed in between copper and lead but again more values were observed in copper exposed

mussels and difference in means was 0.929±0.655 (n=20). The combined metals exposure

induced very low damaging in DNA as showed in all observed parameters.

121

Vincent-Hubert et al. (2011) compared the haemocytes and gills of mussels for best results in

comet assay study and concluded that the gills have more sensitivity for the contaminants as

compared to the haemocytes. Freshwater mussels were used in this study because as being

filter feeder these are the best candidates to assess the contamination in ambient water.

Bivalve mussels are sensitive organisms for the environmental pollutants and preferably used

in bio-monitoring studies (Viarengo and Canesi, 1991; Chase et al., 2001). Our results

resemble to previous study where the mussels were exposed to the Nano and ionic forms of

copper and significant difference was observed in copper exposed mussel as compared to

controls and it was also stated that the higher damage was observed in mussel exposed to

ionic form of Cu, reported by Gomes et al. (2013).

Bolognesi et al., (1999) also showed similar results where the mussels were exposed to

different concentrations of Cu and high level of DNA damage was observed at low

concentration of Cu (40 µg L-1). Al-Subiai et al. (2011) reported the effect of copper on

marine mussels and it was stated that the high dose of Cu was toxic to animal and 100%

mortality was observed at 100 µg L-1 which is contradictory to our results where no mortality

was observed in freshwater mussels at any dose of copper. Our finding resembles to one of

previous study where mussels were exposed to low and high doses of polycyclic aromatic

hydrocarbon and higher level of DNA strands breaks was observed in mussels which were

exposed to the low doses of polycyclic aromatic hydrocarbon (Large et al., 2002). Freshwater

mussels were exposed to various doses of Pb to evaluate the genotoxic effects and

significantly high DNA strands breaks was observed at low dose 50 µg L-1 of lead and very

little DNA damage was found at high dose of 500 µg L-1 (Black et al., 1996).

122

Chromium and combined treatments (Pb + Cu + Cr) showed the same trends for all

the parameters at all doses. The combined exposure of metals showed very low value in

DNA damage than the all metals which showed some synchronized effects of metals. It is

unclear why the combined treatment of heavy metal induce low level of DNA damage but

one of the previous study also showed the similar results where the mussels were exposed to

the a combination of Cu, Cd and Hg and low level of DNA damage were observed in mussels

that were exposed to the combination of metals, studied by Bolognesi et al. (1999). Heavy

metals induced changes in the metabolism of organism and enhanced the reactive oxygen

species which generated oxidative stress and induced DNA damage (Gaetke and Chow,

2003). It is previously mentioned that the metals and nanoparticles have direct effect on

DNA in nucleus and occasionally during cell division which can cause DNA damage (Bhatt

and Tripathi, 2011; Singh et al., 2009; Karlsson, 2010).

Many previous studies had been conducted on freshwater and marine mussels to

assess the genotoxic effect of heavy metals and found deleterious effects. Chromium is the

major industrial discharge that put adverse effects on animal tissues (Wahlberg and Skog,

1965). The large concentration of chromium was observed in aquatic environment and it can

greatly accumulate in tissues of aquatic animals which causes may defects at molecular level

(Eneji et al., 2011; Fatima and Usmani, 2013; Taweel et al., 2011). The sufficient amount of

lead also accumulated in aquatic animals even when its amount may be very less in aquatic

environment (Vinodhini and Narayan, 2008; Abdel-Baki et al., 2011). Lead is noxious even

in very small concentration because lead can mimic other essential elements such as calcium,

magnesium and zinc which put deleterious effects on enzyme activity (Jennette, 1981) and it

was suggested to be carcinogenic (Fracasso et al., 2002). The genotoxic effect of lead on

123

freshwater mussels was also reported by Black et al. (1996). Emmanouil et al. (2007)

reported that the chromium has more adverse effect and it has been found to cause damaging

in DNA strands with the tissue concentration of ≥2.70 μg/g wet weight under laboratory

condition. Chromium has significant correlation with the DNA strand breaks in mussels

collected from the wild condition as described by Rank et al. (2005). Many previous studies

reported the significantly higher level of DNA damaging in aquatic organism collected from

contaminated water with metals (Frenzilli et al., 2001; Nacci et al., 2002; Steinert et al.,

1998).

This is in accordance with our finding, as the DNA damage responses detected with

the Comet assay seem to correlate with the comparative heavy metal concentrations in

surrounding environment. Al-Subiai et al. (2011) reported the similar idea that showed the

effect of copper on the DNA strand breaks of bivalve mussels was found to be more

genotoxic. Induction of DNA damaging has also been reported in numerous other studies

where animals were exposed to different heavy metals such as mercury, cadmium,

chromium, copper and lead (Hartmann and Speit, 1994; Hayashi et al., 2000). Several

previous studies also reported that the 50% or higher level of DNA strand breaks in cells of

aquatic animals including mussels was linked to the different chemicals exposed in

laboratory or in polluted environment (Frenzilli et al., 2001, 2004; Regoli et al., 2005;

Machella et al., 2006; Gorbi et al., 2008). Heavy metals are harmful for aquatic life and in

this study we used the different approach to know the specific effect of metals at the specific

concentrations on sub cellular level of bivalves.

124

8. Conclusions and recommendations

Freshwater mussels (Anodonta anatina) were analysed for bioaccumulation of metals,

proximate composition and mineral elements analysis. Amongst the studied metals, Cu

showed more accumulation than Pb and Cr; the highest metal bioaccumulation was observed

when mussels were exposed to higher doses of metals. Although the bioaccumulation of Cr

was lower than other metals, it was still higher than the control mussels receiving no added

metals in water. Some fluctuations were observed in mineral elements and chemical

composition of mussels following their exposure to different doses of heavy metals. Amongst

the studied mineral elements, Ca was significantly higher in all the tissues; its concentration

was greatly increased after exposure to various doses of Pb, while the values of Na and K

were surprisingly lowered by the exposure to Cu. Protein was found to be greater in the foot

portion than the other tissues. Thus the mussel foot is suggested to be a more nutritious part

for the consumers. Gills, rather than foot and mantle, were the main site for the accumulation

of metals perhaps due to their direct contact with their immediate environment containing

metals.

In our study, the values of nutrients were also investigated in tissue of freshwater mussels

exposed to different doses of metals. The levels of protein were significantly increased in the

Pb, Cu and Cr exposed mussels at the low dose (120 µg/L), and then decreased at the high

doses (360 µg/L) of heavy metals, thus the lower dose may be more effective for protein

formation in mussels. This perhaps is the first study where the interaction between metal and

dietary components is reported. Therefore, there is a need to further explore a synchronized

effect of metal exposure of mussels on body composition reflecting their nutritional value for

125

safe human consumption. Overall, Anodonta anatina seems to be a good candidate for the

assessment of aquatic health. It appears that the tissue specific investigation of metals,

mineral elements and chemical composition is more effective than using the whole animal

for similar investigations in the future.

In the wild experiment, it was concluded that the freshwater mussels especially the Anodonta

anatina from the Chashma Lake was rich source of nutrients. Foot of freshwater mussels was

the best nutritive portion as it had the maximum value of protein among the studied parts.

Most of the macro and micro minerals were found adequately in foot and mantle so

recommended for the human diet. Ca was one of the most prevalent elements in soft tissues

of fresh water mussels as compared to the other metals. The concentration of Pb was slightly

higher than the recommended value that could be the risk to human health. There is need to

sort out the point and non point sources of pollution in Chashma Lake Pakistan. In

biomonitoring study gills found to be the best accumulator part as compared to foot and

mantle so it made the freshwater mussels Anodonta anatina a sentinel animal to assess the

aquatic health. The foot of freshwater mussels (Anodonta anatina) is a good source of

protein, thus this animal could play a vital role to cope the challenge of nutritional deficiency

in developing countries. Furthermore, it is believed that the mussels are potentially a

desirable choice to evaluate aquatic health by assessing metal loads in their soft tissues.

In assessment of genotoxicity, it was observed that the Cu and Pb induced higher

levels of DNA damage as compared to Cr and combined treatments. The value of tail DNA

percentage, tail length and olive tail moment decreased from lower to higher doses of metals

but still the values at high doses were significantly higher than the control except the Cr and

combined treatments which returned to the control level. Furthermore our finding showed

126

that the low dose (120 µg L-1) of metal concentrations have more genotoxic effect as

compared to the medium (240 µg L-1) and high (360 µg L-1) doses. Gills are suggested to be

the best target organ to assess the genotoxic effect of metals and freshwater mussels

(Anodonta anatina) are suggested as one of the key species for bio-monitoring studies.

In present study, freshwater mussels were used due to their ecological and nutritional

importance. These playing a vital role in aquatic environment and said to be the “ecosystem

engineers” because they can transform the aquatic environment, making it more favorable

not only for their own survival but also for the other aquatic organism. In Pakistan, this

imperiled fauna yet not studied well so there is need to explore the ecological role of bivalves

in future studies to make betterment in aquatic environment.

127

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