CHEMICAL, MICROBIOLOGICAL AND TOXICOLOGICAL …prr.hec.gov.pk/jspui/bitstream/123456789/1209/1/2012S.pdf · chemical, microbiological and toxicological screening of tannery effluent

CHEMICAL, MICROBIOLOGICAL AND TOXICOLOGICAL

SCREENING OF TANNERY EFFLUENT WASTEWATER

Lubna Shakir

2007- VA- 03

A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF

THE REQUIREMENT FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

IN

PHARMACOLOGY & TOXICOLOGY

UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES,

LAHORE

2012

To, The Controller of Examinations,

University of Veterinary and Animal Sciences, Lahore.

We, the supervisory committee, certify that the contents and form of the thesis, submitted by

Mrs. Lubna Shakir Regd. No. 2007-VA-03 have been found satisfactory and recommend that

it be processed for the evaluation by the external examination (s) for award of the degree.

Supervisory Committee

----------------------------- Supervisor: Prof. Dr. Muhammad Ashraf

------------------------------ Member: Dr. Aqeel Javeed --------------------------------- Member: Dr. Aftab Ahmad Anjum

IN THE NAME OF ALLAH,

THE COMPASSIONATE, THE MERCIFUL

All Praises and thanks are for

Almighty ALLAH,

The source of all knowledge and wisdom

Endowed to mankind, Who guides us in

Darkness and helps us in difficulties

And all respects are for His last

Holy Prophet

HAZRAT MUHAMMAD

(Peace Be Upon Him)

Who enabled us to recognize our Creator

WISDOM IS THE PART AND PARCEL

OF MY RELIGION,

KNOWLEDGE MY DRESS,

PATIENCE MY WEAPON,

FAITH MY DIET,

AND SINCERITY

MY COMPANION

(HADIS-E-NABVI)

DEDICATION

To my dear parents, husband, sister, brothers

Who dedicated their lives for me

Dearest family members,

I know and understand, you actually gave me

More than one life, my own and yours.

So much of what I have become is just because of you.

I can only show you my extreme appreciation for your

Support by being true to all the ideals and values that

You tried to teach me,

Thank you forever for standing by me.

Today my definition of happiness is being with you

Lubna Shakir

I would like to pay all my praises and humblest thanks to most Gracious, Merciful and

Almighty ALLAH who bestowed me with potential and ability to contribute some material to

the existing knowledge in the field of Pharmacology & Toxicology and made every thing

possible for me to complete my M. Phil leading to Ph D degree. I offer my humblest thanks

from the core of my heart to the HOLY PROPHET HAZRAT MUHAMMAD (P.B.U.H)

who is forever a torch of guidance and knowledge for humanity as a whole.

I deem it as my utmost pleasure to avail this express the heartiest gratitude and deep

sense of obligation to my venerated supervisor Prof. Dr. Muhammad Ashraf, Chairman

Department of Pharmacology & Toxicology, Dr. Aqeel Javeed, Assistant Professor in

department of Pharmacology and Toxicology, and Dr. Aftab Ahmad Anjum, Associate

Professor in department of Microbiology, University of Veterinary and Animal Science,

Lahore. Their skillful guidance, unfailing patience, masterly advice and inspiring attitude made

it very easy to undertake this work and to write this manuscript.

I have the honor to express my deep sense of gratitude and profound indebtedness to Dr

Sohail Ejaz, Post-doctroral Research Scientist in department of Clinical Neurosciences,

Neurology, Unit, Addenbrookes Hospital, University of Cambridge, UK for his generous

guidance, expert advice and skillful suggestions during the entire course of my study.

I am grateful to Dr. Nisar Ahmad, SI Centre of Advance Studies in Physics,

Government College University, Lahore who granted me the permission to work in

Accelerator Laboratory and supported my work a lot, Dr. Noureen Aziz Qureshi, Dean

faculty of Fisheries and Wildlife, University of Veterinary and Animal Sciences, Lahore for

her kind assistance throughout the study and Dr. Imran Altaf, Dr. Ovais Omer for his great

support. In addition, I want to say deep thanks to Badir Munir, Assistant Controller

Examination who guided and supported me a lot. Special thanks and deep love are extended to

my friends, for their lovely company and help.

Indigenous PhD 5000 Fellowship Program of HEC (Higher Education Commission)

of Pakistan is an enormous support for postgraduate studies and I was able to win this HEC

indigenous scholarship under batch IV in 2007. I am really thankful to HEC for providing me

financial support throughout my Ph D studies. Without HEC help it was unfeasible for me to

complete my research work in time. I also appreciate their supported for visiting University of

Cambridge UK under International Research Support Initiative Program.

At the end, it is customary to say that all errors and omissions are of me alone.

LUBNA SHAKIR

[email protected]

i

TABLE OF CONTENTS

S. No. Chapter Page No.

01. Introduction 01-10

02. Review of Literature 11-58

03. Materials and Methods 59-78

04. Results 79-143

05. Discussion 144-168

06. Summary 169-170

07. Literature Cited 171-202

08. Annexures 203-210

ii

LIST OF FIGURES

Figure No.

Title of Figure Page No.

01. Flow chart describing steps of tanning operations.

60

02. Map (Kasur tannery area) indicating the locations of sample collection. Black dots are pointing the location of domestic tanneries and shallow tubewells while red dented circles are indicating the position of deep tubewells.

62

03. PIXE spectrum of tannery effluent wastewater (TEW9).

82

04. Graph (A) presenting detected concentration of various elements and dendogram (B) illustrating the percentage similarities between all TEW samples.

84

05. PIXE spectrum of ground water of shallow tubewell (GWS7).

86

06. Graph (A) presenting detected levels of various elements and dendogram (B) illustrating the percentage similarities between all GWS samples.

88

07. PIXE spectrum of ground water of deep tubewell (GWD1).

89

08.

Graph (A) presenting detected concentration of various elements and dendogram (B) illustrating the percentage similarities between all GWD samples.

91

09.

Dendogram illustrating the relationship between all the three types of water samples (TEW, GWS and GWD).

92

10. Graphical percentage representation of detected bacterial isolates.

104

11.

Graphical outline demonstrating comparison among diameter of blood vessels in different groups.

124

12. Graph illustrating comparison between different roughness 124

iii

parameters of all groups.

13.

The 1, 24, 48 and 72 hrs mortality charts of marine shrimps. Marine shrimps have followed concentration and time dependent mortality.

132

14.

Graph showing the inhibition of root elongation of maize seeds.

134

15.

Comparative weight gain in Wistar rats of all groups (A) and vital organs (B) at the end of three months.

136

iv

LIST OF PLATES

Plate No.

Title Page No.

01. Large pool of tannery effluent wastewater (A), samples

collected from tannery area (B) and hand pump located near pool of stagnant tannery water (C).

63

02. Tannery effluent wastewater samples before preconcentration (A), dried residue after evaporating at 70oC and air dried smear of TEW (C).

64

03. Pelletron Accelerator 6SBH located at CASP, GCU Lahore. 65

04. Pictorial presentation illustrating detailed procedure of CAM assay.

72

05. Hatching setup for marine shrimps.

75

06. Rats receiving TEWD1. 78

07.

Plates having different colonies of viable bacteria in serial dilutions (A=10-1, B=10-2, C=10-3, D=10-4, E=10-5, F=10-6) of collected tannery effluent wastewater sample.

95

08.

Two way streaking (for purification) of bacterial culture. Single isolated colony can be seen as pointed by arrows.

96

09.

Microscopic representations of Gram positive bacillus at 100X.

97

10.

Representative of Lactose fermenting bacteria (A) and Lactose non fermenting bacteria (B) on MacConkey’s Agar.

98

11.

Interpretation of the results for Indole production test using Tryptophan Broth (A:+ve and B: -ve).

99

12.

Interpretation of the results for VP test using Glucose Phosphate Buffered Saline (A:+ve and B: -ve).

100

v

13.

Interpretation of the results for MR-test using Glucose Phosphate Buffered Saline (A:+ve and B: -ve).

100

14.

Interpretation of the results for Citrate utilization test using Simmon Citrate Agar (A:+ve and B: -ve).

101

15.

Interpretation of the results for Urease production test using Urea Broth (A:+ve and B: -ve).

101

16.

Interpretation of the results for H2S production test using TSI Agar slants (A:+ve and B: -ve).

102

17.

Representative identification plates for Bacillus azotoformans.

103

18.

Plates presenting the tolerance levels of Bacillus subtilis for increasing concentration of chromium sulphate.

108

19.

Macroscopic evaluation of chicken CAM on day 6 of incubation.

111

20.

Topographic explanation of CAMs depicting variations in blood vessel branching pattern.

113

21.

Colored convoluted topographical images of CAMs. 115

22.

3D micrographs, illustrating surface activities of CAMs in different groups.

116

23.

Abbott curve measurement’s graphical outline on CAMs of different groups.

118

24.

Angular spectrum’s graphical outlines of CAMs of different groups.

119

25.

Graphical representation of dimensions of blood vessels on CAMs of different groups.

120

26. Scan probing image processing software based measurement of the diameter of blood vessels on CAM.

122

27. Microphotographs showing variable capillary plexus formation in CAMs of different treatment groups.

127

vi

28. Severe macroscopic/gross lesions observed in chicken embryos.

129

29. Microscopic view of hatched marine shrimps.

130

30. Zero germination observed in the D1 of TEW (A), PDC (B) and CHC (C).

133

31. Lung microphotographs of rats exposed to TWED1 (A), TEWD2 (B), TEWD3 (C), CHC (D), PDC (E) and Control (F).

139

32. Liver microphotographs of rats exposed to TWED1 (A), TEWD2 (B), TEWD3 (C), CHC (D), PDC (E) and Control (F).

140

33. Kidney microphotographs of rats exposed to TWED1 (A), TEWD2 (B), TEWD3 (C), CHC (D), PDC (E) and Control (F).

141

34. Heart microphotographs of rats exposed to TWED1 (A), TEWD2 (B), TEWD3 (C), CHC (D), PDC (E) and Control (F).

142

35. Brain microphotographs of rats exposed to TWED1 (A), TEWD2 (B), TEWD3 (C), CHC (D), PDC (E) and Control (F).

143

vii

LIST OF TABLES

Table No.

Title Page No.

01. A brief description of different groups with respective

treatments.

71

02. PIXE results for standard sample (IAEA soil 07). 80

03. Detected concentrations of various elements present in the tannery effluent wastewater samples.

83

04. Detected concentrations of various elements present in the ground water samples of shallow tubewells.

87

05. Detected concentration of various elements present in the ground water of deep tubewells.

90

06.

Total number of viable bacteria in different tannery effluent wastewater samples.

94

07.

Total number and types of isolated and identified bacterial isolates.

105-106

08.

Table presenting the tolerance levels of isolated bacteria for various salts of chromium

109

09.

Table presenting the percentage mortality with respective treatments.

128

10. Table presenting the weight gain by rats.

135

viii

LIST OF ANNEXURES

Annexure

No. Title Page

No.

1. Blood Agar

203

2. Nutrient Agar

204

3. Crystal violet

205

4. Gram’s Iodine

205

5. Safranine

206

6. MacConkey’s Agar

206

7. Tryptophan Broth (For indole production test)

207

8.

Kovac's Reagent 207

9.

Glucose Phosphate Buffered Saline (For MR and VP tests) 208

10.

Voges-Proskauer Reagents 209

11.

Simmons Citrate Agar 210

ix

LIST OF ABBREVIATIONS

S. No. Abbreviation Description

1. BV Blood Vessel

2. CAM

Chorio Allantoic Memberane

3. CHC Chromium Chloride

4. GWD Ground Water of Deep tubewell

5. GWS

Ground Water of Shallow tubewell

6. PBS

Phosphate Buffer Saline

7. PIXE

Proton Induced X ray Emission

8.

PDC Potassium Dichromate

9.

TEW

Tannery Effluent Wastewater

10.

TEWD1

First dilution of Tannery Effluent Wastewater

11.

TEWD1

Second dilution of Tannery Effluent Wastewater

12.

TEWD3

Third dilution of Tannery Effluent Wastewater

13.

CPs Capillary Plexuses

14.

Cr (III) Trivalent chromium

15. Cr (IV) Hexavalent chromium

Introduction

1

The introduction of contaminants or chemical compounds in to the environment

by human that endanger health, cause scathe to the living resources and ecological

systems is referred to as pollution. The number and level of hazardous substances such as

agrochemicals, environmental pollutants and sewage wastes have dramatically increased

in the recent years (Maduka and Hugh 2006). Water pollution is the direct or indirect

addition of tainting molecules into the pure water resources, which make the water

incongruous for drinking, bathing and general use (April et al 2003). Strikingly, water

pollution linked illness generated million dollars oppress on public per year (Dwight et al

2005). Continuous irrigation of agricultural land with polluted water is increasing

sodicity and salinity of prolific land (Alvarez et al 2006). The risk of communicable

diseases and infections is massive riddle with the use of industrial wastewater

(Varadarajan et al 1991).

Tanning process transmutes the animal skins into staunch and non decomposable

leather. Ammonium (ammonium sulfate/ammonium chloride), chloride (sodium

chloride), chromium (chromium sulphate) and sulfide (chromium sulphate) salts are

commonly used for this course of action. Besides these, natural and/or synthetic biocides

(Casacide T100/Casacide T300), tannins, metalorganic dyes, sulfonated oils, surfactants

CHAPTER-1

INTRODUCTION

Introduction

2

(sodium dodecylbenzenesulfonate and dodecyl trimethylammonium bromide), acrylic

resins are being utilized too (Costa et al 2008). In Pakistan, Chrome (chromium sulphate

and Namak (sodium chloride) are mainly applied for tanning purpose.

For last few decades, a brisk ascension in leather yield was seen in developing

countries (Sajjad et al 2008). Semi-finished leather’s growing demand by world market

has urged an increase in the number of tanneries in Pakistan. As per report of Ministry of

Industries and Production, around 650 registered leather industries dwell in whole

country (Naseem et al 2007) while approximately 223 tanneries are operational in Kasur.

“Kasur district” which is located 55 Km southeast of Lahore city approaching the

Indian border, is well known for its tannery industry. About half million people rely on

tannery industry for their earnings. Strikingly, around 9,000 cubic meters of the effluent

waste is being discharged on daily basis, which is chief source of water pollution in this

area.

Tanneries are a major source of effluent wastewaters and solid wastes (Costa et al

2008). Wastewater of unique and complex composition is being released by them

because they have marked variations (based upon type of raw chemicals, their quantity

used for tanning and animal skin type) in their production methodologies (Sajjad et al

2008). Apart from this, different tannery effluents come on different times because

tanning procedures are run in a batch form. Effluent runoffs have high levels of

Introduction

3

chromium, organic matter, solids, sulfates and sulfides (Sajjad et al 2008). Furthermore,

it contains Cr, Ca, Cd, Co, Fe, Ni, K, Mg, Mn, Pb and Zn (Tariq et al 2005).

It was observed that local residents are being seriously affected from these parlous

chemicals and suffer occupational diseases such as asthma, dermatological problems and

ulcers (Srinivasa et al 2008). Chromium based tanning is one of the most common

methods for hide processings. Solid and liquid wastes produced during chromium tanning

contain almost 30 to 40% of the chromium, which is a major contributor to

environmental pollution (Abass et al 2005).

Naturally, chromium exists in many oxidation states (ranging from +2 to +6). The

trivalent chromium (Cr III) is considered essential trace element whereas hexavalent

chromium (Cr VI) is non vital, corrosive, toxic and results in dermatitis, allergic

reactions, exasperation of gastrointestinal tract, ulcers (Laborda et al 1986),

neurotoxicity, genotoxicity, immunotoxicity (Hesham et al 2010) and has carcinogenic

potential (Tagliari et al 2004). High levels of Cl are a possible cause of hyperchloremic

acidosis (Eisenhut 2006), disproportion in immune reactions and impaired

neurobehavioral functions (Kilburn 2009). The Cl along with Na and K is a well known

cause of hypertension (Jia et al 2007; Kim et al 2007), vascular lesions (Jia et al 2007;

Wang et al 2006) and kidney failure. While high amount of K may give rise to

hypertension (Braschi and Naismith 2008), lung injury, cardiovascular disease (Cook et

al 2008), reduction in urinary albumin (He et al 2010), renal damage and interruptions of

consciousness. The extended Fe surplus produces cardiac disease, diabetes, cirrhosis,

Introduction

4

hepatic fibrosis and hepatocellular cancer (Hershko et al 1998). Likewise, Ni is a

potential neurotoxic pollutant (Xu et al 2010) associated with lymphocyte toxicity (Chen

et al 2003). Increased quantity of Si has been accounted for a cause of skin irritation,

transient eye irritation, corneal endothelial amendments and retinal toxicity (Green et al

1994).

Current research project was undertaken for assessment of toxic burden of tannery

effluent wastewater (TEW). Various methods have been used for the chemical

characterization of TEW such as procedures described in Standard Methods for the

Examination of Water and Wastewaters was used for characterization of TEW by Sajjad

et al (2008). In addition, Jar test method can also be used too (Esmaeili et al 2005).

Sequential solid phase extraction followed by high performance liquid chromatography

fractionation/automated multiple development thin layer chromatography fractionation

was utilized. Then finally collected toxic fractions were determined by gas

chromatography mass spectroscopy (Reemtsma et al 1999).

Chromium content can be evaluated calorimetrically (by utilizing the

diphenylcarbazide detection method) with spectrophotometer (Hesham et al 2010).

Besides this, a simple extractive separation method based on the extraction of chromium

(as its ionpair with tribenzylamine) was used by Kalidhasan et al (2009). While high

temperature molecular absorption spectrometry can be used for the quantification of Cl in

water samples (Parvinen and Lajunen 1999). For the analyzing sulphate and sulphite, a

simple ion chromatographic method was devised (using an ODS column dynamically

Introduction

5

coated with0.5 mmol 1−1 phthalate 0.01% triethanolamine 5% methanol and

cethylpyridinium bromide) (Michigami et al 1996).

My entire research project was divided into four major phases. Phase I has been

designed for the characterization of TEW of Kasur using accelerator based analytical

method (PIXE analysis). Ground water of shallow tubewells (GWS) was analyzed along

with TEW samples of domestic tanneries. Likewise, ground water of deep tubewells

(GWD) was also scrutinized for the presence of toxic elements. The working principle of

PIXE (proton induced X ray emission) analysis is that, protons (2-3 MeV proton beam)

from an electrostatic accelerator are used to bombard the sample under investigation and

the characteristic X-rays emitted by the elements which constitute that sample are

analyzed by an energy dispersive solid state Si-Li detector. The method followed for

PIXE analysis of water samples is: first each water sample was evaporated till dried

residue was obtained. Then a smear was made from the dried residue using Yuttrium

Nitrate Solution, afterwards it was air dried and irradiated. In the end, concentrations of

heavy metals were measured from obtained spectrum (Johansson and John 1988). We

have preferred PIXE analysis over atomic absorption spectroscopy because of its

numerous advantages: as it has high level of sensitivity (detection limit as low as 20

µg/L), fastness (takes about 10-25 minutes), nondestructive nature, multi-elemental

analysis competence and little sample preparation requirements.

High levels of chromate in the environment have an inhibitory effect on most

microorganisms. In response to its exposure, microorganisms resistant to antibiotics and

Introduction

6

tolerant to metals appear. Such microbial resistance to metal ions and antibiotics is a

potential health hazard as such traits are frequently associated with transmissible

plasmids (Verma et al 2001). Apart from this, potential of several chromate reducing

bacteria to detoxify hexavalent chromium has also been suggested (Shukla et al 2007).

So, I have assessed the heavy metal tolerant status of those microorganisms which reside

in the effluent wastewater of Kasur tanneries. Microbes present in TEW were counted

through viable count method, isolated through sub-streaking and then identified

following Bergy’s Manual of Determinative Bacteriology (Krieg et al 1984). In addition,

tolerance level of isolated bacteria was determined against various chromium compounds

such as chromium sulphate, chromium chloride, chromium oxide and potassium

dichromate (Basu et al 1997).

In addition, I have appraised the dicey burden of TEW on various bioassays too,

as a full view picture of pollutant’s bioavailability can be drawn through bioassays.

Moreover, a highly catholic understanding of joint toxic hazards of harmful chemicals

can be acquired. Laboratory methods based on the use of different experimental models

(whole organism) can over generalize obtained results for estimating the level of exposed

risks (Arias-Barreiro et al 2010).

For development, responding to ischemia, reproduction and wound repairing

angiogenesis (development of new blood vessels) is an integral physiological process (Li

et al 2000). Pathological angiogenesis (neovascularization) is associated with disease

conditions such as arthritis, cancer, psoriasis and retinopathies (Folkman, 1995). There

Introduction

7

are many methods for evaluating angiogenesis (chicken chorioallantoic membrane assay,

cell proliferation and chemotatic assay, cornea pocket assay, coculture angiogenesis

assay, matrigel subcutaneous air sac model, embryoid body angiogenesis assay, plug

assay, micro carrier based angiogenesis assay, leech angiogenesis assay, rat aortic ring

model, miniature ring supported assay, rodent mesenteric window angiogenesis assay,

placental fragment assay and tube formation angiogenesis assay). The chick embryo’s

chorio allantoic membrane (CAM) is an extra embryonic membrane which work as a gas

exchange exterior and its function is assisted by a profound capillary network. Because of

its extensive vascularization and serene accessibility, CAM has been vastly utilized for

studying the morpho-functional aspects of the angiogenesis (in vivo) and for

investigating the mechanism of action and efficacy of antiangiogenic and proangiogenic

natural and synthetic substances. CAM assay is a worthwhile model for assessment of

angiogenesis and vasculogenesis (Ribatti et al 2001). In third phase of my study, I have

screened hazardous load of three dilutions of TEW (TEW1, TEW2 and TEW3), solutions

of chromium chloride (CHC), potassium dichromate (PDC) and control (PBS, phosphate

buffer saline) on angiogenesis through CAM assay. I have also assessed the acute risk of

TEW to developing embryos. For conducting CAM and embryotoxicity assay, albumin

was removed first and sample solution was dispensed on developing CAM and then

sealed with sterile parafilm. Following 24 hrs incubation, CAMs and embryos were

separated, macroscopically and microscopically evaluated for assessment of any changes

in their development (Ejaz et al 2006).

Introduction

8

For the estimation of acute toxicity profile of TEW, I have used marine shrimps

mortality assay (Artemia bioassay) which was first time proposed by Michael et al. and

later on developed by Vanhaecke et al. and Sleet and Brendel. Now it is a valuable tool

for precursory assessment of toxicity (Carballo et al 2002). I have followed method

described by Tzong-huei et al (1999). Larvae within one day of hatching were exposed to

five dilutions of TEW (D1 to D5), CHC, PDC and control. At the end of specified

incubation period, the number of dead shrimps was recorded. In past, marine shrimps

have been largely utilized in research for the investigation of sources of toxicity (in

chemical mixtures) and environmental samples such as cyanobacteria toxins, fungal

toxins, pesticides and plant extracts (Carballo et al 2002; Kokkali et al 2011; Persoone

and Wells 1987). Marine shrimps have been preferred as they could be cultured under

laboratory conditions more easily, have a small body size, short life cycle and found

receptive to a wide range of pollutants (Bu-Olayan and Thomas 2006). Additionally,

marine shrimps are continuously available around the year in the form of dry cysts, cost

effective, have no feeding requirements during the assay and large offspring production

(Hadjispyrous et al 2001; Kokkali et al 2011). These inherent features have turned them

into an appropriate organism for use in toxicology studies (Nunes et al 2006).

Phytotoxicity assay (root elongation inhibition test) generally uses toxicological

endpoints (such as percentage of seed germination and seedling growth). In terrestrial and

aquatic ecosystems, the US EPA has recommended several plants as biomarkers for

toxicity assessment. The maize is one of them (Lopez-Luna et al 2009). The use of maize

plants to evaluate toxicity provide enough information for a plant (requirements) to

Introduction

9

develop under unpropitious conditions (Calheiros et al 2008). TEW has been reported for

causing the reduction in Cicer arietinum germination and growth. It has also caused the

diminution in chlorophyll synthesis of sugar beet (Javaid et al 2000). In addition,

excessive amount of chromium can also cause the plant growth blockage such as it leads

to poor root development and stunted shoot growth. Furthermore, it can also cause leaves

chlorosis, diminished photosynthesis, decreases enzyme activity, membrane damage,

tissue necrosis and changing of chloroplast (Jun et al 2009). In current research project, I

have estimated the toxic outcomes of TEW on maize’s root elongation. Javaid et al.

(2000) was followed for the assessment of TEW, CHC and PDC impacts on maize root

growth. Total ten maize seeds were sown in each petriplate and after five days, the length

of roots of germinated seeds was measured. Inhibition rate of root length was expressed

as percentage inhibition.

In the last phase of my project, I have explored deleterious effects of TEW after

three months chronic exposure (ad libitum) to Wistar rats. At the end of particular time

length (three months), vital organs (lung, liver, kidney, heart and brain) were carefully

removed, weighed and processed for histological investigation and were stained with

haematoxylin eosin (Silva et al 2006). Although chromium is known to be essential for

the growth as microelements (in traces) but intakes of higher concentrations is toxic,

particularly to the liver and kidney of experimental animals (Barthwal and Kummar

1991).

Introduction

10

Aims & Objectives:

1. Characterization of the tannery effluent wastewater (TEW), ground water of

shallow tubewells and ground water of deep tubewells using accelerator based

analytical method (PIXE analysis).

2. Isolation and identification of bacteria from TEW. Determination of tolerance

level of isolated bacteria for various salts of chromium.

3. Acute toxicity profiling of TEW through CAM, embryotoxicity, marine shrimps

mortality and phytotoxicity assay.

4. Assessment of chronic toxicity torment of TEW samples.

Review of literature

11

2.1. Chemical Analysis of Tannery Effluent Wastewater

Various techniques have been established in past for the characterization of

tannery effluent wastewater (TEW). Chromium, chloride, phosphate, nitrate, nitrite,

ammonia, sulphide and sulfate have been quantified in TEW by Cooman et al. (2003)

according to standard chemical methodologies. The detected levels are 230-35,200 mg/L

for sulphate, 1813-16,500 mg/L for chloride and 8600-87,100/L for total solids.

Similarly, sulphate and chlorides were analyzed by Krishanamoorthi (2009) and the

detected levels were: chloride (5000-6000 mg/L), chromium (80-100 mg/L) and sulphate

(1800-2000 mg/L). Respirometric techniques and an activated sludge model have also

been used for the analysis of TEW. The traditional respirometric tests have been further

modified based on specific operating conditions, solid liquid separation technology and

the complexity of wastewater (Munz et al 2008).

For the quantification of chromium in TEW, a simple extractive separation

method has been developed (based on the extraction of hexavalent chromium as its ion

pair with tribenzylamine). While chromium concentration in the organic phase was

measured spectrophotometrically (Kalidhasan et al 2009). Large chromium contents

CHAPTER-2

REVIEW OF LITERATURE


12

(with an average value of 68 mg/L) and sulfate and sulfide concentrations were found

(with an average value of 1240 and 156 mg/L) in TEW of Saddiq Leather Work (Haydar

and Aziz 2009).

The objective of first phase of my research project was the evaluation of

composition of water samples. For this purpose, I have utilized PIXE (Proton Induced X

ray Emission) analysis technique. For PIXE analysis, a target was placed in the way of a

proton beam (produced by 3 MeV Van de Graaff accelerator). Thus, atoms of the target

get excited and emitted characteristic X rays which were then captured by solid state

detector. Afterward X rays were counted by a pulse height analyzer and thus an energy

spectrum was obtained. There was little impedance from bremsstrahlung and X ray

production cross section is larger for PIXE analysis as compared to conventional X ray

fluorescence spectroscopy (Lochmuller et al 1974). Raith et al. (1977) have applied PIXE

to environmental problems using thin targets. Most of work was done in the area of water

pollution. For trace element analysis, PIXE has turned out as a useful tool. Comparatively

thin target analysis was done much more easily. Broad range of the PIXE technique and

its praiseworthy sensitivity has made it an attractive method for the multi element

analysis of water (Rickey et al 1979).

In 1981, highly sensitive PIXE technique has been utilized for large scale

monitoring of household tap water (for trace elements) and lead contamination was

observed (Fou 1981). For the analysis of rainwater (from Tallahassee), a PIXE based

procedure has been developed and tested too. Results unveiled that insoluble fraction


13

contained Al, Cr, Si and Ti while soluble portion has Ca, Cr, Cu, K, Ni, S and Zn. But Pb

and Fe were present among the both fractions. The surface water samples from rainfall

runoff of two shopping centers and a lake (Tallahassee area) have demonstrated similar

partitioning of elements among the two fractions (Tanka et al 1981). Later on, method for

utilizing PIXE along with the procedure of spotting for quantitative and qualitative

multielemental analysis of drinking water (in eight cities of Jordan) was discussed by

Saleh (1982). Twenty metals were detected in finest fraction of suspended matter in

stream water when analyzed with PIXE (Carserud 1983).

For enhancing PIXE sensitivity, a preconcentration method has been contrived in

1984 for metal analysis in of seawater. A complexing agent was used for the extraction of

trace elements and then extracted ones were re-dissolved in a small volume of nitric acid.

This acidic solution (about a drop) was placed on much thin carbon foil and evaporated

then. Mostly for elements heavier than Ti, the detection limit of about 1ppt was achieved

with this carbon foil which gave virtually no background (Eva-Marta and Sven 1984).

Savage et al. (1995) have used dried algae as a novel preconcentration methodology for

characterization of water samples by PIXE. Linear responses were observed for Ag, Ba

and Cd (10 ng/g - 1 µg/g), Cu and Pb (10 ng/g - 5 µg/g) and Hg (10 ng/g -10 µg/g) when

C. uulgaris was used on mixed metal solutions. But when S. bacilluris was used, linear

responses were obtained for all of the metal cations (10 ng/g - 10 µg/g). Another

preconcentration technique, based on use of nebulizer was devised within this year.

Water was sprayed to a droplet aerosol through use of nebulizer and then air dried. The

dry aerosol molecules having diameter of about 1 pm were made deposit on a thin


14

polystyrene foil (in multijet impactor). For this proposed setup, best detection limits of

about 0.1 ppb were achieved for 10 ml sample (Hans-Christen et al 1984). About 250 ml

municipal water (Dhaka, city) has been mixed with 200 mg of cellulose powder, then

evaporated on steam bath in a platinum dish. Standard pellets were made from residues

and were excited by 2.5 MeV beams from accelerator. Current methodology has made

possible the quantitative analysis of (10 to 15 different elements) Br, Ca, Cr, K, Ti, Mn,

Fe, Ni, Cu, Sr, Pb, Zn etc (Ali et al 1985). 1n 1986, one more new preconcentration

technology based on the extraction of the metals as carbamates for determination of metal

concentrations in natural water has been utilized by Cecchi et al (1986). The treatment of

large sample volumes has been tested for checking the capability of this methodology in

low concentrations for PIXE measurements. During the analysis procedure of solutions

containing 1000, 500, 100, 50 and 10 ppt of Co, Cd, Hg, Cu, Fe, Ni, and Pb as internal

standard, a linear behavior (of experimental result versus the nominal concentration) have

been observed. Rainwater was dried onto polystyrene films for preconcentration. The

concentrations of As, Br, Ca, K, Fe, Mn, Ni, P, Pb, S, V and Zn were considerably

greater than the cheniical blank and detection limits. Thus PIXE analysis could be used

routinely. But significant meliorations (in detection limits) can be achieved by increasing

analysis time and decreasing the contents of the blank. In addition, PIXE technique

proved auspicious for the other elements such as Co, Cr, Cu, Ga, Ge, Rb, Ti, Se and Sr

(Hans-Christen et al 1988).

The values much higher than those referred to be in bibliography for oceanic

water were obtained for the seawater of the subtidal zone (between Leirosa and Pedrogao,


15

on west coast of Portugal) through PIXE analysis (Costa et al 1988). About fifty two

elements were discerned in drinkable water through the use of PIXE. A methodology for

the quantification of Al was also developed on the basis of an appropriate combination of

PIXE and Instrumental Neutron Activation Analysis technologies (Blasi et al 1990).

Trace metal levels in water and sediment samples (collected in the Venice Lagoon,

northern Italy) have been determined by PIXE for the investigation of metal distribution

mechanisms and pollution progress. Besides this, metal enrichment in suspended

particulates has been evaluated too (along the stream of an inflowing river and at its

mouth) for studying effect of water mixing and retarded hydrodynamics (Ghermandi et al

1991). Anoxic waters of a soft water lake were filtered through 1.2 pm pore size filters

and collected black particles were characterized by a scanning proton microprobe which

quantitatively analyzed element through PIXE and Rutherford backscattering. A uniform

distribution of sulfur across the filter was noticed and Mn was localized in 5 pm diameter

clusters to a smaller extent (Davison et al 1992). Heavy water samples (from the

moderator of a power reactor) and unused heavy water have been evaluated by using

heavy ion beams of PIXE. Cu, Fe and Zn in variable concentrations were detected in both

types of samples. While only moderator samples have Tc and Xe. A very high sensitivity

below the ppb limit was achieved (Ozafrh et al 1992).

Concentration of trace elements in water (Chaliyar River) has been analyzed

through PIXE analysis. During summer and rainy seasons, samples were collected from

various sites along the course of the Chaliyar (Malabar, India). Preconcentration

methodology has been used during preparation of samples. The 2 MeV protons were


16

utilized for the analysis purposes (Kennedya et al 1998). Next year, especially for

measuring heavy metals in same water Kennedy et al. (1999) have again used PIXE.

During summer and rainy seasons, samples were collected from three different depths.

Now, operational conditions were 2 MeV proton beam using a 3 MV tandem pelletron

accelerator. Concentrations of heavy metals like Hg, Pb and Zn were more than the

prescribed limits because of industrial wastes pollution.

In 2000, Nishiyama et al. (2000) have investigated preparation methods of sample

supporting film (backing foil) for PIXE analysis of natural water. New apparatus for

making a polyvinylformal film on a quartz glass plate was evolved. Then a water drop

was dried on that film as target for analysis. Moreover, measuring conditions were also

optimized. Then few natural water samples (mineral water, water for sake brewing and

rain water) were analyzed. Minimum detection limit less than 0.1 ng/ml for Z < 20 and

less than 0.05 ng/ml for Z > 20 were achieved. Drinking water samples which have been

collected (in 1999) from nineteen points located in the western part of China have been

screened through PIXE technique. Total hardness (permanent hardness and temporary

hardness) were quantified with determined Ca and Mg levels (Matsuda et al 2001). For

the purpose of microanalysis of selenium in water, PIXE analytical method was studied.

Polycarbonate was concluded to be the best backing material for dropping samples as it

had littlest level of background. The proposed method was found sufficiently applicable

to 0.01 mg/L, which was standard for wastewater from business firms. The PIXE

analytical method was found to be the most germane for the examination of many

samples in a short time, because it could measure a sample in about one hour (Takeshi et


17

al 2003). Contamination of river water and sediment samples have been assessed by

PIXE too. In every spring season since 1998, river water and sediment samples were

continuously collected. The elemental contents (Ca, Cr, Cl, K, Mn, Fe, Pb and Zn) in

river water gradually declined with the rising tide as evident from PIXE spectra (Zhang et

al 2003).

For surveillance of As levels in river basin, an embellished sample preparation

method for PIXE study was devised. Dissolved As was oxidized to pentavalent state

(with permanganate ions) and was then adsorbed by ferric hydroxide colloids. Later on

these colloids adsorbed As ions, were collected on Nuclepore filter (0.2 µm) and were

irradiated for 5 to 10 minutes by 3 MeV proton beam having 0.7 to 2 nA beam currents.

For As, 0.3 ppb detection limit was attained (Yamazaki et al 2004). Sao Francisco river

water was examined through PIXE analysis for determing its trace element composition.

Apart from this, worthy information about the levels of metallic ions pollutants has been

collected too. Monthly, from five locations along the course of river (from July 2003 to

April 2004) water samples were collected. Yttrium (11.6 mg/L) was added to 10 ml of

the water as an internal standard. The PIXE analysis was done at the Materials

Laboratory by using a proton beam. For collection of spectra, Si (Li) detectors were used.

Up to fifteen elements have been quantified due to the excellent detection limits of the

PIXE. The highest total content of As, Cu, Cr and Zn in river water were far above the

recommended limits of the environmental legislation (Espinoza-Quinones et al 2005).

Similarly, water samples (of Boroo River) have been examined which were collected

from the mining area of Mongolia. Preconcentration sample preparation method


18

comprised of following steps: samples has been treated to form metal

dibenzyldithiocarbamate complexes, collected on nuclepore tracketch membrane filter

and irradiated by 2.5 MeV proton beam (Oyuntsetseg et al 2006). While Kennedy et al.

(2007) have utilized PIXE technique for the evaluation of mean elemental levels (of

various size ratios in particulate phase) of the urban storm water (derived from areas with

different land use characteristics). Considerably elevated heavy metal contents were

noted (for samples collected from commercial land and industrial areas).

PIXE analysis was preferred for measuring heavy and light element (ranging from

Al to Pb) concentrations in various polluted and unpolluted liquid samples as well as soils

(collected from different phosphate factory sewers). The impression of wastes of

phosphate industry wastes on concentrations of both radioactive and non radioactive

elements of samples was also investigated in El Jadida Safi Atlantic coastal zone,

Morocco (Erramli et al 2008).

In 2010, Donghui et al. (2010) have studied sampling behavior of multielements

in the stream sediment matrix (with sample sizes in a range of 9 orders of magnitude) by

a combination of PIXE, INAA (Instrumental Neutron Activation Analysis) and SR

XRF(Synchrotron Radiation excited X-ray fluorescence analysis). For sample sizes that

cannot be accurately weighed (< 1 mg), PIXE and SR XRF were used and the effectual

sample sizes were estimated.


19

For enhancing analytical sensitivity of PIXE technology, water samples (treated

tap water, untreated ground water and natural mineral water) were preconcentrated

through evaporation (at 50°C under atmospheric pressure). A 2.0 MeV proton beam was

used for exciting dry dissolved solids. For drinking water quality, results were within the

permissible limits (reported by Iraqi standards, WHO guidelines and European

standards). While levels of Al, As, Br, Ca, Cl, Cr, Fe, K, Mn, Rb, Si, Sr, V and Zn have

presented marked variation from one city to other depending on the geographical

locations (Al- Bedri 2010). Recently, Thomyasirigul et al. (2011) have developed a

sample preparation method for the quantification of chromium in water through PIXE.

The proposed methodology has allowed the separation and determination of chromium

with a significant accuracy and precision.

From literature review, it is evident that PIXE analysis has not been yet used for

characterization of TEW samples, so I have selected this technique for analysis of water

samples (TEW, GWS and GWD). Tannery industries are mostly operational in

developing countries. Pakistan has more than 650 tanneries (Naseem et al 2007). In India

more than 3000 tanneries are present and the tannery clusters are mainly positioned in

four states of India (West Bengal, Uttar Pradesh, Tamilnadu and Punjab).While

Hazaribagh tannery area (having about 90% of the total tanneries) at the southeast part of

Dhaka, is the largest tanning area of Bangladesh. In China, the major tannery industry

areas are Zhejiang, Hebei, Shandong and Guangdong. About 65 tanneries are operational

in Mashhad (Iran). In Turkey, 1300 are dispersed over Tuzla (İstanbul), Menemen

(İzmir), Manisa, Bursa, Uşak, Gönen (Balıkesir) and Çorlu (Tekirdağ) while medium


20

firms are found in Çanakkale, Denizli, Isparta and Niğde. More than 300 tanneries exist

in Egypt (Hesham et al 2010).

The short description of the method applied for PIXE analysis of collected water

samples (TEW, GWS and GWD) of current research project is described as follows:

Total 100 ml of each water sample was separately evaporated till dried residue was

obtained. Then from dried residue, a smear was made with Yuttrium Nitrate Solution, air

dried and irradiated in target holder. From the obtained spectrum of each sample,

concentrations of heavy metals were obtained through use of GUPIX software. Johansson

and John (1988) method has been used as reference method. The advantages of PIXE

which make it valuable include: (1) PIXE is highly sensitive technique with the detection

limit as low as 20 µg/L. (2) It is non-destructive technique because analyzed samples are

not damaged. (3) It yields multielemntal analysis with atomic number greater than 10. (4)

Very little preparation is needed by PIXE for most of the samples and any kind of object

under analysis. (5) The size of object varies from a single cell to a large painting. (6) It is

fast analytical technique (takes about 10 to 25 minutes). On the opposite side, the PIXE

analysis is costly technique because of running and maintenance cost. It is run under

strict supervision of highly trained staff.

2.2. Microbial Evaluation of TEW

Along with the chemical characterization of TEW, I have also enumerated the

microbiological profile of TEW during the 2nd phase of my research project. During last


21

decades, many scientists have isolated microorganisms from the TEW, such as methane

producing bacteria, Methanogenium bourgense (irregular coccoid, nonmotile and used

H2CO2 and formate as methanogenic substrate) was isolated from a tannery byproducts

enrichment culture (inoculated with sewage sludge). For its growth, acetate was required

while trypticase peptone and yeast extract were found significantly stimulatory. Optimum

temperature for its growth was 37°C. It has grown well through the pH range (from 5.5 to

8.0) while the optimum pH was 6.7 (Ollivier et al 1986). Effluents from tanneries contain

chrome salts in excess than maximum permissible limit. Sludge deposition from such

discharges provides a natural environment for fortification of chromium resistant

bacteria. The status of chromium resistant bacteria in the tannery effluent sediments of

Calcutta based tanning industries have been investigated by Basu et al (1997). Similarly,

Verma et al. (2001) have also scrutinized the occurrence of metal tolerant and antibiotic

resistant organisms in tannery effluents. Seventy seven isolates comprising of

heterotrophs (41) and coliforms (36) which were found tolerant to chromate levels of

>50 µg/ml were selected for further study. The majority of coliforms were resistant to

higher concentrations of chromate (upto 200 µg/ml) while around 3% of the heterotrophs

were resistant to chromium at a level of > 150 µg/ml.

Bacterial strains having capability of bioaccumulating Cr (VI) were enumerated

from treated tannery effluents of a common effluent treatment plant. Two strains,

Bacillus megaterium and Bacillus circulans were able to bioaccumulate 32.0 and 34.5 mg

Cr/g dry weight, respectively. Strikingly in 24hrs, these have brought the residual

concentration of Cr (VI) to the permissible limit whereas initial concentration was 50 mg


22

Cr (VI)/L (Srinath et al 2002). S. marcescens (chromate resistant strain) was separated

from tannery effluents too. It was able to reduce hexavalent chromium to trivalent

chromium. Around 80% of chromate was removed from the medium and reduction seems

to occur on the cell surface. Particles were deposited on outside of bacterial cells as

depicted through transmission electron microscopy. Thus immobilized S. marcescens can

be used in processes for industrial waste treatment (Mondaca et al 2002).

Viti et al. (2003) have differentiated previously isolated bacterial strains (from

chromium contaminated soil) on the basis of Gram reaction and biochemical

characteristics. Besides this, chromate reduction capability, multiple chromate MICs,

heavy metal tolerance, and antibiotic susceptibility were also tested for each isolate.

Except one, all strains were Gram +ve and resistant to chromate high levels. Maximun

number of hexavalent chromium resistant isolate was Corynebacterium hoagie.

All bacterial strains separated from tannery effluents have resisted very high

levels of potassium chromate (25 mg/ml in nutrient broth and 40 mg/ml on nutrient agar).

For temperature range (42 to 24°C) and pH range (9 to 5) they have grown very well.

They have demonstrated antibiotics (ampicillin, chloramphenicol, kanamycin,

streptomycin and tetracycline) and multiple metal (Co, Cu, Mn, Ni, Pb and Zn)

resistances (Faisal and Hasnain 2004).

In 2004, Chowdhury et al. (2004) have estranged bacterial strains even from

tannery soil after enrichment in minimal medium with gallic acid or tannic acid which


23

has served as single carbon source. Interestingly, a bacterium has been separated and

characterized from lake water that is having capability of aerobically degrading 2-

Methylisoborneol. Light microscopy and transmission electron microscopy revealed that

this strain is a spore forming, flagellated bacterium that is bacilloid in shape (Lauderdale

et al 2004).

From industrial saline wastewater (contaminated with chromium), mixed cultures

have been obtained after using enrichment with 50 mg/L chromium (VI). From mixed

cultures, eleven isolated bacterial strains have demonstrated huge chromium

bioaccumulation in molasses media having 100 mg/L chromium (VI) to have more

efficiently to bioaccumulate chromium than mixed cultures (Donmez and Kocberber

2005). From a tannery waste contaminated soil, RNP4 was isolated and identified as

Pseudomonas sp. RNP4 and has tolerated up to 450 mg Cr (VI)/L on a Luria Bartani agar

medium. It has also reduced significant quantity of Cr (VI) to Cr (III) (Luria Bartani

liquid medium). Moreover, in the presence of Cr (VI), it has also promoted growth of

pearl millet, blackgram and indian mustard (Rajkumar et al 2005).

Surprisingly, from plant’s rhizosphere (Canna indica, Typha latifolia and

Phragmites australis) growing in that area which are receiving discharges of tannery

industries, few bacteria have been screened out. The relative proportions of sulfate

reducing, denitrifying and aerobic bacteria were calculated. Most abundant were aerobic

bacteria. From mixed cultures, six bacteria have been isolated which were proficient to

utilize tannic acid (as sole carbon source) in axenic culture. These isolates were found


24

closely similar to Herbaspirillum chlorophenolicum, Klebsiella oxy toca, Pseudomonas

putida, Stenotrophomonas maltophilia and taxa Serratia on base of 16S ribosomal DNA

sequence analysis (Franco et al 2005).

Several chromate tolerant bacteria have been separated from tannery effluents

emanating from Common Effluent Treatment Plant (UP, India). Maximum tolerant

strains (NBRIP1, NBRIP2, NBRIP3 and NBRIP4) were characterized further. These

strains have exhibited multiple metal and antibiotic resistances. At higher chromium

levels, growth of these strains was declined. Chromium accumulation by these isolates is

a great potential for recovery and detoxification of chromium from effluents (Shukla et al

2005). The metal resistant microorganisms were sampled and identified from treated oil

mill industry effluent wastewater samples. They have further shown values of minimum

inhibitory concentration towards metals (Cd, Cr, Ni and Pb) ranging from 100 to 800

mg/L level. Isolate BC15A, potent metal resistant organism was identified as

Pseudomonas sp. The detailed morphological, biochemical analysis and 16S rDNA

sequence of this isolate revealed that it is much similar to Pseudomonas aeruginosa.

Within 48 hrs, it has absorbed Cr (30%), Cd (50%), Ni (93%) and Pb (65%) from the

medium having 100 mg/L of each heavy metal (Raja et al 2006).

Besides bacteria, five morphologically different fungi have been separated from

leather tanning effluents. Among them, Aspergillus sp. and Hirsutella sp. had highest

potential for chromium removal. The maximum chromium was removed at pH 6;

temperature 30 °C, yeast extract (0.1%) and sodium acetate (0.2%). After 3 days, around


25

70% chromium was removed when Aspergillus sp. was applied in 21bioreactor for

removing chromium (Srivastava et al 2006).

From consortia, eight isolates have been recognized as Gram positive and four

were belonging to genus Cellulomonas (based on membrane fatty acid composition

and16S rRNA sequence homology). In less than 3 days fermentative growth on D xylose,

two strains have decreased Cr (VI) levels from 0.04 to 0.002 mM. Surprisingly even after

four months, they have retained this reduction capability (Viamajala et al 2007).

From tannery effluents, biogas slurry, mine soil, sewage, pulse rhizosphere and

paddy rhizosphere, sulphur oxidizing bacteria were separated. Total fourteen out of

twenty eight isolates were further screened based on their ability of reducing pH of

growth medium (from 8.0 to ≤ 5.0). The selected isolates were found related to genus

Thiobacillus (Vidyalakshmi and Sridar 2007). Similarly, from pulp paper mill waste, total

seven aerobic bacterial strains have been isolated and screened for pentachlorophenol

tolerance (on pentachlorophenol supplemented mineral salt agar medium). The ITRC S7,

isolated strain has degraded up to 90.33% of 1.127 mM of pentachlorophenol (Singh et al

2007).

In 2008, chromium tolerant microorganisms were isolated from solid waste and

liquid effluents of an electroplating industry. Obtained nine isolates have tolerated

chromium level up to 700 mg/L. Within 8 to 12 hrs, they have reached the stationary

phase and within 4 to 10 hrs, they biosorbed 95% of initially added (200 mg/L)


26

concentration of chromium. The amino and carboxylate groups (in biomass) have taken

part in biosorption process as indicated through Fourier Transform Infra Red analysis of

the chromium exposed biomass. The two most active organisms were identified as

Bacillus marisflavi and Arthrobacter sp. by 16S RNA results (Mishra and Doble 2008).

KUCr1 (chromium resistant bacterial strain) have exhibited varied degree of resistance to

different heavy metals. In complex medium, Minimum Inhibitory Concentration of

chromium was about 950 mM (under aerobic culture condition). It has also exhibited Cr

(VI) reducing capability under in vitro aerobic conditions. Under culture condition, the

factors which have affected Cr (VI) reduction were evaluated too. At 35 oC and pH 8 to

10, maximum Cr (VI) reduction was observed. The reduction was slowed down by higher

Cr (VI) concentration but reduction was related to growth supportive condition (in terms

of carbon, nitrogen and phosphorous supply in wastewater fed with tannery effluent).

Furthermore, with longer incubation time all metal could be reduced. But Zn and Cd have

inhibited reduction. Biochemical characterization and 16S rDNA sequence analysis, this

strain was identified Bacillus firmus (Sau et al 2008).

Zahoor et al. (2009) had assessed the potential of Staphylococcus capitis and

Bacillus sp. (JDM21) for reducing hexavalent chromium to its trivalent form. JDM21

have tolerated 4800 μg/mL while S. capitis have tolerated tolerate 2800 μg/mL of Cr

(VI). After 96 hrs, from the medium JDM21 and S. capitis have reduced 85% and 81% of

hexavalent chromium. After 144 hrs, they have reduced hexavalent chromium 86% and

89% from the industrial effluents. Cell free extracts of S. capitis and JDM21


27

demonstrated 70% and 83% reduction at concentration of 10 µg Cr (VI) /mL,

respectively.

In 2009, for the isolation of microorganisms from tannery effluent water samples

(Baluchara, Chittagong) were inoculated in Luria Bertani medium having hexavalent

chromium (K2Cr2O7). Total 35 isolates belonging to species of Alcaligenes (3.5%),

Bacillus (11.43%), E. coli (13.3%), Enterobacter (11.3%), Hafnia alvei (2.45%),

Moraxella (14.3%), Salmonella (12.3%), Staphylococcus (5.7%) and Streptococcus

(25.72%) have been chosen as potential organism for further evaluation. All selected

isolates have tolerated 500 mg/L of hexavalent chromium at least. Two isolates have

reduced 38% and 32% of hexavalent chromium (which was added to the medium).

Besides this, seven isolates have exhibited (18 to 22%) hexavalent chromium reducing

capacity (Fakruddin et al 2009).

Based on 16S rRNA gene sequence analysis, CSCr 3, bacterial strain with

elevated Cr (VI) reducing capacity was cut off from a chromium landfill and recognized

as Ochrobactrum sp. It was Gram negative, rod shaped and motile. CSCr 3 has

demonstrated tolerance up to 800 mg/L of hexavalent chromium. It was also competent to

reduce various chromate and dichromate under a wide range of pH (11 to 7) and

temperatures (40 to 25oC). Strikingly, a spectacular increase in hexavalent chromium

reduction was noticed for addition of glucose. Metal (Mn, Co and Cu) addition has also

considerably motivated hexavalent chromium reduction while nitrate or sulfate yielded

no influence (He et al 2009).


28

From tannery wastes and agricultural soils (Central Thailand), twenty seven high

arsenic resistant isolates had demonstrated minimum inhibitory concentrations for

arsenate and arsenite of > 400 mM and ≥ 40 mM respectively. Cultural, physiological,

morphological, biochemical characteristics, principal ubiquinone componency and 16S

rRNA gene sequence have confirmed one isolate of Enterobacter, nine isolates each of

Acinetobacter and Klebsiella and four isolates each of Comamonas and Pseudomonas sp.

As determined by silver nitrate staining (of arsenite agar plates), one isolate (A3-3, genus

Comamonas) was found capable of oxidizing arsenite to arsenate (Chitpirom et al 2009).

In 2009, from delimed and limed tannery fleshings, lactic acid bacteria species

were isolated and evaluated for their fermentation efficiency and antibacterial properties

by Rai et al (2009). Tannery fleshings have been proficiently fermented by lactic acid

bacterial isolates and yielded fermented products having antioxidant properties. On the

basis of various molecular and biochemical tests, a proteolytic isolate has been identified

as Enterococcus faecium HAB01. The ability of Staphylococcus capitis and Bacillus sp.

for reducing hexavalent chromium to trivalent form has been assessed by Zahoor and

Rehman (2009). Staphylococcus capitis has tolerated 2800 μg/ml while Bacillus sp. has

tolerated 4800 μg/ml of hexavalent chromium. These organisms have resisted Cd, Cu,

Hg, Ni, Pb and Zn too. Optimum growth was noticed at pH 6 to 7 and 37°C temperature.

S. capitis and Bacillus sp. have reduced 81% and 85% of hexavalent chromium (from the

medium) after 96 hrs while 89% and 86% of hexavalent chromium was reduced after 144

hrs from the industrial effluents. S. capitis and Bacillus sp (cell free extracts) have

demonstrated reduction of 70% and 83% at 10 μg/ml hexavalent chromium concentration


29

respectively. For the incidence of ammonia oxidizing bacterial communities, Wang et al.

(2010) have investigated activated sludge of total eight wastewater treatment plants.

Prominent ammonia oxidizing bacteria was found related to Nitrosomonas sp. In all the

samples, members of Nitrosomonas communis and Nitrosomonas oligotropha were

identified, while in some systems, members of Nitrosomonas europaea were present too.

From denitrification, nitrification and settling tanks of treatment plant of Elmo

Leather AB tannery (Boras, Sweden), wastewater samples have been screened for the

presence of bacteria. These samples were first cultured on nutrient agar with optimal

dilution (10-2). Colony morphology, Gram reactions, growth on triple sugar iron agar

slants, phenylethanol media, MacConkey, oxidase and catalase tests have been utilized

for biochemical and phenotypic identification of bacteria from collected samples. From

the nitrification and denitrification tanks, isolates were recognized as Spingomonas

wittichii (1%), Azoarcus sp. (3%) and Paracoccus denitrificans (67%) while from the

settling tanks, Bacillus cereus (1%), Corynebacterium freneyi (20%) and Paracoccus

denitrificans (22%) were isolated (Desta et al 2010).

Four different chromium resistant bacteria which were sampled from tannery

effluents (Alexandria, Burgelarab, Egypt) have demonstrated different levels of chromate

reduction under aerobic conditions. Two of them, were identified as Pseudomonas and

Acinetobacter. For Acinetobacter sp. minimum inhibitory concentration was 160 mg/L

and for Pseudomonas sp. it was 200 mg/L (Farag and Zaki 2010). Similarly, Halophilic

bacteria which were segregated from ‘drained soak liquor’ were found enormously


30

pleomorphic, aerobic, motile Gram -ve organisms. These organisms have demonstrated

slower growth outlines at 37 oC comparable to E. coli. Pleomorphic Gram -ve nature and

optimum salinity of media of halophiles were the causative agents for the insensitivity of

antimicrobials (Ghosh et al 2010). In addition, the chromate resistant bacteria have been

enumerated from soil, mud and rhizospheres of the Carduus acanthoides L. and

Potamogeton natans L. The microbial population was subjected to various potassium

dichromate levels (1000, 300, 100 and 40 mM). At 1000 mM chromium (in the medium),

nearly 45% of bacteria from rhizospheres and 25% from soil were found resistant. Only

Pseudomonas sp., Bacillus stearothermophilus and Serratia fonticola within 24 hrs have

reduced 50 μM potassium dichromate. While following 72hrs incubation, they have

reduced up to 500 μM Cr (VI) levels in medium. (Raicevic et al 2010). In 2010, the

capability of Alcaligenes faecalis, Bacillus pumilus and Staphylococcus sp. to reduce

hexavalent chromium into trivalent chromium has been evaluated by Shakoori et al

(2010). Alcaligenes faecalis, Bacillus pumilus and Staphylococcus sp. have tolerated

about 1.4, 2 and 1.6mg/ml of hexavalent chromium respectively. Strikingly within 24hrs,

Alcaligenes faecalis, Bacillus pumilus and Staphylococcus sp. have reduced 97, 95 and

91 percent of hexavalent chromium to trivalent chromium from medium having 100 μg

Cr (IV) /ml.

For better understanding of heavy metal tolerance of native bacterial flora of the

Palar river basin (Vellore District), chromium tolerant strains have been separated from

contaminated sediments, water and effluents of various tanneries. Total sixty eight

chromium tolerant bacteria were separated. The tolerance concentration rang of these


31

isolates was 100 to 3300 mg/L. Besides this, eighty percent isolates have demonstrated

resistance to Fe, Ni, Pb and Zn (100 mg/L) while fourty five percent isolates have

depicted resistance to Cd. In addition, these isolates have tolerated up to 9% of NaCl

(Sundar et al 2010).

From tannery effluents, IFR3 and IFR2 (chromium tolerant bacteria) were

separated and were further identified as Pediococcus pentosaceus and Staphylococcus

aureus respectively. On Luria Bertani medium having 2000 mg/L of potassium

dichromate, they have grown well. The isolated strains were competent to reduce Cr (VI)

to Cr (III). From 7.0 to 8.0 and 35 to 40oC were the optimum pH and temperature range

for their growth and reduction. Moreover, for both isolates, Cr (VI) reduction was found

growth associated (Ilias et al 2011). Recently, total fifteen bacterial strains were isolated

from tannery effluent sludge sediment core (UP, India) by Tewari et al (2011). Out of

these fifteen isolates, only eight strains have evinced pentachlorophenol (a preservative

being used by leather industry) degrading aptitude and were further biochemically and

morphologically characterized. The strains have revealed similarities with Arthrobacter

species, Bacillus species, Proteus species and Pseudomonas species. Highest

pentachlorophenol degrading potential was demonstrated by Arthrobacter species

(degraded 55% in 30 days) while lowest for Proteus species (only 38%). Bacillus species

and Pseudomonas species have degraded 44 and 47% respectively. Furthermore, from

samples of a tannery waste treatment plant (having prolonged application of nonylphenol

polyoxyethylene), OPQa3, bacterial strain having capacity for using nonylphenol

polyoxyethylene (as single source of carbon) has been screened in 2011. Preliminarily,


32

OPQa3 has been recognized as Brevundimonas sp. based on morphological

characteristics, physiological biochemical tests and resemblance of 16S rDNA gene

sequence. Within 120 hrs, the degradation rate of nonylphenol polyoxyethylene was

84.5% by OPQa3 as determined by degradation test while optimum pH was around 7 and

temperature was 30oC (Yan et al 2011). Likewise, MPC1 bacterial strain has been

separated from tannery effluent waste water sample (Trichy, India). On the basis of

bacterial 16s rRNA gene sequence phylogeny, this isolated bacteria was found closely

related to members of Pseudomonas aeruginosa Sp (Senthil et al 2011).

During the second phase of my research project, Krieg et al. (1984) have been

followed for enumerating the microbial load of collected TEW. Ten fold serial dilutions

of TEW samples were done for viable count. Post incubation, total number of bacteria

was then counted from only those plates having 30-300 colonies. With reference to

Bergy’s Manual of Determinative Bacteriology, the colony, microscopic and biochemical

characteristics were recorded for identification of isolated bacteria. Method utilized by

Basu et al. (1997) was used as reference methodology for the assessment of toxic metal

tolerance. Isolated bacterium’s fresh overnight peptone water broth cultures were

inoculated on nutrient agar plates supplemented with chromium compounds (ranging

from 600- 2600 µg/ml). Growth of bacteria following incubation was marked as either

positive or negative. The advantages of microbial these microbial assays include: (1) cost

effectiveness, (2) reproducible results, (3) individual test can be repeated. While

disadvantages include (1) time taking nature, (2) laborious, (3) chances of contamination

are more.


33

2.3. Toxicological Evaluation of TEW through CAM and

Embryotoxicity Assay

2.3.1. Toxicological Evaluation of TEW through CAM Assay

During embryo development, chick chorioallantoic membrane (CAM), a very

simple extra embryonic membrane serves numerous functions: location for respiratory

gaseous exchange, reabsorb ion and water from allantoic fluid, maintain acid base

homeostasis within embryo and transport Ca from the eggshell. The epithelia of CAM

carry out all above stated functions (Gabrielli and Accili 2010). In 1989, biological and

non-biological matters which were being used clinically (as temporary skin substitutes or

hemostyptica, as vascular prostheses) were implanted on the CAM during 9 to 14th days

of incubation. On the basis of outcomes of these experiments, CAM (in vivo) assay could

be recommended for selection of materials for their connective tissue reaction and

biocompatibilities (SpaneI-Borowski 1989).

Angiogenesis that is growth of new blood vessels involves planned alterations in

endothelial cell connections with nearby cells and with underlying basement membrane

components. Matrix metalloproteinases activity is required for angiogenesis (Haas and

Madri 1999). The CAM of Anas platyrhynchos (mallard duck) have three distinct layers

(from 12th to 24th day of incubation) that is allantoic epithelium, mesoderm and chorionic

epithelium as assessed through light and transmission electron microscopy. After 12th day

of incubation, allantoic epithelium is composed of only one layer of overlapping flattened

cells while chorionic epithelium comprised of two layers of elongated flattened epithelial


34

cells. The mesoderm consisted of a loose matrix having collagen fibrils and mesenchymal

cells (Lusimbo et al 2000).

A new approach for testing the biomaterials (such as a cotton thread, endotoxin

LPS and a Silastic tubing) through use of CAM assay, as a replacement to the traditional

mammalian models has been reported by Valdes et al (2002). At 4th day of incubation, a

small window was made in shell of fertilized chicken eggs. Several test materials were

applied to CAMs after seven days of incubation (cotton thread, LPS and Silastic tubing).

After eight days, the tissue reaction to materials applied was then appraised through

histological, gross and scanning electron microscope evaluations. The geometry and

structure of test materials deeply affected the absorption of samples in CAM.

Along with the associated disciplines for quantifying angiogenesis, image probing

has presented massive potential for surface characterization of CAM, detailed

quantification of blood vessels and exact measurement of very small blood vessels (Ejaz

et al 2004).

In 2004, Hasan et al. (2004) have reviewed that CAM assay is a well established

assay with its countless advantages. While the most comprehensive information on

angiogenesis could be provided by histological analysis. Moreover, Tufan et al. (2005)

have also highlighted the importance of CAM assay among all presently used

angiogenesis assays. Advantages, uses and limitations of the CAM assay have been

described by them as well. For precise quantification of the vasculature of CAM from 4th


35

to 13th day of incubation, a new 3D model was proposed by Ejaz et al (2006). At 6th day

of incubation, highly significant increase in surface roughness values was noted. For

visualizing CAM’s 3D microvascular architecture, image probing technique offers a

helpful modality. It can reveal the hidden information and amplify the fine details for

precise quantification of angiogenesis. The CAM is a complete tissue as it is composed of

veins, arteries and capillaries. By observing adverse changes in CAM, the potential

irritancy of any compound may be detected too. Six agrochemicals have been directly

poured onto the CAM. The results obtained from HET CAM have good correlation with

those based on the Draize eye test (Tavaszi and Budai 2006). CAM treated for 24hrs with

50 and 100 μg of koetjapic acid (seco a ring oleanene triterpene) has displayed distorted

architecture in the vasculature. The number of blood vessels was considerably decreased

too. Thus, it has inhibited the development of new blood vessels (Nassar et al 2011).

CAM assay has been used extensively in literature because it offer under

mentioned advantages: (1) simplicity, (2) less expensive nature, (3) easier to handle, (4)

effortless access, (5) could be utilized with small restrictions, (6) various quantitative

and semi-quantitative methods could be used for evaluation of anti-angiogenesis and

angiogenesis levels, (7) because of continuous visualizing of the test materials through

the shell window, (8) similarities among the tissue reactions of CAM and mammalian

model, (9) massive vascularisation, (10) can be performed promptly in any laboratory

settings (Kleinmann et al 2003; Ribatti et al 1995; Ribatti 2010; Valdes et al 2002). While

chances of contamination is the main disadvantage.


36

2.3.2. Embryotoxicity Assay

For conducting CAM and embryotoxicity assay, applied methodology is as

follows: albumin was removed by making a window in the eggshell according to Ejaz et

al (2006). After 24hrs, 200µl of each sample solution was dispensed on developing

CAM. Following incubation, CAMs and embryos were separated and macroscopically

evaluated for assessment of any changes in their development. The main advantages of

embryotoxicity assay is compound can be administered by injection while disadvantage

is large number of eggs is required for getting reliable results (Clegg 1964).

The motivational force for evaluating the effects of the different elements on

developing chick embryos was started by the conspicuous effects of thallium sulphate on

developing embryo of four day age (Ridgway and karnofsky 1952). Around 0 to 20

percent malformations were observed after injecting styrene and styrene oxide into the air

space of fertilized chicken eggs (Vainio et al 1977). The teratogenic potential of five

aliphatic chlorinated hydrocarbons and toluene (injected into the air space of 2, 3 and 6

days fertilized chicken eggs) diminished in following order: 1,1,1-trichloroethane,

trichloroethylene, methylene chloride, tetrachloroethylene, 1,1,2-trichloroethane, toluene

and olive oil (Elovaara et al 1979). In addition, toxic potential of total eighty chemicals

(0.50 - 75 mg/egg) have been assessed after their application to developing chicken

embryos. Results revealed that chicken embryo test is competent enough for investigating

teratogenic effects of various chemical compounds (Verrett et al 1980).


37

For screening of potential embryotoxic effects, seven different industrial effluents

and seven heavy metal, organic solvent, and petroleum solutions have been externally

exposed to Mallard eggs (at 3rd and 8th day of incubation). Tannery effluent, mineral

pigment, scouring effluent and sludge resulted significant embryonic growth reductions

(David et al 1981).

For evaluating chemical toxicity on the chicken embryo, a test protocol was

developed in 1982. On day three of incubation, eggs were injected with the chemical and

the test was continued till 14th day of incubation for a nonstop monitoring of the

developing embryo especially for those ones that died (before maceration). Early deaths

(first 2 day after injection), late deaths (with non malformed embryos), late deaths (with

malformed embryos) and malformed survivors were recorded. The seventy percent

observed late deaths were with malformations. Eye defects and open coeloms were most

common types of the malformations. While dead embryos mostly have miscellaneous

malformations (Korhonen et al 1982).

For the assessment of embryotoxicity and teratogenicity of 50 and 25 percent

Buckley's formocresol, white Leghorn chick embryos have been utilized by Friedberg

and Gartner (1990). At 48 hrs of incubation, embryos were injected with test compounds

and then on 9th day of incubation they were sacrificed. Total 100 and 40% mortality was

noted for 50 and 25% formocresol respectively. It has proven embryotoxic and

teratogenic as it caused gross morphological abnormalities such as facial alterations,

cranial hematomas, eye and beak deformities and variations in feather germ appearance.


38

Effects of heavy metals (arsenic, cadmium, copper, iron, cobalt, indium,

molybdenum and manganese) on chick embryogenesis was evaluated by dissolving salt

of each metal in normal saline and then on second day of incubation, were injected in air

sacs. On 14th day of incubation, live embryos were separated from the eggs. Embryos

were thoroughly examined for gross abnormalities. Reduced body size, twisted neck,

micromelia, everted viscera, microphthalmia and hemorrhage were present. Among all

the tested heavy metals, Co and As proved more teratogenic (Gilani and Alibhai 1990).

Liver tissue of embryo has shown retarded growth along with decrease in its

weight when exposed to contaminated seawater. Exposed fetuses have also depicted

bulges in the lungs as determined through histological examination. Besides this,

significantly obstructed capillaries formation was also prominent. Consequently,

malformed fetuses were developed as a result of seawater contamination (Hatano and

Hatano 1992).

After 3 days incubation, 0.1 and 0.5 mg of lead nitrate was injected in the eggs.

Decreased body weight (of developing embryos) and elevated mortality rate was noticed.

Furthermore, the hatchability and weight of hatching youngs was declined. Macroscopic

abnormalities on all body especially the beak, eyes head, neck, and hind limbs along with

retarded growth were observed significantly. A considerable destructive effect on the

kidney’s nephric units (proximal, distal and collecting tubules) was also prominent. In

addition, abnormally small and compact glomeruli were evident in such kidney (El-

Shabaka et al 1993).


39

Chicken embryo lethality, dose dependent malformations, liver lesions and

oedema were observed after 3, 3', 4, 4', 5 pentachlorobiphenyl (0.5 to 12.0 µg/kg)

exposure (Zhao et al 1997).

The low level of chemical mixture (arsenic, trichloroethylene, benzene, lead and

cadmium) has increased embryonic mortality percentage. Egg production and egg weight

was considerably decreased too. Hens have proven sensitive to hazardous effects of

contaminated drinking water (Vodela et al 1997).

Potassium dichromate solutions (1 to 100µg/egg) were injected into chicken eggs

before incubating for assessment of their embryotoxic and teratogenic effects. When

examined on 7th and 14th day of incubation, concentration dependent mortality of

developing embryos was observed in all groups (Asmatullah et al 1998A). Heavy metals

being components of tannery effluents do cause carcinogenic, teratogenic and

embryotoxic effects in large biota (Asmatullah et al 1998B). Even potassium dichromate

(250 and 500mg/Kg feed) ad libitum feeding for thirty two weeks has significantly

declined hatchability (Asmatullah et al 1999).

The effects of chromium, cadmium and lead (low levels equivalent to amounts in

Hungarian polluted surface waters) were studied on the embryogenesis, viability and

hatching success of the mallard eggs. Prior to incubation, eggs were treated through

immersion/injection. All the tested metals proved lethal by causing an increase in

developmental anomalies and mortality but chromium proved most teratogenic. Highest


40

percentage of mortality was caused by cadmium. Besides this, cadmium has also caused

massive reduction in hatching success (Kertész and Fáncsi 2003).

Chickens, American kestrels and Mallards embryonic survival was recorded after

air cell delivery of poly chlorinated biphenyl congener126 and pentabrominated diphenyl

ether mixture. Poly chlorinated biphenyl congener126 has decreased survival in kestrel

and chicken embryos while Mallards embryos proved less sensitive (Mckernan et al

2009).

In 2011, embryotoxicity of weathered crude oil (Gulf of Mexico) using mallard

ducks (Anas platyrhynchos) has been determined by Finch et al (2011). Through

paintbrush 0.1 to 99.9 mg weathered crude oil was offered to fertilized eggs on 3rd day of

incubation while on 7th day mortality has occurred.

2.4. Marine Shrimps Mortality Assay

I have determined acute toxicity of TEW to marine shrimp larvae by using the

method described by Tzong-huei et al (1999). Larvae within one day of hatching were

exposed to five dilutions of TEW, CHC and PDC for various exposure times (1, 24, 48

and 72 hrs) at 24oC in photoincubator. At the end of incubation, the number of dead

shrimps was recorded.


41

The impact of dilutions of selected TEW, CHC and PDC on marine shrimps

(Artemia franciscan) lethality or mortality has been explored in current research project

because of following advantages: (1) cost effectiveness, (2) small body size, (3) short life

cycle, (4) around the year availability, (5) no feeding requirement during the assay, (6)

large offspring production, (7) could be cultured under laboratory conditions more easily,

(8) found receptive to a wide range of pollutant, (9) no permanent maintenance of stock

culture is required, (10) marine shrimps are well recognized for their aptitude to continue

to exist under prolonged dry periods (dormant encysted state) and its extensive saline

range, (11) for their emergence and continuing life cycle, (12) reactivation require just

water and suitable conditions of pH and temperature (Bu-Olayan and Thomas 2006;

Hadjispyrous et al 2001; Kokkali et al 201; Okasako and Siegel 1980; Vanhaecke et al

1980). While instar period of the nauplii, duration of test, sensitivity among different

stages of nauplii, cysts storage conditions, differences among different batches of

Artemia and geographical strains put question mark on the credibility of marine shrimps

mortality assay (Vanhaecke et al 1980).

Several species of the genus Artemia have been extensively used over last decades

in various scientific areas such as applied toxicology and research, ecotoxicology,

ecology, physiology, aquaculture and genetics (Nunes et al 2006). In 1956, Corner and

Sparrow have studied the toxicity of copper and mercury compounds on Artemia larvae.

Potassium salts were found significantly toxic to marine shrimps. In solutions of

potassium nitrate, potassium chloride and potassium benzene sulphonate, marine shrimps

become adynamic within 30 minutes of contact while 5% nitric acid has caused mortality


42

of marine shrimps in only 2 to 3 minutes. Besides these, dilute solutions of silver nitrate

were proven highly toxic (Corghan 1958). Impact of heavy metals on the mortality and

growth rate of marine shrimp has been evaluated in past too. Descending toxicity order

for heavy metals was Pb, Zn, Fe, Cd, Cu and Hg. The Pb and Zn have caused massive

suppression of marine shrimp growth while Cu has caused slight repression. Larvae of

marine shrimps proved highly sensitive to Zn and Cu solutions (Brown and Ahsanullah

1971). Larvae and adults of marine shrimps have demonstrated significant tolerance to

copper sulphate (1mg/L) but the tolerance decreased following successive generations.

Moreover, inhibition of growth rate and an unfavourable effect on reproduction was also

observed (Saliba and Ahsanullah 1973).

Artemia salina is most well situated test organism for toxicity studies. Mostly in

literature, toxicity assays have been performed with its larvae. For incubation of cysts,

firmly manage the environmental temperature for the start of hatching and also during

entire hatching period. Furthermore, toxicity experiments should be conducted with the

identical life stage of nauplii as earlier stage larvae are notably more responsive to

pollutants than older larvae. Sensitivity response to pollutants alters based on

geographical location on strain of Artemia (Sorgeloos et al 1978). Now, for the

evaluation of effect of pollutants on (marine and fresh water) ecosystem, standardization

of the toxicity assay based on aquatic organisms is a vital requirement. For the freshwater

environment, simple standardized tests based on marine shrimps are now very close to

get adopted at the international level (Okasako and Siegel 1980). In 1987, heavy metals

impact on the relative hatching of Artemia salina was studied by Liu and Chen (1987).


43

Marine shrimp cysts were hatched in seawater containing various heavy metal

concentrations (Cd2+, Co2+, Cr3+, Cu2+, Fe2+, Hg2+ , Mn7+, Mn4+, Mn2+, Ni2+, Pb2+ and

Zn2+). The number of hatched nauplii was counted at the end of 48 hrs. Between heavy

metal exposed levels (except copper) and hatching rate of marine shrimps, a negative

linear relationship was developed. Mn2+ has proven least toxic while Cu2+ established as

most toxic among all tested heavy metals.

Acute toxicity of Cr, Cu oil (zarzaitine type tunesian crude oil) and oil dispersant

(finasol OSR 2) found in nearshore polluted waters have been evaluated on 25 days old;

3.5 to 4.5 mm long Artemia salina. Moreover, individual and /or joint action

(combinations of two, three of four chemicals) was assessed by determination of the 48hr

LC50. The Cr+oil, Cr+Finasol and Cu+Finasol mixtures strict additivity have proved that

these pollutants when in mixtures of two can act independently to marine shrimps.

Independent toxic action of chromium in mixtures of two pollutants has been indicated. A

relatively strict additive joint action was noticed (ranged from -0.016 to -0.53) in all

solutions containing chromium (Verriopoulos et al 1987). Seven trace elements (Cu, Cd,

Fe, Mn, Ni, Pb and Zn) toxicity has been evaluated on 48 hrs cultured nauplii of marine

shrimps. Additive (Cd and Pb, Cu and Zn, Ni and Fe) and synergistic effects of all the

heavy elements have been investigated as well. After 24 and 48 hrs exposure, the median

lethal concentration of tested heavy metals was calculated by probit analysis. The degree

of toxicity was higher for compound bioassays as compared to individual simple tests.

The order of the metals toxicity to marine shrimps was Pb > Cd > Cu > Ni > Zn > Fe >

Mn. The 24 and 48 hrs variations obtained in LC50 values were considerably different


44

(Gajbhiye and Hirota 1990). Likewise the influence of four metals, cupric sulfate, lead

nitrate, nickel sulfate and zinc sulfate on emergence and hatching of marine shrimp has

been estimated in 1991. Cu and Pb were proved equally toxic, reducing the rate and

extent of shrimp’s development (at or below concentrations of 0.1µg/L). Zn was less

toxic than Cu and Pb, while Ni proved the least toxic. The emerging marine shrimps were

highly susceptible to metals than larvae and adults. Furthermore, the toxicity of Pb was

high for prelarval stages in contrast to results obtained with larvae and adults. In short,

for studying metal pollution in coastal marine waters, use of early stages of marine

shrimps is an alternative to the examination of slower growing animals (MacRae and

Pandey 1991).

The short phase toxicity of potassium dichromate and sodium lauryl sulphate on

instar II-III nauplii populations of marine shrimps was investigated in 1998. The

experiment was found suitable for acute toxicity standardization on the basis of

comparisons among obtained results and the ARC test (Artemia Reference Center)

protocol (Togulga 1998). Similarly the effects of potassium dichromate, cadmium

chloride, dibutyltin diacetate, dimethyltin dichloride and trimethyltin chloride was

gauged on Artemia franciscana mortality. The toxicity ranking of all the tested chemicals

was trimethyltin chloride, potassium dichromate, dimethyltin dichloride, dibutyltin

diacetate and cadmium chloride (Hadjispyrous et al 2001). Bioactivity of the

isopropanolic extracts (of six species of macroalgae and fourteen species of marine

invertebrates) has been assessed through marine shrimp’s lethality and hatchability assay

(inhibition of hatching of the cyst). In order to test the sensitivity of the marine shrimp


45

assays to identify cytotoxic activity, the extracts have also been tested for cytotoxicity

against two human cell lines (lung carcinoma A549 and colon carcinoma HT29). One

tunicate (Polyclinum laxum), two sponges (Dysidea sp. and Hyatella sp.), two gorgonians

(Pacifigorgia adamsii and Muricea sp.) and three echinoderms (Pseudoconus californica,

Holothuria impatiens and Pharia pyramidata) presented a strong cytostatic and cytotoxic

effect. The hatchability assay demonstrated a significant activity in four of the species

(Dysidea sp., Hyatella sp, Pacifigorgia adamsii and Muricea sp.) active against the tested

two human cell lines. The marine shrimp’s lethality assay also showed a high lethality in

four of them (Muricea sp., Pacifigorgia adamsii, Polyclinum laxum and Pharia

pyramidata). Results were found consistent with the correlation previously established

between marine shrimp lethality and cytotoxicity in plant extracts (Carballo et al 2002).

The complex effluents discharged to coastal regions (Turkey and Greece) were

assessed for crustacean mortality assay (Artemia franciscana). All the tested discharges

were found to be acutely toxic to A. franciscana. An intensive ecotoxicological

monitoring programme (that incorporates the most befitting bioassays and biomarkers) is

necessary for the maintenance and upgrading of the ecological quality of coastal waters

(Okay et al 2005).

Toxicity and combine toxicity of Cd, Cu, Mg, hydrargyrum and plumbum to

marine shrimp’s nauplius were evaluated using the dynamic living water method. The 48

hr LC50 of these heavy metals to the marine shrimp’s nauplius were 0.023, 1.31, 2.10,

12.15, 28.91 mg/L. The order of toxicity of cadmium, hydrargyrum, plumbum, cuprum


46

manganese for marine shrimp’s nauplius was Hg2+, Pb2+, Cu2+, Mn2+ and Cd2+. Cu-Mn

and Hg-Cu have shown antagonism while Mn-Cd, Pb-Cu and Pb-Mn established

synergism (Yuxin et al 2006). Higher levels of cadmium (CdCl2.2H2O) beyond 100 mg/L

have shown observable synergistic toxic effects with zinc sulphate on marine shrimps

lethality assay. On the contrary, low doses of cadmium, 50 mg/L have resulted in a

significant decrease in mortality to marine shrimps (Novakova et al 2007).

Toxic effects of vanadium and nickel compounds on Artemia urmiana and

Artemia franciscana have been studied under laboratory conditions. The physical and

chemical characteristics of water (such as temperature, pH, soluble oxygen, hardness and

electric direction) were controlled during experiment. The control group received no

metal while the seventeen treatment groups were exposed to different concentrations of

vanadium and nickel. The LC50 of vanadium and nickel was 0.01146 and 0.007201 mg/L

for Artemia urmiana and 0.01158 and 0.0107 mg/L for Artemia franciscana. Vanadium

and nickel had shown toxic effects on the both species of marine shrimps. Interestingly

vanadium on Artemia urmiana has expressed more toxic response than nickel. While on

Artemia franciscana, toxicity levels of vanadium and nickel were identical (Bani et al

2007).

Ecotoxicological endpoints such as immobilization of Artemia salina were

assessed for retaining chemicals (Southern Italy). Marine organisms use has been

proposed for assessment of impact of tannery chemicals and in tannery real effluents

(Lofrano et al 2008). Invertebrate crustacean (Artemia salina) has been used for the


47

evaluation of a set of water samples collected from wastewater treatment plants.

Exposure to these samples was for a period 0 to 24 hrs and respiration rates (1 hr) were

then assessed. This screening platform proved considerably appropriate for monitoring of

environment and potentially hazardous samples selection (Zitova et al 2008). In addition,

Artemia salina has been used as an indicator to measure the water quality of Hangzhou

Section of Beijing Hangzhou Grand Canal because physical and chemical tests alone are

not sufficient enough for the quantification of potential hazardous effects on aquatic

organisms. Marine shrimps were receptive in detecting toxicity in water quality (Lu et al

2010).

An improved “marine shrimp larvae lethality microwell test method” has utilized

a simply designed connecting vessel adjusted with alternative photo-period. Artemia

parthenogenetica nauplii have been effortlessly cultured and harvested with huge

concentrations (about 100 to 150 larvae/ml). Because of abolition of artificial pointless

disturbance, natural mortalities have been declined to around zero. Various reference

toxicants (four heavy metals salts, two pesticides, five antitumor agents, three organic

pollutants) have been used for the validation of its sensitivity by determination of 24 hr

LC50. Most of the tested reference toxicant (except for bleomycin and mitomycin) has

presented 24hr LC50 between 0.07 to 58.43 mg/L (Zhang et al 2011).

Recently, digital image processing has been used for the evaluation of effect of

toxic metal’s aqueous solutions on the mobility of marine shrimp nauplii. The instrument

was composed of a dark chamber, a light source, a laptop computer and a camera with a


48

macro lens. Four marine shrimp’s nauplii were inserted into a macro cuvette which

contained cadmium, copper, iron and zinc ions (at various concentrations). Then marine

shrimps nauplii were filmed (inside the dark chamber) for two minutes. Motion tracking

algorithm that can estimate mobility has processed the video sequence. This system

demonstrated significant sensitivity (instead of small number of tested animals) in

quantifying the mobility of the nauplii, which lead to significantly lower EC50 values as

compared to mortality assay. Moreover, parts per trillion concentrations of toxic

compounds could be measured for some metals (Kokkali et al 2011).

2.5. Phytotoxicity Assay

In a further attempt, I have examined the hazardous burden of collected TEW

through phytoxicity assay (root elongation inhibition test) in accordance with Javaid et al

(2000). Total ten maize seeds were sown in each petriplate and after five days, the length

of roots of germinated seeds was recorded. Inhibition rate of root length was expressed as

percentage inhibition. Advantages of phytotoxicity assay include: (1) comparative cost

efficiency, (2) simplicity, (3) sensitivity, (4) little or no maintenance, (5) long shelf lives,

(6) biomarker for toxicity assessment, (7) provide sufficient data for a plant growth under

adverse circumstances. For effluent monitoring, phytotoxicity assay proved most suitable

as a routine test based upon afore mentioned qualities (Calheiros et al 2008; Lopez-Luna

et al 2009; Wang and Williams 1990).


49

In past, precarious effects of effluents from a tannery treatment plant were

evaluated for seed germination and early seedling growth with two varieties of the

freshwater marsh plant Palisot de Beauvois (Poaceae) and Echinochloa crusgalli

(Linneaus). Rooted marsh plants have detected toxicity of a range of pollutants in water

and sediment and were found sensitive to pollutants (Walsh et al 1991).

Similarly, TEW (with different treatment levels) has been investigated for its

toxic effects on two wetland plants, Typha latifolia and Phragmites australis which are

being used for water treatment. Trifolium pretense (an indicator) was included as a

control. At effluent concentration of 50 %, germination of Trifolium pretense was

completely restrained (Calheiros et al 2008).

The various dilutions of TEW (100, 50, 25 and 10 %) and Cr6+ (10, 5.0, 2.0 and

0.5 mg/L) have exhibited abundant reduction in germination percentage, seedling growth

(number of lateral roots, plumule and radicle length, fresh and dry weight) and pigments

concentration (pheophytin, chlorophyll and carotenoids) with increase in concentrations.

The lower doses of Cr6+ (0.5, 2 and 5 mg/L) and TEW (10%) have slightly increased the

pigments concentration (Nath et al 2009). The effect of soil amended with tannery sludge

(8000, 4000 2000, 1000, 500 and 0 mg Cr kg−1 soil), potassium dichromate (500, 200,

100, 50, 25 and 0 mg Cr kg−1 soil) and chromium chloride (2000, 1000, 500, 250, 100,

1000 mg Cr kg−1 soil) was assessed on oat, sorghum and wheat plants. Root growth was

found highly sensitive assessment of Cr toxicity (P < 0.05). A significant correlation

between chromium accumulation (in dry tissue) and toxic effects on seedling growth was


50

observed. All the three chromium sources had different accumulation and mobility

patterns. The tannery sludge was less toxic (for all three plant species) followed by

chromium chloride and potassium dichromate (López-Luna et al 2009).

In another study, seeds of six pulses have been exposed to seven concentrations of

chromium (0 - 3.2 mM) for assessment of its ecotoxicological effects. Lablab purpureus

and Glycine max have proven most sensitive to Cr2+ as germination percentage, root and

coleoptile length were considerably lower than other tested species. While Lathyrus

odoratus and Dumasia villosa were found most resistant species (Jun et al 2009).

Toxicity of chromium, arsenic, cadmium, cobalt, copper, lead, nickel and zinc

(0.01–1 µg/L) was tested by standard ecotoxicity test on 23 cultivars of flax (Linum

usitatissimum L.). After 72 hrs of incubation, length of root length was measured. Heavy

metal toxicity declined in following order: As3+ > As5+ > Cu2+ > Cd2+ > Co2+ > Cr6+ >

Ni2+ > Pb2+ > Cr3+ > Zn2+ (Soudek et al 2010). TEW were also collected for phytotoxicity

studies from Hazaribagh tannery area of Dhaka City. Raw wastewater effluent displayed

considerable acute toxicity to Lactuca sativa in a 5 day root elongation inhibition test.

While water samples from upstream from discharging site (on River Buriganga) and

downstream the discharging sluice gate has not shown significant toxicity in the tested

bioassay (Arias-Barreiro et al 2010).


51

2.6. Chronic Toxicity Testing

In the last phase of my research project, I have judged the chronic toxic effects of

TEW on various organs of Wistar rats in a 90 days study. Many researchers have reported

parlous effects of chromium in different time based studies ranging from days to years. In

1977, Mathur et al. have documented that hexavalent chromium (2 mg/kg BW/day,

intraperitoneally for 1.5 months) exposed rabbits have shown very significant histological

changes in kidney, myocardium and brain as compared to trivalent chromium. For these

organs, no definite correlation was established between the degree of histological

alterations and level of exposed chromium. Like wise, trivalent and hexavalent chromium

(0.9 and 0.6 mg/m3 BW/day, for 4 to 6 weeks) aerosols exposed rabbits have

demonstrated some pathological alterations. Alveolar macrophages nodular

accumulations were prominent, indicating that they were directly stimulated by

chromium (Johansson et al 1986).

Intraperitoneally injected chromium chloride and sodium chromate (2mg

chromium/kg BW/day) have resulted in many hepatoxotic and nephrotoxic damages to

Wistar albino rats. The damages were progressive and became sever with time. Reported

kidney adaptations were quite analogous for both the chromium salts (Laborda et al

1986).

Levy et al. (1986) have tested total twenty one chromium containing compounds

(using intra bronchial pellet implantation system) for their carcinogenic potential in a two


52

year study. Considerable lung tumors related to offered treatments were reported for

sparingly soluble chromate compounds. In another study, Kostial et al. (1981) have

studied effluents of coal gasification plant offered for sixteen months for screening of

their risk on health. Animals exposed to 100 percent effluents had an increased daily

intake of various inorganic elements (Cd, Cr, Fe, Cu, K, I, Se, As, F, Pb and Hg). The

treatment exposed and control animals had the equivalent mortality rate. It was concluded

that elevated intake of various inorganic material has caused no significant amendments

in parameters such as hematological results, urinary protein secretion, trace element

levels in liver, kidneys and femur bone and histological findings.

For the evaluation of interactive toxicity of potassium dichromate and ethanol,

young female Wistar rats were dosed with 25 mg/L BW/day chromium + 10%EtOH or

10% EtOH through water for twenty two weeks. No change in body weight of (chromium

and EtOH + chromium treated) rats was observed. In the centrilobular and periportal

area, altered hepatic structures with enlarged sinusoidal spaces, necrosis and vacuolation

of hepatocytes (more pronounced) were noticed in the histological sections of liver of

chromium treated rats. Furthermore, chromium + EtOH offered rats illustrated regular

damages in both centrilobular and periportal areas. In kidneys of all treatments, more

significant diffused rather than compact harm to Bowmans capsule and renal tubules was

found because of degeneration of basement membrane (Chopra et al 1996).

On the contrary, no toxicity was reported for chromium chloride in Harlan

Sprague Dawley rats which were fed diet containing100, 50, 25, 5 and 0mg of Cr/kg


53

BW/day. After twenty four weeks feeding, animals were sacrificed. For the Kidney and

liver (receiving 100 mg/kg BW/day of chromium chloride), histological findings were

normal and were quite similar to control (Anderson et al 1997).

In a thirteen week (5days per week) inhalation study of soluble basic chromium

sulphate (168, 54 and 17mg/m3 BW/day) and insoluble chromium oxide (168, 15 and 4.4

mg/m3 BW/day) dusts, decreased body weights and difficult breathing was reported for

high exposure of basic chromium sulfate. Additionally cellular debris, increased

neutrophils, lactic dehydrogenase and protein were seen after examination of

bronchoalveolar lavage fluids. More intense and dispersed effects such as chronic and

granulomatous inflammation, infiltration of alveolar macrophages, accumulation of

foreign material and septal cell hyperplasia in nasal cavity, larynx and lungs were evident

for basic chromium sulphate (Derelanko et al 1999).

Effects of single administration of a new Cr (V) complex [1Cr(V)-BT](2-)], a

stable compound at neutral pH, on histology of mice liver have been judged by Das

Neves et al (2002). Reversible hepatic damage was noticed with a time dependent

behavior. Toxic effects of Cr (V) have developed more promptly than Cr (VI). Effects of

chromium picolinate (8 µg/ml in water) on histological and functional alterations of rats

(having streptozotocin induced diabetes) have been evaluated after six weeks subchronic

supply. Histology microphotographs of liver and kidney have shown declined intensity

and prevalence of cellular infiltration, vacuolations, and hypertrophy. Results revealed


54

that chromium picolinate is devoid of hepatotoxic or nephrotoxic actions (Shinde and

Goyal 2003).

Lesions such as, goblet cell hyperplasia, squamous cell carcinoma, squamous

metaplasia and carcinoma in situ/dysplasia were seen in many rats after nine months

insertion of strontium chromate pellet. Intensity of chromium accumulation in bronchial

lesions of rats was also calculated by using a microscopic x ray fluorescence analyzer.

Strikingly, chromium accumulation was enormously increased with progression of

malignant changes in bronchial epithelium (Takahashi et al 2005).

In contrast to chronic administration, even acute exposure of potassium chromate

to mice kidney has portrayed some toxic effects too. Mice have been subcutaneously

injected with a daily dose of 0.3ml potassium chromate (30mg in 0.9% NaCl /kg

BW/day) for six successive days. After the last injection, mice have been sacrificed at 2,

4 and 6 weeks. In proximal convoluted tubules, mostly cellular degeneration was

observed while in lumen of the renal tubules hyaline casts were present (Oliveira et al

2006).

Female Wistar rats which have received 300 or 500 mg/Kg BW/day of trivalent

chromium for four months have shown some pathological adjustments in liver.

Pericentrilobular, midzonal and periprotal zones with parenchyma cells have expressed

different levels of vacuolation. Besides this, overstuffed hepatocytes and broken nuclei

were also present. The dilation and congestion was present in centrilobular vein.


55

Sinusoidal spaces were swollen along with erythrocytes. Fibrosis was evident in the

portal area (Silva et al 2006). For nine months, tannery wastes (chrome shavings) have

been offered to quail chicks at different levels (5 and 2.5%) by replacing animal proteins

in commercially available chick feed. Histopathological divergences like degeneration,

necrosis, pyknosis, splitting of muscle, vacuolation, and loss of striation in heart were

recorded. These pathological lesions have followed dose and time dependent fashion

(Riaz et al 2006).

Male Wistar rats were sub chronically exposed to the mixture of toxic metals

(chromium, cadmium, arsenic, lead, mercury, nickel, manganese and iron) through

drinking water for three months. The exposed levels were 100, 10, 1 and 0 times the

mode quantities of the individual metals occurring in environment. Another group was

offered to the mixture at levels equal to the MPL (WHO) in drinking water. The 10 time

and 100 time doses have increased weights of liver, brain and kidneys while body weight

has declined along with water consumption. These mixture levels have led to

development of dose dependent necrotic, vascular and degenerative alterations in liver,

brain and kidney. This subchronic exposure to toxic metal mixture has affected male rat’s

general health by changing the structural and functional integrity of liver, brain and

kidney (Jadhav et al 2007).

Similarly, a range of doses of hexavalent chromium (potassium dichromate),

gentamicin and mercury were injected to Male Sprague Dawley rats. Following 72 hrs

treatment, about 50% necrosis involving proximal tubules was apparent (Zhou et al


56

2008). For six months, five groups of chickens (each comprising of 10 chickens) have

been treated orally with different dosage levels of chromium (VI) for elucidation of its

impact on histology of kidneys. Significant alterations in interstitial areas, tubules and

glomerulus were evident at the end of specified time period (Gashi et al 2008).

The hepatocytes of male Sprague Dawly rats treated with chromium picolinate

(0.8 and 1.5 mg/100g feed) have shown some degenerative adaptations in liver such as

degenerating nuclei and swollen cells while some cells have presented regeneration (by

division of their nuclei) too. Methyl green pyronin staining has demonstrated fewer

amounts of DNA within nuclei. Long term consumption of chromium picolinate has

highly encouraged numerous damages to liver (Mahmoud et al 2009). Likewise, male

obese Zucker rats have been offered feeds lacking or having 10 and 5 mg/kg of

chromium picolinate for twenty weeks. But the histopathological examination of kidney

has revealed no damages (Mozaffari et al 2009).

Teleost fish (Channa punctatus) has been subjected to chronic exposures of sub

lethal levels of potassium dichromate (4 and 2 mg/L). Histology of vital organs such as

gill, kidney and liver has revealed distinct lesions. Edema was prominent in the gill

lamellae. Moreover, reduction in tubular lumens and epithelial cellular hypertrophy of

renal tubules were observed. In the liver, nuclear pyknosis, increased sinusoidal spaces

and hepatocyte vacuolization and shrinkage were seen. Thus, chronic hexavalent

chromium exposure could weaken the vital functions (such as respiration, metabolic

regulation and excretion) (Ashish et al 2009). The Goldfish were exposed to different


57

quantities of hexavalent chromium in a 96hrs static renewal bioassay. They have

experienced oxidative stress which was characterized by significant morphological

alterations in kidney and liver, modulation of enzyme behavior, initiation of DNA smash

up. Microscopic study of organ morphology has shown necrosis of central vein and

degeneration of liver tissue. While high chromium levels have caused necrosis of renal

tubular epithelial cells and tubules (Velma and Tchounwou 2010).

The forty eight Wistar rats have been fed for more than two months with a

standard AIN93 diet and then replaced partly with 50, 37.5, 25 or 0% of the chromium

tanned leather residue in natura or similar levels of residue after chromium extraction. In

animals, large chromium levels have demonstrated inhibition of weight put on. Greater

number of histopathological lesions in kidneys was seen for chromium extracted

treatment offered rats (Silva et al 2010).

The histopathological effects for 5, 4 and 3mg/L of basic chromium sulphate and

9, 8 and 7mg/L of nigrosine black (tannery chemicals) were observed after 1 week,

96hrs, 48hrs, and 24hrs on gills of the fresh water fish (Catla catla). Epithelial lifting and

hemorrhage (primary and secondary lamellae), shortening (of the secondary lamellae)

and degeneration of cells (with increasing exposure), edema, lamellar fusion and

desquamation were noticed for both chemicals (Daksh and Capoor 2011).

Likewise, post chronic exposure (30 days) to chlorides of chromium, zinc and

nickel have caused some histopathalogical deviations particularly degenerative ones


58

(necrosis, vacuolation of hepatocytes, disordered hepatic cords, compression of

cytoplasm, enlarged nuclei and blood congestion in sinusoids) were reported in liver of

Labeo rohita (Bhatkar 2011).

In this research project Wistar rats after procurement were weighed and divided

into six groups. Test compounds and control were offered to them for a period of three

months. Then vital organs (lung, liver, kidney heart and brain) were carefully removed

and weighed. The tissues were processed for histological investigation and were stained

with haematoxylin eosin in accordance with Silva et al (2006).

Advantages are (1) cheapness, (2) resemblance of vital organs with human. While

continuous monitoring of animals is main disadvantage.

Materials and Methods

59


3.1.1. Sample Collection

First phase of current research project has been planned for characterization of

tannery effluent wastewater (TEW) samples. Kasur (the selected study area) is situated

between the river Ravi and river Satluj and occupies an area of 3,995 km2. Its total

population is 290,728. Pre-tanning comprises of following steps: curing, soaking, liming,

deliming, pickling, depickling as described in figure 01 (Amir et al 2008).

PIXE technique has been selected for analyzing TEW because of its various

advantages. Beside TEW, GWS (having 100 to 300 ft borehole) and GWD (having about

600 ft borehole) have been subjected to investigation as well. TEW samples have been

CHAPTER-3

MATERIALS AND METHODS

3.1. Chemical Analysis of TEW (PIXE analysis) 3.2. Microbial Characterization

3.3. CAM and Embryotoxicity Assay

3.4. Marine Shrimps Mortality Assay 3.5. Phytotoxicity Assay



60

collected randomly from the side line drainage of different domestic tanneries of Kasur as

pointed out in map (figure 02, plate 01).

BEAM HOUSE OPERATIONS

TANNING OPERATIONS

RETANNING AND DRYING

SHAVING

FLESHING AND

SPLITTING

RAW HIDESOAKINGUNHAIRING

DELIMING PICKLING TANNING

UNHAIRINGFINISHING

DRYING AND

FINISHING

WASTE WATER

WASTE WATER

Figure 01. Flow chart describing different steps of tanning operation.

The sampling was carried out in “peak production time” of the tannery industry,

i.e., May–July. Total 26 water samples, consisting of ten TEW, ten GWS and six GWD

were included in this investigation. Only one water sample from each location was

collected at single time.


61

For sample collection, glass bottles were first washed with detergent to remove

any dirt and particles, and then these were rinsed with ethanol thoroughly. After drying,

these bottles were autoclaved (Eyela, Japan) at 121°C and 15 pound pressure for 20

minutes. Effluent wastewater has been collected randomly from 10 different tanneries of

Kasur (figure 02). TEW which is being released by local tannery industries into the side

stream drains has been collected in the previously sterilized glass bottles and stored at

4°C till further use. Ground water of shallow tubewells of those tanneries (whose

effluents were collected) has been collected too for PIXE analysis (plate 01). Besides

this, municipal corporation implanted deep tubewell’s water was also taken for chemical

characterization.


62

Figure 02. Map (Kasur tannery area) indicating the locations of sample collection. Black dots are pointing the location of domestic tanneries and shallow tubewells

(100 to 300 ft) while red dented circles are indicating the position of deep tubewells (upto 600 ft).


63

Plate 01. Large pool of tannery effluent wastewater (A), sample collected from side drainage line of tannery area (B) and hand pump located near pool of stagnant

water (C).

A

B C


64

3.1.2. PIXE Analysis

For preconcentration purpose, one hundred milliliter of each water sample was

evaporated at 70°C in hot air oven (Heraeus, USA) under controlled environmental

conditions. Dried residues (plate 02B) were collected and weighed. Amount of dried

residue ranged from 0.96 to 6.63 gm for TEW samples while for GWS samples, the rang

of dried residue was 57 to 825 mg. GWD samples collected dried residue were very less

as they have range of 18.2 to 40.7 mg. Accurately weighed 5 mg residue of each water

sample was mixed with 10 microlitre of Yuttrium Nitrate Solution, Y (NO3)3 (Alfaeasr,

Johson Mathey Company) having 1.16 mg of Yuttrium as internal standard and was

spread on a 100 µm thick Mylar sheet. A smooth smear of about 10 mm was made in the

center and air dried (plate 02C).

Plate 02. Tannery effluent wastewater samples before preconcentration (A), dried residue after evaporating at 70oC. Air dried smear of TEW (C).

A B

C


65

An accelerator based analytical method for detecting various heavy metals in

water samples was devised, optimized and validated in the Center of Advance Studies in

Physics, Government College University, Lahore using 2MV tandem Pelletron

Accelerator 6SBH (6SDH-2, National Electrostatics Corporation, USA) (Plate 03 )

(Johansson and John 1988).

Plate 03. Pelletron Accelerator 6SBH located at Center of Advance Studies in Physics, Government College University Lahore, Pakistan.


66

The air dried slides were mounted on the target holder (plate 03) for irradiation

purpose. The target was maintained at an angle of 45o with respect to the proton beam.

The collimated proton beam (3.8 MeV) has irradiated each dried water sample. The

diameter and current of beam was about 3 mm and 20 nA respectively. At this current,

the dead time of detector was around 5%. Every sample was irradiated for 20 to 30

minutes to obtain reasonable counts for each element.

Si (Li) energy dispersive detector (with 30mm2 area and energy resolution of 138

eV) was utilized for the detection of characteristic X rays emitted by the elements. It was

energy calibrated using L X rays of Au. The placement of detector was at right angle to

the incident proton beam (distance of approximately 15 cm from the target). PIXE

electronics has processed signals from the detector and were displayed by multichannel

analyzer as energy (channels) versus counts (peak height) of spectrum.


67

3.2. Microbial Characterization

During second phase, I have enumerated collected TEW samples for the presence

of microorganisms within 5 to 6 hrs of collection, using a serial dilution technique in

accordance with Bergy’s Manual of Systemic Bacteriology (Krieg et al 1984).

3. 2. 1. Sample Preparation and Microbial Load

The aseptically collected total ten TEW samples in previously washed and

autoclaved glass bottles were processed in Microbiology Laboratory, University of

Veterinary and Animal Sciences, Lahore as follows: Under aseptic conditions, 1 ml of

TEW was taken with sterile syringe from sample bottle and was diluted with 9 ml of

normal saline to prepare 1st dilution (10-1) and this process was continued till 10th dilution

(10-10) was obtained (Verma et al 2001). Then, 1 ml from each prepared dilution was

mixed with 20 to 25 ml of Nutrient Agar (Lab M Limited, UK) in sterilized glass bottles

at 50oC and then it was poured in petriplate. After solidification of nutrient agar,

petriplates were incubated for 24 hrs at 37°C in incubator (ISUZU, Japan).

3. 2. 2. Isolation and Identification

The isolation and identification of bacteria has been accomplished in accordance

with the Bergy’s Manual of Determinative Bacteriology (Krieg et al 1984) following the

under mentioned steps.


68

3.2.2.1. Primary Cultivation

For primary cultivation, nutrient agar (Annexure 02) plate was used. Before

inoculation of the TEW sample, sterility of plates was also verified. After one way

streaking, the plate was incubated at 37°C. After 24 hrs, the nutrient plate was observed

for the growth of bacteria.

3.2.2.2. Purification of Bacteria and Colony Characterization

From different colonies which have appeared on plate (primary culture of

bacterium) following incubation, completely isolated colony of bacteria was selected for

further purification by two or three way streaking.

After isolation of pure colonies of various isolates of bacteria, the macroscopic

characteristics of those colonies such as color, size, and margin types were observed.

3.2.2.3. Microscopic Characterization

Gram’s staining and spore staining was also done for identification of bacteria

(Krieg et al 1984).

3.2.2.4. Biochemical Characterization

Along with staining, various biochemical tests were performed for identification


69

of isolates of bacteria (catalase test, coagulase test) too (Krieg et al 1984). No test kit was

used.

3. 2. 3. Toxic Chemicals Tolerance Assessment

The isolated bacteria were also screened for their tolerance to various compounds

of chromium. Four chromium compounds: chromium sulphate, chromium chloride,

chromium oxide and potassium dichromate (Merck, Germany) have been used for the

assessment of bacterial isolate tolerance. Aseptically, the fresh overnight peptone water

broth culture of the isolated bacteria was inoculated on nutrient agar plates supplemented

with chromium compound ranging from 600- 2600 µg/ml. Following 24 hrs incubation,

the growth of isolates was assessed visually as positive or negative (Basu et al 1997).


70


The objective of third phase was the determination of any perilous effects of TEW

on angiogenesis and embryonic development. The selected TEW9 sample having highest

concentration of chromium (Table 01) has been selected for this assay and was serially

diluted. Total three dilutions (TEWD1, TEWD2 and TEWD3) of selected TEW, solutions

of chromium chloride and potassium dichromate (Merck Germany) and control (PBS)

has been used in this assay. Sample solution’s pH was adjusted at 7±0.5, were filtered

with syringe filters (Orange Scientific Gyro, endotoxin free sterile) having pore size of

0.2µm and was kept in refrigerator at 4ºC till further use. The average weight of eggs

was sixty seven grams.

3. 3. 1. Preparation for CAM and Embryotoxicity Assay

Fresh fertilized eggs were purchased from local hatchery. 70% ethanol

(Vivantisd, USA) was used cleaning them and then air-dried. Three groups (A to C)

served as experimental groups and group D and E were +ve control while group F was –

ve control. Each group comprised of five eggs. Three experimental groups received three

dilutions TEW (TEWD1, TEWD2 and TEWD3) and positive control received chromium

chloride and potassium dichromate solutions. One group was subjected to PBS.

Incubation of eggs was maintained at 37°C and at 60 to 70% humidity.


71

Table 01. A brief description of different groups with respective treatments.

At day 4 of incubation, 4 to 5 ml of albumin was removed with the help of syringe

(plate 05) by removing shell and shell membrane and was sealed with sterile parafilm

(Parafilm® M dispenser, SPI Supplies). At day 5 of incubation, 200 µl of each sample

solution (table 01) was dispensed with micropipette (Orange scientific) on developing

CAM. Again eggs were sealed with sterile parafilm and were kept in incubator for 24 hrs

at 37oC. Post incubation, CAMs and embryos were separated and macroscopically and

microscopically evaluated for assessment of any changes in their development (Ejaz et al

2006).

No Group name No of eggs/ rats per

group

Solution applied

1 A

5 TEWD1

2 B

5 TEWD2

3 C

5 TEWD3

4 D

5 CHC

5 E

5 PDC

6 F

5 PBS


72

Plate 04. Pictorial presentation illustrating detailed procedure of CAM assay. Wiping the surface of egg with 70% ethanol (A), making hole at the broader end of egg (B), removing shell and shell membrane (C), extracting albumin (D) and applying the test dilution on the

surface of CAM.

A B C

D E


73

3. 3. 2. Image Acquisition and Image Probing System

By using high resolution Lebeca cam built in CMOS image sensor

supporting high quality VGA resolution (with 24bitRGB color), a novel system has been

build. The cam lens consisted of 5 glasses and has partaken for amelioration of picture

quality. With respect to x, y, and z proportions, serial images were taken for an objective

3 D measurement (of angiogenesis on CAMs). Ten percent skim milk was injected into

the CAM for enhancing image contrast by using 30 gauge needle.

All the obtained images were then imported to “Scan Probing Image Processing”

software (IBM Denmark). After that, x, y and z dimensions of each image were uploaded

for determination of different parameters that compute angiogenesis. By using calibration

and measurement command length and diameter of different blood vessels were

measured. 3 D surface roughness (one of the main parameters in 3D image analysis) was

also calculated for precise quantification of angiogenesis (on the surface of the CAMs).

Furthermore, vascular areas were also determined (Ejaz et al 2006).

3. 3. 3. Histological Evaluation of CAM

Very carefully CAMs were separated, fixed in 10% PFA/formaldehyde solution

(Scharallaus, Spain) and processed for histological study. The CAM tissues were then

embedded in paraffin wax and sectioned at thickness of 3 µm. Then, they were mounted

on slides and were stained with haematoxylin eosin (H & E) for usual light microscopy

(Will, Germany). The slides were investigated for elusive variations in CAMs matrix and

capillary plexus (CP) formation.


74


On the day of experiment, selected TEW9 was serially diluted in artificial

seawater. Commercially available marine shrimp, Artemia franciscan cysts were

purchased from Artemia International LLC, USA and were hatched in illuminated

incubator/photo-incubator (Eyela, Japan) in Microbiology Laboratory, University of

Veterinary and Animal Sciences, Lahore. Cysts were hatched in filtered, disinfected

artificial seawater (3% [wt/vol] artificial sea salt in H2O) which was prepared on the same

day (Wood and Ayres 1977) as shown in plate 05. Acute toxicity of TEW to marine

shrimp larvae was determined as described by Tzong-huei et al (1999). Larvae within one

day of hatching were exposed to five dilutions (D1 to D5) of TEW in 96 well cell culture

plates (10 to 15 larvae per well in 100 µl of each sample dilution) for various exposure

time (1, 24, 48 and 72 hrs) at 24oC in photoincubator. Similarly, five dilutions of CHC

and PDC have been used along with TEW dilutions for comparison purpose. The number

of dead shrimps was recorded post incubation. Tests were run in triplicate.


75

Plate 05. Hatching setup arranged for marine shrimps


76


For assessment of the dicey effect of selected TEW9 on plants, phytotoxicity (five

day maize root elongation inhibition test) assay was used. Ten maize seeds (Zea mays)

were sown in each petriplate and incubated for five days at 22 ± 2oC. Tape water was

used for making dilutions and also served as control. Total five dilutions of TEW, CHC

and PDC were used. Three replicates were run. Following specified time period, the

length of roots of germinated seeds was measured with ruler. The non germinated seeds

accounted as 0 cm for root length. Inhibition rate of root length compared to the water

control at day 5 was expressed as percentage inhibition, no of germinated seeds/ total no

of seed X100 (Javaid et al 2000).


77


For chronic toxicity evaluation purpose, Wistar rats (n=30) were purchased from

the Department of Physiology, University of Veterinary and Animal Sciences, Lahore.

At the start of the experiment, all Wistar rats were weighed and divided into total 6

groups. Each group was comprised of 5 animals, housed in same cage and having rats of

single sex and female rats were non-pregnant (OECD guidelines). Three dilutions of

TEW (TEWD1, TEWD2 and TEWD3), CHC, PDC and water (control) were offered to

rats for a period of three months (table 01). Only moderately toxic doses were used not

toxic one were offered according to OECD guidelines.

Prior to conducting the study, testing laboratory was considered for all information

regarding identity, chemical structure, physicochemical properties and the results of any

other toxicity studies of the toxicants under investigation (OECD guidelines). The rats

were housed in steel cages in a room maintained at 23 ± 2oC and alternating 12 hrs cycle

of darkness and light according to OECD guidelines (plate 07). They were provided feed

(Pre-broiler starter crumbles [High Tech Feed], supplemented with 0.25% milk,

5%vegetable oil and 1% berseem meal) and sample water ad libitum (OECD guidelines).

At the end of 3rd month, the rats were starved for 24 hrs and then sacrificed by

decapitation; vital organs (lung, liver, kidney, heart and brain) were carefully removed

and weighed. Any gross lesions observed were recorded (OECD guidelines). The tissues

were fixed in 10% PFA (formaldehyde solution) and processed for histological

investigation. The tissues were embedded in paraffin wax, sectioned for 3 µm thickness,


78

mounted on slides and were stained with haematoxylin eosin for routine light

microscopy. The slides were investigated for any deleterious effects of the TEW samples

(Silva et al 2006).

Plate 06. Rats receiving TEWD1.

Results

79


4. 1. 1. Calibration of Accelerator for Analysis of Water Samples

The accelerator was calibrated for K X-rays using known salt mixture having 12

elements between Z 11 to Z 38 along with Y (NO3)3 as internal standard (because its inert

compound) and for L X-rays using Ba, Hg and Pb salts. The sensitivity curve relative to

Yttrium was plotted and sensitivity values for all elements were tabularized to ensure the

accuracy of our calibration, a target, IAEA SOIL 7 (reference material) was prepared by

again using yttrium nitrate as the internal standard in the same way as described earlier. The

results obtained for the soil sample 7 are presented in table 02.

Concentrations of most of the elements are within or close to the range specified by

International Atomic Energy Agency except for Sr, Na and Mg. Error for S, K, Ca Mg, Al,

Ti and Fe elements are less than 7 percent and for the elements Na, Mn and Sr error is 11 to

42 percent. This has validated the experimental method used for the analysis of water

samples.

CHAPTER-4

RESULTS

Results

80

Table 02. PIXE results for reference material (IAEA soil 07)

Element

Accelerator lab results

(mg/kg)

n=3

International Atomic

Energy Agency information value

(mg/kg)

Mg 5060 ± 330 11000 – 11800

Al 51600 ± 2830 44000 – 51000

Si 160400 ± 8660 169000 – 201000

Ca 211260 ± 11408 157000 – 174000

K 12300 ± 713 11300 – 12700

Mn 740 ± 140 604 – 650

Ti 3340 ± 240 2600 – 3700

Fe 27400 ± 1510 25300 – 26300

Sr 2300 ± 966 103 – 114

Results

81

Typical PIXE spectrum of the tannery effluent wastewater (TEW) is shown in figure

03. Peaks generated by Yttrium (internal standard) are evident on right side of spectrum.

Prominent peak of chromium can also been seen in the spectrum of TEW9 sample.

From the PIXE spectrum, the concentration/ levels of heavy metals were calculated

by using GUPIX software. The detailed elemental composition of all TEW samples is

presented in table 03. The diversity in composition of TEW samples is quite prominent (table

03), TEW9 which was collected during curing stage of tanning, has high levels of the

detected elements as compared to rest of samples. TEW1 and TEW8 (liming stage) varies in

their composition too while others were quite similar as shown in dendrogram in figure 04B.

Results

82

Figure 03. PIXE spectrum of tannery effluent wastewater (TEW9).

Results

83

Table 03. Detected concentrations of various elements in the tannery effluent wastewater samples (n=3).

Concentrations are in mg/L.

TEW1 TEW2 TEW3 TEW4 TEW5 TEW6 TEW7 TEW8 TEW9 TEW10 Mg 109.34

± 5.50 1309.03 ± 51.95

59.07 ± 2.95

444.91 ± 22.24

345.24 ± 17.3

104.86 ± 5.24

184.42 ± 9.22

1161.98 ± 58.10

710.49 ± 35.52

126.54 ± 6.33

Si 93.52 ± 5.6

N.D

264.46 ± 15.90

318.96 ± 19.14

143.72 ± 8.62

119.73 ± 7.18

255.57 ± 15.33

N.D

272.33 ± 16.34

191.10 ± 11.46

S 1697.25 ± 203.70

9079.07 ± 1089.50

1784.56 ±214.15

2586.31 ± 310.36

846.40 ± 50.80

983.70 ± 10.04

4814.89 ± 577.80

10691.20 ± 1282.90

8160.64 ± 979.30

364.55 ± 43.75

Cl 1821.9 ± 91.10

13901.78 ± 695.10

12643.50 ± 632.20

19215.88 ± 96.10

5041.83 ± 252.10

4232.10 ± 211.60

269.10 ± 13.45

22245.13 ± 1112.30

25827.83 ± 1291.40

3991.20 ± 199.60

K 57.85 ± 2.90

44.52 ± 2.23

125.44 ± 6.30

100.59 ± 5.03

65.88 ± 3.30

80.672 ± 4.03

68.102 ± 43.05

16.04 ± 0.80

216.84 ± 10.84

153.10 ± 7.70

Ca 170.66 ± 4.31

468.60 ± 18.74

1311.54 ± 52.50

645.50 ± 25.82

221.37 ± 8.90

25.39 ± 1.02

321.40 ± 12.90

1457.80 ± 58.30

788.67 ± 31.55

79.65 ± 3.98

Cr 529.99 ± 95.40

21.054 ± 3.80

3.14 ± 0.60

314.04 ± 56.50

20.70 ± 3.73

4.03 ± 0.73

1206.60 ± 184.80

1213.81 ± 218.50

1266.85 ± 228.03

4.39 ± 0.80

Sr 27.405 ± 5.50

150.90 ± 30.20

40.76 ± 8.20

63.75 ± 12.75

50.40 ± 10.10

55.99 ± 11.20

123.32 ± 24.70

97.39 ± 19.50

400 ± 80

N.D

Mn 5.87 ± 3.52

N.D

N.D

N.D

N.D

N.D

12.90 ± 7.40

N.D

34.162 ± 20.50

0.59 ± 0.35

Fe 6.98 ± 0.69

4.062 ± 0.41

4.56 ± 0.46

N.D

N.D

0.12 ± 0.011

13.26 ± 1.33

21.26 ± 2.10

16.21 ± 1.60

0.894 ± 0.09

Ni 0.07 ± 0.05

N.D

N.D

N.D

N.D

N.D

3.00 ± 2.3

1.61 ± 1.21

1.68 ± 1.30

N.D

Zn 1.06 ± 1.24

N.D

N.D

4.79 ± 2.63

N.D

N.D

N.D

2.46 ± 1.35

5.71 ± 3.14

0.48 ± 0.30

Results

84

TEW7TEW8TEW9TEW6TEW10TEW5TEW4TEW3TEW2TEW1

80.11

86.74

93.37

100.00

Tannery effluent wastewater samples

Sim

ilarit

y

DendrogramSingle Linkage, Correlation Coefficient Distance

Figure 04. Graph presenting detected concentration of various elements (A) and dendogram illustrating the percentage similarities between all TEW samples (B).

B

A

Results

85

The range for detected concentration of chromium in tannery effluent samples is 3.14

to 1266.8 mg/L. Highest chromium concentration was present in TEW9, which was collected

during drum stage of tanning cycle because 20 - 40% of applied chrome is released into the

environment as such (Fahim et al 2006). As salts of chromium, ammonium, chloride, and

sulfide are used in tanning industry commonly (Amir et al 2008). Mg, Si, S, Cl, K, Ca, Mn,

Fe, Ni, Zn and Sr have been detected in all samples of tanneries which were in accordance to

those results reported by Tariq et al (Tariq et al 2005).

Worldwide, the quality of ground water is a growing interest these days (Midrar et al

2005). So, we have analyzed ground water of shallow tubewells of same tanneries too.

Surprisingly, water from these tubewells have presented chromium from 0.13 to 0.68 (table

04). The concentration of chromium in GWS has demonstrated a relationship with their

depth. Depth of shallow tubewells varied from tannery to tannery (100 to 300 ft). Lower was

the depth, higher concentration of chromium was determined.

But when ground water samples from deep tubewells was analyzed for the presence

of chromium, it was almost absent in them, only one outlet has presented chromium (table

05) which may be merely because of corroded pipe lines or leakage of pipes in that area.

Spectrum of ground water of deep tubewell without chromium is shown in figure 07.

Results

86

Figure 05. PIXE spectrum of ground water of shallow tubewell (GWS7).

Results

87

Table 04. Detected concentration of elements present in the ground water of shallow tubewells (n=3).

GWS1 GWS2 GWS3 GWS4 GWS5 GWS6 GWS7 GWS8 GWS9 GSW10

WHO/ EPA limits mg/L

Mg 544.73 ± 27.24

59.12 ± 2.96

128.21 ± 6.41

172.97 ± 8.65

41.82 ± 2.10

17.79 ± 0.89

43.96 ± 2.20

46.15 ± 2.31

12.69 ± 6.34

114.20 ± 5.71

150

Si 508.60 ± 30.52

67.73 ± 4.10

38.86 ± 2.33

1814.99 ± 108.90

28.88 ±1.73

25.73 ± 1.54

24.82 ± 1.49

13.44 ± 0.81

20.89 ± 1.25

181.85 ±10.91

4

S

5626.60 ± 675.20

229.78 ± 27.60

381.26 ± 45.80

185.20 ± 22.22

85.61 ± 10.30

94.88 ± 11.40

76.17 ± 9.14

61.75 ± 7.40

50.15 ± 6.02

273.52 ±32.82

500

Cl 4184.30 ±209.22

380.33 ±19.02

1383.13 ± 69.16

2188 ± 109.4

655.40 ± 32.80

174.74 ± 8.74

390.72 ± 19.54

255.41 ±12.77

37.18 ± 1.86

635.79 ±31.79

250

K 169.97 ± 8.50

59.70 ± 2.98

32.21 ± 1.61

49.52 ± 2.48

51.32 ± 2.57

14.13 ± 0.71

28.62 ± 1.43

4.26 ± 0.21

6.71 ± 0.34

53.49 ± 2.67

20

Ca 533.24 ± 21.33

131.51 ± 5.26

90.76 ± 3.63

67.41 ± 2.69

10.21 ± 0.41

29.33 ± 1.17

12.09 ± 0.48

29.25 ± 1.17

26.46 ± 1.06

144.52 ± 5.78

200

Sc N.D

N.D

N.D

N.D

0.92 ± 0.14

N.D

0.49 ± 0.07

0.652 ±0.09

N.D

N.D

NA

Cr 0.81 ± 0.00

0 .35 ±0.06

0.68 ± 0.12

0.71 ±0.128

0.29 ±0.05

0 .21 ±0.04

0.39 ± 0.07

0.36 ± 0.07

0.13 ±0.02

0.25 ±0.05

0.1

Mn N.D

0.34 ±0.21

N.D

N.D

N.D

N.D

N.D

0.09 ±0.06

0.04 ±0.03

N.D

0.5

Fe 6.74 ± 0.67

0.98 ±0.09

0.37 ± 0.04

N.D

0.21 ±0.02

0.19 ± 0.02

0.134 ± 0.01

0.001 ±0.0001

N.D

0.36 ± 0.04

0.3

Ni N.D

0.21 ± 0.16

N.D

N.D

N.D

0.05 ± 0.03

0.10 ± 0.07

0.03 ±0.010

N.D

0.14 ± 0.11

0.02

Cu N.D

N.D

0.16 ± 0.11

N.D

N.D

N.D

0.06 ± 0.04

N.D

0.03 ±0.02

N.D

NA

Zn N.D

0.28 ±0.15

N.D

N.D

N.D

N.D

0.00 ± 0.00

0.09 ±0.05

0.08 ±0.042

0.33 ± 0.20

2


Results

88

GWS6GWS4GWS9GWS7GWS5GWS8GWS3GWS10GWS2GWS1

88.09

92.06

96.03

100.00

Ground water of shallow tubewells

Sim

ilarit

y


Figure 06. Graph (A) presenting detected levels of various elements and dendogram (B) illustrating the percentage similarities between all GWS samples.

B

A

Results

89

Figure 07. PIXE spectrum of ground water of deep tubewell (GWD1).

Results

90

Table 05. Detected concentration of elements present in the ground water of deep

tubewells (n=3).

GWS1 GWS2 GWS3 GWS4 GWS5 GWS6 GWS7 GWS8 GWS9 GSW10

Mg 544.73 ± 27.24

59.12 ± 2.96

128.21 ± 6.41

172.97 ± 8.65

41.82 ± 2.10

17.79 ± 0.89

43.96 ± 2.20

46.15 ± 2.31

12.69 ± 6.34

114.20 ± 5.71

Si 508.60 ± 30.52

67.73 ± 4.10

38.86 ± 2.33

1814.99 ± 108.90

28.88 ±1.73

25.73 ± 1.54

24.82 ± 1.49

13.44 ± 0.81

20.89 ± 1.25

181.85 ±10.91

S

5626.60 ± 675.20

229.78 ± 27.60

381.26 ± 45.80

185.20 ± 22.22

85.61 ± 10.30

94.88 ± 11.40

76.17 ± 9.14

61.75 ± 7.40

50.15 ± 6.02

273.52 ±32.82

Cl 4184.30 ±209.22

380.33 ±19.02

1383.13 ± 69.16

2188 ± 109.4

655.40 ± 32.80

174.74 ± 8.74

390.72 ± 19.54

255.41 ±12.77

37.18 ± 1.86

635.79 ±31.79

K 169.97 ± 8.50

59.70 ± 2.98

32.21 ± 1.61

49.52 ± 2.48

51.32 ± 2.57

14.13 ± 0.71

28.62 ± 1.43

4.26 ± 0.21

6.71 ± 0.34

53.49 ± 2.67

Ca 533.24 ± 21.33

131.51 ± 5.26

90.76 ± 3.63

67.41 ± 2.69

10.21 ± 0.41

29.33 ± 1.17

12.09 ± 0.48

29.25 ± 1.17

26.46 ± 1.06

144.52 ± 5.78

Sc N.D

N.D

N.D

N.D

0.92 ± 0.14

N.D

0.49 ± 0.07

0.652 ±0.09

N.D

N.D

Cr 0.81 ± 0.00

0 .35 ±0.06

0.68 ± 0.12

0.71 ±0.128

0.29 ±0.05

0 .21 ±0.04

0.39 ± 0.07

0.36 ± 0.07

0.13 ±0.02

0.25 ±0.05

Mn N.D

0.34 ±0.21

N.D

N.D

N.D

N.D

N.D

0.09 ±0.06

0.04 ±0.03

N.D

Fe 6.74 ± 0.67

0.98 ±0.09

0.37 ± 0.04

N.D

0.21 ±0.02

0.19 ± 0.02

0.13 ± 0.01

0.001 ±0.0001

N.D

0.36 ± 0.04

Ni N.D

0.21 ± 0.16

N.D

N.D

N.D

0.05 ± 0.03

0.10 ± 0.07

0.03 ±0.01

N.D

0.14 ± 0.11

Cu N.D

N.D

0.16 ± 0.11

N.D

N.D

N.D

0.06 ± 0.04

N.D

0.03 ±0.02

N.D

Zn N.D

0.28 ±0.15

N.D

N.D

N.D

N.D

0.00 ± 0.00

0.09 ±0.05

0.08 ±0.042

0.33 ± 0.20


Results

91

GWD1GWD6GWD3GWD5GWD4GWD2

95.35

96.90

98.45

100.00

Ground water of deep tubewells

Sim

ilarit

y


Figure 08. Graph (A) presenting detected concentration of various elements and

dendogram (B) illustrating the percentage similarities between all GWD samples.

B

A

Results

92

Dendrogram shown in figure 09 is presenting the percentage similarity among TEW,

GWS and GWD. Level of similarity of all types of water samples composition is up to 85%

which is further strengthening, a strong impact of tannery effluent wastewater pollution on

underground water table of Kasur city.

TEW7

GWS6

GWS4

GWD1

GWS1

0GW

D6GW

D3GW

S1GW

S9GW

D2GW

S2GW

D5GW

D4TE

W8GW

S8TE

W9GW

S3TE

W6GW

S7GW

S5

TEW

10TE

W5TE

W4TE

W3TE

W2TE

W1

86.07

90.71

95.36

100.00

Water samples

Sim

ilarit

y


Figure 09. Dendogram illustrating the relationship between all the three types of water samples (TEW, GWS and GWD) Kasur.

All water samples (TEW, GWS and GWD) were compared statistically by applying

Analysis of Variance Technique at 95% confidence interval, for chromium concentration

significant (P=0.018) differences was obtained.

Results

93

4.2. Microbial Characterization

Collected TEW samples were also enumerated for bacteria within 5 to 6 hrs of

collection using a serial dilution technique in accordance with Bergy’s Manual of

Determinative Bacteriology (Krieg et al 1984).

4. 2. 1. Microbial Load

Under aseptic conditions, collected TEW sample was serially diluted using tenfold

serial dilution in normal saline. Then each dilution was mixed with molten nutrient agar at

45oC and was poured in sterilized petriplate. After solidification of the nutrient agar, the

plates were incubated at 37oC in incubator.

Following 24hrs, the total number of viable bacteria in individual sample was

determined as CFU/ml (Verma et al 2001). No of bacterial colonies gradually decreased with

increase in dilution factor. Plates having colonies 30 to 300 were selected for counting.

Colonies were counted by using colony counter. The total no of viable bacteria in all TEW

samples is presented in table 06.

Results

94

Table 06. Total number of viable bacteria in tannery effluent wastewater samples

Sample

Name

Dilution number

having 30-300

colonies

Total number of

bacterial colonies in

selected plate

No of viable bacteria in

water sample

(CFU/ml)

A 104 239 2.39 X 106

B 103 150 1.5 X 105

C 105 300 3.0 X 107

D 103 75 7.5 X 104

E 105 55 5.5 X 106

F 104 60 6.0 X 105

G 105 75 7.5 X106

H 104 132 1.32 X 106

I 105 80 8.0 X 106

J 104 95 9.5 X 105

Control

(Nestle)

N.D N.D N.D

Results

95

Plate 07. Plates presenting different types and number of colonies of viable bacteria in serial dilutions (A=10-1, B=10-2, C=10-3, D=10-4, E=10-5, F=10-6) of collected TEW

sample.

A B

C D

E F

Results

96

4. 2. 2. Isolation of Bacteria

The isolation and identification of bacteria was accomplished in accordance with the

Bergy’s Manual of Systemic Bacteriology (Krieg et al 1984) following the under mentioned

steps.

4.2.2.1. Purification of Bacteria

For purification of bacterial culture, all types of colonies were selected from the

primary mixed culture plate (plate 07). Nutrient agar plates were used and before inoculation,

sterility of plates has been verified (through overnight incubation). Sample from an isolated

single colony of each type was inoculated (two-way streaking) on nutrient agar plate and

incubated at 37oC. Purified isolated single colony of each bacterium was then obtained (Plate

08).

Plate 08. Two way streaking (for purification) of bacterial culture. Single isolated

colony can be seen as pointed by arrows.

Results

97

4. 2. 3. Identification of Bacteria

All the isolated bacteria were identified on the basis of various characteristics.

4.2.3.1. Colony Description and Microscopic Characterization

The macroscopic characteristics of isolated purified colonies (such as color, size,

elevation and margin type) were recorded.

Gram’s staining was done for microscopic identification of the bacterial isolates and

results were observed under the oil immersion objective (100X) of compound microscope

(Plate 09). Moreover spore staining was done for identification of bacteria.

Plate 09. Microscopic representations of Gram positive bacillus at 100X

Results

98

4.2.3.2. Biochemical Characterization

Biochemical tests were also performed for identification of isolated bacteria such as.

Lactose Fermentation Test

Lactose fermentation test was performed on MacConkey’s agar and results were

recorded after 24 hrs of incubation. Pink colonies of lactose fermenting bacteria and yellow

or colorless colonies of lactose non fermenting bacteria (Plate 10) were observed.

Plate 10. Representative of Lactose fermenting bacteria (A) and Lactose non fermenting bacteria (B) on MacConkey’s Agar

A B

Results

99

Indole Production Test

Indole test was performed on all the purified bacterial isolate and results were

recorded. Pink to wine colored ring after addition of reagent was observed showing positive

results and pale yellow colored ring was observed in case of negative results ( Plate 11).

Plate 11. Interpretation of the results for Indole production test using Tryptophan

Broth (A: +ve and B: -ve)

Voges Proskauer Test

VP test was also performed for the identification of bacterial isolates and results were

noted down. Pink or red color at the surface of the medium if the sample was positive and

yellow or copper color at the surface of the medium was produced by the VP-negative

isolates (Plate 12).

AB

Results

100

Plate 12. Interpretation of the results for Vogus proskauer test using Glucose Phosphate Buffered Saline (A: +ve and B: -ve)

Methyl Red Test

MR test was performed on purified bacterial isolates and results were observed. Pink

to red color was the indication of positive result while Pale yellow color indicated the

negative results (Plate 13).

Plate 13. Interpretation of the results for MR-test using Glucose Phosphate Buffered Saline (A: +ve and B: -ve)

A B

A B

Results

101

Citrate Utilization Test

Citrate utilization test was performed on Simmon’s Citrate Agar and results were

recorded. Development of Blue color after incubation time was positive result (Plate 14).

Plate 14. Interpretation of the results for Citrate utilization test using Simmon Citrate

Agar (A: +ve and B: -ve)

Urease Production Test

Urease production test was performed on bacterial isolates and results

were recorded. Positive results were indicated by appearance of pink color. Negative

results were indicated by no change in color (Plate 15).

Plate 15. Interpretation of the results for Urease production test using Urea Broth (A:+ve and B: -ve).

A

A

B

B

Results

102

Triple Sugar Iron (TSI) Agar

The production of H2S by the bacteria was confirmed by the

appearance of black color on the slant. Bacterial cultures were inoculated and results were

recorded (Plate 16).

Plate 16: Interpretation of the results for H2S Production test using TSI Agar slants (A:+ve and B: -ve)

A B

Results

103

4. 2. 4. Identification Profile for Bacillus azotoformans

Bacillus azotoformans was identified after the application of biochemical tests and

results were presented in Table 07. Representative identification plates for Bacillus

azotoformans are presented at Plate 17.

Plate 17. Representative identification plates for Bacillus azotoformans. Colony characteristics (A), microscopic view (B), starch utlization; -ve (C) catalase test (D) and

citrate utilization test (+ve)

A B

C D E

Results

104

On the basis of results of biochemical tests, colony characteristics, staining results

following bacterial isolates were identified: Corynebacterium kutsceri (5.3%), E. coli

(13.2%), Micrococcus varians (7.9%), Staphylococcu aureus (13.2%), Staphylococcus

epidermidis (10.5%), Bacillus subtilis (18.4%), Bacillus cereus (5.3%), Bacillus

azotoformans (8%), Bacillus megaterium (13.2%), Bacillus laterosporus (5.3%) as shown in

figure 10.

Figure 10. Graphical percentage illustration of detected bacterial isolates.

Results

105

Table 07. Total number and types of isolated and identified bacterial isolates from TEW samples.

Sample

Name

Total number

of viable

bacteria

Total types of

isolated and

purified bacteria

Identified species of isolated

bacteria

A 2.39 X 106 04 1. Corynebacterium kutsceri

2. E. coli

3. Bacillus megaterium

4. Bacillus subtilis

B 1.5 X105 05 1. Staphylococcus epidermidis

2. Bacillus azotoformans

3. Staphylococcu aureus

4. Bacillus laterosporus

5. Micrococcus varians

C 3.0 X 107 03 1. Bacillus megaterium


3. E. coli

D 7.5 X 104 04 1. Bacillus subtilis

2. Corynebacterium kutsceri



E 5.5 X 106 04 1. Micrococcus varians



4. E. coli

Results

106

F 6.0 X105 04 1. Bacillus subtilis

2. E coli

3. Staphylococcu epidermidis

4. Bacillus cereus

G 7.5 X 106 03 1. Bacillus laterosporus



H 1.32 X 106 03 1. E. coli



I 8.0 X 106 04 1. Staphylococcus epidermidis

2. Bacillus cereus.



J 9.5 X 105 04 1. Bacillus megaterium

2. Staphylococcus epidermidis


4. Micrococcus varians

Control/

Nestle

water

N. D N.D N.D

Results

107

4. 2. 4. Toxic Chemicals Tolerance Assessment

The isolated and identified bacteria were further screened for their tolerance towards

various toxic chromium compounds (chromium sulphate, chromium chloride, chromium

oxide and potassium dichromate). The plate 18 is presenting growth response of Bacillus

subtilis to exposed levels of chromium sulphate (600, 1000, 1400, 1800, 2200 and 2600

µg/ml).

Corynebacterium kutsceri has proven most sensitive while assessing the chromium

tolerance as it has tolerated 1800 µg/ml of chromium sulphate, 1400 µg/ml of chromium

chloride and chromium oxide and 600 µg/ml of potassium dichromate. After it,

Staphylococcus epidermidis has tolerated 1800 µg/ml of chromium sulphate and chromium

chloride, 2200 µg/ml chromium oxide and 1000 µg/ml of potassium dichromate. While

Micrococcus varians and Staphylococcus aureus have tolerated almost same levels of

chromium compounds that is 2200 µg/ml of chromium sulphate and chromium chloride,

1800 µg/ml chromium oxide, 1000 µg/ml (for Micrococcus varians) and 1400 µg/ml (for

Staphylococcus aureus) of potassium dichromate. All the Bacillus species and E coli have

tolerated high levels of chromium compound such as 2600 µg/ml of chromium sulphate,

2200 µg/ml of chromium chloride, 2200 µg/ml of chromium oxide (except for Bacillus

azotoformans, 2600 µg/ml) and 1800 µg/ml of potassium dichromate (except for Bacillus

megaterium, 2200 µg/ml).

Results

108

Plate 18. Plates presenting the chromium tolerance response of Bacillus subtilis for

increasing concentration of chromium sulphate (A:600 µg/ml, B:1000 µg/ml, C:1400 µg/ml, D:1800 µg/ml, E:2200 µg/ml and F:2600 µg/ml).

D C

F E

B A

Results

109

Table 08. Table presenting the comparative tolerance levels of isolated bacteria for four salts of

chromium.

Maximum tolerated level of chromium compounds

Bacterial isolate Chromium

sulphate

µg/ml

Chromium chloride

µg/ml

Chromium oxide

µg/ml

Potassium dichromate

µg/mlCorynebacterium

kutsceri

1800 1400 1400 600

E. coli

2600 2200 2200 1800

Micrococcus varians

2200 2200 1800 1000

Staphylococcus aureus

2200 2200 1800 1400

Staphylococcus epidermidis

1800 1800 2200 1000

Bacillus subtilis

2600 2200 2200 1800

Bacillus cereus

2600 2200 2200 1800

Bacillus azotoformans

2600 2200 2600 1800

Bacillus megaterium

2600 2200 2200 2200

Bacillus laterosporus

2600 2200 2200 1800

Results

110


In current phase, I have examined the effect of TEW9, which has presented highest

level of chromium (table 03) on angiogenesis. I have recorded changes in vascular

organization of CAM and structural deformations in CAM by the application of three

dilutions of TEW (TEWD1, TEWD2 and TEWD3), CHC and PDC with reference to control

group treated with Phosphate Buffer Solution (PBS).

4.3.1. Visual Architectural Alterations in Vascular Organization of

CAM

Macroscopic and microscopic evaluations have been utilized to screen structural

deformations caused by application of TEWD1, TEWD2, TEWD3, PDC and CHC. The

CAM of control (PBS) has intensive meshwork of vasculature and having a tree like

branching pattern with equal distribution, covering the whole area of the CAM. The vascular

architecture of the CAM appeared originating from the main “Y” branch of blood vessel,

which was further differentiated into primary, secondary and tertiary branches (Plate 19).

Highly aggravated antiangiogenic effects were observed following application of the

TEWD1, TEWD2, CHC and PDC. These have caused marked disturbances and disorganized

vascular architecture with decrease in number of vessels in the normal branching pattern of

the blood vessels. There was also thinning of major blood vessels and fading of tertiary blood

vessels, thus distressing the complete CAM vascular network. Moreover, the total area of the

treated CAMs was remarkably reduced (plate 19).

Results

111

Plate 19. Macroscopic evaluation of chicken CAM on day 6 of incubation. Note the well defined architecture of CAM blood vessels in control group with well developed area of CAM (F), while CAM

treated with various dilutions of TEW resulted in extensive reduction in the total area of CAM representing extensive antiangiogenic activities (A, B and C). Area and total number of blood vessels was

also less in CAM which was offered CHC (D) and PDC (E).

A

B

C

D

E

F

Results

112

4.3.1.1. Computerized Evaluation of the Structural Malformations in

CAM

“Scan Probing Image Processing” software was used for the quantification of any

changes in the developing blood vessels of CAMs of all groups.

After the application of TEWD1, tree like branching pattern of CAM blood vessels

was severely dislocated. The distance between secondary blood vessels was not uniform as

compared to control. Blood vessels are located at far distances from neighboring blood

vessels leaving some area uncovered (plate 20A). Likewise uneven blood vessels distribution

is also evident in the CAM of PDC (plate 20E). Long thin blood vessels lacking many

branches were present on CAMs treated with TEWD2 (plate 20B) and PDC (plate 20D).

CAM exposed to TEWD3 is somewhat analogous to control. While the blood vessels of

control are organized in a regular pattern and uniformly covering the entire area of CAM

(plate 20F).

Results

113

Plate 20. Topographic explanation of CAMs depicting variations in blood vessel branching pattern. Parallel and progressively long blood vessels formation like tree branches is present in control (F). While blood vessels branching pattern is highly disturbed in TEWD1 (A) and PDC.

The TEWD2 (B) TEWD3 (C) and PDC (E) was comparatively better.

A

B

C

D

E

F

Results

114

4.3.1.2. Tertiary Blood Vessels Development

The maximum number of tertiary blood vessels is quite prominent in the convoluted

colored image of control CAM (plate 21F) and 3D image (plate 22F). In contrast the number

of tertiary blood vessels is tremendously less or almost absent in TEWD1 (plate 21A, 22A)

and PDC (plate 21E, 22E) offered CAMs. Only primary and secondary blood vessels are

evident. While in the CAMs of TEWD2 (plate 21B, 22B) and CHC (plate 21D, 22D) the

number of tertiary blood vessels is also low. The convoluted image of TEWD3 (plate 21C,

21C) is comparable to control (plate 21F, 22F).

Results

115

Plate 21. Colored convoluted topographical images of CAMs indicating the presences of large number of tertiary blood vessels in control (F). Number of tertiary blood vessels is significantly

lower in TEWD1 and PDC CAMs. CAM of TEWD3 is comparable to control. TEWD1 (A), TEWD2 (B), TEWD3 (C) and PDC (E) was comparatively better.

A

B

C

D

E

F

Results

116

Plate 22. 3D micrographs, illustrating presence of tertiary blood vessels on CAMs of different groups. Maximum number is present in control CAM (F) while significantly less is present on

TEWD1 (A) and PDC (E).

A

B

C

D

E

F

Results

117

4.3.1.3. Decrease in Bearing Area of CAMs

Total area of the CAM was drastically reduced for the CAMs treated with TEWD1, PDC,

CHC and TEWD2 (plate 19). For holistic quantification of angiogenesis, angular spectrum

(graph presenting angular arrangement of blood vessels on CAM) and abbot curve (graph

illustrating height of blood vessels on CAM) were also calculated. In control, height of abbott

curve was 47 mm which was higher than all other treatment groups along with maximum

bearing area (plate 23F). The lowest height i-e., 20 mm (plate 23A) was noted for the CAM

blood vessel that was offered TEWD1. Height of abbott curve for the blood vessels of CAM

treated with PDC was also considerably low that is 22 mm (plate 23E). While the height of

abbott curves of CHC (plate 23D), TEWD2 (plate 23B) and TEWD3 (plate 23C) is 28, 39

and 44 mm respectively.

4.3.1.4. Disturbances in Angular Distribution and Dimensions

Angular spectrum was calculated for the determination of angular distribution of the

blood vessels on CAMs of all groups. Blood vessels on CAM of control were evenly

distributed observing maximum area coverage with amplitude of 16mm (plate 24F). The

amplitude obtained for TEWD1 (plate 24A), PDC (plate 24E), CHC (plate 24D), TEWD2

(plate 24B) and TEWD3 (plate 24C) are 4, 6, 8 10 and 12mm respectively. Considerable

reduction in amplitude was noticed only for TEWD2 and PDC. The dimensions of blood of

control is uniform (plate 25F) while among other treatment groups, uneven dimensions was

obtained for TEWD1 (plate 25A), TEWD2 (plate25B), CHC (plate 25D) and PDC (plate

25E).

Results

118

Plate 23. Abbott curve measurement’s graphical outline on CAMs of different groups. Note the impediment in height among different treated groups. TEWD1 (A), TEWD2 (B) TEWD3 (C),

CHC (D), PDC (E) and control (F).

A

B

C

D

E

F

Results

119

Plate 24: Angular spectrum’s graphical outlines for CAMs of different treatment groups. Note the variations in amplitudes for TEWD1 (A), TEWD2 (B) TEWD3 (C), CHC (D), PDC |(E) and

control (F).

A

B

C

D

E

F

Results

120

Plate 25. Graphical representation of dimensions of blood vessels on CAMs of different groups.

Note the deviations particularly in TEWD1 (A), TEWD2 (B), CHC (D) and PDC (E) offered groups. While dimensions of Control (F) and TEWD3 (C) is quite comparable.

A

B

C

D

E

F

Results

121

4.3.1.5. Decrease in Diameter of Primary, Secondary and Tertiary

Blood Vessels

For measuring diameter of all types of blood vessels (primary, secondary and tertiary)

growing on CAM, Scan probing image processing software was utilized. Significant

reductions (P = 0.015) in the diameter of primary, secondary and tertiary blood vessels were

observed for treated CAMs (figure 11).

Results

122

Plate 26. Scan probing image processing software was used for the measurement of the

diameter of blood vessels on CAM. Photomicrograph explanation of quantification of primary, secondary and tertiary blood vessel diameter of CHC exposed CAM.

Results

123

4.3.1.6. Quantification of Different Parameters of 3D Surface Roughness

Total five roughness parameters that is Sa (average roughness of surface), Sq (root mean

square values), Sz (maximum height of the surface), Sk (core surface roughness) and Spk (reduced

summit height) have been calculated for the evaluation of topographic information of CAMs

of all groups (figure 12) and significant reduction was observed for TEWD1 and PDC

(P=0.002).

Results

124

Figure 11: Graphical outline demonstrating comparison among diameter of blood vessels in different groups. Note a significant reduction (P = 0.015) in the diameter of blood vessels treated with TEWD1 and PDC. Concentration of chromium in TEW1= 0.663mg/ml,

TEW2= 0.0663 and TEW3= 0.0063mg/ml.

Figure 12. Graph illustrating comparison between different roughness parameters of all groups. Sa (average roughness of surface), Sq (root mean square values), Sz (maximum height

of the surface), Sk (core surface roughness) and Spk (reduced summit height). Highly ssignificant reduction (P = 0.002) for roughness parameters was observed for TEWD1.

Concentration of chromium in TEW1= 0.663mg/ml, TEW2= 0.0663, TEW3= 0.0063mg/ml.

0

0.5

1

1.5

2

2.5

TEWD1 TEWD2 TEWD3 CHC PDC PBS

Dia

met

er o

f B

loo

d V

esse

ls (

mm

)

Primary BV

SecondaryBV

Teritary BV

Results

125

4.3.1.7. Histological Evaluation of CAMs

The H & E stained microphotographs of CAMs of all groups are presented in plate

27. CAM belonging to control group has a twice layered ectoderm and was well stabilized

with sandwiched bunch of capillary plexuses (directly adhered to basal lamina) which help in

exchange of waste material and gas at air interface of CAM. Fibroblast and mesodermal

collagen fiber’s mash work was quite evident. The mesodermal blood vessel appeared

strongly intact (with exterior smooth muscle sheet and interior linings of endothelial cells). A

dense endoderm was completely casing the interior (plate 27F).

CAMs exposed to TEWD1 and TEWD2 were of considerably small size and have

demonstrated significant reduction of ectodermal membrane which has resulted in less

capillary plexuses (CPs) generation on peripheral surface. Besides this, mesodermal collagen

mesh work was somewhat destroyed too (plate 27A and 27B). While TEWD3 offered CAM

was somewhat identical to control CAM except the number of CPs was less. Network of

mesodermal collagen was less compact than control (plate 27C). Treatment with CHC has

also caused development of fragile ectoderm with depreciating CPs mash work. Extracellular

matrix of mesoderm was considerably destroyed too. Crumpled blood vessel and endodermal

thinning was also noticed (plate 27D). While harsh changes were also clear in PDC treated

CAM, illustrating less number of CPs and small sized CAM (plate 27E).

Results

126

Plate 27. Light microphotographs showing variable capillary plexus (CP) formation in

CAMs of different treatment groups (magnification: X10). In control (F), numerous CPs formed along the ectoderm. Blood vessel of considerably small lumen size is seen TEWD1 (A) and TEWD2 (B): small sized blood vessel and few capillary PCs can be seen in TEWD3 (C): formation of scanty CPs in CHC (D) offered group: distorted CAM matrix with sparse CP formation beneath the ectoderm and small sized CAM with reduced number of CPs was

observed in PDC (E).

A

B

C

D

E

F

Results

127

4.3.2. Mortality and Macroscopic Lesions in Embryos

Developing 5 day old embryos have been offered 200 µl of three dilutions of TEW

(TEW1, TEW2 and TEW3) and solutions of two salts of chromium (PDC and CHC) and

PBS (control) for 24 hrs. Post incubation, the no of dead embryos were counted and

presented in table 09. Different mortality percentages were observed for all the experimental

groups. Highest morality (80%) was observed in PDC receiving group. Prominent gross

lesions such as hemorrhages (near neck) can be seen in the first two dilutions of TEW (Plate

28A and 28B). While the PDC has also caused severe hemorrhages in the head, neck and

ventral side of abdominal regions of embryo (Plate 28E) but not as significant as TEWD1.

While moderate lesions are evident on the neck of that embryo who have received TEWD3

(Plate 28C) and abdomen of that embryo which was offered CHC (Plate 28D). While the

control embryo has no such hemorrhagic lesions (plate 28F).

Results

128

Table 09. Table presenting the percentage mortality with respective treatments.

No Group name

Total no of

eggs

Solution applied

Chromium Concentration

Percentage Mortality

1 A

5 TEWD1 0.663 mg/ml 70%

2 B

5 TEWD2 0.0663 mg/ml 30%

3 C

5 TEWD3 0.00663 mg/ml 10%

4 D

5 CHC

0.663 mg/ml 40%

5 E

5 PDC 0.663 mg/ml 80%

6 F Control

5 PBS 0 mg/ml 0%

Results

129

Plate 28. Severe macroscopic/gross lesions observed in chicken embryos exposed to TEWD1 (A) and PDC (E). Moderate lesions were present TEWD2 (B), TEWD3 (C) and CHC (D) offered

embryos. While embryo of control group is without any hemorrhagic lesions (F).

B

F

A C

ED

Results

130


Hatched zero day old marine shrimps (plate 29) were exposed to five dilutions (D1 to

D5) of TEW, CHC and PDC for various exposure times such as 1, 24, 48 and 72 hrs. At the

end of specified time period, the no of dead marine shrimps were counted. The 1, 24, 48 and

72 hrs mortality charts of marine shrimps are presented in figure 13. For all the four exposure

times, the differences among mortality caused by the dilutions of TEW, CHC and PDC is

insignificant (P= 0.964).

Plate 29. Microscopic view of hatched marine shrimps.

Results

131

1hr Mortality

After 1hr incubation, for TEW treated group, mortality percentage in the D1 and D2

was 23.3 and 16.7% respectively. PDC offered group presented mortality percentage was 20

and 13.7% for the D1 and D2 respectively which is less than TEW treated group. While for

CHC offered group, mortality percentage in the D1 and D2 was 13 and 10%. No mortality

was observed for D3, D4, D5 and control.

24hr Mortality

Following 24 hrs incubation exposure, D1, D2 and D3 of TEW offered group have

presented 100% mortality while for D4 and D5 it was more than 60%. Among dilutions of

PDC treated group, only for D1 and D2, marine shrimps lethality was 100% while in next

dilution it up to 60%. Similarly, D1 and D2 of CHC treated group have presented 100%

mortality while the order of mortality in next three dilutions was 73, 60 and 53%. No

mortality was observed in control of this group.

48hr Mortality

Following 48 hrs incubation period, first four dilutions (D1, D2, D3 and D4) of TEW

treated group have demonstrated 100% death of marine shrimps. For D5 it was more than

90%. Almost similar percentage of mortality was recorded for dilutions of PDC. In CHC

treated group, the mortality was 100% for D1, D2 and D3. There was gradual fall in the

mortality of D4 and D5 that is 87 and 83%.

72hr Mortality

Results

132

After 72 hrs incubation, all the dilutions of TEW, PDC and CHC (except D5 of CHC)

have shown more than 100% mortality. In control mortality was 6.7%.

Figure 13. The 1, 24, 48 and 72 hrs mortality charts of marine shrimps. Marine shrimps have demonstrated concentration and time dependent mortality. Concentration of chromium in

D1= 0.663, D2= 0.317, D3= 0.158, D4= 0.079 and D5= 0.04 mg/ml.

1hr Mortality Chart

0

10

20

30

D1 D2 D3 D4 D5

Control

Concentration of Chromium (mg/ml)

Perc

enta

ge M

orta

lity

TEW

PDC

CHC

24hr Mortality Chart

020406080

100120

D1 D2 D3 D4 D5

Control


Perc

enta

ge M

orta

lity

TEW

PDC

CHC


050

100150

D1 D2 D3 D4 D5

Control


Perc

enta

ge M

orta

lity TEW

PDC

CHC


020406080

100120

D1 D2 D3 D4 D5

Control


Perc

enta

ge M

orta

lity TEW

CHC

PDC

Results

133


Maize seeds have been offered dilutions of TEW, PDC, CHC and control. No seed

has germinated for first dilution (D1) of TEW, PDC and CHC (plate 30). The length of roots

(cm) is presented in the phytotoxicity chart (figure 14). Dilutions of PDC have proved most

toxic as compared to TEW and CHC. The variations among length of all the three treated

groups is insignificant (P= 0.581).

A

B C

Results

134

Plate 30. Zero germination observed in the D1 of TEW (A), PDC (B) and CHC (C).

Figure 14. Graph showing the inhibition of root elongation of maize seeds which were irrigated with dilutions of TEW, PDC and CHC. Concentration of chromium in D1= 0.663, D2=

0.317, D3= 0.158, D4= 0.079 and D5= 0.04 mg/ml.

Phytotoxicity Chart

0

2

4

6

8

D1 D2 D3 D4 D5 Control


Leng

th o

f roo

t (cm

)

PDC

TEW

CHC

Results

135


Noteworthy reduction in weight gain was observed in those Wistar rats (figure 15A)

and their vital organs (figure 15B) which have been offered TEWD1 for three months (P=

0.966). Weight gain by the rats receiving TEWD2 and TEWD3 was comparatively more.

PDC has also caused reduced weight gain than CHC. Comparative weight gain by control

group rats was very high. The rats have consumed sample water ad libitum.

Table 10. Table presenting the weight gain by rats.

TEWD1

gm

TEWD2

gm

TEWD3

gm

CHC

gm

PDC

gm

Control

gm

Rat

weight

191.2

453

499

293

245.1

567

Heart

0.7

1.4

1.9

0.9

0.8

2.9

Kidney

0.8

2.2

2.9

1.2

0.9

4.1

Brain

1.7

2.1

3.4

1.9

1.6

3.9

Lung

1.9

5.8

6.5

2.5

2.1

8.6

Liver

9.2

27.8

31.9

14.4

11.3

36.6

Results

136

0

100

200

300

400

500

600

700

TEWD1 TEWD2 TEWD3 CHC PDC Control

we

igh

t (g

m)

0

5

10

15

20

25

30

35

40

45

TEWD1 TEWD2 TEWD3 CHC PDC Control

we

igh

t (g

m) Heart

Kidney

Brain

Lung

Liver

B

A

Results

137

Figure 15. Comparative weight gain in Wistar rats (A) and vital organs (B) of all groups at the end of three months.

The microphotographs of lung, liver, kidney heart and brain of Wistar rats are

presented in plate 31, 32, 33, 34 and 35 respectively. The most severe damages were

observed in TEWD1 and PDC offered rat’s vital organs. The histopathological alterations

observed in the rats which were subjected to TEWD2 and CHC were of relatively moderate

intensity. The microphotographs of TEWD3 taking rat is analogous to control.

Severe peribronchiolitis, congestion, emphasema (enlargement of alveolar spaces),

and infiltration of leukocytes in periarticular area were observed in lungs of TEWD1 offered

Wistar rats (plate 31A). Intensity of peribronchiolitis was also high in lung’s

microphotograph of rats taking PDC. Besides these, moderate emphesema, atelectasis,

compressive hyperplasia, congestion and infiltration of lymphocytes were also evident (plate

31E). Moderate peribronchiolitis, leukocyte infiltration, congestion and emphesema were

present in TEWD2 taking rats (plate 31B). CHC receiving rats’s lung has shown the presence

of fibrinous material in bronchiole and infiltration of lymphocytes and peribronchiolitis

(plate 31D). The degree of emphesema, peribrochilitis and leukocyte infiltration is

comparatively low in TEWD3 drinking rats (plate 31C). No such pathological lesions were

seen in control (plate 31F).

Liver microphotographs of TEWD1 offered rats have demonstrated the presence of

very severe liquifactive necrosis, accumulation of cellular debris, congestion, reduced

sinusoidal spaces due to swelling of hepatic cords, infiltration of leukocytes and dilated

Results

138

central vein (plate 32A). The intensity of hepatic necrosis has gradually declined with

progressive dilutions of TEW. It was moderate for TEWD2 (plate 32B) while mild for

TEWD3 (plate 32C). Focal hepatic necrosis due to toxicosis, hepatocyte swelling and

congestion is also evident in the liver microphotographs of CHC (plate 32D) and PDC (plate

32E) receiving rats. Control (plate 32F) liver was regular with radiating hepatic cords with

regular sinusoidal spaces, round nuclei and homogenous cytoplasm.

Renal histological section of the group which was subjected to TEWD1 has shown

the development of intensive congestion, necrosis, cellular swelling, degeneration of tubules,

formation of proteinous cast and activation of Bowmans capsular epithelial cells (plate 33A).

Similar lesions but of lesser intensity were also present in PDC drinking rats (plate 33E).

Extent of congestion, necrosis, cellular swelling and renal tubular degeneration is almost

same for both TEWD2 (plate 33B) and CHC (plate 33D) offered groups. While mild

congestion is evident in TEWD3 (plate 33C) receiving rats and the control (plate 33F) has

exhibited ordinary pattern with no abnormal alterations in renal cells (plate 33F).

Strong myocarditis, degeneration and fragmentation of cardiac muscle, loss of

sacroplasm and infilteration of leukocytes is evident in the cardiac morphological sections of

TEWD1 (plate 34A) and TEWD2 (plate 34B) drinking Wistar rats. But only degeneration

and fragmentation of cardiac muscle is present in microphotographs of rats belonging to PDC

(plate 34E), CHC (plate 34D) and TEWD3 (plate 34C) group. Control was without such

pathological adaptations (plate 34F).

Results

139

Microphotographs of brain of all treatment groups were comparable to control and no

neuronal loss was seen in any group (plate 35).

B E

C F

A D

Results

140

Plate 31. Lung microphotographs of rats exposed to TWED1 (A), TEWD2 (B), TEWD3 (C), CHC (D), PDC (E) and Control (F).

A D

B E

C F

Results

141

Plate 32. Liver microphotographs of rats exposed to TWED1 (A), TEWD2 (B), TEWD3 (C),

CHC (D), PDC (E) and Control (F).

A D

B E

C F

Results

142

Plate 33. Kidney microphotographs of rats exposed to TWED1 (A), TEWD2 (B), TEWD3 (C),

CHC (D), PDC (E) and Control (F).

A D

B E

C F

Results

143

Plate 34. Heart microphotographs of rats exposed to TWED1 (A), TEWD2 (B), TEWD3 (C), CHC (D), PDC (E) and Control (F). Fragmentation and generation is quite prominent in A, B,

C, D and E. While prominent lymphocyte infiltration was present only in A and B.

A D

B E

C F

Results

144

Plate 35. Brain microphotographs of rats exposed to TWED1 (A), TEWD2 (B), TEWD3 (C),

CHC (D), PDC (E) and Control (F). No pathological changes were seen in any treatment group.

Discussion

144


The PIXE analysis of tannery effluent wastewater (TEW) samples (table 03) has

revealed some variations in their composition. These differences among the concentration

of heavy elements belonging to different domestic tanneries of Kasur city may be

attributed to the stage of tanning process, as each step of tanning cycle use different raw

chemicals (figure 01). The amount and source of raw chemicals which are being used by

local tanneries significantly varies too. So mostly the effluent coming from one tannery

doesn’t have same levels of toxic chemicals as others have. These results are in

accordance with those previously presented by Calheiros et al (2008). They have also

documented broad irregularity in the composition of TEW samples. Quantities of

detected elements mostly depend upon level of production cycle (Haydar and Aziz 2009).

Similar results were reported by Sajjad et al (2008). They emphasized that stage of

leather production cycle has profound impact on detected concentrations of all elements

and composition of TEW deviates from tannery to tannery. The results of my research

project are also in agreement with those described by Tariq et al. (2005) who have

quantified same toxic elements in tannery effluents and related soil.

CHAPTER-5

DISCUSSION

Discussion

145

Moreover, the results of current project have also highlighted that large portion of

the ground water (shallow tubewells with depth 100 to 300 ft) of Kasur has been tainted

with unacceptably high levels of various toxic elements particularly chromium (table 04).

The situation is even more worse especially for those ground water sources which are

particularly closer to tannery area. Strikingly, almost all shallow tubewells were found

contaminated with chromium concentrations far above than WHO recommended limits

(0.05 mg/L). The main contributing reason for chromium contamination of underground

water sources of Kasur may be the pounds of TEW in the surroundings of tannery area

(plate 01). These pounds especially spread out their areas during rainy/moonsoon season.

Second reason may be that, the sewage drainage system of tannery area is not carpeted

with cement. So toxic metals may seeps through the ground and get mixed with

underground water and polluting it. Apart from this, TEW can also pollute other surface

water reservoirs at far distance because mostly wastewater of tanneries get discharged

untreated into rivers and lakes (Khan et al 2004).

Municipal Corporation implanted deep tubewells have an average depth of about

600 ft. So the absence of chromium in the GWD samples (table 05) can be linked to their

greater depth than shallow tubewells. The other possible root cause seems that they are

present at far distance from tannery area while shallow tubewells were implanted within

those domestic tanneries from where TEW samples have been collected for PIXE

characterization.

Discussion

146

In short, the amounts of toxic elements in ground water samples were found

dependent on the distance of tubewells from domestic tanneries location (figure 02) and

as well on the depth of tubewell. Higher was the level of detected toxic elements, lower

the depth of tubewell. This spotlights the increased soil permeability of heavy elements

like chromium.

Huge quantities of chromium salt (chrome i-e., chromium sulphate) is being

utilized by local tannery industry which ultimately generates high incursion of chromium

in environment and natural water reservoirs (Sirajuddin et al 2007) because of release of

large quantities of TEW and chromium containing sludge exclusions (Pham et al 2010).

About 40% of applied chromium is released as such in the environment (Fahim et al

2006). Some elements which are considered essential for health exist in water but their

massive intake may result in severe health problems (Midrar et al 2005). Trivalent

chromium is required for normal functioning of insulin and also for metabolism of

carbohydrate, protein and fat but hexavalent chromium is a well established toxicity.

Chromium easily infiltrates down through soil and cause contagion of

underground water table. Results of my research project have also intricated the

movement of chromium from TEW to GWS (tables 03 and 04). This chromium

contaminated GWS is evolving as the most dangerous source of chromium ingestion in

Kasur city.

Discussion

147

Although trivalent chromium is much less lethal but have excessive

permeability/solubility (Essahale et al 2010) and get transported to disposal sites

effortlessly and oxidized to hexavalent chromium (Sirajuddin et al 2007). Hexavalent

chromium [Cr (IV)] is a well established toxin, carcinogen and mutagen (Bagchi et al

2002). Likewise, compounds of chromium (particularly hexavalent ones) are corrosives,

delayed contact sensitizers and carcinogens i-e., human lung carcinogen (Gad 1989; Wise

et al 2002). Cr (IV) also builds up in the hypothalamus and pituitary giving rise to a drop

in prolactin levels (Quinteros et al 2007). Small doses of Cr (IV) may also cause some

cell mortality (Carlisle et al 2000). Various point mutations in DNA and chromosomal

damage are evident on exposure to Cr (IV) (Dayan and Paine 2001). Besides this, Cr (IV)

exposure (through drinking water) has resulted in the embryo and fetotoxic effects.

Number of resorptions, pre-implantation and post-implantation loss was also increased

under experimental conditions (Kanojia et al 1996). Chromium caused alteration in

gametes physiology, so enhanced the risk of reproductive failure (Chowdhuri et al 2001).

Chronic chromium exposure by developing a reversible oxidative stress (seminal plasma

and sperm) has caused reduced motility of live sperm and sperm death (Subramanian et al

2006).

Despite of production cycle, it was noticed that the concentrations of Ca, Cl, Mg,

Fe, K, S, Si and Sr in TEW (Table 03) were significantly higher than GWS (Table 04)

and GWD (Table 05) which also exceeded the WHO limits. Huge quantity of different

salts, i.e., salts of chromium, chloride, ammonium and sulfide (Amir et al 2008) is

commonly employed during tanning operations (figure 01), which generates trace

Discussion

148

elements of different levels in TEW. Therefore, an increase in the concentration of Cr,

Ca, Cl, Mg, Fe, K, S, Si and Sr was evident in all TEW samples. These findings

obviously point up that TEW contains huge amounts of diverse toxicants, which have

great capacity to directly pollute the natural water assets.

PIXE analysis of GWS strikingly revealed significant increase in the

concentrations of major toxic elements, i.e., Cr, Cl, K, Ni, Fe and Si (Table 04) while

increased concentrations of Cr and Si were also revealed in various GWD samples (Table

05), which points up obvious relocation of toxic substances from TEW to majority of

freshwater sources. It has already been documented that TEW have great prospective to

pollute the GWD in and around a tannery for a distance of 5 km and makes it unfit for

human and animal consumption (Srithar and Mani 2004).

The information described in table 04 revealed that there was a substantial

increase in the concentration of Cl in majority of GWS samples, which could be greatly

referred as the level of significant Cl toxicity. An increase in the amount of Cl is a

potential source of hyperchloremic acidosis (Eisenhut 2006), which may lead to increase

in urinary plasma alanine, aspartate aminotransferases and reduction in blood sugar

(Fisher et al 1983). Increase intake of Cl may also lead to disparity in immune responses

and impaired neurobehavioral functions (Kilburn 2009). Besides these, excess Cl has got

capacity of soil degradation and crop destruction (Parker et al 1983).

Discussion

149

The Cl in combination with some closely allied compounds (present in TEW) has

strong capability to produce highly toxic end products entirely dissimilar from the

original chemicals. The Cl in amalgamation with other compounds may form cyanogen

chloride and dichloramine which are toxic for aquatic life (Bidleman et al 1993; Brungs

1973; Grimwood and Dobbs 1995; MacCrehan et al 1998; Pasternak et al 2003). While

Cl in combination with Na and K is a popular cause of hypertension (Jia et al 2007; Kim

et al 2007), vascular lesions (Jia et al 2007; Wang et al 2006) and kidney failure.

Thus, increase amount of K, as discovered by PIXE analysis of GWS, may

produce assorted health impacts on people including hypertension (Braschi and Naismith

2008), reduction in urinary albumin, lung injury (He et al 2010), renal damage,

disturbance of consciousness and cardiovascular disease (Cook et al 2008).

The findings of my research project have also identified increase prevalence of Ni

and Fe in some GWS samples, which is beyond WHO limits (Table 04). The chronic Fe

overloads in human develop attained disorders, which may produce clinical consequences

of diabetes, hepatic fibrosis, cardiac disease, hepatocellular cancer and cirrhosis (Hershko

et al 1998). Elevated levels of Fe is generally observed to be a hazardous aspect for an

increasing number and diversity of disease including neuronal disorders (Liang et al

2008), alzheimer’s disease, arteriosclerosis (Brewer 2010), abnormal brain iron

homeostasis (Park et al 2011), aging muscle atrophy, rosacea, viral replication and

pulmonary alveolar proteinosis (Weinberg 2009).

Discussion

150

Similarly, Ni is a potential neurotoxic toxicant (Xu et al 2010) connected with

lymphocyte toxicity (Chen et al 2003). It has the aptitude to cross human placental barrier

and lead to development of embryotoxicity and teratogenesis (Chen and Lin 1998).

Furthermore, it is a toxic element that can damage many fluvial ecosystems (Cloran et al

2010) by causing alterations in water hardness (Deleebeeck et al 2009) and soil

composition (Li et al 2010) which successively damage roots of plant (Seregin and

Kozhevnikova 2009).

My data has also pinpointed existence of abundant amount of Si in GWS and

GWD (Tables 04 and 05). Despite the reality that Si is known to own a low scale of

toxicity, consuming the water having high levels of Si has been documented to cause skin

irritation, transient eye irritation, corneal endothelial changes and retinal toxicity (Green

et al 1994).

5.2. Microbial Evaluation of TEW

The detected viable count range for all collected TEW samples (10) is from 7.5 X

104 to 3.0 X 107 CFU/ml (table 06, plate 07). This variation between the viable counts of

collected TEW samples may be again attributed to the stage of tanning cycle (at sample

collection time). Maximum number of bacterial isolates was obtained for those effluent

samples which were collected during soaking and fleshing stage (figure 01).

Discussion

151

Moreover, I have isolated the bacteria from mixed culture plates by sub-culturing

on nutrient agar and then identified them from their colony characteristics, staining

results and outcomes of biochemical tests. On the basis of percentage

occurrence/detection, following ranking of bacterial species (figure 10) is obtained,

Bacillus subtilis (18.4%), Bacillus megaterium (13.2%), Staphylococcus aureus (13.2%),

E. coli (13.2%), Staphylococcus epidermidis (10.5%), Bacillus azotoformans (8%),

Micrococcus varians (7.9%), Bacillus cereus (5.3%), Bacillus laterosporus (5.3%) and

Corynebacterium kutsceri (5.3%). Most of my detected species of bacteria are in

agreement with those which had been described previously by many researchers. As,

Bacillus megaterium was isolated from treated tannery effluents by Mondaca et al. (2002)

and Viti et al (2003). In 2010, Desta et al. (2010) have alienated Bacillus cereus from

treatment plant of Elmo Leather AB tannery. Staphylococcus aureus was screened from

tannery effluents by Ilias et al (2011). Similarly species of Bacillus and Corynebacteria

have been separated by Viti et al (2003). Among screened bacterial isolates, 11.4, 13.3

and 5.7% were found belonging to Bacillus, E. coli and Staphylococcus respectively

(Fakruddin et al 2009).

I have also determined the tolerance limits of all isolated bacteria towards four

salts of chromium (chromium chloride, chromium sulphate, chromium oxide and

potassium dichromate) ranging from 600 to 2600 µg/ml (table 08). Corynebacterium

kutsceri was proven the most sensitive because it has tolerated 1800 µg/ml of chromium

sulphate, 1400 µg/ml of chromium chloride and chromium oxide and 600 µg/ml of

potassium dichromate. After it, Staphylococcus epidermidis has tolerated 1800 µg/ml of

Discussion

152

chromium sulphate and chromium chloride, 2200 µg/ml chromium oxide and 1000 µg/ml

of potassium dichromate. While Micrococcus varians and Staphylococcus aureus have

tolerated almost same levels of chromium compounds [2200 µg/ml of chromium sulphate

and chromium chloride, 1800 µg/ml chromium oxide, 1000 µg/ml (for Micrococcus

varians) and 1400 µg/ml (for Staphylococcus aureus) of potassium dichromate]. All the

Bacillus species and E coli proved resistant as they have tolerated high levels of

chromium compounds such as 2600 µg/ml of chromium sulphate, 2200 µg/ml of

chromium chloride, 2200 µg/ml of chromium oxide (except for Bacillus azotoformans,

2600 µg/ml) and 1800 µg/ml of potassium dichromate (except for Bacillus megaterium,

2200 µg/ml). Tolerance levels of bacterial isolates similar to my recorded results have

been reported previously, such as exposed 250 μg/ml of PDC was resisted by 22.22%

isolates (Basu et al 1997). Bacillus, E. coli and Staphylococcus have tolerated even 500

mg/L of hexavalent chromium (Fakruddin et al 2009). Elevated level up to 40 mg/ml of

PDC on nutrient agar was resisted by all strains (Faisal and Hasnain 2004). Shukla et al.

(2007) have documented PDC’s minimum inhibitory concentration for four different

isolates, 1200 µg/ml: NBRIP2, 1400 µg/ml: NBRIP1, 1800 µg/ml: NBRIP3, and 2100

µg/ml: NBRIP4 which are in agreement with our obtained results. On the contrary,

Verma et al. (2001) stated that majority of heterotrophs was found tolerant only to 50 ±

100 µg/ml of chromate.

Discussion

153

5.3. Toxicological Evaluation of TEW through CAM and

Embryotoxicity Assay

5.3.1. Toxicological Evaluation of TEW through CAM Assay

In this research project, I have used a variety of bioassays for toxicological

screening of TEW because bioassays are mandatory for the integrated evaluation of water

pollution (Lu et al 2010). Moreover, for the better appraisal of imposed risk of any

chemical compound, use of battery of toxicity tests is a best approach (Hernando et al

2005).

The main object behind performing CAM assay was to check the hypothesis that

chromium content of TEW may or may not inhibit normal angiogenesis. However, no

study has assessed toxicological lumber of TEW on angiogenesis yet. Outcomes of my

project have described significant dicey impacts of TEWD1 and PDC on developing

blood vessels of CAM. All three dilutions of TEW, PDC (hexavalent chromium) and

CHC (trivalent chromium) have constrained various parameters like pattern of blood

vessel origination (plate 20), tertiary blood vessel development (plate 21 and 22), total

area of CAM (plate 23), angular distribution (plate 24), dimensions of blood vessels

(plate 25), diameters of blood vessels (figure 11) and CP formation (plate 27) to different

extents. It was observed that normal orientation of blood vessels (tree branches pattern)

was tremendously sensitive to the application of TEWD1 and PDC (plate 20A, E).

Furthermore, the number of tertiary blood vessel was markedly suppressed as well (plate

21 and 22). The diameter of primary, secondary and tertiary blood vessels has been

Discussion

154

diminished significantly (figure 11) when quantified (plate 26) using the scan probing

image processing software (P ˂ 0.05). The antiangiogenesis was also found related to

decrease in number of total blood vessels and the number of bifurcations. For getting

comparable parameters, the sample size, magnification, image field were kept constant

while analyzing CAMs of all the groups. Besides these, I have compared roughness

parameters (Sa, Sq, Sz, Sk and Spk) of all groups (figure 12) because their measurement

is easy and standards are available for explanations. Such measurements of topographies

provide precise data regarding different activities of angiogenesis and scan probing image

processing automatically quantify all the parameters (Ejaz et al 2006).

In the CAM, the respiratory exchange is carried out through a large number of

CPs. In the early stages, CP develops bordering to chorionic ectoderm and later on they

get inter-digitated in between ectodermal cells of the chorion (Melkonian et al 2002).

After the application of dilutions of TEW, solutions of CHC and PDC, the number of CP

has decreased as evident from the photomicrographs (plate 27) of CAMs. This fall in

number of CP led to a reduced gas exchange. Similar CP formation inhibition was

already reported by Ejaz and Woong (2006) after application of cigarette smoke

condensate and total particulate matter. As activities of CAM is comparable to placenta

of human, so can be argued that consumption of chromium contaminated water can have

adverse effects to developing foetus. In addition, the CAM which has received

application of CHC has demonstrated uneven meshwork of mesoderm. This

reorganization and degradation of extracellular matrix is mostly responsible for vascular

Discussion

155

remodelling that is any stable alteration in size and/or composition of adult blood vessels

(Streuli 1999).

Findings of CAM assay have illustrated that TEWD1 has largely disrupted the

process of angiogenesis. Another important point is that PDC appeared more lethal than

CHC with respect to impacts on angiogenesis. So it may be suggested that TEW either

contains more toxic elements mixture which are absent in PDC and CHC solutions.

While assessing impact of TEW on blood vessel development, CAM assay was proven an

economical, quicker, semi-quantifiable assay (Ribatti 2008) and has been extensively

used for studying antiangiogenesis (Ribatti 2010). Furthermore, the image acquisition and

processing system proved a useful device while quantifying angiogenesis because of easy

application, rapid throughput and reproducible outcomes.

5.3.2. Toxicological Evaluation of TEW on Developing Embryo

I have investigated the dicey effects of dilutions of TEW, PDC and CHC on

developing chicken embryo because invasive investigation of animals is required for the

use of mammalian embryos and may involve damage to tissues and aggravate ethical

considerations (Datar and Bhonde 2005). Now the testing of detrimental effects on

developing embryos has become a fundamental part of toxicological studies (Ejaz et al

2010). In literature, chicken embryos have been extensively used for studying the

toxicological effects of various chemical and physical agents (Friedberg and Gartner

1990). Recently the chicken embryo toxicity studies have been preferred because of

Discussion

156

sensitivity, inexpensiveness, parallelism to morphological mammalian development

(Korhonen et al 1982), large number of chicken embryos can be kept in a small space for

observation and over a short span of time (McLaughlin et al 1963).

The highest percentage mortality (table 09) of developing embryos was observed

in groups treated with PDC (80%) and TEWD1 (70%). Mortality percentage was

comparatively low for other groups while no mortality was recorded for control group

(PBS). Similarly, severe haemorrhages were observed at the end of incubation in

embryos of TEWD1 (plate 28A) and PDC (plate 28E) group. In the case of treatment

with TEWD1 (plate 28A), the whole body was covered with lesions. Moderate

haemorrhagic lesions were seen in embryos of TEWD2 (plate 28B) and CHC (plate 28D)

in comparison to control. During early developmental stages, embryotoxic effects were

also observed post injecting trace mineral solutions (Richards 1997). Similarly, drinking

water contaminants (As, Cd, Pb, Benzene and trichloroethylene) at high levels have

resulted in elevated embryonic mortality (Vodela et al 1997). Embryonic growth (Mallard

eggs) was extensively inhibited/reduced by TEW external exposure at 3rd and 8th day of

incubation (David et al 1981).

The huge mortality induced by PDC administration is agreement with the reported

deaths of those developing embryos that were treated with hexavalent chromium

(Ridgeway and Karnofsky 1952). Chromium a major component of TEW is well known

for its embryotoxic effects. Additionally, heavy metals being component of tannery

effluents have caused embryotoxic effects in large biota. Embryo lethality in Japanese

Discussion

157

Meadaka, Oryzias latipes was associated with the presence of heavy metals (Cooper and

McGeorge 1991). Reduced body size and haemorrhages were resulted in chick embryos

by heavy metals (Asmatullah et al 1998A, B; Asmatullah et al 1999; Gilani and Alibhai

1990). Besides this, injections of heavy metal compounds have resulted in huge

intoxication to developing embryos.

On the contrary, 50 µg/L of chromium has not induced an increase in mortality

(Kertész and Fáncsi 2003). Apart from chicken embryos, the embryotoxic effects of

chromium compound have been reported in other species of animals as well. In mice,

chromium (maternal) administration via drinking water has caused large number of

embryonic deaths (Trivedi et al 1989) and proved embryo toxic in hamster (Gale 1982) as

well. Hexavalent chromium was declared embryotoxic and fetotoxic by Junaid et al.

(1996) because elevated incidence of dead foetuses was seen after PDC administration

through drinking water to mice. I have obtained similar results embryotoxic effects.

5.4. Marine Shrimp Toxicity

Dilutions of all the three test toxicants (TEW, PDC and CHC) have displayed a

time and concentration dependent toxicity in marine shrimps mortality assay. Percentage

mortality of marine shrimps has significantly increased with time (figure 13). It can be

concluded that exposure time of any perilous chemicals has strong effect on mortality as

it was highest after 72 hrs exposure. In short, marine shrimps were proven much more

responsive to TEW than CHC and PDC. Among the TEW and salts of chromium, PDC

Discussion

158

has been established more toxic than CHC as previously reported by Hadjispyrous et al

(2001). Similar results were reported by Eisler (1986) that marine shrimps are most

susceptible to PDC as determined by 96 hrs LC50 values. Additionally, Artemia

franciscana is more sensitive for detecting toxicity (Vanhaecke and Persoone, 1984).

Marine shrimps proved reliable organism for detecting toxicity of industrial

effluents as determined by marine shrimps mortality assay in this current research

project. Shrimps have several advantages as a marine test organism for investigating the

toxicity of effluents and chemical compounds (Hadjispyrous et al 2001).

5.5. Phytotoxicity

Based on the data of phytotoxicity assay, it is concluded that PDC is more toxic

than CHC and TEW. It has caused massive toxicity by extensively reducing the maize

seed germination and root length. Even no seed germination was reported for PDC’s first

five dilutions (D1 to D5) (plate 30, figure 14). My reported results of this assay are in

accordance with those previously documented by López-Luna et al (2009). They have

also reported greater toxic effects for PDC on wheat, oat and sorghum seedlings.

Germination of seed is variable in all the three experimental groups, lowest for PDC

followed by TEW and CHC. In 2008, Calheiros et al. (2008) have also reported that

highly concentrated tannery wastewater (100, 70 and 50%) with low level of treatment

has caused complete inhibition of germination of T. Pratense. While in my project for

first three dilutions of TEW (D1, D2 and D3), no seed has been germinated. It is also

Discussion

159

clear from phytotoxicity chart that there is remarkable difference between the PDC and

CHC offered maize seed germination and root length. In past, similar difference has also

been reported by Soudek et al. (2010), as very huge differences in toxicity for cultivars

was observed between hexavalent and trivalent chromium. In the end, we can sum up that

phytotoxicity/root elongation inhibition assay is very sensitive assessment for toxicity of

chromium polluted samples.

5.6. Chronic Toxicity

In last phase of my research study, I have determined the hazardous effects for

dilutions of TEW, solutions of CHC and PDC on vital organs of Wistar rats after three

months oral administration. Histopathological investigation was done to see any

transformation caused by toxic chemicals ingestion. For the evaluation of the toxic

effects of contaminants, microscopic examination of organ (for assessing early effects on

its morphology) has become fundamental tool. In past, it has been utilized as a biomarker

for evaluation the toxicity of a variety of pollutants (Velma and Tchounwou 2010).

At the end of chronic toxicity study (three months), the weight of Wistar rats was

recorded and compared. Massive reduction in weight gain (rat body weight and vital

organs weight i-e., heart, kidney, liver, lung and brain) was observed for TEWD1 (figure

15). Among other treated groups, weight gain by PDC, TEWD2 and TEWD3 was also

less. Similar negative impact on rat’s weight gain by high levels of chromium was

described by Silva et al (2010).

Discussion

160

After chronic exposure, many morphological changes were observed in the vital

organs of rats in past. Likewise, chromium toxicity in animals has been documented in

few studies (Silva et al 2010). Cr (IV) was found to have hepatotoxic, nephrotoxic

(Laborda et al 1986; Silva et al 2010; Tagliari et al 2004; Tandon et al 1979; Wedeen and

Qian 1991), cardiotoxic potential (Tandon et al 1979) and also caused damage to lungs

(Laborda et al 1986). Besides this, hexavalent chromium exposure to industrial

workers/animals has lead to development of hepatic and renal toxicity (Asmatullah et al

1998B).

Severe emphysema, peribronchiolitis and congestion in the lung

photomicrographs of TEWD1 drinking rats (plate 31A) were observed. While

emphysema of moderate and light intensity was seen in TEWD2 (plate 31B) and TEWD3

(plate 31C) offered rats respectively. Emphysema was also present in rats of PDC (plate

31E) and CHC (plate 31D) group rats. Massive infiltration of leukocytes was seen

particularly in PDC group rats. Similar to my research outcomes, Derelanko et al. (1999)

have reported alveolar spaces packed with cellular debris, lymphocytes, macrophages and

neutrophils for basic chromium sulphate. Lamellar degeneration and oedema was seen in

primary and secondary lamellae with basic chromium sulphate (5, 4 and 3 mg/L)

treatment. While one week treatment has led to development of haemorrhage in

secondary and primary lamellae (Daksh and Capoor 2011). No such haemorrhages and

oedema was observed in rats receiving dilutions of TEW.

Discussion

161

Liver of TEWD1 group rats have demonstrated significant congestion, enhanced

sinusoidal spaces, liquifactive necrosis, accumulation of cellular debris and infiltration of

leukocytes (Plate 32A). Similar dilated sinusoids (containing erythrocytes) were observed

in female Wistar rats after four months chronic exposure of 300 or 500 mg/L trivalent

chromium by Salvia et al (2006). They further described that most of hepatocytes were

ballooned, the nuclei were lysed and centrilobular vein is dilated and congested.

Outcomes consistent with my obtained results such as, chromium induced loss of normal

architecture of liver, extensive vacuolization in hepatocytes, fatty changes and necrosis

have been reported by many scientists (Das and Mukherjee 2000; Parvathi et al 2011).

Same morphological hazards to liver after few months oral administration of chromium

picolinate in rats was documented by Mahmoud et al (2009). Degenerative changes in

hepatocytes (swollen cells and degenerating nuclei) were prominent. While the high dose

of chromium picolinate has resulted in abnormal liver architecture and between the

swollen hepatocytes, the blood sinuses were lessened. In addition, vacuolated cytoplasm

and faint nuclei appeared.

Even acute exposure of hexavalent chromium (40 mg/L) has caused hepatocyte

shrinkage along with enhanced sinusoidal spaces (Mishra and Mohanty 2008) and

chronic PDC treatment has given rise to development of altered hepatic architecture with

necrosis, vacuolation and increased sinusoidal spaces (Asmatullah et al 1999; Chopra et

al 2008; Velma and Tchounwou, 2010) which are analogous to my documented results of

PDC supply. Similarly, thirty days treatment of CHC has lead to condensation of

cytoplasm, enlarged nuclei, disarrayed hepatic cords, elongations of blood vessels,

Discussion

162

degeneration and necrosis (Bhatkar 2011) which are in agreement with my findings of

CHC group. Likewise, intraperitoneally injected CHC to Wistar albino has caused

enlargement of central veins and sinusoids (Laborda et al 1986).

Several damages such as intensive congestion, necrosis, cellular swelling,

degeneration of tubules, formation of proteinous casts and activation of Bowmans

capsular epithelial cells were observed in light microphotographs of kidney of those rats

which were offered dilutions of TEW, CHC and PDC. TEW dilutions have caused

congestion development which was particularly high in TEWD1 group (plate 33A),

modest for TEWD2 (plate 33B) and insignificant for TEWD3 (plate 33C). Moderate

congestion was also seen in CHC (plate 33D) and PDC (plate 33E) drinking rats. Above

stated histopathological lesions of rat’s kidney are comparable with those reported by

many researchers. Silva et al. (2010) have illustrated the existence of huge number of

similar severe lesions in the rats which have received chromium extracted tannery residue

materials. Mild to severe interstitial fibrosis, lymphoplasmacytic infiltration and

multifocal epithelial cell regeneration in proximal convoluted tubules was quite clear. In

the 37.5 and 50% chromium extracted tannery residues offered groups, lesions were more

serious because chromium ingestion was high thus spotlighting a dose dependent effect.

Similar concentration dependent congestion is also apparent in photomicrographs of three

TEW dilutions. Likewise to my reported renal necrosis, Laborda et al. (1986) have

demonstrated presence of necrosis (proximal tubular) in rats which were treated with

trivalent chromium. Similarly, rabbit kidneys after three week exposure to chromium

have presented significant congestion, tubular necrosis along with mononuclear cells

infiltration and extravasations of RBCs in intertubular spaces (Mathur et al 1977; Zhou et

Discussion

163

al 2008). Even acute exposure of PDC has yielded adverse effects similar to my reported

outcomes such as cellular degeneration and hyaline casts formation, tubular necrosis in

the outer and inner cortex (Oliveira et al 2006), hypertrophy and vacuolization of renal

tubular epithelial cells and declined tubular lumens (Mishra and Mohanty 2008).

Subcutaneously injected dichromate causes a considerable nephrotoxicity than

intraperitoneally injected one (Kim and Na 1991). PDC treatment for 5.5 months has

given rise to significant damages to renal tubules in the form of syncitial appearance of

its epithelial cells, degeneration and diffused Bowmans capsule (Chopra et al 2008).

Short term heavy exposure of chromium (hexavalent) has induced necrosis of renal

tubules and infiltration of lymphocytes (Mathur et al 1977; Wedeen and Qian 1991)

which agrees with my renal findings of PDC group. Similarly, total 2 ml/kg subcutaneous

injection having 20, 10, 5, 2.5 and 1 mg/Kg PDC in 0.9% saline to male sprague dawley

rats has resulted in dilatation of the tubules, degeneration and regeneration of the

proximal convoluted tubules, epithelial cellular necrosis, interstitial lymphocytic

infiltration, presence of protein casts in tubular lumina and vacuolization of glomerulus

were seen after 72 hrs (Zhou et al 2008). Again these lesions are consistent with my

obtained results. Similar changes such as eosinophilic protein casts, minimal interstitial

chronic inflammation, cystic tubular formations, tubular dilation and dispersed patchy

foci of interstitial fibrosis were observed microscopically in rats (lean Zucker and obese)

who have received 10 or 5 mg/kg of chromium picolinate for 6.5 months (Mozaffari et al

2009). Thus chromium has large attraction for kidney and chromates are established

promoter of intensive renal injury (Oliveira et al 2006).

Discussion

164

Photomicrographs of cardiac tissues have shown some pathological alterations

such as fragmentation, degeneration and infiltration of leukocytes (plate 34). But severe

degeneration was observed in cardiac tissues of those rats which have been offered

TEWD1 (plate 34A) and PDC (plate 34E) as compared to control (plate 34F) which have

presented regular arrangement of cardiac muscles. But infiltration of leukocytes was seen

only in those rats which have consumed dilutions of TEW. Similar degenerative

amendments in muscle fibres and significant congestion were described by Mathur et al.

(1977) in those animals, which have been exposed to trivalent chromium for six weeks.

Besides this, mononuclear cells have infiltrated in the interstitial tissues too. Same

pathological changes were prominent in hexavalent chromium exposed rats. Findings

alike to my reported cardiac transformations were seen in the heart of chickens which

have consumed 5 and 2.5% chromium like splitting of muscles, destruction and loss of

striation, dislocation of nuclei and brown atrophy (Riaz et al 2006). No neuronal loss was

observed in any treatment group (plate 35).

This multi approach research project was designed to a give massive contribution

about tannery effluents risk. During the toxicity screening, TEW sample proved much

more toxic than PDC and CHC. The toxicities caused by PDC was far higher than those

caused by CHC in CAM assay (Figure 11-12, plate 19-25), embryotoxicity assay (table

09, plate 28), marine shrimps mortality assay (figure 13, plate 30), phytotoxicity assay

(figure 14) and chronic toxicity study (figure 15, Plate 31-35) because PDC (hexavalent

chromium) is roughly 500 times more toxic than the CHC (trivalent chromium) (Bagchi

et al 2002). This difference in toxicity of both salts of chromium is mainly attributed to

Discussion

165

ready absorption or penetration of hexavalent chromium in cells and poor/lower cellular

absorption of trivalent chromium (Mathur,et al 1977; Silva et al 2010; Zayed and Terry

2003; Tagliari et al 2004). In rats, the absorbed quantity of CHC through gastrointestinal

tract was less than 0.5% in case of oral supply (Visek et al 1953) and less than 3% in case

of single dose by gavage (Mertz et al 1965) and stomach tube (MacKenzie et al 1959).

Henderson et al. (1979) determined that hamsters have absorbed less than 1.5% of an

administered oral dose of trivalent chromium. Furthermore, Mertz et al. (1965) reported

that absorption in rats was independent of the administered dose and dietary chromium

status (deficient or supplemented in chromium) of the animals. Cr (III) was found to be

better absorbed in fasted than in fed rats (MacKenzie et al 1959).

Additionally, the hexavalent chromium significantly differs from trivalent

chromium in many biological properties (Asmatullah et al 1998B). Trivalent chromium

becomes toxic only at enormously high doses (Silva et al 2006). For mice LD50 of

hexavalent chromium is 5 mg/kg body weight and of trivalent chromium is 260 mg/kg

body weight (Eisler 1986).

Large sample sized prospective studies can determine health effects of chromium

in drinking water of Kasur. For minimization of heavy metal contamination particularly

in tannery area (with extremely high chromium), appropriate interventions are required.

In addition to this, I may conclude on the basis of the results of this project that ground

water sources (especially shallow tubewells) are not safe in tannery area of Kasur and

extensive cautions are necessary to use this water for drinking and cooking purposes.

Discussion

166

Following conclusions may be drawn from the collected results of current

research project:

1. Level of chromium is significantly high in GWS indicating the massive impact of

TEW on underground water table of Kasur.

2. The detected total viable range for collected TEW is 7.5 X 104 to 3.0 X 107 CFU/ml.

Besides this, several strains of chrome tolerant bacteria were isolated and identified.

Most of the isolated bacteria were found tolerant to high level of chromium salts.

3. Dilutions of TEW have presented level dependent antiangiogenic effects as assessed

through CAM assay. These dilutions have also caused precarious affects to

developing chick embryos.

4. TEW dilutions have also demonstrated concentration dependent toxic effects in

marine shrimps mortality and Phtyotoxicity assay.

5. In chronic toxicity study, TEW lead to development of several hazadous changes in

vital organs of Wistar rats.

6. Effluent wastewater being discharged by tanneries is a real menace for local

inhabitant of Kasur because of its high content of chromium.

For amelioration of health status of people of Kasur, I am presenting following

recommendations:

1. A non toxic substitute of Chrome (chromium sulphate) must be searched for tanning

process.

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167

2. Shallow tubewells must be implanted up to the depth of 500ft or more for minimizing

the level of chromium in underground water.

3. Awareness campaigns should be encouraged for enhancing health status of Kasur

inhabitants.

4. More treatment plants should be implanted for treating TEW, as one plant is not

sufficient for whole district.

5. The sewerage drain system in Kasur district must be cemented/tiled.

6. The use of domestic water filtration units must be encouraged and should be provided

free of cost to the local residents.

7. Most of the isolated strains of bacillus (Bacillus megaterium, Bacillus azotoformans,

Bacillus laterosporus, Bacillus cereus and Bacillus subtilis) were found tolerant of

high level of chromium salts, their potential for removal of chromium from tannery

effluents must be explored.

8. At the early stage of the wastewater treatment, technology such as “Chemically

Enhanced Primary Treatment” (which makes use of coagulants for pollutants

removal) should be used for effectual results.

9. Government research departments should explore all possible means such as

chemical, physical and biological or the combination of these in their research centers

(e.g. PCSIR) for their possible use in detoxification of TEW.

10. Routine methodologies such chemical precipitation, reverse osmosis membrane

processes and adsorption should be used in treatment plant of Kasur under strict

monitoring. Apart from these, advanced treatment techniques, such as ion exchange,

Discussion

168

reverse osmosis, electro dialysis and membrane filtration should be used for removal

of heavy metals such as chromium.

11. In addition, biological removal may present an appropriate cost effective means for

heavy metal removal from TEW.

Summary

169

Over the last decade or so the chromium based tanning industry has shown rapid

growth in Pakistan. However the rule and regulations promulgated by the government are

not strictly followed for the processing of effluent discharged by the tanneries.

Consequently tannery effluents have become a great source of water pollution in

surrounding area. This project was designed to evaluate the hazardous effects of tannery

effluent wastewater (TEW) through various bioassays.

During the first phase of the project, composition of the TEW samples was

determined by PIXE analysis. Besides this, we have also investigated the impact of TEW

on trace element content of ground water in Kasur tannery area. The ground water from

shallow tubewells (100 to 300 ft) in the area has shown very high content of chromium

while the ground water from the deeper tubewells (upto 600 ft) generally does not contain

the toxic elements except for one outlet of the water supplied by the Muncipal

Corporation. This could be due to corroded pipes in the tannery area.

Microbial load was determined during second phase of this research project by

viable count method. The detected viable count was 7.5 X 104 to 3.0 X 107CFU/ml.

CHAPTER-6

SUMMARY

Summary

170

Various strains of chromium tolerant bacilli were isolated and they were found tolerant

up to 2600 µg/ml supplemented chromium sulphate.

During the third phase of this research plan, dilutions of TEW were evaluated for

their effects on angiogenesis using CAM assay. TEWD1 and potassium dichromate were

found highly anti-angiogenic. Moreover, dilutions of TEW and potassium dichromate

have demonstrated significant toxicity when assessed through marine shrimps mortality

assay and phytotoxiciy assasy.

Chronic toxicity study on Wistar rats was conducted in the last phase. Chronic

exposure of TEW for three months to rats leads to the development of various lesions in

lung, liver, kidney and heart of rats.

In short, TEW and contaminated ground water of Kasur is imposing a great threat

not only to local inhabitants of the city but also to the population of far distance.

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Annexures

203

Annexure: 0l. Blood Agar

Formula (g/L)

Peptone 10 grams

Lab-Lemco powder 10 grams

Sodium chloride 05 grams

Agar 15 grams

Final pH 7.3 ± 0.2

All the above mentioned ingredients were dissolved in 1000 ml. of distilled water. Mixed

it well, autoclaved, cooled and then 7% sterile defibrinated sheep blood was added.

CHAPTER-8

ANNEXURES

Annexures

204

Annexure: 02. Nutrient agar

Formula (g/L)


Peptone 5 grams

Yeast extracts 2 grams

Lab-Lemco powder 1 gram

Agar 15 grams

Final pH 7.4 ± 0.2

All the above mentioned ingredients were dissolved in 1000 ml. of distilled water. Mixed

it well, autoclaved, cooled and then poured in sterilized petri plates.

Annexures

205

Annexure: 03. Crystal Violet

Deionized water 800 mililiters

Ethanol denatured 200 mililiters

Amonium oxalate 08 grams

Crystal violet 20 grams

Annexure: 04. Gram’s Iodine

Deionized water 1000 mililiters

PVP (poly vinyl pyro iodine) 110.60 grams

Iodine 18 grams

Potassium iodine anhydrous 19 grams

Annexures

206

Annexure: 05. Safranine

Ethanol 10%v/v

Methanol less than 1% v/v

Safranin O 0.25% w/v

Annexure: 06. MacConkey’s Agar

Formula (g/L)

Pancreatic Digest of Gelatin 17.0 grams

Pancreatic Digest of Casein 1.5 grams

Peptic Digest of Animal Tissue 1.5 grams

Lactose 10.0 grams

Bile Salts 1.5 grams

Sodium Chloride 5.0 grams

Agar 13.5 grams

Neutral Red 0.03 grams

Crystal Violet 1.0 miligrams

Final pH 7.0 ± 0.2

AOAC (1995)

All the above mentioned ingredients were dissolved in 1000ml. of distilled water. Mixed

it well, autoclaved, cooled and then poured in sterilized petri plates.

Annexures

207

Annexure: 07. Tryptophan Broth (For indole production test)

Formula (g/L)

Peptone 10 grams


Bromothymol blue (0.2%) 2 grams

Mixed in distilled water and autoclaved.

Annexure: 08. Kovac's Reagent

Composition: P-aminobenzaldehyde 10 grams/150 mililiters of isoamylalcohol Hydrochloric acid (concentrated) 50 mililiters (adding slowly) MacFaddin (1980)

Annexures

208

Annexure: 09. Glucose Phosphate Buffered Saline (For MR and VP

tests)

Formula (g/L)

Peptone 7 grams


Dipotassium hydrogen phosphate 5 grams

Glucose 5 grams

Final pH 6.9 ± 0.2

Mixed in distilled water and autoclaved.

Annexures

209

Annexure: 10. Voges-Proskauer Reagents

(α-NAPHTHOL, 40% POTASSIUM HYDROXIDE & CREATINE)

Formulation per 100 ml α-Naphthol Reagent α-Naphthol 5.0 grams

Potassium Hydroxide (40%)

Potassium Hydroxide 40.0 grams Sterile De-ionized Water 100.0 mililiters

Creatine Reagent (0.5%)

Creatine 0.5 grams Sterile De-ionized Water 100.0 mililiters Voges and Praskauer (1898)

Annexures

210

Annexure: 11. Simmons Citrate Agar

Formula (g/L)

Ammonium Dihydrogen Phosphate 1.0 grams

Dipotassium Phosphate 1.0 grams

Sodium Chloride 5.0 grams

Sodium Citrate 2.0 grams

Magnesium Sulfate 0.2 grams

Agar 15.0 grams

Bromthymol Blue 0.08 grams

Final pH 7.2 ± 0.2

Koser (1923)

All the above mentioned ingredients were dissolved in 1000ml. of distilled water. Mixed

It well, autoclaved, cooled and then poured in sterilized Petri plates.

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