chemical, microbiological and toxicological...
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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
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
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
Review of literature
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
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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,
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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
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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
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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
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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.
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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
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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
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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
Review of literature
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
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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
Review of literature
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
Review of literature
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)
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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
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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).
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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
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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
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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
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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,
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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.
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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
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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
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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.
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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).
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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.
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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).
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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
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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.
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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
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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).
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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
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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
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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
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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
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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
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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).
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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
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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).
Review of literature
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
Review of literature
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
Review of literature
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
Review of literature
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.
Review of literature
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
Review of literature
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
Review of literature
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
Review of literature
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. Chemical Analysis of Tannery Effluent Wastewater
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
3.6. Chronic Toxicity Testing
Materials and Methods
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.
Materials and Methods
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.
Materials and Methods
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).
Materials and Methods
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
Materials and Methods
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
Materials and Methods
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.
Materials and Methods
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.
Materials and Methods
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.
Materials and Methods
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
Materials and Methods
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).
Materials and Methods
70
3.3. CAM and Embryotoxicity Assay
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.
Materials and Methods
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
Materials and Methods
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
Materials and Methods
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.
Materials and Methods
74
3.4. Marine Shrimps Mortality Assay
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.
Materials and Methods
75
Plate 05. Hatching setup arranged for marine shrimps
Materials and Methods
76
3.5. Phytotoxicity Assay
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).
Materials and Methods
77
3.6. Chronic Toxicity Testing
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,
Materials and Methods
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. Chemical Analysis of Tannery Effluent Wastewater
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
Concentrations are in mg/L.
Results
88
GWS6GWS4GWS9GWS7GWS5GWS8GWS3GWS10GWS2GWS1
88.09
92.06
96.03
100.00
Ground water of shallow tubewells
Sim
ilarit
y
DendrogramSingle Linkage, Correlation Coefficient Distance
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
Concentrations are in mg/L.
Results
91
GWD1GWD6GWD3GWD5GWD4GWD2
95.35
96.90
98.45
100.00
Ground water of deep tubewells
Sim
ilarit
y
DendrogramSingle Linkage, Correlation Coefficient Distance
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
DendrogramSingle Linkage, Correlation Coefficient Distance
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
2. Staphylococcu aureus
3. E. coli
D 7.5 X 104 04 1. Bacillus subtilis
2. Corynebacterium kutsceri
3. Bacillus megaterium
4. Staphylococcu aureus
E 5.5 X 106 04 1. Micrococcus varians
2. Bacillus azotoformans
3. Bacillus subtilis
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
2. Bacillus subtilis
3. Bacillus megaterium
H 1.32 X 106 03 1. E. coli
2. Staphylococcu aureus
3. Bacillus subtilis
I 8.0 X 106 04 1. Staphylococcus epidermidis
2. Bacillus cereus.
3. Staphylococcu aureus
4. Bacillus azotoformans
J 9.5 X 105 04 1. Bacillus megaterium
2. Staphylococcus epidermidis
3. Bacillus subtilis
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
4.3. CAM and Embryotoxicity Assay
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
4.4. Marine Shrimps Mortality Assay
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
Concentration of Chromium (mg/ml)
Perc
enta
ge M
orta
lity
TEW
PDC
CHC
48hr Mortality Chart
050
100150
D1 D2 D3 D4 D5
Control
Concentration of Chromium (mg/ml)
Perc
enta
ge M
orta
lity TEW
PDC
CHC
72hr Mortality Chart
020406080
100120
D1 D2 D3 D4 D5
Control
Concentration of Chromium (mg/ml)
Perc
enta
ge M
orta
lity TEW
CHC
PDC
Results
133
4.5. Phytotoxicity Assay
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
Concentration of Chromium (mg/ml)
Leng
th o
f roo
t (cm
)
PDC
TEW
CHC
Results
135
4.6. Chronic Toxicity Testing
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
5.1. Chemical Analysis of Tannery Effluent Wastewater
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.
Discussion
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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Abass E, Reza AM, Vazirinejad. 2005. Chromium removal and recovery from tannery
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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)
Sodium chloride 5 grams
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
Sodium chloride 5 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
Sodium chloride 8 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.