chemical microbiological and toxicological …

CHEMICAL MICROBIOLOGICAL AND TOXICOLOGICAL

EVALUATION OF PHARMACEUTICAL EFFLUENT

WASTEWATER

BY

ALI SHARIF

(2011-VA-266)

A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE

REQUIREMENT FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

IN

PHARMACOLOGY & TOXICOLOGY

UNIVERSITY OF VETERINARY & ANIMAL SCIENCES,

LAHORE

2016

IN THE NAME OF ALLAH, THE MERCIFUL, THE

COMPASSIONATE

To,

The Controller of Examinations,

University of Veterinary & Animal Sciences,

Lahore.

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

submitted by Ali Sharif, Registration number 2011-VA-266 have been found satisfactory and

recommend that it be processed for the evaluation by the External Examiner for the award of the

degree.

Prof. Dr. Muhammad Ashraf ___________________________

SUPERVISOR

Dr. Aqeel Javeed ___________________________

MEMBER

Prof. Dr. Aftab Ahmad Anjum ___________________________

MEMBER

I

DEDICATION

ALI SHARIF

I would like to dedicate my thesis to

My parents

Muhammad Sharif and Surriya Soofia

who loves me unconditionally and endorsed my decisions

My loving wife Bushra Akhtar

who encouraged and supported me during this whole journey

and to My Mentor

Professor Dr. Muhammad Ashraf

for his kind guidance throughout my career.

II

ACKNOWLEDGEMENTS

I am thankful to the most Gracious, Merciful and Almighty ALLAH who gave me the

health, thoughts and the opportunity to complete this work. I bow before my compassionate

endowments to HOLY PROPHET MUHAMMAD (P.B.U.H) who served a torch of guidance

and knowledge for humanity as a whole.

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

and a deep sense of obligation to my reverend supervisor, Prof. Dr. Muhammad Ashraf (T.I),

Ex-Dean, Faculty of Biosciences, Ex-Chairman, Department of Pharmacology & Toxicology, for

his keen interest, valuable guidance, enlightened views, worthy suggestions, constructive criticism

and inspiring attitude during my studies, research project, and writing of this manuscript. .

I have been honored to express my heartiest gratitude to the member, supervisory

committee Dr. Aqeel Javeed, Associate professor, Department of Pharmacology & Toxicology

for his constructive criticism, moral support during the course work and finalizing this manuscript.

I gratefully acknowledge the invaluable help rendered by my member, supervisory

committee Prof. Dr. Aftab Ahmad Anjum, Department of Microbiology. He gave me time and

valuable suggestions during this research project.

It is a greater pleasure for me to express my heartiest gratitude and a deep sense of

appreciation to Higher Education Commission (HEC) for providing me financial support during

my whole Ph.D.

I gratefully acknowledge the invaluable help during my research Dr. Imran Altaf, Mr.

Mateen Abbas and Mr. Abdul Muqeet Khan, QOL-WTO and Dr. Saif-ur-Rehman Kashif

Department of Environmental Sciences University of Veterinary and Animal Sciences, Lahore.

They have helped me in my research work.

I am also appreciative to all the personal staff of post-graduate laboratories, Department of

Pharmacology and Toxicology, University of Veterinary and Animal Sciences (UVAS), Lahore

for providing their capabilities and keen interest which help me in conducting this research.

I am thankful to my friends, especially Muhammad Furqan Akhtar, Sajid Ali Chishti,

Sohaib Peerzada, Shehzada Khuram, Khaleeq Anwar, Irfan Hamid, Abdul Rehman and Moosa

Raza, for their moral support and encouraging opinions regarding my research work.

At the end, I would like to extend my thanks and compliments to my loving wife Bushra

Akhtar, kids Aroosh Ali and Ayesha Ali, brothers Ahmed Sharif and Usman Sharif for their

great care, affection and prayers which enabled me to continue and complete my studies

successfully.

Ali Sharif

[email protected]

mailto:[email protected]

III

TABLE OF CONTENTS

DEDICATION……………………………………………………………………………….... (I)

ACKNOWLEDGEMENTS…………………………………………………………………… (II)

TABLE OF CONTENTS……………………………………………………………………... (III)

LIST OF TABLES…………………………………………………………………………...... (IV)

LIST OF FIGURES…………………………………………………………………………... (VII)

LIST OF ABBREVIATIONS…………………………………………………………………. (IX)

SR. NO. CHAPTERS PAGE NO.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 7

3 MATERIALS AND METHODS 16

4 RESULTS 52

5 DISCUSSION 98

6 SUMMARY 111

7 LITERATURE CITED 113

8 APPENDICES 123

IV

LIST OF TABLES

TABLE

NO.

TITLE PAGE

NO.

3.1 Activation mixture composition 38

3.2 Different dilutions of pharmaceutical wastewater used for MTT assay 42

4.1 Toxic metal content in the effluent of pharmaceutical industries 53

4.2 Components found in pharmaceutical effluent PEW1 55

4.3 Compounds identified in pharmaceutical effluent waste water PEW1

using GC-MS

58

4.4 Total number of viable bacteria in pharmaceutical waste water sample

(PEW1)

61

4.5 Morphological and biochemical characteristics of bacterial isolates 62

4.6 Total number of isolated and identified bacteria from pharmaceutical

waste water (PEW1)

63

4.7 Maximum tolerable concentration of heavy metals against PEW 1

bacterial isolates

64

4.8 Antibiotic sensitivity and resistant activity of isolated bacterial isolates of

PEW1

65

4.9 Mean diameter and tail length of different pharmaceutical waste water

samples

67

4.10 Percentage fragmentation, Damage index and genetic damage index of

different pharmaceutical waste water samples

68

V

4.11 Mean diameter and tail length of different concentrations of

pharmaceutical wastewater sample (PEW 1)

73

4.12 Percentage fragmentation, damage index and genetic damage index

detected with increasing dilutions of pharmaceutical effluent (PEW 1)

74

4.13 Mean diameter and tail length of different concentrations of

pharmaceutical waste water sample (PEW 6)

75

4.14 Percentage fragmentation, damage index and genetic damage index

detected with increasing dilutions of pharmaceutical effluent (PEW 6)

76

4.15 Revertant colonies and mutagenic index obtained by exposure to

pharmaceutical waste water (plates with 30-300 colonies were selected)

80

4.16 Revertant colonies and mutagenic index obtained by exposure to

pharmaceutical waste water (PEW1) at different levels of dilutions

81

4.17 Dose dependent decrease in revertant colonies observed on exposure to

Pharmaceutical effluent waste water sample (PEW 6)

83

4.18 Quantification of viable cells in BHK-21 cell line prior to assay 85

4.19 Mean optical density (OD) and cell survival percentages against various

concentrations of PEW1 after incubation of 48 hours in MTT assay using

BHK-21 cell line

87

4.20 Mean optical density (OD) and cell survival percentages against various

concentrations of PEW6 after incubation of 48 hours in MTT assay using

BHK-21 cell line

89

4.21 Effect of vitamin E on SOD activity in rat plasma/blood, kidney and liver

caused by chronic exposure to PEW1 at different levels of concentration

92

VI

4.22 Effect of vitamin E on CAT activity in rat plasma/blood, kidney and liver

caused by chronic exposure to PEW1 at different levels of concentration

93

4.23 Effect of vitamin E on H2O2 activity in rat plasma/blood, kidney and

liver caused by chronic exposure to PEW1 at different levels of

concentration

94

VII

LIST OF FIGURES

FIGURE

NO.

TITLE PAGE

NO.

3.1 Schematic representation of chemical characterizations 18

3.2 Schematic representation of microbiological evaluation 22

3.3 Schematic representation of procedure adopted for comet assay 26

3.4 Schematic representation of procedure adopted for comet assay 33

3.5 Schematic representation of procedure adopted for MTT assay 43

4.1 Box whisker plot of heavy metal concentrations in different samples

of Pharmaceutical waste water.

54

4.2 Mass spectra of basic fraction of PEW 1 56

4.3 Mass spectra of lidocaine using DB1 column 56

4.4 Mass spectra of neutral fraction of PEW 1 57

4.5 Mass spectra of digitoxin using DB1 column 57

4.6 Mass spectra of dipyrone using DB-35ms column 59

4.7 Mass spectra of trimethoprim using DB-35ms column 59

4.8 Comets appeared in sheep lymphocytes when exposed to

pharmaceutical effluent wastewater (PEW 1)

69

4.9 Tail lengths of damage induced in sheep lymphocytes at an altered

concentration of PEW 1 (%v/v)

70

4.10 Comets appeared in sheep lymphocytes when exposed to

pharmaceutical effluent wastewater (PEW 6)

71

VIII

4.11 Tail lengths of damage induced in sheep lymphocytes at an altered

concentration of PEW 6 (%v/v)

72

4.12 Potential of mutagenicity of PEW1 in TA-100 strain in presence and

absence of metabolic activation mixture

82



82



84



84

4.16 Percentage survival of cells (CSP) against different log

concentrations of PEW1 on BHK-21 cell lines

88

4.17 Cell survival percentage (CSP) against different log concentrations of

PEW6 in MTT assay on BHK-21 cell lines

90

4.18 Effect of PEW1 on SOD activity in rat plasma, kidney and liver 95

4.19 Effect of PEW1 on CAT activity in rat plasma, kidney and liver 95

4.20 Effect of PEW1 on H2O2 activity in rat plasma, kidney and liver 95

4.21(a) Effect of pharmaceutical waste water on kidney 97

4.21 (b) Effect of pharmaceutical waste water on Liver 97

4.21 (c) Effect of pharmaceutical waste water on lungs 97

4.21 (d) Effect of pharmaceutical waste water on intestine 97

4.21 (e) Effect of pharmaceutical waste water on heart 97

IX

LIST OF ABBREVIATIONS

2AA 2-aminoanthracycline

AAS Atomic Absorption Spectrophotometer

BHK-21 Baby hamster kidney cell line

CAT Catalase

CuSO4 Copper Sulfate

DCM Dichloromethane

DMEM Dulbecco’s modified eagle cell culture medium

DMSO Di-methyl sulfoxide

DNA Deoxyribonucleic acid

EA Ethyl acetate

EDTA Ethylene-diamine-tetra-acetic acid

GC-MS Gas chromatograph mass spectrophotometer

GM Glucose Minimal

H2O2 Hydrogen peroxide

HCl Hydrochloric acid

IPA Isopropyl alcohol

K2Cr2O7 Potassium dichromate

X

LMPA Low melting point agarose

MCF McFarland unit

MDA Malondialdehyde

MTT Methyl thiazol tetrazolium

NaCl Sodium chloride

NaOH Sodium hydroxide

NMPA Normal melting point agarose

NOEC No observed effective concentration

PCP Pentachlorophenol

PEW Pharmaceutical effluent wastewater

ROS Reactive oxygen species

SCGE Single cell gel electrophoresis

TMP Trimethoprim

T-SOD Total superoxide dismutase

VB Vogel-Bonner

1

CHAPTER 1

INTRODUCTION

Water pollution is the direct or indirect addition of contaminating substances into the pure

water resources, which make the water inappropriate for drinking, bathing and/or for noncontact

recreation (Clark et al. 2003). During the past few years level of environmental pollutants,

agrochemicals and sewage waste has increased dramatically (Maduka 2006). Medicinal agents

have been used globally. The quantity of drugs existing in atmosphere is less, but constant input

may contribute to the potential risk to different life forms (Klavarioti et al. 2009).

There is a rapid rise in water contamination, the reason after this is a unprecedented population

growth, urbanization and industrialization (Ma et al. 2009). An agricultural country like Pakistan

has been facing the serious encounter of water contamination. Establishing of industrial

components and their makings have been tangled so there is a rise in intensity of water

contamination. Moreover the carelessness of responsiveness in guarding the environment and

improper organization of arrangements has increased the problems related to pollution of water

bodies (Zaroual et al. 2005).

Toxic chemicals such as Lead (Pb), Copper (Cu), Zinc (Zn), Mercury (Hg), Cyanide (CN)

got released into open water bodies caused mortality of fish and other beings present in water along

with lesions in humans even if present in lesser amounts.

2

An Epidemic of Minimata was linked to mercury poisoning associated with fish usage due

to discharge from mercury from a plastic industry setup. It was the first major outbreak of mercury

associated poisoning (Okoye 1992).

Different antibiotics and antiepileptic drugs were detected and their concentrations were

measured in waste waters and surface waters. The study was conducted in the Po Valley, Italy

where a waste water treatment plant was selected and the investigation was conducted in the

effluent and influent. Three sampling sites were selected and predicted concentrations were

compared with measured concentrations. Predicted and measured concentrations for ciprofloxacin,

azithromycin, trimethoprim and carbamazepine were accurately measured, whereas difference

between measured and predicted concentrations were very high for other compounds (Verlicchi et

al. 2014)

Dutch water reservoirs were studied for the detection of nine different drugs which are

metformin, metoprolol, sotalol, valsartan, losartan, irbesatran, hydrochlorothiazide, diclofenac and

carbamazepine. The predicted concentrations were compared with waste water concentrations,

removal in sewage treatment plants and recovery in regional surface water. It was suggested that

consumption data from local sources provided useful information regarding the selection of drugs

for monitoring. Metformin was found in highest concentrations in waste waters. Guanylurea which

is a biodegraded product of metformin was also detected in effluents and surface waters at concentrations

of 39-56 µg/mL and 1.8 to 3.9 µg/mL respectively (Oosterhuis et al. 2013)

Ciprofloxacin, tamoxifen and cyclophosphamide are anticancer agents frequently used in

hospitals. These agents might get released into the hospital effluents and in open environments. It

was established that concentration were low but data was not present regarding eco-toxicological

impact. Genotoxicity and cell viability was determined using comet and cell proliferative assays.

The results were compared with the standard algaltoxkit F and microtox tests. The results

3

suggested that that non-monotonic dose response was observed when cell viability was measured

using hepatic cell line HepG2. Single drug did not induced any DNA break whereas mixture of

drug combination was able to induce DNA breaks which was also confirmed by standard microtox

assay (Mater et al. 2014)

Hundreds of tons of drugs are emitted into the environment as such or their metabolites.

Most of pharmaceuticals have been reported at trace levels (hundreds of nanograms per liter) in

the rivers and lake water. It indirectly substantiates capability to persist in surface waters.

Complete data about adverse toxic effects on simple living organisms at the little amounts at which

pharmaceutical molecules are present in the environment are still lacking. The xenobiotic nature

of pharmaceuticals would suggest to impede their release to the environment by confining the

sources of pollution (Andreozzi et al. 2004). Different types of drugs and their metabolic products

have been reported in water bodies, especially water emitting from sewage treatment plants

(Halling-Sørensen et al. 1998), (Kümmerer 2009), (Sarmah et al. 2006).

The occurrence of drugs in municipal sewage was first stated in America. Drugs and

organic compounds have been reported in 139 streams of America from 1999 to 2000 (Kolpin et

al. 2002). Acidic and neutral drugs have been reported in Canada and Ontario emphasizing that

large portion of these drugs is removed partially from treatment plants (Carrara et al. 2008). This

has also been recognized as an environmental issue in Germany, where pharmaceuticals were

found in water bodies. Even the entire removal of many of these pharmaceuticals cannot be

possible with conventional sewage treatment (Brun et al. 2006).

Marine and life present on earth can be affected by components which are possibly

hazardous, when present in reduced quantities. Pharmaceuticals are lipophilic and non-

4

biodegradable along with their biological activities. These facts make them a concerning factor

when talking about the environment (Velagaleti and Burns 2006).

The concentration of individual drugs in effluent might be low, but the effect of combined

drugs with same mechanism known as additive effect and the drugs having different modes of

action called as synergism might have significant eco-toxicological effect (Brain et al. 2004). It is

suggested to evaluate the toxicity in a combination to inspect the collective consequence (Kolpin

et al. 2002), (Gros et al. 2007). Additive and synergistic effects of different drugs were observed

several times when concentration addition was investigated (Cleuvers 2003, Cleuvers 2004).

A compound alone may produce a little effect, but in mixture of compounds the effect may

be hazardous, which on the other hand might be underestimated. The compounds which are present

below the no observed effective concentration (NOEC) might contribute to the eco-toxicological

effect of mixture and a realistic picture can therefore be sketched through the analysis of mixture

of pharmaceuticals (Fent et al. 2006).

A current research project was undertaken for assessment of toxicity associated with

pharmaceutical wastewater. The entire Project was divided into three phases. Phase 1 was designed

for the characterization of pharmaceutical effluent wastewater (PEW) using atomic absorption

spectrophotometer and Gas Chromatography Mass Spectrophotometer.

During the 2nd phase microbiological evaluation was performed. Bacterial isolates were

separated from PEW and identified using different biochemical tests. Heavy metal tolerance and

antibiotic resistance were checked against different isolates identified.

During the 3rd phase toxicological evaluation was done using different biological assays.

Ames test was performed for the determination of mutagenicity, MTT assay was performed for

5

the determination of cell viability against different PEW samples. Geno toxicity was evaluated

using the alkaline comet assay.

During last stage oxidative stress was evaluated by exposing Wistar rats with PEW for 2

months and later estimating the enzyme levels spectrophotometrically using a kit method and

further evaluated for histopathological effects.

Exposure to inhabitants with untreated pharmaceutical wastewater is a serious threat. Very

few studies are available highlighting this issue. There is no data available reporting the toxic

effects of untreated pharmaceutical wastewater in Pakistan. None of previous studies regarding the

toxic potential of Pharmaceutical wastewater were performed on pure samples as such, all the

studies demonstrated some pretreatments to detect organic pollutants. Present study evaluated the

toxic effect of PEW as such. A comprehensive way was selected to determine the toxicological

profile of PEW. A comprehensive chemical characterization was performed to determine the actual

picture of organic and inorganic pollutants present in the PEW. Microbiological characterization

and the link between heavy metal tolerance and antibiotic resistance with PEW was not evaluated

before in Pakistan. None of the study previously reported the use of PEW for the assessment of

mutagenicity, genotoxicity and cytotoxicity. It was also tried to determine the effect of PEW in

animals when exposed chronically in order to establish a correlation of the toxic potential of PEW

with the emergence of diseases that are caused by imbalance of antioxidant enzymes. The

histopathological effects caused by chronic exposure of PEW were investigated. This study

evaluates certain approaches that may be applicable when assessing potential health risks

associated with direct or indirect exposure to pharmaceutical effluent wastewater.

6

Aims and Objectives

Chemical characterization of pharmaceutical effluent wastewater

Isolation and identification of bacteria from pharmaceutical effluent wastewater along with

investigation of their tolerance level for various chemicals.

Toxicological evaluation of pharmaceutical effluent wastewater using in-vitro and in-vivo

toxicity models

Review of Literature

7

CHAPTER 2

REVIEW OF LITERATURE

2.1 Organic compound

Pharmaceuticals and their metabolites enter into the environment through a variety of ways,

among them are elimination of human source, agriculture water, waste water from treatment

facilities and direct drainage in drains without treatment which is a serious cause of toxicity

(Crouse et al. 2012).

About 200 active pharmaceutical ingredients have been detected in aquatic environment

usually referred as micro pollutants, but several anthropogenic influences on aquatic environment

can be determined by using pharmaceutical compounds as tracers (Müller et al. 2012).

Physico-chemical analysis alone is not sufficient to estimate the toxicity associated with

composite water mixtures because of the synergistic, additive and antagonistic effects that might

produce in mixtures. Substitution methods for the assessment of toxicity/genotoxicity are

biological tests which have proven to produce global responses without previous knowledge of

mixture composition (Žegura et al. 2009). Both human and environmental health is endangered if

their effluent is being wrongly handled and disposed (Bakare et al. 2009), (Fick et al. 2009).

A large number of drugs detected were antibiotics, especially the fluoroquinolones, which

were expected toxic to bacteria present in water. It was a matter of serious concern that high levels

of fluoroquinolones led to the development of antibiotic resistance and production of multi

resistant pathogens al. (Li D 2008).

Production of drugs at a massive level has been identified as a source of pollution in an

environment containing active pharmaceutical ingredients. It was investigated that sewage


8

treatment plant of Patancheru, near Hyderabad, India showed a high concentration of drugs

(mg/mL). This plant receives water from 90 bulk drug manufacturers of India. (Larsson et al.

2007).

Pharmaceuticals are used by people and after their application in different setups they are

entered into the environment by different pathways. The most common methods of entrance in the

environment is excretion following the ingestion of medicines and the disposing of drugs in the

form of wastewater. Sewage treatment plants do not degrade pharmaceuticals completely resulting

in discharge of significant concentrations in treated effluents. This effluent then becomes a source

of contamination in rivers and sometimes of ground water and drinking water. Sewage sludge also

proves to be a source of contamination in soil when it is applied via agricultural fields along with

manure application and its runoff which leads to a source of pharmaceuticals in agriculture fields.

The point of concern is not the high production of drugs rather their existence in the atmosphere

and biological activities (Fent et al. 2006).

Pharmaceuticals have been recognized as potential pollutants because they have similar

physicochemical properties. The least amount of pharmaceuticals (ng/L or μg/L) present in the

environment is capable of inducing toxic effects, e.g. antibiotics and steroids are capable of

inducing bacterial resistance in the natural bacterial flora and endocrine disrupting effects

(Hernando et al. 2006). Incorporation of contaminating substances, directly or indirectly in the

pure water makes the water unsuitable for any type of use (Clark et al. 2003).

Significant amounts of drugs have been found in wastewater emitting from hospitals,

wastewater from pharmaceutical units and leaching from landfill sites (Holm et al. 1995). Different

kind of medicines are being produced during any given period so the waste streams of

pharmaceutical wastewater are not always uniform (Houk 1992).


9

2.2 Inorganic compounds

Water pollution in the water bodies have been aroused due to various industrial effluents

contributing serious concerns toward the environment. Drugs usually poorly absorbed when taken

orally in humans and animals excreted out via urine and feaces along with 25 to 75 % added

excipients (Krifa et al. 2013).

Pharmaceutical industries waste water may contain different heavy metals besides some

organic pollutants or phenolic compounds which are posing threat to water reservoirs (Anyakora

et al. 2011). A number of antioxidants have the capacity of chelating metal ions and reduces their

ability to form reactive oxygen species (Jomova and Valko 2011). Some elements which are toxic

to organisms might become beneficial under some other conditions (Lane et al. 2005), (Singh et

al. 2011). It has been found that redox metals like chromium, copper and iron including some other

metals have the property to induce toxic radicals. Metal ion disturbance homeostasis can lead to

generation of stress, which is characterized by the generation of toxic radicals, which can overcome

body antioxidant potential and results in DNA damage, lipid peroxidation, protein modifications

and other effects which are indicative to certain diseases which include severe disorders of heart,

metabolic disorders and central nervous system. The reason for these disorders might be attributed

to the generation of free radicals (Jomova and Valko 2011).

Heavy metals have been considered most abundant and toxic inorganic pollutants of the

environment. Metals cannot be degraded unlike many resistant pollutants which are organic in

nature but heavy metals do have a potential to accumulate in the food chain. Their genotoxic and

mutagenic effects have also been investigated in humans and characterized as priority

environmental pollutants (Martín-González et al. 2006).


10

There is a strong emphasis that chronic toxic effects cannot be excluded. Individual toxicity

study of most drugs has been conducted but in the environment as a mixture of different drug

classes very few investigations were found. It is suggested and found necessary by many authors

to investigate the toxicity potential of pharmaceutical waste water mixture (Gros et al. 2007),

(Kolpin et al. 2002).

Metals bind with proteins and other structures in the body and affect the membranous

structures leading to generation of oxidative biomarkers leading to a number of diseases of various

organs (Valko et al. 2005),(MatÉs et al. 1999),(Mates 2000).

Organisms can be damaged by excessive levels of metals. Mercury and lead are not

considered vital and have no beneficial effect on organisms. Heavy metal pollution has many

negative consequences on the hydrosphere. Heavy metals have been considered most abundant

pollutants in sewage and industrial wastewaters. Certain heavy metals like Ni and Zn are

considered necessary for the growth of microorganisms in trace amounts but damages human

health at higher concentration. Heavy metals emissions might arise from casting of metals, fuel

burning, and using of antiseptics and disinfectants (Filali et al. 2000). Varying amount of heavy

metals is required by living organisms (Lane and Morel 2000). All metals are lethal at higher

concentrations (Chronopoulos et al. 1997).

2.3 Microbiology

Pharmaceutical industry being a major contributor to this risk have been involved in severe

heavy metal and drug associated toxicity. Effluent being a complex mixture is capable of altering

composition, distribution, diversity of microorganisms (Bisht et al. 2012)


11

Resistant of bacteria to heavy metals have been reported previously, which have been

obtained from soils, waters and sediments. Bacterial resistance to heavy metals has been associated

with plasmid mediated mechanism which also encodes resistance to antibiotics (Filali et al. 2000).

Development of anti-biotic resistance bacteria and potential to accumulate antibiotics have

produced serious environmental concerns. Extensive usage of antibiotics in humans practice and

use as growth promoter has been involved in the generation of resistant pathogenic microbes.

Resistance of microbes to heavy metals and antibiotics is a serious concern because the mechanism

involved is plasmid mediated (Ramteke 1997). Genes responsible for heavy metals and antibiotics

resistance share same location on plasmids so it is necessary that resistant isolates should be

investigated for heavy metal tolerance (Novick et al. 1979).

2.4 Comet assay

DNA damage has been associated with a lot of substances. The reason behind the DNA

damage might be oxidation of bases, which will eventually lead to breakage of strands. DNA

damage can be accessed by comet assay. The method has been devised for the detection of damage

in DNA using different organisms (Azevedo et al. 2011).

It has become a normal practice to screen normal and wastewater for the detection of

mutagens. -Allium cepa root used to estimate the genotoxic effect in various areas of Croatia.

Sampling was performed for a three month period. Different physicochemical characters have been

studied during the study. Chromosomal aberrations, growth inhibition of roots and different

modifications in morphology have been evaluated during the study (Radić et al. 2010).

Genotoxicity of different pesticides in fresh water have been studied using two different

tests, including comet assay and micronucleus test. Micronucleus assay was conducted using blood

cells collected from different peripheral areas. (Kumar et al. 2010).


12

Flame atomic absorption has been used for detecting heavy metals. High levels of heavy

metals were confirmed when samples taken from Etremoz lake waters in Northeastern Brazilian

coasts at various time intervals. The findings of the study confirmed that the quality of water has

been deteriorating in Etremoz lake due to heavy metals (Barbosa et al. 2010).

Comet assay was reported to be a delicate method for the determination of genetic damage

in the area of environmental toxicity studies as compared to micronucleus assay. It has been

influenced by the experimental conditions present in the laboratory because of its high sensitivity

(Frenzilli et al. 2009).

Metals have been associated with the production of tumors that is why genotoxicity of

these heavy metals is also necessary. Chromium is a documented genotoxic agent and exhibits its

mechanism via breakage of strand and several other mechanisms. Different streams in Brazil have

been investigated for checking the potential to induce genotoxicity with the help of comet assay

and a micronucleus assay in erythrocytes. Tannery waste water has been associated with the

greatest level of genotoxic effect. Mutagenic potential of the samples was also investigated with

the help of onion as an experimental model in the root tip test. The results validated that chromium

is the causative metal which has also been linked to mutagenicity (Matsumoto et al. 2006).

Unicellular protozoan has been evaluated in comet assay to detect the genotoxicity of

wastewater samples. Tetrahymena thermophile has been used in the assay with slight

modifications. A short term exposure was given to T. thermophila using hydrogen peroxide,

formaldehyde and phenol as controls. Waste water was checked for the genotoxic ability. The

results indicated that the effluent was less toxic than influents and treatment procedures decreased

the ability of waste water to induce genetic damage (Lah et al. 2004).


13

Fish and earth worm have been the experimental subjects in determining the genotoxicity

of the waters of Noyyal River. The erythrocytes of fish were measured for the damage induced by

waste water and a ratio of damaged DNA length to width was observed. It was suggested that with

the increase of the time of exposure the damaged induced was also increased. (Rajaguru et al.

2003).

Human lymphocytes were exposed to different quantities of organic agents. These organic

agents were extracted from water samples of Tirpur collected from 12 different locations. It was

projected that all samples have a mutagenic potential and induces DNA damage in human

lymphocytes. It was further proposed that aromatic amines were responsible for this effect

(Rajaguru et al. 2002).

Silk coloring industry waste water was examined for the induction of mitotic abnormalities

in Allium cepa root system. Cytotoxicity of the effluent was studied by treating Allium cepa roots

with different concentrations of effluent (25, 50, 75, and 100%) for different durations (6, 12, 24,

and 48 h). Cell division was inhibited. A strong concentration dependent effect was evident from

a decline in the mitotic index. A wide range of mitotic abnormalities was also induced.

Abnormalities were appeared which included stickiness of chromosomes, fragments, bridges,

laggards, bi-nucleate cells and vacuolated nuclei. The results presented that silk dyeing industry

effluents act as potential mutagens (Sudhakar et al. 2001).

2.5 Ames test

Waste water was screened using different strains TA-98 and YG1041 for the mutagenic

potential. It was observed that rats pretreated with different concentrations of waste water exhibit

lesion in the colon and responded positively to both the tester strains (de Lima et al. 2007).


14

The SOS chrome test was used to investigate potential of genotoxicity of waste water

collected in Rouen area. The test was performed on Escherichia coli. Also, different strains of

Salmonella typhimurium TA98, TA100 and TA102 were evaluated for mutagenicity. The

experiment was conducted with and without rat liver extract (Jolibois and Guerbet 2005).

Hospital waste water was investigated for the presence of mutagenicity using a chrome test

of mutagenicity and Ames fluctuation analysis using different strains of Salmonella typhimurium.

Sampling was performed at random for the whole day and this method was adopted for a period

of three months. It was suggested that mutagenic effect was different for samples collected at

different time intervals (Jolibois et al. 2003).

Wastewater samples were screened for mutagenicity before and after treatment. The

sampling was performed in summer and winter. Extraction was done using C18 .Wastewater

samples were collected before and after disinfection in summer and in winter. Alliumcepa test and

Tradescantia/micronuclei test was employed to access the damage induced by waste water. It was

suggested that mutagenicity was produced in experimental organisms when treated with

disinfectants especially ClO2 and ozone. (Monarca et al. 2000).

A 20 month study was conducted to check for the mutagenicity of water samples collected

from Cai River. Different strains (TA98 and TA100) were used to screen samples. The study was

conducted with and without metabolic activation system. The study concluded that TA 98 was

much more sensitive than TA100 (Vargas et al. 1993).

2.6 Cytotoxic evaluation

Two different fish cell lines PLHC-1 and RTG-2 were used to evaluate cytotoxicity of

different drugs having different modes of action. The values of effective concentration EC50 were


15

calculated. The results indicated that PLHC-1 cell line produced more sensitive results than RTG-

2 cell (Caminada et al. 2006).

Fish hepatocytes were exposed to nine different drugs and cell viability was evaluated with.

MTT assay. 7-ethoxyresorufin-o-deethylase (EROD) was employed for the determination of

interaction with cytochrome P4501A (CYP1A) enzyme and di-chloro fluorescein (DCFH-DA)

assay was used to detect oxidative stress (Laville et al. 2004).

2.7 Oxidative stress

It has been found that metals have the property to yield radicals such as superoxide-

dismutase (SOD) and nitric oxide (NO) in living systems. Disturbance of metal levels can alter

oxidative biomarkers, which is characterized by the generation of reactive oxygen species (ROS)

which can overwhelm body capacity of antioxidant ion and resulted in DNA damage, protein

modifications and other effects that are indicative to certain diseases. The reason behind diseases

is the generation of superoxide associated radical, hydroxyl radical, and other ROS leading to

mutagenicity and carcinogenicity. Antioxidants like vitamin C, vitamin E and glutathione (GSH),

can chelate heavy metals and help reduce oxidative stress (Jomova and Valko 2011).

It is also found that metal-mediated formation of free radicals causes various modifications

to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. These

processes leads to a number of diseased states including Alzheimer’s, cancer, ischemia, failures in

immunity and endocrine dysfunctions (Valko et al. 2005),(MatÉs et al. 1999),(Mates 2000).

CHAPTER 3

MATERIALS AND METHODS

3.1. Study Area

Lahore, is the second largest city of Pakistan. It is the capital of Punjab province. It is

situated on river Ravi. Lahore has been categorized among top 30 populated urban areas of the

world. The population is greater than 10 million. The weather is tremendously hot during summer.

An average rainfall of 735 mm has been reported. (Shakir et al. 2012). Lahore is located in

northeast of Pakistan and with northern India. It is the second largest city of Pakistan (Ghauri et

al. 2007). It is a metropolitan city with a population of more than 10 million and is growing at a

rate of 4% (Ali and Athar 2010).

3.2. Sampling

Samples were collected in summer season from April 2013 to June 2013. The industries

were selected at random and sampling was performed for ten pharmaceutical industries. Composite

sample was made by collecting waste water 8 times during a 24 hour time interval. It was assured

that the sample bottles did not touch the side and foot of stream flow. Samples were placed in an

ice bath during the collection procedure.

All the samples were gathered in autoclaved amber colored acid washed bottles. The

samples were used to rinse the bottles before collection. An air gap was left in each bottle at the

top. Samples were placed at 4°C. First pharmaceutical waste water sample PEW1 was divided into

two sets for the evaluation of polar contaminants (drugs) and nonpolar pollutants which are heavy

metals.

Materials and Methods

17

All the ten samples were used as such for the evaluation of toxicity using different

bioassays. However sample PEW1 and PEW6 were further diluted two fold to check for the effect

of diluted concentrations of PEW on experimental models.

Sample collection sites


18

3.3. Chemical characterization

Figure 3.1 Schematic representation of chemical characterizations


19

3.3.1. Sample preparation for nonpolar pollutants

Digestion was performed in a fume hood with concentrated HNO3. 250 mL of each sample

was subjected to digest with acid at a temperature if 150°C and then raising the temperature to

250°C. Prior to digestion, samples were placed overnight for the removal of bubbles. The end point

for digestion was considered a transparent greenish color. After cooling of samples each of the

sample was diluted to 25mL using concentrated HNO3. All the samples were analyzed after

filtration.

3.3.2. Standard preparation

Solutions were purchased from Fluka Analytical TraceCERT Reg. and Fisher Scientific

UK limited, Bishop Meadow road Leicestershire. The strength of different standards were 1000

mg. L-1. Calibration curves were drawn for all heavy metals to be analyzed. Calibration curves

were prepared from 1 mg.L-1 to 5 mg.L-1 for zinc (Zn) and Iron (Fe). Copper (Cu), Chromium (Cr),

Lead (Pb), Arsenic (As) calibration curves were made within the range of 0.2 mg.L-1 to 1 mg.L-1.

Whereas Cadmium (Cd) calibration curves were drawn in range of Serial dilutions were made in

the range of 0.02 to 0.1 mg.L-1. The apparatus used was Z-8230 Zeeman atomic absorption

spectrophotometer.

3.3.3. Sample preparation for polar pollutants

A micro filter (0.22 µm) was used to filter all the samples before analysis. Fractions of the

sample were prepared using liquid liquid extraction. (Aleem and Malik 2005) (Seiler et al. 1999).

Acidic, basic and neutral fraction of samples was prepared for GC-MS analysis as drugs exist in

these three forms.

Acidic fraction was prepared by taking 2 mL of sample mixed with 0.1 mL (1 M, NaOH)

and then incubate it at 25 °C for 10 minutes. pH was adjusted to 3 with phosphate buffer of pH 3


20

(Europe 2004) and the sample was extracted with 5 mL dichloromethane (DCM). The sample was

centrifuged at 5000g for 5 minutes and the DCM layer was dried at 50°C and used for analysis by

using DCM as solvent.

Neutral drugs fraction was prepared by taking 5 mL sample mixing it with 3 mL phosphate

buffer pH 6.

Basic fraction was prepared by taking 9 mL of sample and mixed with 2mL 0.5M Na2CO3

having a pH of 9. Extraction was performed with a mixture of DCM and isopropyl alcohol (IPA)

3:1 v/v. The organic layer was collected by centrifugation at 1500g for 5 minutes. Layer was dried

and further used for analysis by incorporating ethyl acetate.

GC-MS was conducted for the three fractions previously prepared. GC system, Agilent

Technologies 6890N was used to perform analysis, which is connected with a mass selective

spectrophotometer 5975 inert XL. The apparatus was equipped with an injector 7683 B series.

Different columns were used for the analysis. The column used were DB-1-1022, DB35-ms 122-

3832 and DB-5-122-5032 (Nikolaou et al. 2007). Comparison of mass spectra was performed

using the NIST library (Alam et al. 2009).

The method used for the column DB-1-1022 was 40°C as the initial temperature

maintained for 1 min, the temperature was increased to 290 °C by raising it at a rate of 25 °C/min,

maintained 40°C for 0.38 minutes and 290°C for 13.09 minutes. Helium was used as the carrier

gas at 25cm/s measured at 40°C. Split injection was used at a ratio of 40:1 at 250 °C along with

MSD detector at 230 °C. The Full scan run was performed at m/z 30-500 for finding of polar

contaminants.

Program conditions with the column DB35-ms 122-3832 were initial temperature at 50°C

for 1 min, raising to 50-100°C with a rate 25 °C/min and further increasing to 100-300 °C with a


21

rate of 5°C/min and reaching at a temperature of 300 °C for 5 minutes. Helium was used again as

the carrier gas at 35cm/s maintained at a temperature of 50°C. Split less injection at 250 °C was

used along with the MSD detector at 300 °C. The full scan run was directed at m/z 50-500 for

detecting polar contaminants.

A third method adopted with the column DB-5-122-5032 was a 80 °C hold for 1 minute, 80

to 280 °C at a rate of 10 °C/min, 280 °C for 9 min. Split injection 1: 40 at 250 °C was used with

helium as a carrier gas and MS detector at 230 °C with full scan at m/z 30-500.

3.4. Microbial load

Microbial loads of the pharmaceutical waste water samples were determined on tenfold

dilutions prepared under aseptic conditions. Each of these dilutions were mixed with molten

nutrient agar at a temperature of 45°C. All of these dilutions of pharmaceutical waste water were

incubated in sterilized Petri plates at a temperature of 37°C for 24 hours. Total number of viable

bacteria were counted as colony forming units per ml cfu/ml. Plates with 30 to 300 colonies were

selected for bacterial load.


22

Figure 3.2 Schematic representation of microbiological evaluation


23

3.4.1. Bacterial isolation and identification

Bacteria were isolated and identified according to Bergey’s manual of systemic

bacteriology (Goodfellow et al. 2012). All types of colonies from each plate (previously counted)

of pharmaceutical wastewater were poured on sterilized nutrient agar plates and incubated for 24

hours. Two way streaking was performed for isolation purposes. Identification was performed by

observing macroscopic characteristics such as color, size and shape, gram staining and using a

series of biochemical tests.

3.4.1.1 Glucose fermentation test

Isolated colonies were transferred to sterile tubes containing phenol red broth. The tubes

were incubated at 37°C for 24 h. Change of color from red to yellow was recorded as positive

result which was due to change in pH of media to acidic environment indicating bacteria had

fermented glucose.

3.4.1.2 Mannitol fermentation test

Isolated colonies were transferred to sterile tubes containing phenol red mannitol broth

(nutrient broth with 0.5 to 1 % mannitol). The tubes were incubated at 37°C for 24 h. Change of

color from red to yellow was recorded as positive result which was due to change in pH of media

to acidic environment indicating bacteria had fermented glucose.

3.4.1.3 Indole test

Indole test was performed on purified bacterial isolates and results were recorded. Pink to

wine color ring after addition of reagent was observed showing positive results and pale yellow

color ring was observed in case of negative results


24

3.4.1.4 Oxidase test.

A filter paper was soaked with the substrate tetra methyl-p-phenylenediamine di-

hydrochloride. It was moistened with DW. Colony was picked with platinum loop and smeared on

filter paper. Deep blue to purple color was recorded for positive result. No color was observed in

case of negative results.

3.4.1.5 Catalase test

Bacterial colony was poured on a clean dry glass slide using a sterile loop and a drop of

3% Hydrogen peroxide was poured over it. Bubbling with rapid evolution of oxygen was recorded

as positive result whereas no bubbling was exhibited for the negative result.

3.4.1.6 Coagulase test

Staphylococci colony was emulsified with a drop of water on two clean glass slides. A drop

of rabbit’s plasma was added in one slide and mixed gently. Clumping was observed within 10

seconds. No plasma was added on the second suspension which served as a control to differentiate

granular appearance and true coagulase clumping.

3.4.1.7 Starch hydrolysis test

Bacterial inoculum was streaked on agar plate’s containing starch. Inoculated plate was

incubated at 37°C for 24 h. Later iodine reagent was added. Presence of clear halos surrounding

colonies were recorded as positive result.

3.4.1.8 Mannitol salt agar test

Purified isolates were streaked on mannitol salt agar plates containing 7.5% sodium

chloride to inhibit growth of other bacteria except staphylococcus and phenol red indicator. Plates

were incubated for 24 h and change of color to yellow was recorded as positive result as mannitol

was fermented due to decrease in pH and converting neutral medium to acidic.


25

3.4.2. Tolerance assessment

The isolated and identified bacteria of pharmaceutical waste water were assessed for their

tolerance against potassium dichromate (K2Cr2O7) and Copper Sulfate (CuSO4) with

concentrations ranging from 25µg/mL to 500µg/mL with an increment of 25µg/mL. Nutrient agar

was poured in Petri plates and the required volume of metal from stock solutions were added and

allowed to solidify. Isolates were streaked on the plates with metal salts and placed in incubator at

37°C for 48 h. Maximum tolerated concentration was defined as the highest concentration of heavy

metal that allows growth of bacteria after 48 h (Bauer et al. 1966, Samanta et al. 2012)

3.4.3. Bacterial resistance

Strains isolated were analyzed for the sensitivity against different antibiotics. The selected

antibiotics were vancomycin 30 µg/disc, co-trimoxazole 35 µg/disc, methicillin 10µg/disc,

chloramphenicol 25µg/disc, streptomycin 10µg/disc and levofloxacin 5µg/disc. Mueller-Hinton

agar plates were prepared and were checked for the sterility by placing overnight in an incubator.

Bacterial isolates were spread on the plates and anti-biotic discs were placed on the agar, sealed

plates were placed in an incubator at 37°C for 24 h (Hudzicki 2009). Zones of inhibition were

calculated and isolated strains were categorized as resistant or sensitive. All experiments were

conducted in triplicate.

3.5. Comet Assay

Single cell gel electrophoresis is a valuable tool for the determination of DNA damage

induced by toxic compounds on an individual cell. It consists of a series of steps, each step is

equally vital for the successful processing of experiment. The basic processing involves the

embedding of suspension of a single cell on a cavity slide coated with agarose gel. The cells

undergo lysis in a detergent and the exposed DNA is electrophoresed. Fragments of DNA being


26

negatively charged move toward anode. The slides were stained with a fluorescent dye e.g.

ethidium bromide and observed under electron microscope.

Figure 3.3 Schematic representation of the procedure adopted for the comet assay

3.5.1. Instrumentation

Electrophoresis system, centrifuge machine, refrigerator, fluorescent microscope,

micropipette, microwave oven, tips, magnetic stirrer, pH meter, aluminum, foils, cavity slides,

steel tray with lids, pipette, coverslips, beakers, flasks, syringes, capped test tubes, test tube stands,

Eppendorf tubes, pipette, conical flasks.


27

3.5.2. Chemicals

Lymphocyte separation media, RPMI 1640, agarose, boric acid, methanol, phosphate,

sodium chloride, Trizma base, di sodium EDTA, sodium hydroxide, di-methyl sulfoxide (DMSO),

Triton X, , ethidium bromide.

3.5.3. Reagents preparation

Reagents were prepared just before the start of the experiment and all the accessories like

gloves, face mask, goggles and lab coat were used while handling the chemicals.

3.5.3.1. Phosphate buffer solution

PBS tablets commercially available were solubilized each tablet in 1000 mL of distilled

water. pH of the buffer was checked and stored at 4°C.

3.5.3.2. Normal melting point agarose

Dissolve 1000mg in 100 mL warm water. Heat it until a clear solution is formed.

3.5.3.3. Low melting point agarose

Dissolve 1000 mg of agarose in 200 mL hot distilled water and heated till it becomes a

clear solution. Before use it was placed in microwave oven and then placed in a water bath to

stabilize the temperature.

3.5.3.4. Lysing solution

1000 mL lysing solution was prepared by dissolving 146.1 gm sodium chloride (NaCl),

37.2 gm ethylene-diamine-tetra-acetic acid (EDTA) and 1.2 gm trizma base in 700 mL of distilled

water respectively. Each ingredient was added after complete dissolution of first ingredient. pH of

the solution was adjusted to 10 by using sodium hydroxide (NaOH) or hydrochloric acid (HCl).

Volume was made 890 mL using distilled water. Finally, 10% of total volume, i.e. 100 mL of

DMSO and 1% of total volume 10 mL triton-X was added.


28

3.5.3.5. Alkaline buffer solution

10 N NaOH solution 20gm in 50mL distilled water (DW) and 200mM EDTA 0.73gm in

10mL of DW were prepared. 30 mL NaOH solution and 5 mL EDTA solution were mixed in 1 L

flask and final volume was adjusted 1000 mL. The pH of the solution was raised to 13.

3.5.3.6. Electrophoresis buffer solution

Trizma base 10.8 gm, EDTA 0.93 gm and boric acid 5.5 gm were mixed in a flask by

adding in a sequential manner after complete dissolution of the first ingredient on a hot plate using

a magnetic stirrer and final volume was made up to 1000mL.

3.5.3.7. Neutralization buffer solution

48.5 gm of Tris base was dissolved in 700 mL of DW. pH of the solution raised 7.5 with

10M NaOH and volume was increased to 1000 mL with DW.

3.5.3.8. Staining solution

10 mg of ethidium bromide was dissolved in 50 mL of distilled water. It was enveloped in

aluminum foil. 10X solution of ethidium bromide was prepared by diluting 1 mL of 10X in 9 mL

DW and was used for staining of slides.

3.5.4. Experimental procedure

The sample was diluted with distilled water and six different concentrations were prepared.

Dimethyl sulfoxide 20% (DMSO) is positive control. Negative control used in the experiment was

distilled water. Comet assay was implemented based on the protocol described in (Tice et al.

2000).


29

3.5.4.1. Preparation of base slides

Slides after being sprayed with ethanol, blazing was done to remove any oily, greasy

material or dust particles. Labelling was done with the slides according to sample number. Slides

were dipped in normal melting point agarose (NMPA). In a manner that one third remained

undipped and placed on a tray after wiping off the lower area. Slides were placed in the refrigerator

for 12 h.

3.5.4.2. Procedure for cell separation

Peripheral blood of a healthy sheep was collected in a heparinized tube. 3mL of sheep

blood was layered over a 5 mL of lymphocyte separating media (LSM) in a capped glass tube

gently so that blood and LSM did not get mixed. It was centrifuged at 8000g for 45 minutes at 25

°C. Three layers were formed. Lymphocytes were present in the middle layer the Buffy coat. Cells

were sediment in the deepest layer and the plasma layer as formed on the top. Sheep was used as

an experimental organism in this assay because the experiment was standardized using sheep blood

during pilot project before conducting the study. The experiment was carried out following the

experimental protocol in agreement with the Institutional Guide Lines for Care and Use of

Laboratory Animals of University of Veterinary and Animal Sciences, Lahore, Pakistan

Buffy coat was extracted into a 5 mL of RPMI 1640. Centrifugation was performed at 300

g for 10 min until the formation of lymphocyte pellet. The lymphocytes pellet was again suspended

in RPMI 1640. Lymphocytes were counted using hemocytometer and final cell count adjusted was

2x104 cells per 100 µL.


30

3.5.4.3. Preparation of test dilutions

Ten pharmaceutical effluent samples were used as such and two samples were diluted to

different concentrations to check the effect of different dilutions on genotoxicity based on comet

assay. Two fold dilution of samples was made using distilled water.

3.5.4.4. Cell exposure to test samples

1 mL of test sample and 100 µL of cell suspension were added in an Eppendorf tube, the

tubes were inverted and placed in incubation for a period of 3 h. The cells were centrifuged at 3000

rpm for 5 minutes and lymphocyte pellets were settled down at the bottom. Test chemical was

aspirated and discarded using a 1cc syringe and pellets were re suspended in RPMI 1640.

3.5.4.5. LMPA Layering

10 µL treated lymphocyte were mixed with 65 µL LMPA. It was dropped on the base slide,

the slides were refrigerated for 10 minutes so that the agarose hardens. A third layer of LMPA 90

µl was placed and again refrigerated.

3.5.4.6. Lysis solution

Lysing solution was prepared freshly in the tray along the sides gently as to minimize the

disturbance to the slides. The tray was covered and placed in refrigerator for 10 h.

3.5.4.7. Exposure to alkaline buffer solution

Lysing solution was aspirated from the tray and the freshly prepared alkaline buffer was

poured in the tray pH >13. The slides were allowed to remain there for 20 minutes, after which the

buffer solution was aspirated. An alkaline buffer causes the unwinding of DNA and damage was

expressed.


31

3.5.4.8. Electrophoresis

Electrophoresis buffer was poured into a horizontal tube of apparatus. 5 slides were

electrophoresed at one time. The power supply was given to apparatus with a 24 V and 300 mAmp

current. Slides were electrophoresed for a 30 minute period.

3.5.4.9. Neutralization process

The slides were placed in dry tray after electrophoresis were completed. Neutralization

solution was poured gently into the tray and remained there for 10 minutes, aspirated and then this

process was repeated three times.

3.5.4.10. Fluorescence dye stain

1-X solution of ethidium bromide was prepared. 70 µl of ethidium bromide was fell over

the cavity slides using a micropipette and remained there for 5 minutes. After that chilled water

was used for removing excessive stain. The slides were washed for three times.

3.5.4.11. Visualization

The slides were observed under a fluorescent microscope at the 40-X lens. All the slides

were observed immediately after processing. Intensity of light emitted was directly proportional

to the amount of due bound to DNA.

3.5.4.12. Scoring of a comet

Each slide was observed under a microscope and 50 images of comet per slides were taken.

The comets were analyzed using a software image j. Length of head diameter and migrated DNA

was documented. Percentage fragmentation and damage index of all the dilutions were calculated.

3.5.5. Controls

3.5.5.1. Negative control

1% DMSO was used as negative control (1 mL)


32

3.5.5.2. Positive control

DMSO 20 % was used as positive control (1 mL)

3.5.6. Interpretation of results

Result of come assays was interpreted using following criteria and indices.

3.5.6.1. Classification of comets

The comets were classified into following 4 categories based on tail length

Class 0 unharmed cells

Class 1 tail length was smaller than or equivalent to head diameter

Class 2 tail length larger than head diameter, but lower than double of the head diameter

Class 3 tail length bigger than double of the head diameter

3.5.6.2. Damage index

3.5.6.3. DNA fragmentation

3.5.6.4.Genetic damage index

3.6. Bacterial reverse mutation assay

Dr. Bruce Ames developed Ames assay in 1970 for the detection of mutagenicity. An

auxotroph strain of bacteria is used in this model for the determination of mutagenic probability

of test substance in which bacteria are unable to synthesize required nutrients. If test substance

proves to be mutagenic it means it has caused back mutation.


33

Figure 3.4 Schematic representation of the procedure adopted for Ames assay


34


Petri plates, test tubes (10 mL, 20 mL), glass pipette (5 mL, 10 mL), capped test tubes,

autoclave, incubator, horizontal laminar flow hood, deep freezer, drying hot air oven, water baths,

analytical balance, Eppendorf tubes, syringes ( 1cc, 10 cc, 20 cc), micro filters (0.22 µm), Burner,

glass flasks, beakers, platinum wire loop, glass spreader, aluminum foil, face mask, magnetic

stirrer, sterile test tubes, test tube rack, micropipette.

3.6.2. Chemicals required

Distilled water, phosphate buffer, salmonella Shigella agar, purified agar, magnesium

sulfate monohydrate, nutrient agar medium, potassium phosphate dibasic anhydrous, sodium

phosphate di hydrate, dextrose.

3.6.3. Test organism

Salmonella typhimurium strains with mutations were used. These are genetically modified

and mutations are induced in different genes of histidine operon.

3.6.4. Strains used

Two strains TA 100 and TA 102 of bacterium Salmonella typhimurium were purchased

from Environmental Bio Detection Products Incorporation (EBPI) Canada.

3.6.5. Preparations of media and reagents

3.6.5.1. Nutrient broth preparation

5gm of nutrient broth was mixed with 200mL DW. The mixture was stirred to solubilize

the components. Nutrient broth was divided into four test tubes and autoclaved. Both strains TA

100 and TA 102 were grown in the test tubes for overnight in the incubator.


35

3.6.5.2. Nutrient agar preparation

Nutrient agar (1.5 gm) was dissolved in distilled water (100mL) in a flask. Solubilize it in

water and autoclaved. The medium was allowed to cool till the temperature reaches 65°C and

media was poured in petri plates. The plates were incubated at 37°C for 24 hours to validate the

sterility of media. The plates were placed in refrigerator upside down till next use.

3.6.5.3. Vogel-Bonner salt preparation

65 mL of DW was taken in 1000 mL flask and ingredients were added in the following

manner such that next salt is added after the complete dissolution of the first one on a hot magnetic

plate with continuous stirring. Magnesium sulphate 1 gm, citric acid monohydrate 10 gm,

potassium phosphate di basic anhydrous 50 gm and sodium ammonium phosphate 17.5 gm were

added according to the said order. The solution was divided into 20 mL portions in glass tubes and

autoclaved. Then they were allowed to cool, tighten up and stored at 25 °C in a dark place.

3.6.5.4. Preparation of glucose solution 10 % v/v

The solution was prepared by mixing dextrose in distilled water. 10 g of dextrose was

dissolved in 70 mL DW, mix it with the help of magnetic stirrer until a clear solution was formed

and the final volume was made up to 100mL. The solution was divided into two portions, 50 mL

each in loosely capped flasks and autoclaved. It was used immediately after cooling (Mortelmans

and Zeiger 2000).

3.6.5.5. Preparation of GM (Glucose Minimal) agar

Purified agar (15 gm), was mixed in 900 mL DW in a glass flask. It was autoclaved and

when the temperature reached to 65 °C, Vogel-Bonner salts (20mL) and glucose solution (50 mL)

were added and mixed .This mixture was uniformly poured in petri plates.


36

3.6.5.6. Preparation of ampicillin solution

pKM 101 plasmid presence in mutant strains of TA100 and A102 was confirmed by

ampicillin disk. Ampicillin solution was prepared by dissolving 80mg in 10mL of water. The

solution was filtered through 0.45µm filter before use.

3.6.5.7. Preparation of crystal violet solution (0.1%)

Crystal violet dye (10mg) was dissolved in 10 mL of DW. The solution was stored in an

amber colored bottle at 4-8 °C.

3.6.5.8. Preparation of biotin solution (0.01 %)

It was used for the enrichment of glucose minimal (GM) agar plates and used for the

detection of strain. The solution was filtered through 0.45 um filter prior to use and stored at 4°C.

1mg was dissolved in 10mL of water.

3.6.5.9. Preparation of histidine solution (0.5 % w/v)

Histidine solution was prepared by dissolving 50mg in 10mL of DW. The solution was

subjected to pass through 0.45µm filter and stored at 4°C. Histidine solution was used to check the

strain of bacteria.

3.6.5.10. Preparation of histidine/biotin solution 0.5mM

It was prepared by adding 9.6 mg of L-Histidine and 12.4mg of D-Biotin in 100mL of

water. Histidine gets dissolved immediately, while biotin dissolved slowly in DW. Histidine/biotin

solution was passed through 0.45 um syringe filter and stored at 4°C.

3.6.6. Metabolic enzyme activation system S9 mixture

S-9 fraction is a rat liver homogenate which contains microsomal supernatant portion along

with S9 cofactors; given in table no 3.1. It was obtained from EMPI Canada. As salmonella

bacteria were deficient in metabolizing capability these cofactors supplied them with the vitality


37

to regenerate the system. The system was prepared freshly and passed through 0.45 µm syringe

filter and stored at 4°C. The activation mixture was prepared as follow.


38

Table 3.1 Activation mixture composition

1. S-9 mixture components

Volume

mL µl

S9-A [Magnesium chloride+ Potassium chloride] 0.40 400

S9-B [Glucose-6-phosphate] 0.09 90

S9-C [Nicotine amide Di-nucleotide Phosphate (NADP)] 0.81 810

S9-D [phosphate Buffer Saline (PBS)] 9.98 9980

S9-E [Sterile Distilled water] 6.72 6720

S9-F [Rat liver extract] 2 2000

Final Volume 20 20, 000

0.5 mL of S-9 mixture was provided to each plate.


39

3.6.7. Preparation of lyophilized bacteria

Salmonella typhimurium TA100 and TA102 were purchased from Environmental Bio

detection Products Incorporation (EBPI) Canada. 1 mL of sterilized broth was used to cultivate

the bacteria. The culture was placed in an incubator for 24 hours. The colony was picked from

culture and streaked on nutrient agar plates.

3.6.8. Tester strain purification

A single colony was picked from plate and incubated in broth and placed in an incubator

for 24 hours. Again a single colony from culture was taken and grown on agar plates. Both TA100

and TA102 strains were purified by repeating this process thrice.

3.6.9. Genetic analysis

Several tests were conducted for the genetic identification of strains TA100 and TA102.

3.6.9.1. Biotin dependence

GM agar plates enriched with histidine were prepared. 0.5%w/v histidine was made and

0.08mL was poured on agar plates. The bacterial strains were streaked on agar plates and growth

was observed after 24 hours.

3.6.9.2. Histidine dependence

GM agar plates enriched with biotin were prepared. 0.1%w/v biotin was made and 0.08mL

was poured on agar plates. The bacterial strains were streaked on agar plates and growth was

observed after 24 hours.

3.6.9.3. Histidine/biotin dependence

GM agar plates were prepared with 0.01%biotin and 0.5%histidine. 0.08mL of each was

poured in the agar plates. The tester strains were placed on agar plates with the said amount of


40

histidine and biotin separately. The plates were placed in an incubator for 24 hours to check the

growth of bacteria.

3.6.9.4. Crystal violet test

GM agar plates augmented with histidine and biotin were streaked with TA100 and TA102

under sterile conditions. A crystal violet soaked disc prepared by dipping filter paper in 0.1 %

solution was placed in the center of both the plates. The plates were incubated to check for the

growth of bacteria.

3.6.9.5. pkM101 plasmid

GM agar plates enriched with histidine and biotin were streaked with both bacteria under

sterile conditions. Ampicillin solution 10µg/mL was placed and filter paper was soaked in the

solution which were placed in each of the two plates of both the strains and incubated at 37 °C. It

was used to access the resistance of mutant strains against Salmonella typhimurium in comparison

with wild type Salmonella typhimurium.

3.6.10. Working suspension of bacteria

Freshly prepared bacteria cultures were prepared before the start of the experiment. It was

ensured that bacteria must be in log phase when the experiment was conducted. A single colony

from each purified plate was inoculated in 5mL of autoclaved broth culture. The broth was later

incubated. The growth was confirmed using spectrophotometer and 0.5 McFarland (MCF) was

considered as standard. It equals 1-2× 108 colonies. The absorbance of bacteria was adjusted to 0.1

with the help of autoclaved broth.


41

3.6.11. Experiment

Mutagenicity testing of pharmaceutical waste water was carried out using the plate

incorporation assay, with and without metabolic activation mixture. Steps below were followed

prior to pre incubation assay.

Inoculation of tester strains was done in sterilized nutrient broth 15-18 hours before the

experiment was carried out to obtain a fresh culture.

All glass equipment was sterilized and labelling was performed according to dilutions.

Metabolic activation system was prepared by mixing and placed on ice till further use. Stock

solutions and dilutions of PEW were prepared. Top agar was prepared according to the method

explained above.

3.6.11.1. Pre incubation assay in the absence of metabolic activation system

The plate incorporation assay was modified and in pre incubation assay test chemical and

bacteria were exposed prior to plating on the GM agar plate. Test dilution (0.5 mL) along with

tester strains Salmonella typhimurium (TA100 and TA102) were added in an autoclaved screw

capped test tube. Test dilution was mixed with mutant strains on a vortex and test tubes were

incubated for 20 minutes. GM agar plates already prepared with 0.5mM histidine/biotin solution

according to the method mentioned above were exposed to test dilution and mutant bacteria

mixture. The agar was allowed to cool until it was solidified. The plates were incubated at 37 °C

in an incubator for 48 hours and revertant colonies were observed and counted. Number of

revertant colonies were counted.

3.6.11.2. Pre incubation assay without metabolic activation system

Xenobiotics might become mutagenic after their metabolism through liver enzymes as their

metabolites could be mutagenic. A metabolic activation system was introduced which was


42

extracted from rat liver and the contained enzymes were provided along with test chemical and

mutant bacteria. The purpose was to obtain results which might concord with in-vivo system. 0.5

mL of a metabolic activation system was introduced in each plate along with mutant bacterial

strain and test dilution of PEW.

3.6.12. Control groups


Positive control for TA 100 was sodium azide (5µg/plate) and to TA 102 control was 35%

hydrogen peroxide. Metabolic activation system was checked using 2-aminoanthracycline (2AA)

as a positive control.


Negative control plate contained distilled water which was used to make dilutions of the

sample.

3.6.12.3. Dilutions preparation

Dilutions were prepared using distilled water (DW). Two fold dilution of pharmaceutical

waste water samples PEW1 and PEW6 were prepared in an autoclaved test tube using a sterilized

syringes.

Table 3.2 Different dilutions of pharmaceutical wastewater used for MTT assay

3.7. Cytotoxicity assay

Methyl thiazol tetrazolium (MTT) assay was performed for the cell viability evaluation

after exposure to PEW. MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) dye

was used in the experiment. This dye is reduced due to the presence of dehydrogenase enzymes in

Dilution 1 2 3 4 5

% v/v 100 50 25 12.5 6.25


43

metabolically active cells as dye crosses cell membrane and got reduced to insoluble purple colored

formazan crystals by the mitochondrial dehydrogenases. Dimethyl sulfoxide (DMSO) was added

to solubilize the cells. The cells were quantified using a spectrophotometer. Live cells have the

ability to reduce the dye which reflects metabolic activity. A confluent monolayer of baby hamster

kidney cells (BHK-21) was obtained in 96 well flat bottom cell culture plate and then treated with

each dilution of PEW.

Figure 3.5 Schematic representation of the procedure adopted for MTT assay


44


Horizontal laminar flow hood, cell culture plates, CO2 incubator, inverted microscope,

Microtiter plate reader/ELIZA reader, Micropipette, refrigerator, sterile pipette tips, sterile test

tubes, pipette aid, syringes (1 cc and 5 cc), water bath, cell culture flasks, Neubauer’s chamber,

magnetic stirrer, syringe filters (0.22 µm), gloves, masks.

3.7.2. Materials and chemicals

3.7.2.1. Cell culture media preparation

Cell culture medium 1.2gm was dissolved in 100 mL of double distilled water. Fetal bovine

serum and maintenance medium were added along with antibiotics. Negative pressure assembly

was used to filter the sample.

3.7.2.2. Cell line

The cell line (BHK-21) was acquired from Quality Operations Laboratory (QOL),

University of Veterinary and Animal Sciences, Lahore.

3.7.2.3. Cells revival

BHK-21 was revived by defrosting the cryopreserved culture. The tubes were

decontaminated with ethanol 70%, the cells were thawed gently in a water bath at 37°C. Sample

tubes containing BHK-21 were decontaminated with 70% ethanol and thawed gently in water bath

at 37°C. Cell culture medium 10mLwas poured in a falcon tube and contents of cells were placed

in the tube. The mixture was centrifuged for 3 minutes. Washing was performed to remove DMSO

traces. 5 mL of cell culture medium was used to re-suspend cells. The flasks were sub cultured

until 80% confluence was reached.


45

3.7.2.4. Quantification

Cells were loaded in a clean hemocytometer. Coverslip was placed over it. 0.4% trypan

blue was mixed with cell suspension. The principle of loading the cells in hemocytometer was

capillary action. The hemocytometer was placed on the microscope and cells were counted. Dead

cells were stained and live cells remained unstained. Viability of cells was expressed using

following formula.

Percentage viability = number of viable cells per mL/ total number of cells/mL × 100

3.7.2.5. Seeding of BHK-21 in the cell culture plate

Plates attaining 90% confluency were selected for cytotoxic evaluation. Cell culture

medium was decanted and rinsed with phosphate buffer (PBS) medium. PBS was aspirated and

0.5 mL of 0.25% trypsin EDTA was added for the detachment of cells. Plates were incubated at

37 °C for 5 minutes. Cells were washed again with cell culture medium and pipetting was

performed to break the clumps. Suspension of cells was centrifuged at 200 rpm in a sterile falcon

tube for 5 minutes. Supernatant was decanted and cells were re-suspended in growth medium.

Concentration was adjusted to 105 cells per mL and 100µL of cell suspension was added each well

of the 96 well plate. Plates were examined under an inverted microscope for the development of

confluency.

3.7.2.6. MTT assay

Plates that have developed 80-90% confluency were used for cytotoxic evaluation of PEW.

Plates were labelled properly with the respective dilution number. Dilutions of PEW were mixed

with cell culture medium and 200µL were added to each well. Each sample dilution was analyzed

in triplicate. Plates with their lids were placed into a CO2 incubator for 72 hours. Cell culture media

was discarded from wells and 20µL of MTT solution was added to each well along with 100µL of


46

fresh media. After 3-h incubation the media was removed and 100 µL of DMSO was added so that

formazan crystals get dissolved. The intensity of the color of live cells, which developed due to

the alteration of yellow MTT dye to purple color was measured using an ELISA reader.

Absorbance was taken at 570nm.

3.7.3. Control


Cell culture media served as a positive control because we are calculating cell survival percentage


20% DMSO was negative control

Cell survival percentage (CSP) of BHK-21 cells were calculated using the following formula

CSP= Mean OD of PEW dilutions-Mean OD of negative control/Mean OD of positive control×100

Inhibitory concentration (IC50) of samples was calculated using Graph Pad Prism. A log dose was

plotted against the calculated cell survival percentage (CSP) and IC50 was calculated using

nonlinear regression fit.

3.8. Oxidative stress

Experimental animals, 90 days old male Wistar rats (130-150 g), were purchased from

Department of Theriogenology, University of Veterinary and Animal Sciences (UVAS) Lahore.

The rats were kept independently in steel wire netting cages, animals were kept in an air

conditioned room (temperature: 21-25 °C. The rats were divided into five groups (n=5) named as

negative control, PEW 100%, PEW 10%, PEW 1% and PEW 100% + Vitamin E respectively. The

negative group was provided with normal tap water, group 2 received pure pharmaceutical

wastewater, group 3 received 10% of pure wastewater, and group 4 was given 1% of pure waste

water, all dilutions were made with the same water used for negative control. Group five received


47

pure wastewater and vitamin E 100mg/kg body weight as an antioxidant El-Demerdash et al, 2004.

All the animals were given 10 days for acclimatization before starting the experiment and kept in

12h day/light cycle.

Vitamin E (D-α-Tocopherol-polyethylene glycol 1000 succinate) the water soluble form

was purchased from SIGMA-ALDRICH. All the rats were provided with same normal diet ad

libitum. After conducting experiments which consisted of 60 days, all rats were fasted 12 h before

sacrifice for blood and tissue collection.

3.8.1. Sample preparation

The animals were housed and cared according to guideline and principles in the Care and

Use of Animals. All the experimental protocols were approved by the Ethical Committee for the

Use of Laboratory Animals (ERCULA) of UVAS, Lahore. Rats were anesthetized (with diethyl

ether) and were fixed on the experimental desk.

3.8.2. Tissue homogenate preparation

Kidney or liver tissue 1-1.5gm weight was taken. Tissue was washed with cold normal

saline to remove adherent blood. Tissue was dried using filter paper. It was weighed to 1gm and

placed in a small beaker. 6mL of normal saline was poured into a beaker containing tissue. By

using scissors, tissue was cut with a scissor to small pieces. These tissue pieces were homogenized

in a tube for 6 minutes. Then tissues were further minced to grind it to 10% homogenates. This

tissue homogenate was centrifuged at 8500rpm for 10 minutes at 4°C and supernatant was

withdrawn for assays. 1mL of supernatant was diluted with 9mL normal saline to prepare 1% tissue

homogenate.


48

3.8.3. Plasma Preparation

2-4mL blood was withdrawn from each rat with a heart puncture in heparinized

vacutainers. Blood was centrifuged at 1000rpm for 10minutes at 4ºC. Plasma as top yellow layer

was carefully removed and store on an ice-pack for further analysis.

3.8.4. Experimental Procedure

After the 60 day period of treatments, blood was collected from rats in heparinized

vacutainers. Plasma was removed from the blood after centrifugation at 1000rpm for 10 minutes

at 4°C and stored on an ice-pack. Tissue homogenates (10%) of liver and kidney were prepared by

centrifugation at 8500rpm for 10 minutes at 4°C in normal saline. Activities of enzymes such as

T-SOD and CAT and H2O2 concentrations were measured using commercial kits.

Activity of T-SOD was measured in tissue homogenates and plasma by Xanthine Oxidase

based assays, the method of which was described previously (H et al. 1994). Activity of T-SOD

was expressed as units/mg protein (U/ mg protein) for tissue homogenates and units/mL (U/ mL)

for plasma.

Activity of CAT was measured in liver and kidney tissue homogenates and blood

spectrophotometrically at 405nm wavelength by NJBI demonstrated method (Zhang et al. 2008).

CAT activity was measured in U/ mg protein and U/ mL of blood.

H2O2 concentrations were measured in liver and kidney tissue homogenates and plasma

spectrophotometrically at 405nm wavelength (Zhang et al. 2008). H2O2 concentrations were

expressed as mmol/L.

Lowry’s method for protein estimation was used in determining the protein content.

Bovine serum albumin served as a standard (Lowry et al. 1951).


49

3.8.5. Procedure for Estimation of T-SOD Activity

Two types of T-SOD activity test tubes were prepared. One test tube was test blank for T-

SOD whereas the other was sample test tube for T-SOD (Luo et al. 2014). Preparation of these

two types of test tubes for estimation of T-SOD activity is illustrated in Table 3.6.

After vortex sample was placed at room temperature for 10 minutes. After this, absorbance

of each tube was measured at 550nm in a glass cuvette. Following formulae were used for

estimation of T-SOD activity in plasma and tissue homogenates.

1. T-SOD activity in plasma was calculated by using the following formulas:

2. T-SOD activity in tissue homogenates was calculated by using the following formula:

3.8.6. Procedure for Estimation of CAT Activity

50µL of rat blood was taken and diluted up to 10 times with double distilled water. 1mL

of tissue homogenate was taken and 9mL normal saline was added to make 1% tissue homogenate.

0.02mL of prepared sample was taken in cuvette and 3mL substrate solution was added (Zhang et

al. 2008). OD value was measured at 405nm as (OD1) and after 1minute OD value was measured

again as (OD2). CAT activity was calculated by following formula.

CAT activity (U

gmHb) = log

𝑂𝐷1

𝑂𝐷2×

2.303

60𝑠× 𝐴 ÷ 𝐵

Where OD1 represents absorbance at 405nm at t = 0

OD2 represents absorbance at 405nm when t = 60 seconds

“A” is the dilution factor of hemoglobin

“B” is the Hemoglobin concentration (gm Hb/mL)


50

3.8.7. Procedure for Estimation of H2O2 Concentration

Three types of test tubes were prepared for measurement of H2O2 which included one blank

test tube, one standard test tube and the third for test samples.

These solutions were mixed well in test tubes and then absorbance was measured at 405nm

for each tube. The following formula was used for estimation of H2O2 concentrations in different

samples.


51

3.9. Tissue pathology

Experimental animals, 90 days old male Wistar rats (130-150 g), were obtained from

Department of Theriogenology, University of Veterinary and Animal Sciences (UVAS) Lahore.

The rats were kept independently in stainless steel wire netting cages, air conditioned room

(temperature: 21-25 °C). The rats were randomized into five groups (n=5) named as negative

control, PEW 100%, PEW 10%, PEW 1% and PEW 100% + Vitamin E respectively. The negative

group was provided with normal tap water, group 2 received pure pharmaceutical wastewater,

group 3 received 10% of pure water, and group 4 was given 1% of pure waste water, all dilutions

were made with the same water used for negative control. Group five received pure wastewater

and vitamin E 100mg/kg body weight as an antioxidant. Rats were provided with feed and sample

water ad libitum for the duration of the experiment. After conducting experiments which consisted

for 60 days, all rats were fasted 12 h before further treatments.

The rats were starved for 12 h, anaesthetized with light ether then sacrificed by

decapitation following the experimental protocol in agreement with the Institutional Guide Lines

for Care and Use of Laboratory Animals of University of Veterinary and Animal Sciences, Lahore,

Pakistan. Vital organs (lung, liver, kidney, heart and brain) were carefully removed and weighed.

The tissues were fixed in 10% PFA (formaldehyde solution) and processed for histological

investigation. The tissues were embedded in paraffin wax, sectioned for 2µm thickness, mounted

on slides and were stained with haematoxylin & eosin (H&E) for routine light microscopy. The

slides were investigated for any deleterious effects or appearance of lesions appeared after

treatment with PEW (Ejaz et al. 2009).

CHAPTER 4

RESULTS

4.1 Heavy metal analysis

Iron (Fe) was present in all the samples at concentrations higher than the WHO or US EPA

safe level and with a concentration range varying from 0.5 mg.L-1 in PEW10 to 4.96 mg.L-1 in

PEW1. Chromium (Cr) was found to be higher than normal permissible limits of WHO and US

EPA and ranged from 0.18 mg.L-1 in PEW5 and PEW10 to 0.43 mg.L-1 in PEW1. Lead (Pb) also

exceeded the normal range with 0.21 mg.L-1 present in PEW1 and 0.16 mg.L-1 present in PEW2.

Arsenic (As) was present in all samples with a wide range of concentration varying from 0.1 mg.L-

1 to 0.83 mg.L-1. Cadmium (Cd) appeared to be higher than standard limits in only three samples.

Copper (Cu) concentration varied from 0.16 to 0.83 mg. L-1 which is less than normal levels. Zinc

(Zn) was present in the normal limits ranging from 0.88 to 2.01 mg. L-1. Table 4.1 represents the

levels of heavy metals present in samples of wastewater collected from different pharmaceutical

industries. Normal limits of the world health organization (WHO) and US Environmental

Protection Agency (US EPA) are also provide in the table 4.1. The results were analyzed through

box whisker plot and represented in fig. 1.

Results

53

Table 4.1 Toxic metal content in the effluent of pharmaceutical industries

Sample

number

Zn

(mg.L-1)

Fe

(mg.L-1)

Cu

(mg.L-1)

Cr

(mg.L-1)

Pb

(mg.L-1)

As

(mg.L-1)

Cd

(mg.L-1)

PEW 1 1.25 4.96 0.5 0.43 0.21 0.83 0.02

PEW 2 0.96 3.76 0.33 0.38 0.16 0.1 0.04

PEW 3 1.4 3.6 ND ND ND ND ND

PEW 4 1.03 1.85 0.16 ND ND 0.15 ND

PEW 5 1.51 1.15 0.83 0.18 ND 0.35 0.01

PEW 6 2.01 1.55 0.33 0.4 ND 0.1 ND

PEW 7 1.46 2.26 0.16 ND ND 0.5 ND

PEW 8 1.05 0.96 0.33 0.36 ND 0.13 ND

PEW 9 1.6 0.71 0.16 0.28 ND 0.41 ND

PEW 10 0.88 0.5 0.16 0.18 ND 0.25 ND

WHO

(ppm)

5 0.3 2 0.05 0.01 0.01 0.003

EPA

(ppm)

5 0.3 1.3 0.1 0.015 0.05 0.005

Results

54

Co

nc

en

tra

tio

n m

g/L

PEW

1

PEW

2

PEW

3

PEW

4

PEW

5

PEW

6

PEW

7

PEW

8

PEW

9

PEW

10

0

2

4

6

Figure 4.1 Box whisker plot of heavy metal concentrations in different samples of

Pharmaceutical waste water.

Results

55

4.2 GC/MS Analysis

Pharmaceutical waste water was subjected to GC-MS analysis to detect the pharmaceuticals present in wastewater. Peaks

obtained were compared with the peaks of fragments in the library. The matches are depicted in table no. 4.2. Different columns and

different methods were used for the screening of PEW. DB-35ms detected more compounds because the method adopted was EPA

8081A according to catalogue provided by manufacturer Agilent series whereas method adopted for DB-1 column was common drug

screening according to manufacture catalogue.

Table 4.2 Components found in pharmaceutical effluent PEW1

Fraction Peak

no.

Retention

time

Area % Compound m/z ratio Column

Basic 5 3.075 2.328 % Toluene 27,38,39,40,43,45,49,51,52,62,63,64,65,66,74,77,86,8

8,89,91,92,93,94

DB-1 9 16.087 0.993 % Caffeine 28,42,55,67,82,94,109,122,137,150,165,179,194

10 17.029 1.063 % Lidocaine 30,42,58,65,77,86,91,105,120,134,148,217, 234

12 17.984 0.732 % Prednisolo

ne

41,55,77,91,122,147,179,225,249,267,

300

Neutral 12 3.918 1.107 % Glycerin 31,43,55,61,74

13 16.075 0.899 % Caffeine 42,55,67,82,94,109,136,165,176,194,207

- 13.6 0.201% Digitoxin 43,113,203,246,339,401

Acidic - - - - -

Results

56

Fig. 4.2 exhibits mass spectra of basic fraction of PEW where peaks represent different

compounds present in PEW with their specific retention times and abundance. Mass spectra of the

lidocaine obtained after screening through GCMS was depicted in fig 4.3.

Figure 4.2 Mass spectra of basic fraction of PEW 1

Figure 4.3 Mass spectra of lidocaine using DB1 column

Results

57

Fig. 4.4 exhibits mass spectra of neutral fraction of PEW where peaks represent different

compounds present in PEW with their specific retention times and abundance. Mass spectra of

digitoxin, dipyrone and trimethoprim obtained after screening of PEW through GCMS are

represented in fig. 4.5, 4.6 and 4.7 respectively.

Figure 4.4 Mass spectra of neutral fraction of PEW 1

Figure 4.5 Mass spectra of digitoxin using DB1 column

Results

58

Table 4.3 Compounds identified in pharmaceutical effluent waste water PEW1 using GC-MS

Fraction Peak

no.

Retention

time

Area

%

Compound m/z ratio Column

Basic - 13.2 - Caffeine 42,55,67,82,94,109,122,136,150,165,176,194 DB –

35ms 7 11.864 2.498

%

Dipyrone 39,42,56,64,77,83,91,98,106,123,217

12 17.247 1.058

%

Trimethoprim 43,53,66,77,81,95,105,123,130,147,161,172,189,

200,215,228,243,259,275,290

13 21.434 1.480

%

Vitamin E 69,83,95,121,165,177,205,226,243,326,364,430

Neutral 6 6.788 1.773

%

Phenol,2,5-bis(1,1-

dimethyl)

41,57,64,73,91,107,115,135,147,163,176,191,206

7 7.984 1.517

%

Tridecene (Z) 41,43,51,55,65,67,69,83,91,97,111,125

8 10.256 3.385

%

E- 14-Hexadecenal 43,5569,83,97,111,125,140,154,168,182,196,203,

210

9 10.492 2.641

%

Tetra decanoic acid,

propyl ester

43,61,69,73,83,102,111,115,125,129,143,157,171

,185,199,211,229

11 11.531 4.936

%

1, 2-

Benzenedicarboxylic

acid, butyl 2-

ethylhexyl ester

41,50,56,65,76,93,104,121,132,149,160,179,191,

205,223

14 15.893 2.389

%

E- 15-Heptadecenal 41,43,51,55,57,61,67,69,79,83,91,97,

111,125,139,149

16 17.482 1.155

%

11-Tricosene 41,43,55,57,69,71,79,83,91,97,105,111,119

- 18.8 - 17- Pentatriacontene 41,43,55,57,67,69,71,79,83,85,91,97,111,125

Acidic - 9.2 - Canthaxanthin 43,55,69,83,119,145,157,173,203,229,255,281,30

7,360,413,472,485.508,549

Results

59

Figure 4.6 Mass spectra of dipyrone using DB-35ms column

Figure 4.7 Mass spectra of trimethoprim using DB-35ms column

Results

60

4.3 Microbiology

Density of microorganisms was expressed in cfu/mL. All the samples were evaluated for

the presence of bacterial load and the results were shown in table 4.4. Plates having 30-300 bacteria

were counted and mean of triplicate was taken. Mean bacterial count was multiplied with the

dilution factor of the tube exhibiting 30 to 300 colonies per plate. All the isolates found in each

plate of PEW1 were further purified and subjected to a series of identification tests. Results are

given in table 4.5 and table 4.6. Isolates of PEW 1 were evaluated for heavy metal tolerance and

antibiotic sensitivity tests. They were found to be tolerant to varying concentration of heavy

metals. Aeromonas sobria tolerated 25 µg/mL of K2Cr2O7 and 50 µg/mL of CuSO4. Micrococcus

varians maximum tolerable dose for K2Cr2O7 was 50 µg/mL and 100 µg/mL for CuSO4.

Staphyoloccus epidermidis responded to a maximum dose of 50 µg/mL of K2Cr2O7 and 400 µg/mL

of CuSO4. Staphylococcus aureus reacted to K2Cr2O7 and CuSO4 at maximum doses of 200 and

400 µg/mL. Bacillus megaterium tolerated a maximum dose of 50 and 350 µg/mL for K2Cr2O7

and CuSO4 respectively. Results are represented in table 4.7. These bacterial isolates also showed

resistance to a number of antibiotics. Staphylococcus aureus and Bacillus megaterium were found

to be resistant against all the antibiotic used. Both the bacteria showed resistance against

vancomycin 30 µg/disc, co-trimoxazole 35 µg/disc, methicillin 10µg/disc, chloramphenicol

25µg/disc, streptomycin 10µg/disc and levofloxacin 5µg/disc. The results are represented in table

4.8.

Results

61

Table 4.4 Total number of viable bacteria in pharmaceutical waste water sample (PEW1)

Sample no. Dilution factor Total number of

colonies in selected

plates

Number of Viable

bacteria CFU/mL

PEW 1 10 6 296 2.96 ×10 8±10

PEW 2 10 3 256 2.56 × 104±7

PEW 3 10 7 112 1.12 × 10 9±11

PEW 4 10 4 160 1.6 × 10 6±13

PEW 5 10 4 230 2.3 × 10 6±14

PEW 6 10 5 231 2.31 × 10 7±9

PEW 7 10 7 286 2.86 × 10 9±10

PEW 8 10 6 194 1.94 × 10 8±6

PEW 9 10 5 243 2.43 × 10 7±12

PEW 10 10 5 213 2.13 × 10 7±10

Control N.D. N.D. N.D.

Results

62

Table 4.5 Morphological and biochemical characteristics of bacterial isolates

Colony

Type/Gram stain

Characteristics

Glucose

Fermentation

Mannitol

Fermentation

Indole

Oxidase Catalase Coagulase Starch

hydrolysis

Mannitol

salt agar

test

Type of

Bacteria

Identified

Yellow G +ve

Bacilli

positive positive negati

ve

negative positive - positive Bacillus

megaterium

Yellow G +ve,

Cocci

positive negative negati

ve

positive positive - - Micrococcus

varians

White Gram –ve

bacilli

Positive with gas positive positi

ve

positive positive - - Aeromonas

sobria

White to creamy

G +ve Cocci

negative negative negati

ve

negative Slight

positive

negative positive negative Staphyoloccus

saprophyticus

Off white G +ve

Cocci

positive with gas negative negati

ve

negative Slight

positive

negative negative negative Staphyoloccus

epidermidis

Cocci round G

+ve curved white

positive positive negati

ve

negative positive positive negative positive Staphylococcu

s aureus

Results

63

Table 4.6 Total number of isolated and identified bacteria from pharmaceutical waste water

(PEW1)

Sample no. Total types of isolated and

purified bacteria

Identified species of isolated bacteria

PEW 1 3 Bacillus megaterium

Micrococcus varians

Aeromonas sorbia

PEW 2 3 Staphylococcus saprophyticus

Stapyhlococcuus aureus

Aeromonas sorbia

PEW 3 3 Micrococcus varians

Aeromonas sorbia

Stapyhlococcuus epidermidis



PEW 5 3 Stapyhlococcuus aureus

Micrococcus varians

Aeromonas sorbia

PEW 6 5 Aeromonas sorbia


Pseudomonas aeruginosa

Bacillus megaterium



Aeromonas sorbia



Staphylococcus saprophyticus

PEW 9 2 Stapyhlococcuus aureus

Bacillus megaterium

PEW 10 2 Aeromonas sorbia

Micrococcus varians

Control N.D. N.D.

Results

64

Table 4.7 Maximum tolerable concentration of heavy metals against PEW 1 bacterial isolates

Isolated bacteria Maximum tolerated dose of

K2Cr2O7 µg/mL

Maximum tolerated dose of

CuSO4 µg/mL

Aeromonas sobria

25

50

Micrococcus varians

50

100

Staphylococcus epidermidis

50

400

Staphylococcus aureus

200

400

*Bacillus megaterium

50

350

Results

65

Table 4.8 Antibiotic sensitivity and resistant activity of isolated bacterial isolates of PEW1

Bacteria Vancomyci

n 30 µg/disc

Trimethoprim/

Sulfamethoxazole

1.25-23.75µg/disc

Methicillin 10

µg/disc

Chloramphenicol 30

µg/disc

Streptomycin 10

µg/disc

Levofloxacin 5

µg/disc

Aeromonas sobria resistant 3 mm resistant 2 mm 1 mm 4 mm

Micrococcus varians 0.1 mm 0.3 mm 0.4 mm 3 mm 1.5 mm 2 mm

Staphyoloccus epidermidis 1.4 mm resistant 0.6 mm 0.2 mm resistant 0.1 mm

Staphylococcus aureus resistant resistant resistant resistant resistant resistant

Bacillus megaterium resistant resistant resistant resistant resistant resistant

CLSI

Standards

Sensitive NS ≥16 NS ≥18 ≥15 ≥17

Resistant NS ≤10 NS ≤12 ≤11 ≤13

CLSI ~ Clinical and Laboratory Standard Guideline Standards

NS ~Not Specified

Results

66

4.4 Comet Assay

Two fold dilution of pharmaceutical waste water was made with sterilized distilled water.

Scoring of damage induced by PEW1 and PEW6 was performed (Ahuja et al. 1999) using image

J software in Fig no. 4.8 and 4.10. Mean DNA tail lengths and head diameters were calculated

(n=50) and results of all samples of PEW analyzed are represented in table 4.9. A concentration

dependent damage of DNA was observed. Comets were analyzed by comparing tail lengths which

appeared after exposure to different concentrations of waste water. One way analysis of variance

was performed to compare means as shown in Fig no. 4.9 and 4.11. The post hoc test applied was

a Dunnett’s multiple comparison test. Result showed significant difference when compared with

control group (p ˂ 0.05). Damage index (D.I) and percentage fragmentation also displayed a

concentration dependent effect. Table 4.10 depicts percentage fragmentation (%age

fragmentation), damage index (D.I) and genetic damage index (GDI) induced by pharmaceutical

wastewater samples collected from different sites (Raja et al. 2016, Ullah et al. 2016).

Results

67

Table 4.9 Mean diameter and tail length of different pharmaceutical waste water samples

Sr. no.

Sample no.

Mean DNA Tail length

um ± SD (n= 50)

Mean Head Diameter um

± SD (n=50)

1 PEW 1 6.11 ± 2.15 3.32 ± 1.33

2 PEW 2 5.24 ± 2.11 3.22 ± 1.36

3 PEW 3 5.15 ± 2.14 3.26 ± 1.30

4 PEW 4 5.37 ± 2.19 3.38 ± 1.36

5 PEW 5 4.35 ± 2.37 4.1 ± 1.67

6 PEW 6 4.94 ± 2.21 3.50 ± 1.26

7 PEW 7 5.00 ± 2.26 3.66 ± 1.50

8 PEW 8 4.96 ± 2.25 3.37 ± 1.3

9 PEW 9 4.79 ± 2.12 3.57 ± 1.46

10 PEW 10 5.08 ± 2.25 3.79 ± 1.66

Positive Control DMSO 20% 8.16 ± 2.55 3.35 ± 2.66

Negative Control Distilled Water 0.06 ± 0.04 4.66 ± 0.77

Results

68

Table 4.10 Percentage fragmentation, Damage index and genetic damage index of different

pharmaceutical waste water samples

Sr. No.

Sample no.

Class

0

Class

1

Class

2

Class

3

Fragmentation

%

Damage

Index

GDI

1 PEW 1 2 8 21 19 96 107 2.14

2 PEW 2 4 8 22 16 92 100 2

3 PEW 3 5 7 27 11 90 94 1.88

4 PEW 4 5 9 21 15 90 77 1.54

5 PEW 5 5 25 17 3 90 68 1.36

6 PEW 6 12 4 28 16 96 116 2.32

7 PEW 7 4 13 23 10 92 89 1.78

8 PEW 8 4 13 21 12 92 91 1.82

9 PEW 9 3 15 22 10 94 89 1.78

10 PEW 10 3 13 26 8 94 89 1.78

Positive

control

DMSO 20% 1 9 10 30 98 119 2.38

Negative

Control

Distilled

water

50 zero zero zero zero zero zero

Results

69

Figure 4.8 Comets appeared in sheep lymphocytes when exposed to pharmaceutical effluent

wastewater (PEW 1).

Results

70

Figure 4.9 Tail lengths of damage induced in sheep lymphocytes at different concentrations

of PEW 1 (%v/v)

Results

71

Figure 4.10 Comets appeared in sheep lymphocytes when exposed to pharmaceutical effluent

wastewater (PEW 6).

Results

72

Figure 4.11 Tail lengths of damage induced in sheep lymphocytes at different

concentrations of PEW 6 (%v/v)

Mean diameter of the head and tail lengths were calculated by exposing lymphocytes to two fold

diluted concentrations of pharmaceutical wastewater and results are represented in table 4.11 and

table 4.13. Whereas percentage fragmentation damage index and genetic damage index were also

calculated for two fold dilutions of pharmaceutical wastewater which are depicted in table no 4.12

and 4.14.

Results

73

Table 4.11 Mean head diameter and tail length of different concentrations of pharmaceutical

wastewater sample (PEW 1)

Sr. No.

Sample no. PEW 1

Mean DNA Tail

length um ± SD (n=

50)

Mean Head

Diameter um ± SD

(n=50)

1 100 % 6.11 ± 2.15 3.32 ± 1.33

2 50 % 5.40 ± 2.26 4.22 ± 0.81

3 25 % 4.74 ± 2.12 4.26 ± 0.83

4 12.5 % 3.52 ± 0.59 3.84± 1.06

5 6.25 % 1.48 ± 0.65 4.85 ± 1.82

Positive Control DMSO 20% 8.16 ± 2.55 3.35± 2.66

Negative Control DW 0.06 ± 0.04 4.66 ± 0.77

Results

74

Table 4.12 Percentage fragmentation, damage index and genetic damage index detected with

increasing dilutions of pharmaceutical effluent (PEW 1)

Sr. No. Sample no.

PEW 1

Class

0

Class

1

Class

2

Class

3

Fragmentatio

n %

Damage

Index

GDI

1 100 % 2 8 21 19 96 107 2.14

2 50 % 2 17 21 9 94 86 1.72

3 25 % 5 19 24 2 90 73 1.46

4 12.5 % 7 30 10 3 86 67 1.34

5 6.25 % 11 34 5 0 78 54 1.08

Positive

Control

DMSO

20%

1 9 10 30 98 119 2.38

Negative

Control

DW 50 0 0 0 0 0 0

Results

75

Table 4.13 Mean head diameter and tail length of different concentrations of pharmaceutical

waste water sample (PEW 6)

Sr. No.

Sample no. PEW 6

Mean DNA Tail

length um ± SD (n=

50)

Mean Head Diameter

um ± SD (n=50)

1 100 % 5.47 ± 2.19 3.24 ± 1.3

2 50 % 4.09 ± 1.46 3.53 ± 0.93

3 25 % 2.52 ± 1.05 3.55 ± 0.86

4 12.5 % 2.28 ± 1.07 3.66± 1.11

5 6.25 % 0.901 ± 0.59 4.03 ± 0.74

Positive Control DMSO 20% 8.16 ± 2.55 4.94± 4.27

Negative Control DW 0.06 ± 0.04 4.66 ± 0.77

Results

76

Table 4.14 Percentage fragmentation, damage index and genetic damage index detected with

increasing dilutions of pharmaceutical effluent (PEW 6).

Sr. No. Sample

no. PEW

6

Class

0

Class

1

Class

2

Class

3

Fragmentation

%

Damage

Index

GDI

1 100 % 12 4 28 16 96 116 2.32

2 50 % 3 24 19 4 94 74 1.48

3 25 % 4 32 13 0 90 58 1.16

4 12.5 % 3 36 10 1 92 53 1.06

5 6.25 % 12 32 6 0 76 44 0.88

Positive

Control

DMSO

20%

1 9 10 30 98 119 2.38

Negative

Control

DW 50 zero zero zero zero zero zero

Results

77

4.5 Mutagenicity

4.5.1 Genetic analysis of Salmonella typhimurium strains

4.5.1.1 Histidine dependence

Plates were incubated at 37°C for 24 h. Both of the strains, i.e. TA 100 and TA-102 showed

no growth on GM agar plates depicting the dependence of these strains on histidine.

4.5.1.2 Biotin dependence

TA 100 and TA-102 showed no growth on GM agar plates showing the dependence of

these strains on biotin.

4.5.1.3 Histidine/Biotin dependence

Plates were incubated at 37°C. Both strains, i.e. TA 100 and TA-102 showed growth on

GM agar plates representing the dependence of these strains on biotin and histidine.

4.5.1.4 Surface marker test

This soaked crystal violet filter paper was placed in the middle of GM agar plates separately

for each strain and incubated for 24h at 37°C. Zone of growth inhibition was shown in both the

plates exhibiting that both the strains are permeable to test chemicals.

4.5.1.5 pKM 101 plasmid (ampicillin resistance test)

A filter paper disc containing ampicillin (10µg/mL) was placed in the center of both petri

plates. Growth was observed after 24h of incubation at 37°C even in the presence of ampicillin

disk. This showed the presence of pKM 101 plasmid because both strains showed resistance to

ampicillin.

Results

78

4.5.2 Result interpretation of bacterial reverse mutation assay

Mutagenic index was calculated using following formula

𝑀. 𝐼 =Number of Revertant colonies per plate with the test chemical dose

Number of natural Revertant colonies of the negative control plate

If the value of mutagenic index is ≥ 2 means test concentration is possible mutagenic

If the value of mutagenic index is ≥ 3 means test concentration is significant mutagenic

If the value of mutagenic index is ≥ 4 means test concentration is very strongly mutagenic

Mutagenic response was considered as positive when the number of colonies were ≥ 2 fold to the

revertant colonies of negative control (Santos et al. 2008, Ullah et al. 2016).

Mutagenic indices of all the samples were shown in table no. 4.15. All the samples were

subjected to Ames assay as mentioned above. Samples of 100% pharmaceutical wastewater were

used for the determination of mutagenic potential.

Samples PEW1 and PEW 6 were subjected to further evaluation of mutagenic potential by

diluting the samples. Two fold dilutions were made and each dilution was evaluated for the

mutagenic potential. Both PEW1 and PEW6 were subjected to chemical characterization of

organic and inorganic components. A detailed evaluation of these chemically characterized

samples was performed. Mutagenic Index (M.I) was calculated using the formula mentioned above

whereas slope (m) was calculated using graph pad prism 5 and applying a linear regression model.

The sample PEW1 was reported as potent mutagenic when evaluated for mutagenicity

against TA100 and TA-102. Slope (m) was drawn for the concentration response using graph pad

prism as shown in figure 4.12 and figure 4.13. Both the strains showed significant effect with and

without metabolic activation system. Slope values obtained were 20.74 and 5.12 for TA 100 in the

presence and absence of S9 fractions respectively. It is also confirmed from M.I which is 17.35

Results

79

and 5.12 for TA 100 with and without S9 respectively. Values of slope obtained for TA 102 strain

were 21.58 and 18.81 in the presence and absence of S9 fractions respectively. Whereas M.I.

calculated from the formula mentioned above was 17.6 and 21.62. It was demonstrated that

increasing the dilution factor decreases the mutagenic index of the waste water. Results are

presented in table 4.16.

Similarly PEW6 also showed a mutagenic potential, slope (m) of response was drawn in

the form of curves figure 4.14 and figure 4.15. Both TA 100 and TA 102 also responded well with

and without liver extract S9 fraction. The values of slope are 14.50 and 1.69 and M.I calculated

was 14.04 and 2020 for TA 100 with and without S9 respectively. Slope for TA 102 were 38.52

and 28.91 and M.I was 28 and 34.71 with and without S9 respectively. Mutagenic index decreased

(table 4.17) with the increasing dilution.

PEW 1 exhibited a strong mutagenic effect against TA-100 when compared with PEW 6.

It was evident from the Mutagenic index as well as the slopes of the linear response curves also

confirmed the results.

Results

80

Table 4.15 Revertant colonies and mutagenic index obtained by exposure to pharmaceutical

waste water (plates with 30-300 colonies were selected)

Pharmaceutical effluent waste water

Sr. No. CONC.

pure

Revertant Colonies per Plate

TA 100 TA 102

-S 9 M.I. +S 9 M.I. -S 9 M.I. +S 9 M.I.

1 PEW 1 149 1.35 225 1.85 294 3.62 315 2.60

2 PEW 2 121 1.1 213 1.76 277 3.41 327 2.70

3 PEW 3 91 0.82 110 0.90 267 3.29 309 2.55

4 PEW 4 95 0.86 104 0.85 268 3.30 375 3.09

5 PEW 5 132 1.2 222 1.83 288 3.55 391 3.23

6 PEW 6 243 2.20 260 2.14 266 3.28 313 2.58

7 PEW 7 171 1.55 209 1.72 281 3.46 379 3.13

8 PEW 8 167 1.51 211 1.74 226 2.79 361 2.98

9 PEW 9 99 0.9 117 0.96 213 2.62 307 2.53

10 PEW 10 129 1.17 191 1.57 209 2.58 311 2.57

11 -ve control 110 121 81 121

12 +ve

control

1340 12.18 3940 32.56 424 5.23 3940 32.56

Results

81

Table 4.16 Revertant colonies and mutagenic index obtained by exposure to pharmaceutical waste water (PEW1) at different

levels of dilutions


Sr. No. CONC.%

v/v


TA 100 TA 102

-S 9 M.I. +S 9 M.I. -S 9 M.I. +S 9 M.I.

1 PEW1-100%

564±2.0

8 5.12

2100±

5.5 17.35

1752±3

.78 21.62

2200

±5.50 17.6

2 PEW1-50% 149±2 1.35

915±4

.5 7.56

1176±4

.58 14.51

1500

±4.58 12

3 PEW1-25%

119±3.0

5 1.08

653±3

.21 5.39

294±2.

64 3.62

820±

1.52 6.56

4 PEW1-12.5% 89±1.52 0.80 225±2 1.85

204±2.

51 2.51

428±

4.72 3.42

5 PEW1-6.25% 77±1 0.7

113±2

.08 0.93

64±2.0

8 0.79

120±

3.05 0.96

6 -ve control 110±2.5

1

121±3

.51

81±5.6

8

125±

2.08

7 +ve control 1341±3.

61 12.19

3941±

5.51 32.57

425±2.

52 5.24

3941

±1.53 31.528

8 m 5.12 20.74 18.81 21.58

m= slope values obtained from linear regression curve using graph pad prism

Results

82

C o n c e n tra t io n (% v /v )

No

. o

f H

is-R

ev

ert

an

ts/p

late

0 5 0 1 0 0 1 5 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

w ith o u t S 9

w ith S 9

Figure 4.12 Potential of mutagenicity of PEW1 in TA-100 strain in presence and absence of

metabolic activation mixture.

C o n c e n tra t io n (% v /v )

No

. o

f H

is-R

ev

ert

an

ts/p

late

0 5 0 1 0 0 1 5 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

w ith o u t S 9

w ith S 9



Results

83

Table 4.17 Dose dependent decrease in revertant colonies observed on exposure to Pharmaceutical effluent waste water sample

(PEW 6)


Sr. No. CONC.%

v/v


TA 100 TA 102

-S 9 M.I. +S 9 M.I. -S 9 M.I. +S 9 M.I.

1 PEW6-100%

243±2.2

4

2.20 1700±

2.98

14.04 2812±2

.68

34.71 3500

±4.2

28

2 PEW6-50%

137±2.3

8

1.24 797±4

.12

6.58 1300±4

.34

16.04 2600

±1.31

20.8

3 PEW6-25%

117±2.3

6

1.06 646±2

.65

5.33 597±1.

23

7.37 630±

2.41

5.04

4 PEW6-12.5%

92±1.36 0.83 532±2

.64

4.39 266±3.

15

3.28 313±

4.14

2.58

5 PEW6-6.25%

83±2.36 0.75 260±2

.48

2.14 152±4.

32

1.87 110±

2.11

0.88

6 -ve control 111±2.5

2

122±3

.52

82±5.6

9

126±

2.09

7 +ve control 1340±3.

60

12.18 3940±

5.50

32.56 424±2.

51

5.23 3940

±1.52

31.52

8 m 1.69 14.50 28.91 38.52

m= slope values obtained from linear regression curve using graph pad prism

Results

84

c o n c e n tra t io n % (v /v )

No

. o

f H

is-R

ev

erta

nts

/pla

te

0 5 0 1 0 0 1 5 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

w ith o u t S 9

w ith S 9



c o n c e n tra t io n % (v /v )

No

. o

f H

is-R

ev

erta

nts

/pla

te

0 5 0 1 0 0 1 5 0

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

w ith o u t S 9

w ith S 9



Results

85

4.6 MTT Colorimetric Cytotoxicity Assay

The methyl thiazole tetrazolium (MTT) assay was executed for assessment of the cytotoxic

potential of pharmaceutical waste water using a twofold dilution method. Cytotoxic effects were

quantified on Baby Hamster Kidney cell line (BHK-21). The BHK-21 cell line was exposed

against each dilution of PEW.

4.6.1 Quantification cell line:

The BHK-21 cell line was accessed for the availability of viable cells at the time of assay.

Hemocytometer/Neubauer was used and viable cells were recognized using a microscope. The

number of dead and viable cells were presented in table 4.18

Table 4.18 Quantification of viable cells in BHK-21 cell line prior to assay

Results

86

Viable cells/mL = Total no. of viable cells/Total no. of squares ×Dilution factor ×104

= 75/5 × 2×104

= 3.0 ×105 viable cells

Total cells/mL = Total no. of cells/Total no. of squares ×Dilution factor ×104

= 81/5 × 2 × 104

= 3.24 × 105 cells

𝑷𝒆𝒓𝒄𝒆𝒏𝒕𝒂𝒈𝒆 𝑽𝒊𝒂𝒃𝒍𝒆 𝑪𝒆𝒍𝒍𝒔 =𝑻𝒐𝒕𝒂𝒍 𝒏𝒐. 𝒐𝒇 𝒗𝒊𝒂𝒃𝒍𝒆 𝒄𝒆𝒍𝒍𝒔/𝒎𝑳

𝑻𝒐𝒕𝒂𝒍 𝒏𝒐. 𝒐𝒇 𝒄𝒆𝒍𝒍𝒔/𝒎𝑳 × 𝟏𝟎𝟎

= 3.0 × 105/3.24 × 105 × 100

=92.9%

Whereas the cell survival percentage was calculated using following formula (Ullah et al. 2016)

𝑪𝑺𝑷 =𝑴𝒆𝒂𝒏 𝑶𝑫 𝒐𝒇 𝒕𝒆𝒔𝒕 𝒄𝒉𝒆𝒎𝒊𝒄𝒂𝒍 − 𝑴𝒆𝒂𝒏 𝑶𝑫 𝒐𝒇 𝒏𝒆𝒈𝒂𝒕𝒊𝒗𝒆 𝒄𝒐𝒏𝒕𝒓𝒐𝒍

𝑴𝒆𝒂𝒏 𝑶𝑫 𝒐𝒇 𝒑𝒐𝒔𝒊𝒕𝒊𝒗𝒆 𝒄𝒐𝒏𝒕𝒓𝒐𝒍 × 𝟏𝟎𝟎

Result of Cytotoxic Potential of Pharmaceutical waste water

The cytotoxic potential of PEW1 was determined using the BHK-21 cell line, ten different

dilutions of PEW were prepared and exposed to cell line. Incubation period given was 72 hours in

a CO2 incubator, cell survival percentages were given in the table 4.19.

Results

87

Table 4.19 Mean optical density (OD) and cell survival percentages against different

concentrations of PEW1 after incubation of 48 hours in MTT assay using BHK-21 cell line.

Serial number Concentration (%v/v) Mean OD ±SD Cell survival

percentage

CSP%

1 100 0.215±0.005 13.63

2 50 0.343±0.024 29.6

3 25 0.449±0.037 42.82

4 12.5 0.533±0.025 53.26

5 6.25 0.634±0.017 66.90

6 3.13 0.675±0.019 70.97

7 1.56 0.682±0.027 71.85

8 0.78 0.689±0.011 72.80

9 0.39 0.696±0.006 73.63

10 0.20 0.692±0.008 73.09

Positive control Cell culture medium - -

Negative control 20% DMSO - -

Results

88

Figure 4.16 Percentage survival of cells (CSP) against different log concentrations of PEW1

on BHK-21 cell lines

X-axis = log concentration of pharmaceutical waste water in (%v/v)

Y-axis = Cell survival percentage (CSP) BHK-21 cell lines against different dilutions of

pharmaceutical wastewater.

The cytotoxic potential of PEW6 was quantified on BHK-21 cell line. A range of ten

different concentrations of PEW6 was subjected to exposure with kidney cell line for a period of

48 hours in CO2 incubator, and cell survival percentages were presented in table 4.20.

Results

89

Table 4.20 Mean optical density (OD) and cell survival percentages against various

concentrations of PEW6 after incubation of 48 hours in MTT assay using BHK-21 cell line.

Sr no. Concentration

(%v/v)

Mean OD ±SD Cell survival

percentage

CSP%

1 100 0.193±0.004 10.93

2 50 0.320±0.001 26.73

3 25 0.413±0.003 38.37

4 12.5 0.541±0.018 54.30

5 6.25 0.679±0.007 71.47

6 3.13 0.684±0.006 72.18

7 1.56 0.686±0.021 72.34

8 0.78 0.687±0.019 72.51

9 0.39 0.690±0.015 72.84

10 0.20 0.689±0.015 72.76

Positive control Cell culture medium - -

Negative control 20% DMSO - -

Results

90

Figure 4.17 Cell survival percentage (CSP) against different log concentrations of PEW6 in

MTT assay on BHK-21 cell lines

X-axis = log concentration of pharmaceutical waste water in (%v/v)

Y-axis = Cell survival percentage (CSP) BHK-21 cell lines against different concentrations of

pharmaceutical wastewater

Results

91

4.7 Oxidative stress

4.7.1 Effect on total-superoxide dismutase (T-SOD) activity in plasma, liver and kidney

The level of T-SOD in plasma decreased significantly when compared to control on

exposure to PEW1 characterized by (p ˂ 0.05). T-SOD level also showed significant difference

when PEW1 100% were compared with control, but 1% PEW1 group did not differed significantly

when compared to control (table 4.21). The vitamin E treated group showed a significant difference

(p ˂ 0.0.5) when compared with 100 %, 10 % and 1 % PEW1 groups. Treatment with PEW1 for

2 months induced severe oxidative stress in the liver and kidney of the samples which is

characterized by a significant decrease in SOD (p ˂ 0.05) level. Vitamin E partially restored the

T-SOD levels when compared with control groups. The results are represented in fig no.

4.7.2 Effect of catalase (CAT) activity in plasma, liver and kidney

Catalase in blood, liver and kidney showed a significant decrease in its levels (table 4.22)

with PEW1 when compared to control (p ˂ 0.05) suggesting the presence of oxidative stress.

Vitamin E was able to reduce the CAT activity in blood (p ˂ 0.05). Levels of CAT were restored

partially in liver and kidney when treated with by Vitamin E.

4.7.3 Effect of hydrogen peroxide (H2O2) in plasma, liver and kidney

Elevated H2O2 concentrations were significantly tapered by Vitamin E in plasma, liver and

kidney. Changes induced by 100% PEW1 were ameliorated by vitamin E (p ˂ 0.05). All the

treatments also showed significant difference when compared with control (table 4.23).

Results

92

Table 4.21 Effect on T-SOD activity in rat plasma, kidney and liver caused by chronic

exposure to PEW1 at different concentrations with and without vitamin E

Treatments Plasma Kidney Liver

Distilled Water 92.54±0.19 65.75±0.15 57.91±0.29

100% PEW1 63.57±0.19a 32.65±0.77a 43.57±0.23a

10% PEW1 80.17±0.38a 57.54±2.53a 51.70±1.14a

1% PEW1 91.78±0.38 64.95±0.6 57.80±0.29

100% PEW+ Vitamin E 70.65±2.44a,b 43.07±0.29a,b 47.36±0.29a,b

PEW: Pharmaceutical effluent wastewater; T-SOD: total superoxide dismutase

Values are mean ± standard error (n=5)

aP ˂ 0.05 versus control

bP ˂ 0.05 100% PEW versus 100% PEW+ Vitamin E (analysis of variance followed by Tukey’s

test)

a b

Results

93

Table 4.22 Effect on CAT activity in rat blood, kidney and liver caused by chronic exposure

to PEW1 at different concentrations with and without vitamin E

Treatments Blood Kidney Liver

Distilled Water 146.5±1.32 130.7±1.75 135±2.95

100% PEW 89.25±2.03a 54.36±1.71a 62.95±2.82a

10% PEW 128.2±0.78a 73.28±4.56a 87.51±2.84a

1% PEW 141.9±0.79 116.7±3.49 89.72±1.65a

100% PEW+ Vitamin E 100.1±0.77a,b 68.11±4.55a,b 72.75±3.26a

PEW: Pharmaceutical effluent wastewaterCAT: catalase; H2O2


a P ˂ 0.05 versus control

b P ˂ 0.05 100% PEW versus 100% PEW+ Vitamin E (analysis of variance followed by Tukey’s

test)

a b

Results

94

Table 4.23 Effect on H2O2 concentrations in rat plasma, kidney and liver caused by chronic

exposure to PEW1 at different concentrations with and without vitamin E

Treatments Plasma Kidney Liver

Distilled Water 220.6±2.67 402.7±2.67 294.3±2.72

100% PEW 68.80±2.67a 243.3±5.11a 162.9±2.67a

10% PEW 107.3±2.67a 330.9±3.5a 210.5±2.67a

1% PEW 182.1±3.5a 397.6±1.75 236.8±8.76a

100% PEW+ Vitamin E 129.5±2.67a,b 279.3±1.75a,b 267.1±1.75a,b

PEW: Pharmaceutical effluent wastewater; H2O2: hydrogen peroxide


a P ˂ 0.05 versus control

b P ˂ 0.05 100% PEW versus 100% PEW+ Vitamin E (analysis of variance followed by Tukey’s

test)

a b

Results

95

Figure 4.18 Effect of PEW1 on SOD activity in rat plasma, kidney and liver

Figure 4.19 Effect of PEW1 on CAT activity in rat blood, kidney and liver

Figure 4.20 Effect of PEW1 on H2O2 activity in rat plasma, liver and kidney

* p ˂ 0.05 versus control

# p ˂ 0.05 100 % PEW versus 100 % PEW+ Vitamin E

Results

96

4.8 Histopathology

The microphotographs exhibiting pathological alterations in tissues of kidney, liver, lungs,

intestine, heart and brain of Wistar rats are presented in figure 4.21(a-e) The most severe damage

was observed in the groups treated with PEW1 100%. The histopathological changes observed in

the rats, which were subjected to PEW1 10% and PEW1 1% were of relatively moderate intensity.

In the kidney coagulative necrosis of renal epithelial cells took place and many tubular

cells were disintegrated after 100% PEW1 exposure. There was cellular swelling in some tubular

cells along with peritubular congestion fig 4.21 (a). Liver was also compromised due to exposure

with PEW1 for 60 days and severe degeneration and cellular swelling as well as coagulative

necrosis in hepatocytes of hepatic cord were observed figure 4.21 (b). Lungs were also affected to

some extent. Lungs showed atekation and emphysema of alveoli. Effects are represented in figure

4.21 (c). There were mild effects of PEW1 on intestine where there was mild degeneration and

sloughing of intestinal epithelial cells depicted in figure 4.21 (d). In a very area in endocardium

there is break down of cells and presence of some inflammatory cells figure 4.21(e). No changes

were seen in the brain (cerebellum) and (cerebrum). There was no change in cell bodies, neutrophil

areas was normal, microglial cells were intact and no obvious pathological change was seen.

Results

97

Fig. 4.21 (a-e) Representative histological images of tissue sections followed oral exposure of PEW for 60 days. a.

kidney section of rat highlighting necrosis of cells along with control group showing intact glomerulus. b. Liver

section of rat highlighting degeneration and swelling of hepatocytes along with control c. Lungs section of rat

representing atekation and emphysema of alveoli d. Intestine section of rat highlighting mild degeneration and

sloughing of intestinal epithelial cells e. Heart section of rat highlighting break down of cells in endocardium and

presence of some inflammatory cells

CHAPTER 5

DISCUSSION

In this study pharmaceutical wastewater was characterized using AAS and GCMS. Various

inorganic and organic compounds were present in PEW. Heavy metals like Fe, Cr and As and Cd

were present in higher concentrations than the normal permissible limits of WHO and US EPA.

Various organic compounds like trimethoprim, prednisolone, dipyrone, caffeine and solvents like

toluene were also detected in PEW. In this study PEW samples were used without any pre

concentration depicting a true picture of both organic and inorganic contaminants present in it.

None of the previous study reported the use pure PEW for evaluation of genotoxic, mutagenic,

cytotoxic potential in different experimental models. Genotoxicity was evaluated using comet

assay on sheep lymphocytes and PEW showed a dose dependent effect exhibiting decrease in GDI

and DF with the increasing dilution factor. Similarly mutagenicity was evaluated using Ames assay

and PEW exhibited a mutagenic potential particularly with TA 100 salmonella typhimurium strain.

Ames assay results also exhibited a dose dependent decrease in M.I with the increasing dilution

factor of PEW. Cell survival percentage was calculated using MTT assay. Effect of PEW on BHK-

21 cell lines were observed. IC-50 was calculated. CSP was increased with the decreasing

concentration of PEW. None of the previous studies reported effect of pure PEW on different

experimental models demonstrating genotoxic, mutagenic and cytotoxic potential. In this study

effect of chronic exposure of PEW was also investigated. Rats were exposed to PEW and levels

of enzymes which cope with oxidation process were measured. It is linked with oxidative stress

generation. This generation of oxidative stress caused certain histopathological changes in rat liver,

kidney, heart, lungs and epithelium. Chronic exposure of PEW is associated with the generation

Discussion

99

of oxidative stress which may induces certain changes in tissues and may be linked with the

degenerative processes in vital organs of the body.

5.1 Inorganic compounds

The investigational values of the Cd were 0.01, 0.02 and 0.04 mg/L and go beyond the

normal allowable values of US EPA and WHO. Cd has been associated with respiratory distress,

spasms, loss of consciousness, breathing complications, vomiting and nausea (Gowd and Govil

2008). Continuous contact may be associated with cardiovascular risks, hypertension and renal

diseases (Robards and Worsfold 1991).

The amount of Iron (Fe) was considered extraordinary in all the tester samples. These

results were in accordance with earlier reports (Singarea and Dhabardeb 2014). Chronic exposure

to Fe can cause gastric and esophageal ulcers both in animals and humans. Various gastrointestinal

disorders are also associated with the higher levels of Fe along with nausea, vomiting and diarrhea

(Javed and Usmani 2013). Fe higher concentrations in PEW might be due to various iron

preparations manufacturing in these pharma industries.

Chromium (Cr) concentrations were higher in all the PEW samples analyzed. Cr (VI) is a

water miscible compound and exhibits a strong oxidizing potential which is extremely lethal for

living systems and tissue layers (Gowd and Govil 2008). Different species of invertebrates can be

affected by chromium to different levels (Moore and Ramamoorthy 2012). Chromium can damage

plants which are characterized by narrowing of leaves, and making the stems, small (Singarea and

Dhabardeb 2014). In human beings, Cr compounds may cause cancer of the kidneys, urinary

bladder, prostate, testis, stomach , brain, and lungs (Costa 1997). Higher concentration of Cr found

in PEW samples advocates’ higher risk of above mentioned diseases in population.

Discussion

100

In comparison with EPA and WHO standards, concentration of Arsenic (As) were

extremely high in all samples. Skin cancer development is the outcome of oral revelation to As.

Numerous internal organs like lungs, kidneys, bladder and liver may develop cancer after exposure

to As (Tchounwou et al. 2003). Cognitive inconsistencies had resulted in the school going age

children due to the presence of Inorganic As in drinking water (Grandjean and Landrigan 2014).

Different types of neurological diseases have occurred in children who were previously exposed

to (As) containing Morinaga milk (Tanaka et al. 2010). These higher concentration of As detected

in this study strongly suggests that population especially children are at higher risk of developing

neurological disorders.

5.2 Organic compounds

GC-MS analysis was used to accomplish the drug characteristics and numerous other polar

compounds which exist in the treatment plants (Yu et al. 2013). Certain organic contaminants were

detected when PEW was subjected to GCMS analysis.

Prednisolone a glucocorticoid was detected in PEW sample. Prednisolone is persistent in

nature and was reported to occur in a number of previous studies (Chang et al. 2007) (Lozano

2013) . Owing to its endocrine disrupting effect the presence of prednisolone might be associated

with hormonal problems which might be attributed to the suppression of hypothalamus pituitary

axis leading to imbalance of hormones released via this pathway. Exposure to endocrine disrupting

chemicals like prednisolone is associated with reproductive health changes in humans including

male infertility, birth defects, breast and testicular cancer (Nikolaou et al. 2007).

Trimethoprim was also detected in PEW. It is a persistent antibiotic (Halling-Sørensen et

al. 2000). Trimethoprim was associated with carcinogenicity and mutagenicity as folate deficiency

can induce entire range of genetic events. Trimethoprim being a folate antagonist might play an

Discussion

101

important role in induction of carcinogenesis and considered a potential environmental genotoxic

agent (Park and Choi 2008).

Lidocaine is a drug which is investigated in the study in PEW sample. This compound was

not extensively investigated in the environment. Lidocaine and its metabolite

monoethylglycinexylidide are persistent in nature and provides a proof for its detection in PEW.

LD50 of lidocaine was 106 mg.L-1 suggesting its potential Eco toxicological influence.

Toxicological studies at environmental concentrations were not reported before so study of this

sample in chronic toxicity study and oxidative stress induction provides with some new

information regarding the toxic potential of drugs in the environment (Rúa-Gómez and Püttmann

2012).

A number of environmental pollutants were detected along with caffeine, which has been

reported as a waste water indicator (Seiler et al. 1999). The combination mixture of different drugs,

exerts a cumulative additive effect. Although concentrations of pharmaceuticals in waste water are

very low but the presence of a large number these compounds might suggest that in combination

they do have eco toxicological influence as reported earlier (Quinn et al. 2008), (Krifa et al. 2013).

5.3 Microbial resistance

A total of 27 isolates were obtained representing 1 aeromonas species with 7 isolates, 1

micrococcus with 4 isolates, 1 bacillus with 6 isolates and 3 staphylococcus species with 10

isolates. Frequency of isolates varied between different sites with maximum isolates obtained from

PEW6. Aeromonas species was detected in PEW samples. This bacterium is responsible for the

occurrence of hemorrhagic septicemia in humans which is a food born disease (Abou-Elela et al.

2009). Bacillus megaterium was also found in the PEW. Presence of bacillus megaterium was

previously reported in the waste water samples. The higher concentration of the bacteria was due

Discussion

102

to higher levels of heavy metals particularly chromium which is in correlation with this study.

Chromium concentrations were found higher than the normal permissible limits of US EPA. This

suggests the use of the bacillus species in biotransformation of hexavalent Cr to less toxic trivalent

form (Thacker et al. 2007). Staphylococcus species are identified in the present study.

Staphylococci are normal micro biota of air, soil, water, humans, other animals and processed food

products. It is suggested that gram positive cocci are highly tolerant to dryness, dehydration and

low water activity which is responsible for its wide spread distribution and persistence in different

environments including PEW (Thacker et al. 2007).

A large number of viable bacteria were found in all PEW samples. The presence of high

number of bacteria in wastewater samples suggests the presence of adaptive mechanisms of

resistance among these bacterial colonies. This study is unique and one of the few investigations

that reports the incidence of heavy metal and antibiotic resistance among different bacterial

species.

The occurrence of pharmaceuticals in the environment has captured attention recently.

During past few years many investigations have been made regarding the persistence of

pharmaceuticals in the waste water, surface water, ground water and soil (Krifa et al. 2013).

Heavy metals are involved in microbial resistance patterns due to development of

detoxifying mechanisms evolved by xopolysaccharides, binding with bacteria, reduction of metals

and efflux of metals from microbes. Plasmid genes are the carriers of resistance and responsible

for transfer of toxic metal resistance from one cell to another cell (Silver 1996).

Wastewater isolates have previously been studied and it was investigated that

Pseudomonas aeruginosa, Klebsiella pneumonia, Proteus mirabilis and Staphylococcus spp. were

found to be resistant to heavy metals and certain antibiotics (Filali et al. 2000). Similar results

Discussion

103

were found by (Sharma et al. 2000) where he explored cadmium resistant Klebsiella species. These

findings support the result of this study in which various bacterial isolates were found resistant to

Cu, Cr and various antibiotics. Based upon findings it is also suggested that Genes encoding for

heavy metals are located together with antibiotics. Bacteria have unspecific resistance mechanisms

which are common to different substances such as heavy metals, biocides and antibiotics. So it is

very likely that pressure, which is selective for one such compound is responsible indirectly for

resistance of other bacteria (Dalsgarrd and Guardbassi 2002).

5.4 Genotoxicity

In an ecosystem, single cell gel electrophoresis (SCGE) are most widely used assays for

detection of DNA damage. Long term contact and after separation resulted in impaired DNA,

chromosomes and exemplifies subsequent effect (Frenzilli et al. 2009). Though the mechanism of

action is unclear, but in humans Cd and As had been reported to be carcinogenic (Waalkes 2003);

(Goering et al. 1999). Metals may cause different sorts of lesions like DNA and DNA cross

connections, double and single DNA strands breaking, base pairing alterations and protein crosses

of DNA. Comet assay identifies the single strand breakage of DNA. Genotoxic agents may cause

breakdowns in DNA. Apurinic sites, which are alkali sensitive, have the ability to tear apart the

strand when electrophoresis are executed at alkaline solution

Previous studies had shown that Cd brings class 3 or 4 damage to DNA in comparison to

control cells. It has been established previously that Cd in 0.02mg/L prompted DNA breakage

(Mourón et al. 2001). It was stated that DNA lesions and cross linking ensued at high As doses

(Gebel et al. 1997). In Denmark, Blue mussels have been related to obtain waste waters. Greater

damage to DNA arisen in gills and Cd, Ni and Cr, present in wastewater, produced a concentration

dependent movement of tail (Rank et al. 2005).

Discussion

104

A chief contributor to genotoxicity is trimethoprim which is detected in the present study.

Trimethoprim (TMP)’s genotoxicity might be accredited to the generation of oxidative stress and

production of reactive oxygen species ROS (Binelli et al. 2009). TMP was reported to prompt

mitochondrial movement of electron transport in a robust fashion, as this movement is a principal

basis of generation of reactive oxygen radicals in cells.

Xenobiotics caused hydrogen peroxide production and oxidative stress generation by

aiding in withdrawal of electrons from scavenging radicals in mitochondria (Gagné et al. 2006). It

was reported earlier that membrane damage leads to active gene expression and proliferation of

cells takes place (Bengtsson et al. 2001). This is considered a marker of DNA damage. A number

of studies have been conducted on waste water samples depicting DNA damage in-vitro and in-

vivo. Toxicity of wastewater samples was attributed to the heavy metal’s presence (TÜRKEZ et

al. 2009).

5.5 Mutagenicity

A significant index for mutagenic properties is Ames plate incorporation assay, used for

mutagenic properties of numerous substances which are present in the surroundings. It is reputable

from the results that samples proved to be potent mutagenic especially when checked against

TA100 and TA102 strain. It was suggested that TA102 strain has been used to identify the

mutagenic behavior associated with reactive oxygen radicals (Tabrez and Ahmad 2011).

The study confirmed that samples from environment contained a mixture of different

hazardous components so different strains were used to access the mutagenic nature of components

present in the mixture as one strain is unable to show the real picture of mutagenic strength (Tabrez

and Ahmad 2011). Present study followed the same pattern as that of older investigations and

confirmed that the mutagenicity of sample is due to the presence of high concentrations of As, Cd

Discussion

105

and Cr along with anti-bacterial drugs and compound that are phenolic in nature. (Fatima and

Ahmad 2006); (Tabrez and Ahmad 2009). The presence of these different hazardous materials has

been confirmed in the initial part of the study. Cd concentrations were found higher than normal

permissible limits of US EPA. Cd can induce mutations by generation of oxidative stress in DNA.

It inhibits the repair of endogenous and exogenous lesions promoting the probability of mutation

that’s why mutation response was significant when PEW was treated with TA-102 strain of

Salmonella typhimurium. Excessive concentrations of Cd is linked with higher incidence of

prostate, lungs and testes cancer in the population when they get exposed to PEW. Cd may affect

cell proliferation, differentiation and do cause apoptosis (Bertin and Averbeck 2006). Higher

concentrations of As in PEW can be a contributing factor in mutagenicity. As may affect the

methylation of DNA in gene promotor regions which is responsible for gene expression along with

modifications in histone tail which is a regulator for the access of transcriptional machinery to the

gene. These two processes are responsible for the As induced mutagenicity (Bustaffa et al. 2014)

It was also proposed that mutagenicity was due to the presence of dissolved components

as compared to particles which are in suspended form. Present study concluded that TA 102 is

much more sensitive than TA100 confirming the generation of toxic radicals, which is in

accordance with studies conducted by (Fatima and Ahmad 2006), (Tabrez and Ahmad 2009).

Addition of liver extracts of rats has resulted in increase and decrease of mutagenicity

confirming that mutagens might undergo metabolism upon addition of a metabolic activation

system and ultimately the response of the components might be altered (Tabrez and Ahmad 2009).

Oxidative stress has been associated with transition mutations and TA102 responded well in this

study confirming alteration in A-T site (Aleem and Malik 2005).

Discussion

106

5.6 Oxidative stress and Histopathology

In the present study role of PEW induced oxidative stress was investigated. Its role in

facilitating renal and hepatic pathology of rats following 60 days of oral treatment with PEW was

discovered. Efficacy of vitamin E in alleviating the toxicity of PEW was also explored. The liver

and kidneys were selected because they considered to be the major target of toxicity. Liver

containing metabolizing enzymes has the ability to biotransform the xenobiotic into less toxic or

active metabolite. Liver has been a site for lipid peroxidation so considered a hallmark for the

oxidative stress tests. The kidneys do perform the same activities along with filtration (Patlolla et

al. 2009). Both kidneys and liver are vulnerable to the stress, including oxidative stress because of

their dependency on osmotic pressure and concentration gradient along with kidney which is

considered to be a site of peroxisomes that can store catalase (Hook 1993).

Cr had a potential to depress the levels of SOD, CAT, GSH-Px in rats epithelial intestinal

cell (Sengupta et al. 1990). Cr (VI) decreases the enzymatic antioxidant levels including SOD and

CAT (Susa et al. 1996). 60 days of drinking waste water resulted in induction of oxidative stress

and generation of reactive oxygen species (ROS) with a resultant decrease in GSH, SOD and CAT.

Glutathione exhaustion had been a result of declining levels of GST, SOD and CAT which are

known to be free radical scavengers (Al-Qirim et al. 2002) (Dickinson et al. 2003). Normally

enzymatic and non-enzymatic anti-oxidant systems exist in the body. Overproduction of free

radicals or diminished scavenging action of enzymes or both mechanisms are involved in lipid

peroxidation. It is a chain process, initial oxidation of few molecules leads to massive tissue

destruction and diseased organs (Zaidi and Banu 2004). Several diseases of CNS Parkinson’s,

Schizophrenia, Alzheimer have been associated with generation of reactive oxygen species and as

a result of failure of the antioxidants defense mechanism (Smythies 1999).

Discussion

107

Pb concentrations found higher than normal limits of US EPA has also been associated

with oxidative stress. Lead nitrate decreases the activities of SOD, CAT and GSH and resultant

tissues were exposed to per oxidative damage. These enzymes are metallo proteins and induct their

function enzymatically detoxifying peroxides (-OOH), H2O2 and O2* (Lakshmi et al. 2013). Pb

toxicity has also been found to be multifactorial. Possible mechanisms may involve enzyme

inhibition, competitively inhibits mineral absorption or binding with sulfhydryl groups.

As higher concentrations has also been found to exert oxidative damage in tissues by the

generation of ROS e.g. superoxide, hydroxyl and peroxyl radicals. Levels of SOD and CAT

decreased significantly in rat’s liver and kidneys when they were exposed to PEW for 60 days

suggesting the generation of oxidative stress metabolic organs. This decrease in levels of SOD

and CAT in liver of rats and increased MDA levels established the fact that rats suffered from

oxidative destruction after chronic exposure to inorganic As due to lipid peroxidation and oxidative

damage (Santra et al. 1999), (Xu et al. 2013).

The present study is in accordance with the previous investigations in which Pb, As and Cr

have been associated with decreasing levels of enzymes and induction of pathological changes in

body organs especially where the xenobiotic got transformed to other metabolites i.e. liver and

kidney (Patrick 2003).

Pharmaceutical residues, sex steroid hormones and personal care products have been

categorized as new emerging pollutants. Prednisolone was detected in the pharmaceutical waste

water. Steroids are associated with deterioration of endocrine and reproductive function. Studies

have proposed increased risk of breast, prostate and testicular carcinomas on exposure to endocrine

disrupting substances like prednisolone (Kudłak and Namieśnik 2008). Phenolic compounds are

considered potent environmental pollutants. Pharmaceutical, petroleum and chemical industries

Discussion

108

have contributed in the discharge of a large amount phenolic compounds. Phenolic compounds led

to the generation of free radicals in organs like liver leading to the generation of phenoxy radicals

and intermediate metabolites resulting in generation of superoxide radicals and hydrogen peroxide

(Michałowicz and Duda 2007). Toluene increases the formation of reactive oxygen species and

decreases oxidative stress markers like SOD, CAT, and Glutathione S-transferase when different

concentrations of toluene were exposed to Drosophila melanogaster (Singh et al. 2009).

Non-enzymatic anti-oxidants such as vitamin A, C and E have been reported to act as

effective anti-oxidants and had a potential to mitigate diseases and degenerative processes which

are known to occur with oxidative stress (Olas and Wachowicz 2002) (Chaudière and Ferrari-Iliou

1999). Administration of Vitamin E to PEW exposed rats raised the levels of SOD, CAT and H2O2.

Vitamin E administered group exhibits significant difference in raising the level of enzymes when

compared with group which was administered with PEW 100% only. Vitamin E had a primary

effect in limiting the oxidative stress in brain (Zaidi and Banu 2004). It is suggested that that liver

toxicity can be protected from oxidative damage by administering Vitamin E. Vitamin E is a

peroxyl radical scavenger and it maintains the integrity of long-chain polyunsaturated fatty acids

in the membranes of cells and maintain their bioactivity. (Onyema et al. 2006), (Rao et al. 2006).

It was in agreement with previous investigations that vitamin E do play some role in relieving

oxidative stress and improving the markers related to stress generation.

Environmental metals like lead, chromium, cadmium are known hepatotoxic agents (Cui

et al. 2004), (Mousa 2004), (Ramm and Ruddell 2005). They have a potential to cause chronic

renal diseases (Shaikh et al. 1999), (Barbier et al. 2005). It was found that a mixture of heavy

metals can induce oxidative stress in liver, kidney, brain and erythrocytes. The mechanism

involved might be lipid peroxidation along with the decreasing ability of antioxidative defense

Discussion

109

systems in male rats (Jadhav et al. 2007a), (Jadhav et al. 2006). The final observation of the study

suggested that mixture of heavy metals may cause liver injury on prolonged exposure and poses a

health risk. Kidney particularly proximal tubules have been a potential first target of toxicity

caused by heavy metals (Barbier et al. 2005), (Madden and Fowler 2000). It was accessed that the

oxidative stress inducing potential of heavy metals was maximum in the kidney as lipid

peroxidation and diminution in enzymatic and non-enzymatic antioxidant status was maximum

(Jadhav et al. 2007b). The results also suggest that the kidney was most sensitive organ making it

a sensitive and potential target and posing a danger of kidney damage both in humans and animals

when they were repeatedly exposed to heavy metals present in the environment.

In-vivo toxicological studies should include animals from both sexes. However, gender

dependent effects may be prominent in some studies. There are strong evidences of higher

antioxidant activity in female than male. Female animals are also less prone to oxidative stress

than male (Chakraborti et al. 2007).

Waste water from industries should be treated before expulsion into the environment or

open water bodies. Currently there is no waste water treatment plant operational in the industrial

areas of Lahore. There should be strict legislation regarding this issue. It is estimated that half of

the waste water produced worldwide is discarded without specific treatment (Chen et al. 2008).

Different methods are available to treat pharmaceutical waste water can be categorized as

biological processes such as aerobic and anaerobic treatments. Some advance treatments which

include membrane technology, activated carbon and membrane distillation processes are also used

to treat PEW. Advanced oxidation processes like ozone treatment, photo catalysis and Fenton

oxidation can be used to reduce the toxic potential of PEW. Heavy metals can be removed using

hybrid technologies from pharmaceutical waste water (Gadipelly et al. 2014). Several methods

Discussion

110

like ozone based processes are available to remove certain pharmaceutical compounds like

caffeine, cyclophosphamide and Diethyl meta-toluamide and it was reported in different

investigations that concentrations of persistent compounds were reduced markedly from waste

water using above mentioned treatment processes (Kim and Tanaka 2010). Biochar derived from

different sources can be used to remove heavy metals and pharmaceuticals from PEW.

It is suggested that the PEW should be treated with the above mentioned processes and

based upon the findings a cost effective method that can reduce toxic potential of PEW should be

employed in countries like Pakistan where it is difficult to install expensive treatment plants to

protect our environment from hazardous effects of Pharmaceutical waste water.

Summary

CHAPTER 6

SUMMARY

Pharmaceutical effluent being a complex mixture of drugs and heavy metals may affect

human health exhibiting a strong potential of mutagenicity, carcinogenicity, cytotoxicity and

oxidative stress induction along with pathological changes in various organs of the body. The

current study was focused to quantify the presence of heavy metals, detection of various drugs,

determining the bacterial load along with isolation and identification of different bacteria and

assessment of the mutagenic and genotoxic, cytotoxic and oxidative stress induction of

pharmaceutical effluent wastewater when exposed to sheep lymphocytes, Salmonella typhimurium

strains, cell lines and rats respectively.

Atomic absorption spectrophotometer was used to quantify heavy metals and showed the

presence of arsenic, chromium, lead and iron in concentrations above the normal limits

recommended by WHO and EPA. Gas Chromatograph mass spectrophotometer analysis shown

the presence of digitoxin, lignocaine, caffeine and trimethoprim and various other organic

pollutants.

Microbiological evaluation showed a high bacterial load in the pharmaceutical waste water.

Several bacteria were also found in PEW in the presence of different drugs and heavy metals.

Aeromonas sobria, Micrococcus varians, Staphyoloccus epidermidis, Staphylococcus aureus,

Bacillus megaterium showed tolerance to potassium di chromate and copper sulphate and

resistance to various antibiotic discs.

Ames assay revealed a strong mutagenic potential with and without the presence of

metabolic activation mixtures. A concentration dependent effect was observed when samples were

tested with increasing dilution factor.

Summary

112

MTT assay and comet assay also showed a concentration dependent effect. The BHK-21

cell line was used to evaluate cytotoxicity and cell viability decreased with increasing

concentration of PEW. Sheep lymphocytes used in comet assay exhibited a concentration

dependent DNA damage.

Different antioxidant enzymes were also evaluated. Rats were exposed to PEW at different

concentrations and following 60 days oral exposure, rats were evaluated for the presence of total

superoxide dismutase, catalase and hydrogen peroxide in kidney, liver and plasma. Exposure to

Pharmaceutical waste water significantly decreased the (TSOD), (CAT) and (H2O2) levels in

plasma, liver and kidney. Treatment with Vitamin E significantly ameliorated the levels of

enzymes.

Exposed rats were also evaluated for any pathological changes. Coagulative necrosis of

renal epithelial cells were observed along with severe degeneration and cellular swelling in

hepatocytes of hepatic cord.

CHAPTER 7

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induced changes in the distribution of H2O2 and antioxidant system of strawberry leaves.

Agric J, 3, 286-291.

Appendices

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CHAPTER 8

APPENDICES

Annexure-1

Mass spectra of neutral fraction of PEW1 using DB-35ms column

Appendices

124

Annexure-2

Mass spectra of acidic fraction of PEW1 using DB-35ms column

Appendices

125

Annexre-3

Mass spectra of basic fraction of PEW1 using DB-35ms column

Appendices

126

Annexure-4

Mass spectra of basic fraction of PEW6 using DB-5 column

Appendices

127

Annexure-5

Mass spectra of acidic fraction of PEW6 using DB-5 column

Appendices

128

Annexure-6

Mass spectra of neutral fraction of PEW6 DB-5 column

Appendices

129

Annexure-7

Comparison of tail lengths of comets using one way ANOVA at different concentrations of PEW 1 (%v/v)

Appendices

130

Annexure-8

Comparison of tail lengths of comets using one way ANOVA at different concentrations of PEW 6 (%v/v)

Appendices

131

Annexure-9

Mutagenic potential of PEW1 in TA-100 strain with and without S9 using nonlinear fit

Appendices

132

Annexure-10


Appendices

133

Annexure-11


Appendices

134

Annexure-12


Appendices

135

Annexure-13

Cell survival percentage (CSP) against different log concentrations of PEW1 in MTT assay on BHK-21 cell

lines using nonlinear fit

Appendices

136

Annexure-14

Cell survival percentage (CSP) against different log concentrations of PEW6 in MTT assay on BHK-21 cell

lines using nonlinear fit

Appendices

137

Annexure-15

Effect of PEW1 on catalase activity in rat blood caused by chronic exposure at different concentrations using

one way ANOVA

Appendices

138

Appendices

139

Annexure-16

Effect of PEW1 on catalase activity in rat kidney caused by chronic exposure at different concentrations

using one way ANOVA

Appendices

140

Appendices

141

Annexure-17

Effect of PEW1 on catalase activity in rat liver caused by chronic exposure at different concentrations using

one way ANOVA

Appendices

142

Appendices

143

Annexure-18

Effect of PEW1 on hydrogen peroxide activity in rat kidney caused by chronic exposure at different

concentrations using one way ANOVA

Appendices

144

Appendices

145

Annexure-19

Effect of PEW1 on hydrogen peroxide activity in rat liver caused by chronic exposure at different


Appendices

146

Appendices

147

Annexure-20

Effect of PEW1 on hydrogen peroxide activity in rat plasma caused by chronic exposure at different


Appendices

148

Appendices

149

Annexure-21

Effect of PEW1 on superoxide dismutase activity in rat kidney caused by chronic exposure at different


Appendices

150

Appendices

151

Annexure-22

Effect of PEW1 on superoxide dismutase activity in rat liver caused by chronic exposure at different


Appendices

152

Appendices

153

Annexure-23

Effect of PEW1 on superoxide dismutase activity in rat plasma caused by chronic exposure at different


Appendices

154

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