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Biology Thesis and Dissertations

2019-09-03

ASSESSMENT OF

CYTOTOXICITYAND GENOTOXICITY

POTENTIAL OF EFFLUENT FROM

BAHIR DAR TANNERY USING ALLIUM CEPA

Tayachew, Admas

http://hdl.handle.net/123456789/9644

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BAHIR DAR UNIVERSITY

COLLEGE OF SCIENCE

DEPARTMENT OF BIOLOGY

ASSESSMENT OF CYTOTOXICITYAND GENOTOXICITY POTENTIAL OF

EFFLUENT FROM BAHIR DAR TANNERY USING ALLIUM CEPA

A Thesis Submitted to the College of Science, Department of Biology, Bahir Dar University, In

Partial Fulfillment of the Requirements for the Degree of Master of Science in Biology

(Genetics)

By

Tayachew Admas Abeje

June, 2019

Bahir Dar, Ethiopia

i

ASSESSMENT OF CYTOTOXICITY AND GENOTOXICITY POTENTIAL OF

EFFLUENT FROM BAHIR DAR TANNERY USING ALLIUM CEPA

A Thesis Submitted to the College of Science, Department of Biology, Bahir Dar University, In

Partial Fulfillment of the Requirements for the Degree of Master of Science in Biology

(Genetics)

By

Tayachew Admas Abeje

June, 2019

ii

Bahir Dar, University

DECLARATION

I declare that this thesis is my original work in partial fulfillment for the requirements for the

degree of Master of Science in Biology (Genetics). All the sources of the materials used for this

thesis and all people and institutions who gave support for thesis work are fully acknowledged.

Name: Tayachew Admas Abeje

Signature: ______________

Date: __________________

College of Science,

Biology Department,

Bahir Dar University

iii

Bahir Dar, Ethiopia

APPROVAL SHEET

As a thesis research advisor, I hereby certify that I have read and evaluated this thesis prepared

under my supervision, by Tayachew Admas entitled as “Assessment of Cytotoxicity and

Genotoxicity Potential of Effluent from Bahir Dar Tannery Using Allium Cepa” I recommended

the paper to be submitted as fulfilling the requirement for the Degree of Master of Science in

Biology (Genetics).

______________ ________________ ____________________

Advisor Signature Date

As members of the board of examiners for the MSc thesis open defense examination, we certify

that we have read and evaluated the thesis prepared by Tayachew Admas and examined the

candidate. We recommended the thesis to be accepted as fulfillment for the requirements of the

Degree of Master of Science in Biology (Genetics).

________________ _______________ ______________

Chair Person Signature Date

__________________ __________________ _______________

Internal Examiner Signature Date

__________________ _________________ _________________

External Examiner Signature Date

iv

TABLE OF CONTENTS

DECLARATION ii

APPROVAL SHEET iii

LIST OF TABLES viii

LIST OF FIGURES ix

LIST OF ACRONOMYS x

ABSTRACT xi

1. INTRODUCTION 1

1.1. Background of the study 1

1.2. Statement of the problem 3

1.3. Objectives 4

1.3.1. General objective 4

1.3.2. Specific objectives 4

1.4. Significance of the study 4

2. REVIEW OF RELATED LITERATURE 5

2.1. Environmental pollution 5

2.2. Effects of industrial effluent on environment 5

2.2.1. Impact on the Soil 5

v

2.2.2. Effects on plants 6

2.2.3. Effects on human health 6

2.3. Tannery industrial wastes 7

2.3.1. Physical and chemical characteristics of tannery effluent 9

2.3.2. Heavy metal effects on living things 10

2.4. Genotoxicity evaluation 11

2.5. Cytotoxicity studies 13

2.6. Chromosomal aberrations 14

2.7. The Allium cepa test 15

2.7.1. Advantages of Allium cepa test 15

2.8. Wastewater treatment technology 16

3. MATERIALS AND METHODS 17

3.1. General description of the study area 17

3.2. Study design 17

3.3. Selection criteria of sampling site and sampling technique 18

3.4. Experimental organism or plant material 18

3.5. Experimental procedures 18

3.5.1. Physicochemical test 18

3.5.2. Heavy metal analysis 19

3.5.3. Colorimetric analysis 19

3.5.4. Planting of onion 20

vi

3.5.5. Root growth inhibition test 20

3.5.6. Root tip preparation for analysis 20

4. RESULTS 23

4.1. Physicochemical parameter test 23

4.2. Root number and inhibition test 24

4.3. Cytotoxicity test 27

4.4. Genotoxicity test 29

4.4.1. Chromosomal aberrations 29

4.4.2. Nuclear abnormalities 31

5. DISCUSSION 33

6. CONCLUSION AND RECOMMONDATIONS 38

6.1. Conclusion 38

6.2. Recommendations 39

7. REFERENCES 40

APPENDIXS 48

vii

ACKNOWLEDGEMENTS

At first, I recall the gratefulness to almighty God for giving me strength to complete this study.

Secondly, I express sincere gratitude and deep appreciation to my advisor Dr. Bizuayehu

Kerisew for his willingness to provide me with adequate of necessary materials and his technical

support, guidance and give heart full comments starting from the beginning of writing proposal

formulation throughout the study period. His careful reading of the draft and give valuable

comments, criticism and constructive suggestions immensely contributed to the improvement of

the thesis work.

I would also like to extend my deepest gratitude to Dr. Minale Shewa Atlabachew for his

willingness to gives permission to use Blue Nile research center at Poly technique college and he

also give directions how to check heavy metals in the tannery effluent sample. I am thankful to

Bahir Dar University, for providing free scholarship chance and all biology department staff

members who support me directly or indirectly during my work.

I am also thankful for my close friends, my colleagues and my classmates who helped me in the

time of research work, who loves me, and support always, by giving care for me. Finally, I

provide special thanks to my family, especially my lovely brother for his support and

encouragement from starting up to the fulfillment of this research paper.

viii

LIST OF TABLES

Table 1: physicochemical characteristics of Bahir Dar tannery effluent after treatment 23

Table 2: The concentration of some chemicals that were revealed at the tannery effluent 24

ix

LIST OF FIGURES

Figure 1: Root growth (%)of Allium cepa at different concentrations of tannery effluent

on 24 hours exposure 26

Figure 2: Root growth (%) of Allium cepa at different concentrations of the tannery

effluent on 48 hours exposure 26

Figure 3: Root growth (%) of Allium cepa in different concentrations of the tannery

effluent on 72 hours exposure 27

Figure 4: Shows Mitotic index (%) of root cells of Allium cepa following treatment for

different concentrations of tannery effluent 27

Figure 5: Shows Mitotic inhibition (%) of root cells of Allium cepa following treatment

for different concentrations of tannery effluent 28

Figure 6: Allium cepa root tip cells grown as a control group (Tap water) 29

Figure 7: Allium cepa root tip cells grown in tannery effluent 31

Figure 8: Allium cepa root cells to indicate genotocicity of tannery effluent on the

morphological structure of nuclei 32

x

LIST OF ACRONOMYS

AAS Atomic Absorption Spectrophotometer

ADLI Agricultural Development Lead by Industrialization

APHA American Public Health Association

BOD Biological Oxygen Demand

CA Chromosomal Aberration

COD Chemical Oxygen Demand

CSA Central Statistical Agency

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

EC Electrical Conductivity

EEPA Ethiopian Environmental Protection Authority

EMA Ethiopian Meteorology Agency

FEPA Federal Environmental Protection Agency

IARC International Agency for Research on Cancer

IPPC Integrated Pollution Prevention and Control

MI Mitotic Index

NA Nuclear Abnormalities

SPSS Statistical Packages for Social Science

TDS Total Dissolved Solids

TSS Total Suspended Solids

WHO World Health Organization

xi

ABSTRACT

Tannery effluent contributes a significant role to increase pollution in the environment;

especially it contains toxic heavy metals which cause toxic effect on plant genetic materials.

Among tannery effluent chemicals chromium and lead have cytotoxicity and genotoxicity

potentials on Allium cepa. This investigation was undertaken to assess the physico-chemical

properties of tannery wastewater and their effect on the genetic materials of Allium cepa.

Effluents physico chemical characteristics were undertaken using digital instruments for direct

measurement and standard methods of atomic absorption spectrophotometer, colorimetric

analysis. A series of six onion bulbs were grown in 0%, 20%, 40%, 60%, 80% and 100%

concentration of wastewater (v/v) ratio and root tips from each onion bulb were cut and

processed for analysis by Aceto-orcein squash technique. Most effluent components were above

the discharge limit standards set by Federal Environmental Protection Agency and Ethiopian

Environmental Protection Authority. The cytotoxicity effect on the root growth showed a

significant reduction at high concentrations. Simple regression analysis showed that the result of

mitotic index were statistically significant (P<0.05) in different concentration. A decrease in

mitotic index with increase concentration of the effluent was observed. The effluent had induced

chromosomal abnormalities such as laggard, fragmentation, stickiness, bridge, micronucleus,

binucleated and morphologically changed nuclei in Allium cepa root cells among others. The

results showed that cytotoxicity, genotoxicity and chromosomal aberrations were induced by the

tannery effluent. Industries shall think of biological waste treatment methods.

Keywords: Aberration, Mitotic Index, Cytotoxicity, Genotoxicity, Genetic material, Allium cepa

1

1. INTRODUCTION

1.1. Background of the study

Industrialization plays a very important role for developing nations. But the removal of

wastewaters has become a universal concern as the industries are associated with the generation

of high volumes of effluents, limited space for land needed for the treatment including high cost

of treatment technologies (Arjun et al., 2013). Indiscriminate discharge of untreated or partially

treated wastewaters directly or indirectly into aquatic environment may render water resources

unwholesome and hazardous to man and other living systems (Bakare et al., 2009; Olorunfemi et

al., 2010). The effect of industrial wastewaters on aquatic and terrestrial environment has drawn

a lot of attention worldwide because of its overwhelming environmental significance

(Olorunfemi et al., 2011).

Industrial effluents and agricultural wastes can increase the contamination ability of surface

water and, consequently, water pollution has a great problem for the health of living things and

humans that interact with these aquatic ecosystems. Genotoxic compounds can contaminate

water resource. It is a worldwide problem (Ohe et al., 2003; Buschini et al., 2004).

In Ethiopia, industrial effluents containing high contents of organic matter, nitrogen and heavy

metals are discharged into inland surface waters with little or no pretreatment. Significant

pollution concerns related to these effluents include dissolved oxygen depletion, toxicity and

eutrophication of the receiving water. This has not only forced the government to formulate

regulations and standards for discharge limits, but also resulted in an increasing interest and

development of methods and systems by which wastewater can be recycled and used sustainably.

The need for technologies for environmentally friendly treatment of industrial wastes such as

tannery wastewaters is therefore obvious (Seyoum Leta, 2004).

2

Like other developing nations, Ethiopia, particularly urban centers of the country, which are the

centers of industrial expansion, is experiencing socioeconomic and environmental problems

resulting from industrial pollution. In Ethiopia, 90% of the industries are releasing their effluents

into water bodies, streams and land without any treatment mechanisms and are the primary cause

of water pollution (Samuel Melaku et al., 2004).

Industrial wastewaters may contain complex chemical mixtures including metals and organic

compounds with the potential of cytotoxic and genotoxic effects. The evaluation of hazardous

wastes and effluents by genotoxicity assays may provide data useful for hazard identification and

comparative risk assessment (Claxton et al., 1998).

Among tannery effluents, the cytotoxicity or genotoxicity observed in streams influenced by

tanning factories has generally been attributed to the heavy metal chromium (Chandra et al.,

2004; Matsumoto et al., 2006). Tannery effluent and Chromium induced various chromosomal

abnormalities in plant cells, thereby severely reducing mitotic index and root growth

(Olorunfemi et al., 2010). Therefore, the untreated wastewater discharged from tanning

industries contains high level of biological oxygen demand, chemical oxygen demand, electrical

conductivity and heavy metals, especially Chromium above permissible limits as recommended

by various regulatory agencies making it potentially toxic (Lal, 2009).

Contamination by heavy metals has been increasing every year (Majer et al., 2002) and the

analysis of the cytotoxic effects has received special attention due to the fact that they are

potentially mutagenic and induce the formation of tumors in experimental organisms and humans

exposed to them (Garcia-Rodriguez et al., 2001).

Higher plants are recognized as excellent genetic models to detect environmental mutagens and

may serve as a warning to other biological systems, since the target of the mutagens is DNA,

3

which is common to all organisms. Among the plant species, Allium cepa (2n = 16) bioassay is

frequently used for in situ environmental monitoring studies of waste, surface water and

groundwater quality assessments (Leme and Marin-Morales, 2009).

It has Allium cepa has an advantage over other short-term tests that require low cost and easily

handled toxicity test. The Allium cepa bioassay facilitates testing different toxicity like root

growth inhibition, mitotic index alterations (Fiskesjo, 1985), chromosome aberrations, nuclear

alterations and micronucleus analysis (Ma et al., 1995).

1.2. Statement of the problem

Industrial effluents have become one of the biggest problems in many developing and developed

countries. It is known that these effluents, when not treated properly, can cause mutagenic or

toxic effects directly or indirectly on humans, resulting in diseases such as cancer, congenital

malformations, and cardiovascular diseases (Grover and Kaur, 1999). Major problems are caused

by tannery wastewater containing the most toxic (heavy metals like Chromium) chemical

impurities and others (Kawser et al., 2011)

Nowadays, Ethiopia has strategies to implement Agricultural Development Lead

Industrialization (ADLI). In this respect the country needs to plan an effective management

system of effluents which are discharged from industries. Otherwise biotas and human being will

be affected with those industrial effluents which contain a complex toxic chemical.

To the best of our knowledge, regardless of the presence of ample evidence for the presence of

mutagenic components in industrial effluents characterized from Ethiopia, there is no work done

regarding to the degree of toxicity of industrial effluents on cells, genes and DNA especially

from Bahir Dar. As the city is becoming one of the industrial park zones of the country,

understanding the consequences of industrialization on life shall be done for preemptive actions.

4

1.3. Objectives

1.3.1. General objective

The main aim of this study was to investigate the physico chemical characterstics, the

cytotoxicity and genotoxicity potential of tannery effluent from Bahir Dar using Allium cepa as a

model plant.

1.3.2. Specific objectives

The specific objectives of this study were to:

assess the physical and chemical properties of tannery effluents from their discharge

point.

examine the cytotoxic effect of tannery effluent on the Allium cepa root tip cells.

investigate the genotoxicity effect of tannery effluent on the Allium cepa root tip cells.

1.4. Significance of the study

This study will give information on the effect of tannery effluent on life using onion (Allium

cepa) as a model plant to evaluate root growth and their combined effect on cells, gene and

chromosome. The study will also give awareness to people about the effect of tannery effluent on

biotic organism and a biotic environment, if it is not properly treated.

The study can help to allow the detection of chemicals with cytotoxic and genotoxic potential of

industrial effluents which are emitted to the environment and surface waters by using standard

techniques to detect DNA damage such as chromosome aberration and micronucleus formation

on higher plants.

The overall result may give insights to the consequences in environmental health and in the

biodiversity loss resulted from the establishment of industries. The study will also be important

for other researchers as baseline information for further study.

5

2. REVIEW OF RELATED LITERATURE

2.1. Environmental pollution

Pollution is a crucial threat to our environment that the increasing discharge of hazardous

chemicals into the environment has affected the balance of natural ecosystems. These

consequently attract the attention of several researchers and government agencies to the health of

living organisms (Leme and Marin Morales, 2009).

Environmental pollution constitutes a great health hazard to human, animals, and plants with

local, regional and global implication. Pollution has adverse effect on land, water and its biotic

and a biotic component (Al- Dulaimi et al., 2012). Among the damage caused by chemical

agents, exposed organisms are under genotoxic and mutagenic effect. These effects have shown

is worrying due to its capacity to induce genetic damage that can lead to several health problems

and also affect future generations, since these alterations can be inheritable (Ribeiro, 2003).

2.2. Effects of industrial effluent on environment

2.2.1. Impact on the Soil

Sometimes, effluent, especially sludge from the wastewater treatment facility are disposed of by

using them as soil amendment, or just indiscriminately to dump sites. When these effluent or

sludge (as the case may be) contains toxic materials and heavy metals, they immediately become

part of the soil; when these toxic materials and heavy metals become ionized (i.e. in soluble

form), they could be picked by the root of the plant and bioaccumulation in the tissues of the

plant (Mura et al., 2013).

These toxic materials and heavy metals may also disrupt the natural activities of both the flora

and fauna components of the soil. The activities of bacterial and other micro-organisms could be

distorted by the presence of these pollutants (Abel, 1996).

6

2.2.2. Effects on plants

Uptake and accumulation of elements by plants may follow two different paths, i.e. through the

roots and foliar surface. Thus toxic metals may be absorbed by vegetables through several

processes and finally enter the food chain. Uptake of heavy metals by crops may be done through

absorption from contaminated soils through roots or by deposition on foliar surfaces. Uptake

through roots depends on many factors such as the soluble content of heavy metals in soil, soil

pH, plant growth stages as well as type of crops, fertilizers and soil (Sharma et al., 2006).

Toxicity of chromium to plants Chromium (VI) is highly toxic to plants, and results in reduced

root growth, phytomass and photosynthetic pigments. Chromium is not biodegradable and tends

to accumulate in living organisms, causing serious diseases and disorders (Wang et al., 2007).

Chromium (III) is toxic only at high concentrations, whereas Chromium (VI) is toxic to

mammals, even at low concentrations, with a potential carcinogenic effect (Mabasa, 2007).

2.2.3. Effects on human health

Unlike organic contaminants, heavy metals and metalloids are generally non-biodegradable,

immutable and persistent in nature. Nevertheless, they can become mobile in soils, sediments

and in water to the extent that a fraction of their total mass can become bioavailable to

organisms, including plants, animals and humans (Adriano et al., 2004).

Many heavy metals and metalloids are toxic and can cause undesirable effects and severe

problems even at very low concentrations (Ali et al., 2013). Some heavy metals may transform

into the persistent metallic compounds with high toxicity, which can be bioaccumulated in the

organisms, magnified in the food chain, thus threatening human health. Toxic metal ions that

enter plant roots and above ground different organs can pose a potential threat to human health.

7

Metal accumulation in edible parts of crop plants represents the principal route of toxic metal

entry into the human food-chain (Clemens, 2006).

Chromium (Cr) toxicity is observed at multiple levels in plants and animals, from reduced yield,

through effects on leaf and root growth, to inhibition on enzymatic activities and mutagenesis. Cr

(VI) has long been recognized as a carcinogen in human and mammalian systems, and Cr (VI)-

containing compounds are genotoxic and can induce gene mutations and DNA lesions (Shanker

and Venkateswarlu, 2011). Since chromium is a known mutagen and carcinogen, the prevalent

pollution laws in most countries require its complete removal from industrial effluents before

discharge (Itankar and Patil, 2014). Chromium (VI) inhibits DNA, RNA, and protein syntheses

in biological systems (Labra et al., 2003).

In addition, Chromium is known to induce apoptosis, a process by which cell death is initiated

and completed in an orderly manner through activation or the synthesis of gene products

necessary for cell destruction (Shanker and Venkateswarlu, 2011).

The International Agency for Research on Cancer (IARC) has classified inorganic lead

compounds as probably carcinogenic to humans. It has been estimated that lead exposure was

responsible, in 2004, for 143 000 deaths and 0.6% of the global burden of disease (expressed in

disability-adjusted life years), taking into account mild mental retardation and cardiovascular

outcomes resulting from exposure to lead. Childhood lead exposure is estimated to contribute to

about 600,000 new cases of children with intellectual disabilities every year (WHO, 2014).

2.3. Tannery industrial wastes

Extra materials are produced by a variation of industries such as tanning, textile, petrochemicals

from unintentional oil spills or consumption of petroleum-based products, insecticides and

pharmaceutical industries and are extremely variable in configuration. Although certain products

8

are disposed of on soil, some have assistances for agriculture and forestry. In addition, various

are possibly dangerous due to their constituents of heavy metals like Chromium, Lead and Zinc

or poisonous organic mixtures and are sometimes, if ever, supplied to land. Some others are

actually less in plant nutrients or have no soil conditioning effects (Sumner, 2000).

Environmental impacts of tanneries originate from liquid, solid and gaseous waste streams and

from the consumption of raw materials such as rawhides, energy, chemicals and water. Only 20

% of the weight of the rawhide is processed to finished leather. The rest of the weight plus the

chemical input ends up as either waste or by-products. The main releases to wastewater originate

from wet processing in the beam house, the tan yard, and the post tanning operations. The main

releases to air are due to the dry finishing processes, although gaseous emissions may also arise

in all other parts of the tannery. The main sources of solid wastes originate from fleshing,

splitting and shaving. A further potential source of solid waste is the sludge from the effluent

treatment plant, but this is not an onsite activity in all tanneries (IPPC, 2003).

Tanning is one of the oldest industries in the world. Tannery effluent is among one of the most

hazardous pollutants of the industry. Major problems caused by tannery wastewater containing

heavy metals, nutrients, toxic chemicals, chloride, lime with high dissolved and suspended salts,

and other pollutants. With the growth of population, the increasing requirement of leather and its

products led to the establishment of large commercial tanneries. Tanneries are typically

characterized as pollution intensive industrial complexes which generate widely varying, high-

strength wastewaters. Nearly 30 m3 of wastewater is generated during processing of one tone of

raw skin/hide (Suthanthararajan et al., 2004).

Wastewater expelled from leather and tannery industries is of great concern and considered to be

one of the ten most harmful to the environment, responsible for extreme pollution of water

9

resources and generating substances, leading to deterioration and death of a wide range of living

organisms (Junior et al., 2007).

Various authors reported the toxicity of tannery wastewater against Allium cepa exposed to

tannery waste, which led to significant decrease in mitosis and root growth and attributed it to

the presence of Cr and Ni compounds in it (Chandra et al., 2004). The genotoxicity of leachates

of tannery solid waste through aqueous and soil medium has been reported in root meristematic

cells of Allium cepa. The experiment was conducted in two sets in which one set of bulbs was

placed over aqueous concentration of leachates (2.5, 5 and 10%), while in another experiment

bulb were exposed to contaminated soil. Root tips, sampled after 48 hours revealed a higher

frequency of aberrations in aqueous medium as compared to locate-contaminated soil. However,

mitotic abnormalities, CA‟s and MI inhibition were observed in both experiments (Chandra and

Gupta, 2002).

In another study, while evaluating the genotoxicity of water contaminated with leather industry

waste water, (Junior et al., 2007), reported evidence of CA‟s in Allium cepa root tip cells. The

water impacted by tannery waste water, also showed significant frequencies of CA‟s and MN in

exposed Allium cepa meristematic cells and the abnormalities were correlated with the presence

of Cr ions (Matsumoto et al., 2006).

2.3.1. Physical and chemical characteristics of tannery effluent

Effluents from tanning units were discharged indiscriminately into natural water bodies or open

lands, resulting in contamination of the surface and ground waters as well as the soil flora and

fauna (Weber-Scannell and Duffy, 2007).Tannery industries consume a considerable amount of

water in their manufacturing processes and serve as a major source of tanned and untanned solid

10

waste and liquid effluents which contain high organic load, salts and chromium, releasing about

40 – 25,000 mg/L of chromium in their effluent (Obgonna et al., 2008).

Most developing countries without any treatment directly discharge tannery effluent into

aqueous system (Farenzena et al., 2005) which is highly polluted in terms of biological oxygen

demand (BOD), chemical oxygen demand (COD), suspended solids (SS), nitrogen, conductivity,

sulphate, sulphide and chromium (Assefa Wosnie and Ayalew Wondie, 2014). Dissolved organic

contents consume a large amount of oxygen and increase BOD level which leads to anaerobic

fermentation and produces organic acids and hydrolysis of these organic acids causes the

decrease in pH values (Ahmed et al., 2011).

High TDS, BOD and COD content cause decrease in DO of the water system creating stress

conditions to the aquatic living organisms (Kambole, 2003). Degradation of dissolved organic

contents generates cations and anions and changes in the ionic composition of water which can

also exclude some species while promoting population growth of others (Weber - Scannell and

Duffy, 2007). Das et al. (2010) have also reported a higher amount of TDS, BOD, COD, EC,

salinity, alkalinity, hardness and lower amount of DO in tannery effluent.

Chromium is a potential pollutant and well known for its mutagenicity (Cheng and Dixon, 1998)

and carcinogenicity (Wang et al., 1999) effects in humans, animals and plants. Soil profile,

surface water bodies (ponds and rivers), human health, fishes and other aquatic biodiversities are

at risk of serious threat due to the extensive use of chromium in tanning industries and discharge

of wastewater (Mohanta et al., 2010).

2.3.2. Heavy metal effects on living things

Discharge of hazardous chemicals into the environment is increasing which affected the balance

of ecosystems. The heavy metals and other pollutants in water bodies and agricultural soils have

11

led to bioaccumulation of metals in crops and accumulated in different parts of crops (Nagajyoti,

2010). The higher levels of heavy metals in plants suppress the metabolism and translocation of

reserve food materials to the growing regions and their subsequent utilization. Heavy metals such

as cadmium, chromium, iron, lead and iron cause carcinogenic in human beings and

epidemiological studies on genotoxic effects of these heavy metals are reported in many

biological systems (Olorunfemi et al., 2011). Toxicity of heavy metals is a widespread global

problem. Excess heavy metals stress the plant causes oxidative damage (Leme and Marin-

Morales, 2009).

Chromium and lead are unique heavy metals occur in the environment in water bodies and

agricultural soils (Chandran et al., 2012). Chromium occurs in several oxidation states as

trivalent and hexavalent states in the environment. Plants do not have specific mechanisms for

chromium uptake and transport (Ambuj, 2012). There are some non-essential metals like lead

have unknown biological or physiological function (Subhashini and Swamy, 2013). However,

hyper accumulation of toxic heavy metal ions by plants dependent on physiological mechanisms

like higher rates of uptake, efficient translocation and deposition in tissue systems, especially in

the growing region (Lasat et al., 2005).The accumulation of heavy metals causes damages,

alterations in the genetic material occurring over a cell cycle (El-Shahaby et al.,2003).

2.4. Genotoxicity evaluation

Genotoxicity of environmental contaminants is of great concern, due to the capability of genetic

damage to cause health problems and affect future generations, since these damages may be

inheritable (Bickham et al., 2000).

The genotoxicity evaluation on the basis of nuclear abnormality endpoint was started in the

beginning of present decade and is characterized by morphological changes in the nuclei during

12

cell division. The Nuclear abnormality include bi-nucleated, multi-nucleated cell, lobulated

nuclei, nuclei carrying nuclear buds, mini cell, vacuolated nucleus, nucleus with nuclear wall

lesions and deformed nuclei. The nuclear abnormality evaluation is a sensitivity analysis to

detect the toxic effects of any mutagen (Iqbal et al., 2019).

The nuclear bud is well known genotoxic alteration and its formation is related to the initiation of

the nuclear envelope formation prior to the total migration of the chromosomes to the opposite

poles and consequently, their incorporation into the nuclei. The nuclear bud may originate as a

result of chromosome breaks, bridges and rearrangements due to the clastogenic action of agents,

which hinder the proper reorganization of the chromatin in the nucleus. The nuclear buds may

also be the result of cellular activities that promote the elimination of the amplified genetic

material (Mazzeo et al., 2011).

Some authors have recently included another endpoint in the Chromosomal aberration analysis in

Allium cepa meristematic cells. Such endpoint refers to nuclear abnormalities. Nuclear

abnormality is characterized by morphological alterations in the interphasic nuclei, as a result

from the action of the agent tested. Generally, these alterations are observed in Allium cepa test

as lobulated nuclei, nuclei carrying nuclear buds, polynuclear cells, mini cells, among others

(Fernandes et al., 2007).

Nuclear abnormality evaluation, along with Chromosomal aberration, has shown to be a sensitive

analysis, for making the investigation of test agent actions even more accurate in relation to their

effects on the DNA of exposed organisms. According to Leme et al. (2008) the presence of

lobulated nuclei and polynuclear cells can indicate a cell death process, since these abnormalities

are not observed in the control group cells of Allium cepa roots.

13

Micronucleus has been considered by many authors as the most effective and simplest endpoint

to analyze the mutagenic effect promoted by chemicals. This is due to the fact that mitotic index

result from damages, not or wrongly repaired, in the parental cells (Ribeiro, 2003), been easily

observed in daughter cells as a similar structure to the main nucleus, but in a reduced size. Thus,

Micronucleus arises from the development of some Chromosomal aberrations, for instance,

chromosome breaks and losses (Fernandes et al., 2007). Micronucleus analysis also enables an

investigation of the action mechanisms of chemical agents (Leme and Marin-Morales, 2008).

2.5. Cytotoxicity studies

The mitotic index is a very important endpoint for the evaluation of toxicity and is based on the

number of dividing cells in cell cycle and mostly researchers used it as a cytotoxicity indicator.

In normal cell division, the mitotic index (MI) must be equal to control and mitotic index lowers

than the control indicates the abnormality in cell division. The higher value of mitotic index is

also an indication of abnormal growth such as cell proliferation and un-control growth versus

negative control (Hoshina and Marin-Morales, 2009). So, the reduction as well as acceleration in

mitotic index is important indicators for the assessment of cytoxicity of contaminants (Leme and

Marin-Morales, 2009).

The inhibition of mitotic index (MI) may be attributed to the effect of environmental chemicals

on Deoxyribonucleic acid (DNA) and protein synthesis of the biological system (Chandra etal.,

2005). A decrease in mitotic index (MI) below 22% versus control causes lethal effects on test

organisms, while a decrease below 50% (cytotoxic limit value) usually has sublethal effects

(Abdel Migid et al., 2007). Therefore, the inhibition of root growth, in fact, is a measure of the

inhibition of cell division, measured as a decrease in mitotic index (Marcano et al., 2004). The

decrease in mitotic index (MI) due to the exposure of wastewater/any other physical or chemical

14

agent indicates the presence of cytotoxic agent and can be used for the estimation of pollution

level in the sample (Smaka-Kincl et al., 1996).

2.6. Chromosomal aberrations

Sensitive genetic endpoints for exposure of mutagens can range from point mutations to

chromosomal aberrations (CA) in cells of different organs and tissues. Chromosomal aberrations

are defined as any departure from the normal in chromosome structure or number (Preston and

Hoffmann, 2007). These chromosomal aberrations may be detected in both mitotic and meiotic

cell divisions (Grant, 1978).

Mutagenic chemicals may encourage many different types of chromosomal aberrations. The

chromosomal aberrations may arise directly from clastogens, mutagens that create DNA strand

breaks with subsequently survival or misrepair of the damage, or indirectly from breaks created

from unraveling, synthesis and repair processes (Bignold, 2009).

Chromosomal aberration is characterized by change in chromosome number or structure

(Fernandes et al., 2007). To evaluate the structural abnormalities due to a toxic agent, the cellular

stages like prophase, anaphase, metaphase and telophase were studied well (Rank, 2003). The

CA is caused due to the DNA breakage, inhibition of DNA synthesis and altered DNA

replication as a result of contact with physical and chemical polluting agents (Albertini et al.,

2000).

The chromosomal aberration (CA) includes chromosome adherence, loss, breakage, bridge,

irregular distribution, distortion, lagged, irregular separation, stickiness, vagrant, rings, late

separation and un-orientation. Additionally, the C-metaphase, polyploidy anaphase, multipolar

anaphase, polar slip, drifting away from the metaphase plate, disturbed telophase, bridge at

telophase, chromatin degeneration, anaphase with multiple bridges, late anaphase stage with

15

double bridge, disturbed telophase, bridge sticky metaphase, sticky prophase and cytokinesis

failure are also the types of CA‟s abnormalities (El-Shahaby et al., 2003; Abdel Migid et al.,

2007; Gupta and Ahmad, 2012).

It is reported that metaphase with sticky chromosomes loses its normal appearance and is seen

with a sticky „„surface‟‟ causing chromosome agglomeration. The presence of such type of

aberration reflects the toxic effect on chromatin, which generally leads to irreversible cell death.

Chromosomes stickiness is attributed to the formation of complexes of toxic agent with

phosphate groups in the DNA, on DNA condensation or on the formation of inter-and-intra-

chromatic crosslinks (El-Ghamery et al., 2003). Additionally, the late segregation of

chromosome, C-metaphases and multiplier anaphase suggest the effect on microtubule assembly.

The microtubules perform a central role during the growth and mitotic cycle, such as

chromosome migration, cell structure and formation of cell wall (Jordan and Wilson, 1998).

2.7. The Allium cepa test

The Allium cepa test is a plant test system used to examine both toxicity and genotoxicity. The

basic steps of the Allium cepa test are a measurement of root length, determination of the mitotic

index (the proliferation status of a cell population, MI) and observation of chromosomal

aberrations of the common onion, Allium cepa, after exposure to a test solution. The root

appearance and root length can be used as measures of toxicity (Fiskesjo, 1985).

2.7.1. Advantages of Allium cepa test

Allium cepa characteristics make it an excellent genetic model to assess environmental pollutants

and can be used for monitoring chemical and physical toxic agents. The application of Allium

cepa is not only due to the sensitivity to detect mutagens, but also to the possibility of assessing

16

several genetic endpoints ranges from point mutation to chromosome aberrations (CA) in

meristematic cells as well as F1 generation (Leme and Marin-Morales, 2009;).

The Allium cepa test is considered an excellent representative of in vivo biological test, where the

roots grow in direct contact with the substance of interest and enabling to detect the possible

damage to the DNA and results can be generalized for diverse animal and plant biodiversity as a

model. Additionally, the structural aberration and numerical chromosomal alterations can be

directly visualized. The cytogenetic tests in plants are relatively inexpensive and can easily be

handled and have shown good correlation with other bio-testing systems easy handling, low-cost

and have ideal size as well as chromosome number (2n = 16). Allium Cepa can be used for the

indication of toxic compounds mutatoxic, cytotoxic and genotoxic (Fiskesjo, 1988).

2.8. Wastewater treatment technology

Adsorption is recognized as one of the most effective purification and separation technique used

in industry, especially in water and wastewater treatment. Although the commercially available

adsorbents are efficient in the removal of heavy metals, they are costly and some cannot be

regenerated and recycled. A number of approaches have been recently studied for the

development of cheaper and more effective adsorbents for metal removal. Many non-

conventional low cost adsorbents, including natural materials, bio-Sorbents, and waste materials

have been studied and proposed by several researchers (Kilonzo et al., 2012).

Adsorption is a user-friendly technique, especially for the removal of heavy metals. This process

seems to be more versatile and effective method for removal of heavy metal (Rao et al., 2007).

The adsorption process is being widely used by various researchers for the removal of heavy

metals (Ahmed et al., 2009).

17

3. MATERIALS AND METHODS

3.1. General description of the study area

This study was conducted at Bahir Dar tannery which is situated in Bahir Dar town, the capital

city of Amhara National Regional State of Ethiopia. The town is situated at 578km North-West

of Addis Ababa, the capital city of Ethiopia. In its absolute location it is found at 11033'15'' and

11036'53'' north latitude and 37021'11'' and 37025'49'' east longitude. The town has been the

capital city of Amhara National, Regional State since 1991. Currently the town covers a total

area of 256.4 . It is a rapidly expanding town with commercial centers, small industries and

residences in all sectors of the town. The total population of Bahir Dar was 356,757 inhabitants

(CSA, 2010).

Bahir Dar lies on a very gentle slope with elevations ranging between 1783m and 1889m above

sea level. It occupies the head stream of the Blue Nile basin. The town is situated at the southern

shore of Lake Tana a freshwater lake with weak seasonal fluctuation. The town experiences a

tropical climate with annual average rainfall of 1409mm and average temperature of 21.3c0. The

area receives a maximum rainfall during the summer season (June to August) and short rainfall

in the spring season (September and October). The rainy season accounts for nearly over 96% of

the total annual rainfall (EMA, 2009).

Bahir Dar tannery is found in Kebele 07; their wastewater is directly discharged into Blue Nile

River and has a lot of problems on living things that makes direct contact with toxic chemicals

present in wastewaters.

3.2. Study design

The study was carried out by using experimental study at laboratory to examine the cytotoxicity,

genotoxicity and chromosomal aberration of tannery effluent on Allium cepa root tip. Allium

18

cepa was a plant used as an indicator or a model organism in this study to generate the primary

data.

3.3. Selection criteria of sampling site and sampling technique

The sampling site was selected based on prior knowledge about the tannery effluent containing

many toxic chemicals compared to other industries in the town. In addition to this, Bahir Dar

tannery (from where the effluent was collected) is directly discharging the effluent in to Blue

Nile River. Sample was collected using a spot sample technique where a single sample was taken

from the outlet of the effluent for analysis.

3.4. Experimental organism or plant material

Allium cepa was selected as a plant model to detect toxicity effects of tannery effluent on the

organisms. Healthy and equal sized bulb onions (Allium cepa L.: 2n=16) was purchased at Bahir

Dar Market. The onions were dried out with sun for two weeks to remove old grown roots and

the dried bulbs were later used for tests to check toxicity effect of effluent (Babatunde and

Bakare, 2006).

3.5. Experimental procedures

3.5.1. Physicochemical test

The tannery effluent was obtained in Bahir Dar city. The effluent samples were collected before

their discharge into the coastal water.

The collected tannery effluent samples were characterized for their physical and chemical

parameters like temperature, pH, conductivity, turbidity; Biological oxygen demand, phosphorus,

sulfide, total dissolved solids and total suspended solid standards were carried out (APHA,

1995). Among those temperature and pH were measured using portable digital meter (Wagtech

model 901p, china), Conductivity and Total dissolved solid were measured using standard

19

portable smart combined meter model MW 802 EC/TDS meter, Canadian and Turbidity using

turbid meter (turbid meter)HACH2100an turbid meter, Canadian. The above all parameters were

measured using digital instruments at Blue Nile research center in poly campus.

Selectively, some heavy toxic metals like Chromium, Lead, Zinc and sulfide, phosphorous

present in the tannery effluent was checked using atomic absorption spectrophotometer (AAS) at

Amhara Design and Supervision Works Enterprise Laboratory Service.

3.5.2. Heavy metal analysis

To determine heavy metals in water sample used the following steps and it was done at Amhara

design and supervision works enterprise laboratory service. According to Srikanth et al. (2013),

First 100ml water sample was added into digestion flask. Next 3ml of concentrated HNO3 acid

was added and then digested at 120 oC for 1 hour. Then cooled down the sample for 5 minutes

and again 3ml of HNO3 acid was added and digest at 180 oC for 40 minutes. After that cooled the

sample for 5 minutes and 10ml of concentrated HCl acid was added and digest at 240 oC for 20

minutes. Finally, the digested sample was filtered by whatman No.2 in 100ml volumetric flask.

After that 10ml from the digested sample was added in the test tube and then heavy metals like

Chromium, Lead, and Zinc was determined by using atomic absorption spectrophotometer

(AAS). The result was expressed in mg/l or ppm.

3.5.3. Colorimetric analysis

It was also done at Amhara design and supervision work enterprise laboratory service using the

following steps. According to Srikanth et al. (2013), First 10ml of water sample was added in to

palintest tube. Next, sulphide 1 tablet was added and crushed for dissolving and sulphide 2 tablet

was added and crushed it for full dissolving for sulphur and used phosphate in the place of

sulphide for phosphorous. Then wait 10 minutes for full color developing. Finally, Photo 33 and

20

29 were selected for reading for sulfur and phosphorous respectively, and the result was

expressed in mg/l or ppm.

3.5.4. Planting of onion

The outer scales of the bulbs and the brownish bottom plate were first removed. The rings of the

root primordial were not being damaged. A series of cleaning small sized bulbs of onions,

(Allium cepa) were first grown in water as described by Friskesjo (1988).Onion bulbs were

germinated primarily with plastic bottles and glass jar and filled with normal water for 24 hours.

After germination, the best in terms of root growth were selected for tests. Six onions were

selected; each onion was placed on the top of containers filled with different concentrations

(20%, 40%, 60%, 80% and 100%) of tannery effluent for 5 days in three replicates and tap water

was used as positive control. The solutions were changed every day during the experiment.

3.5.5. Root growth inhibition test

The root length of onion bulbs from each different concentration were measured on day 2, 3, and

4 of the experiment using a meter ruler. According to Babatunde and Anabuike (2015) report, the

root growth inhibition percentages were calculated by using the formula below.

( ) ( ) ( )

3.5.6. Root tip preparation for analysis

The emerged root tips of the onion bulbs in the different concentration of tannery effluent were

fixed in ethanol glacial acetic acid (3:1) after the second and fourth days of starting the

experiment. The conventional feulgen-squash method (Khanna and Sharma, 2013) was used to

prepare permanent slides of root meristem cells.

The root tips were put in 1 N HCL at 60˚c for five minutes to soften the tissue. The tips were

macerated and stained with Aceto- orcein stain for 10 minutes. The tip of the root was then cut

21

with a sharp blade and placed on a glass slide and covered with a cover slip carefully (Fiskesjo,

1993).

The macerated and stained root tips were covered with a cover slip and squashed and later

viewed in a microscope. The prepared slides were observed under microscope to evaluate

different stages in mitosis and mitotic index, mitotic inhibition was calculated. Each experiment

was repeated three times, and, at least, three slides were prepared for each parameter. The slides

were scored for mitotic index and chromosomal aberration in Olympus DP73 fluorescent

microscope (cell count at 40X and aberration photo taken at 100X) with an attached image

analyzer.

According to Sehgal et al. (2006) the mitotic index can be recorded using the formula:

According to Fiskesjo (1993) mitotic inhibition can be calculated as

The mitotic activity (analyzing for cytotoxicity effect), by looking the morphological change in

the nuclei during cell division (for genotocicity effect), and structural chromosomal aberration in

different concentration of tannery effluent was recorded in different stages of mitosis.

Micrographs of the above all effects were taken.

3.6. Data analysis

Statistical analyses were performed by using Microsoft office excel 2007 and SPSS version 20

software package programs. Data on physicochemical parameters were represented by mean ±SE

using tables. The mean root length of each treatment in each concentration was calculated by

22

dividing the total root length for each concentration to the replications. The root length of the

control was also calculated and the result changed into percentages and plotted on a graph.

The data of root length and mitotic index (MI) were represented in mean ± SE of three scoring in

each different concentration. The percentage of root growth, mitotic index and mitotic inhibition

were calculated and plotted using a simple bar graph and line graph. The result of root growth,

mitotic index and concentration was statistically evaluated at 5% significance level to confirm

the variability of the data and validity of results. Differences between corresponding controls and

exposure treatments were considered statistically significant at (P <0. 05).

The genotocicity and chromosomal aberrations (CA) of the result were represented by using

micrographs taken using Olympus DP73 fluorescent microscope for each phase of mitosis.

23

4. RESULTS

4.1. Physicochemical parameter test

In the present study, most physico chemical parameters listed (Table 1) were above the

discharge limit set by FEPA and EEPA of the effluent. Among the chemical analyzed in this

study chromium and lead were present in high content in the effluent 2.54mg/l and 2.13mg/l,

respectively (Table 2). These chemicals were toxic and had a negative effect to Allium cepa

genetic material.

Table 1: physicochemical characteristics of Bahir Dar tannery effluent after treatment (All

values are Mean of triplicates ± SE)

Checked parameters

for physic chemical

analysis

Present study

(Mean± SE)

Literature value(Mean±

SE), Reference (Assefa

Wosnie and Ayalew

Wondie, 2014)

Discharge limit

FEPA EEPA

Temperature (co) 23.1±0.8 25.5 ± 2.2 <40 40

pH 7.8±0.0 7.15 ± 0.0 6-9 6-9

Conductivity(μs/cm) 382.8±3.7 3953.2±150.3 2500 100

Turbidity (NTU) 18.6±1.5 - - <5

Salinity (ppt) 0.1±0.0 - - -

TSS (mg/l) 304.6±2.0 339±68.6 30 50

TDS (mg/l) 2130.8±14.8 2003.2±74.5 2000 80

BOD5 (mg/l) 252±5.8 342 ± 52.5 50 200

24

Table 2: The concentration of some chemicals that were revealed at the tannery effluent

Chemicals (mg/l) Present study

values (mg/l)

Literature

values (mg/l)

References EEPA

standard

(mg/l)

Lead (Pb) 2.13 - 2.00

Chromium (Cr) 2.54 3.54 Assefa Wosnie and

Ayalew Wondie, 2014

2.00

Zinc (Zn) 0.018 - -

Phosphorus (P) 26 11.5 Assefa Wosnie and

Ayalew Wondie, 2014

10

Sulfides (S) 65 16.05 Assefa Wosnie and

Ayalew Wondie, 2014

1.00

4.2. Root number and inhibition test

The number of roots was nearly the same at the first day of germination with tap water and later,

they were varying at different concentration because all roots may not germinate at the first day.

The numbers of root were (35±6.02, 37.6±5.84, and 38.3±5.66) in the control group at 24, 48, 72

hours respectively, on the opposite side (19.3±2.60, 20.33±9.43, and 18.6±3.17) in 100%

effluent concentration at 24, 48 and 72 hours respectively. This number indicates that high

numbers of new roots were grown in the control and less number in 100% effluent concentration.

This may be due to the negative effects of different tannery concentration on germination of

25

Allium cepa. The growing of new roots was limited by the presence of toxic chemicals in the

effluent and finally, the roots were disappeared at high concentration. It was determined that

100% concentrations of the incoming tannery wastewaters almost completely inhibited the plant

growth in Allium cepa and reduced the formation of mitotic phases. Allium cepa growth

inhibition was vertically related to the duration of time. The percentage of root growth inhibition

was increased with the increasing of duration of time. After 72hours the roots in 100%

concentration of tannery effluent were almost disappeared. These conditions show that there is a

danger for the plant life found in the area where sampling was taken.

There was a maximum root length observed in onions cultivated in tap water (control) (3.8±0.75)

in 72 hours and there were no morphological deformities found. The roots were whitish in color,

thinner, larger, unbroken and straight, while the root growth retardations were observed in all

tannery effluent induced a concentration-dependent significant (p < 0.05) root growth inhibition

in Allium cepa during the 72 hours (Figures1-3).

During the experiment, the root tips of onions cultivated in 40%, 60%, 80% and 100% turned

into greenish brown color. This was observed on day 3 and 4, and the change in root tip color

was most intense in onions cultivated in 60% - 100% concentration. The root morphology in

different concentration was nearly normal during the first day of germination and becoming

fatter, blunt and shorter after the second day, compared to the positive control. On the third day

(72 hours) of planting Allium cepa root length in the control group is 3.8±0.75 and roots grown

in 100% tannery effluent have a length of 0.1±0.00.

26

Figure 1: Root growth (%) of Allium cepa at different concentrations of tannery effluent on 24

hours exposure. The root growth of effluent concentration is significantly different from

the control as determined by simple regression test (p < 0.05).

Figure 2:Root growth (%) of Allium cepa at different concentrations of the tannery effluent on

48 hoursexposure. The root growth of tannery effluent concentration is significantly

different from the control as determined by simple regression test (p < 0.05).

27

Figure 3:Root growth (%) of Allium cepa in different concentrations of the tannery effluent on

72 hoursexposure. The root growth of tannery concentration is significantly different

from the control as determined by simple regression test (p < 0.05).

4.3. Cytotoxicity test

In this study, the highest number (613±3.46) of dividing cells was observed in the control group

and the least (165.33±25.40) in the 100% effluent concentration. The mitotic index decreased as

the concentration of the effluent increased while in figure 5, the mitotic inhibition increased with

concentration, with the 100% concentration showing 73.09% mitotic inhibition, whereas there

was a major down at the 20% concentration which only inhibited mitosis to 9.53%. There was an

inverse relationship between the mitotic index and the mitotic inhibition such that as the

concentration increased, the mitotic index decreased whereas the mitotic inhibition increased. In

the present study, the mitotic index values showed a significant reduction value with the

increasing of tannery concentrations (p<0.05).

28

Figure 4: Shows Mitotic index (%) of root cells of Allium cepa following treatment for different

concentrations of tannery effluent.

Figure 5: Shows Mitotic inhibition (%) of root cells of Allium cepa following treatment for

different concentrations of tannery effluent.

29

4.4. Genotoxicity test

4.4.1. Chromosomal aberrations

In this study, there were no chromosomal aberrations or nuclear abnormalities observed in the

stages of mitosis in the positive control which were grown in tap water. All the prophase

metaphase anaphase and telophase stages were clearly visible with normal position without any

chromosomal alterations.

Figure 6: Allium cepa root tip cells grown as a control group (Tap water): (A) Prophase with

visible chromosomes, (B) metaphase, chromosomes at the equator plate, (C) anaphase,

chromosomes move to opposite pole and (D) Telophase, chromosomes following

cytokinesis.

Unlike to the control group, the chromosomal abnormalities were observed on mitotic stages of

prophase, metaphase, anaphase and telophase of Allium cepa root cells grown with different

(A)

(C) (D)

(B)

30

concentration of the tannery effluent. When the duration of time was increased, proportional

increase to the degree of chromosomal abnormalities was also observed in this study.

(A) (B) (C)

(D) (E) (F)

(G) (H) (I)

31

Figure 7: Allium cepa root tip cells grown in tannery effluent. (A) sticky chromosomes at

prophase, chromosomes were not clearly visible, (B) polar slip, (C) sticky anaphase, (D)

sticky metaphase, (E) disturbed and fragmented anaphase, (F) irregular metaphase, (G)

chromosome mis-segregation and fragmented, (H) telophase with vagrant chromosome,

(I) single chromosomal bridge anaphase, (J) delayed anaphase, (K) lagging chromosome,

(L) irregular metaphase, (M) fragmented prophase, (N) multiple chromosomal bridges at

anaphase, (O) chromosomal breakage

4.4.2. Nuclear abnormalities

In the current study different types of nuclear abnormalities were also observed including cell

without membrane, fragmented nuclei, binucleated cells and micronucleus formation. Also, the

shape and size of cellular alterations (extended cells) were observe (Figure 8). An abnormality

of nucleus in the cell clearly indicates something becomes wrong in cell;this may be due to the

(J) (K) (L)

(M) (N) (O)

32

effects of heavy metals present in the tannery effluent. It indicated that genetic materials like

DNA were easily altered.

Figure8: Allium cepa root cells to indicate genotocicity of tannery effluent on the morphological

structure of nuclei. (A) Binucleated cell, (B) lobulated nuclei, (C) cell with membrane

damaged, (D) micronucleus, (E) and (F) morphological damaged on cell shape and size

and fragmented nucleus, (G) micro nucleus, (H) binucleated and micro nucleated.

(A) (B) (C) (D)

(E) (F) (G) (H)

33

5. DISCUSSION

In the current study, the tannery waste water showed a significant effect on the physico-chemical

parameters of fresh water and it can disturb all life forms, due to the change in Electrical

conductivity, Turbidity, Total dissolved solids, Total suspended solids, Biological oxygen

demand Chromium, Lead, sulfide, phosphorous. Except electrical conductivity, all parameters

were found to be higher than the discharge limits of FEPA and EEPA standard with deviation in

number from a report by Assefa Wosnie and Ayalew Wondie, (2014). This is due to the

production and processing capacity of the tannery industry during the sample collection time.

Similar results were revealed by Dadi et al. (2017) effluents with concentrations above the

legally permissible limits are likely to degrade and destroy local environments directly and

indirectly by affecting the physical and biological environment, such as land, water, and living

organisms and human beings.

The disposal of such type of tannery waste water is not safe to discharge into water bodies

without proper treatment because they alter the physico-chemical properties of the water and

creating high risk facing deleterious health effects even if present at low concentration (Parveen

and Singh, 2017).

Heavy metals have a negative effect on plant genetic material, among those metals; chromium is

well-known to be toxic to living organisms due to their bioaccumulation and non-biodegradable

property. In this study, a chromium concentration above the permissible limit was recorded and

similar result was reported by Assefa Wosnie and Ayalew Wondie, (2014).Similar results was

done by Najeeb et al. (2014) in China, Lead is also toxic heavy metal that disturbs various plant

physiological processes. A plant with high lead concentration suppresses the overall growth of

the plant.

34

The cytotoxicity effect of tannery effluent is evaluated from the root growth inhibition and cell

proliferation data (Babatunde and Anabuike, 2015). In this regard, our study showed that Allium

cepa root growth depended on the tannery effluent concentration. Root growth and tannery

effluent concentrations were inversely related. When the concentration of effluent increases the

growth ability of roots was reduced. This result agreed with the report of Ukaegbu and Odeigah

(2009) in Nigeria.

The root growth inhibition is manifestations of an arrest of a cell division. This indicates that

root growth inhibition is relatively due to the action of apical meristematic activity and cell

elongation in the process of differentiation (Webster and Mcleod, 1996). Similarly, root growth

inhibition occurs as a result of the inhibition of cell division (indicating cytotoxicity) and it is

thus an index for estimating general toxicity. It occurs when roots are exposed to a wrong pH, or

to unsolved substances that may prevent nutrition uptake (Fiskesjo, 1993).

One of the heavy metals recorded above the permissible limit in this study was lead. It has been

known to cause reduction in root growth and the frequency of mitotic cells in the meristematic

zone of onions. It also induces chromosome damage and its disturbance of mitotic processes in

onions (Lerda, 1992).This supports our result of clear defects of mitotic cell division phases in

Allium cepa root cells caused by tannery effluents.

Increasing or decreasing in mitotic index is an important indicator in environmental monitoring

and for the evaluation of toxic sub-stances that have cytotoxic potential (Hoshina, 2002). Mitotic

index is considered as an acceptable measure of cytotoxicity for all living organisms (Smaka-

Kinel et al., 1996) and it is considered as a parameter that allows estimating the frequency of cell

division (Leme and Marin-Morales, 2009).

35

In this study, mitotic index was decreased with increased tannery concentration; this result is in

agreement with the previous work done by Panda (2002); Kwankura (2012); Olorunfemi et al.

(2012). Mitotic index alterations can indicate changes deriving from chemical action in the

growth and development of exposed organisms (Leme and Marin-Morales, 2009).

The exposure to heavy metals preventsplant cells entering cell division phase‟s leads in a

decrease in the mitotic index. The primary action of heavy metal on the mitotic spindle promoted

spindle related chromosomal abnormalities during cell division (Singh, 2015) and all these have

been clearly observed in our results.

The reduced mitotic index compared to the control in our study suggests that the cells have an

inhibitory effect on the biosynthesis of DNA. This restricts the synthesized DNA entering to the

next stage of cell cycle, which is a second gap stage. It is after this stage that cell can enter

mitosis and this is supported by a report of previous study (Ping et al., 2012). Therefore, the

decrease of the mitotic index with the increase in the concentration of the effluent indicates that

high concentration of tannery effluents inhibits cell proliferation.

In this study, the chromosomal abnormalities were observed on different mitotic stages. This is

in agreement with observations in earlier related study by Olorunfemi et al. (2015).

Chromosomal bridges and stickiness were the most frequent abnormalities detected in the

tannery effluent grown root tips of Allium cepa cells. But, chromosomal bridges and stickiness

were commonly observed in anaphase and metaphase stages respectively. The metaphase

stickiness in this study corresponds to a research done by Peter Firbas and Tomaz Amon (2014).

Previous studies have tried to relate the causes for these chromosomal aberrations. The trace

elements recorded as above the permissible limit in this study have been mentioned repeatedly as

potential mutagens supporting our result. Lerda (1992) and Walker et al. (2012) pointed out that

36

the most frequent chromosome aberration for chromium is stickiness and a chromosome break,

for lead chromosome break, chromosome loss and lobulated nuclei and zinc was related with

stickiness and multipolarity. It is reported that aberration such as breaks and fragments are

induced due to formation of DNA–DNA and DNA–protein cross-links and heavy metals are

considered as a major contributor in this regard. Tannery effluent contains several heavy metals,

especially Chromium that is cytotoxic and inhibits cell division in root tips of plants (Chandra

etal., 2005).

Most aberrations in the current study were sticky at metaphase and the chromosomes were

condensed at the equatorial plate. A Similar report was given by Soodan et al. (2014);

Olorunfemi et al. (2015); Ogunyemi et al. (2017).

In this study, the opposite poles of chromatids were tightly linked to each other during anaphase

forming chromosome bridges resulted from adherence and may persist until telophase. This was

also observed in anaphase stages reported by Soodan et al. (2014); Olorunfemi et al. (2015);

Ogunyemi et al. (2017).

Delayed anaphase was also seen as a result of the mutagenic effect of the tannery effluent. In this

study the two anaphasic chromosomal groups were close to each other near the equatorial plate.

They were not fully separated and move towards opposite poles. This aberration was also similar

with the result reported by Soodan et al. (2014). The lagging chromosomes are resulted due to

failure of the chromosomes to get attached to the spindle fiber and to move to either of the two

poles similar result was observed by Nefic et al. (2013); Ogunyemi et al. (2017).

Another result observed in this study was the nuclear abnormalities which are used to predict

genotoxicity and are characterized by morphological alterations in the interphase nuclei. It has

been reported that along with chromosome aberration analysis, nuclear abnormalities evaluation

37

has shown to be a sensitive analysis for making the investigation of test agent actions even more

accurate in relation to their effects on the DNA of exposed organisms (Leme and Marin-Morales,

2009).The micronucleus test has been considered as the most effective and simplest endpoint to

analyze the mutagenic effect promoted by chemicals (Ma et al., 1995). Similar to the current

study result, the micronucleus was reported as composed either of small chromatin fragments,

which arise as a result of chromosomal breakage or the whole chromosomes that do not migrate

during anaphase as a result of spindle dysfunction (Leme and Marin-Morales, 2009).These all

alterations in the morphology of nuclei in the cell indicated that the tannery effluent has a toxic

effect on the gene of the model plant (Allium cepa) and this can be used as a genotocicity

measurement. These nuclei change in the micro nucleated cells were observed in studies reported

by Nefic et al. (2013); Ogunyemi et al. (2017).The presence of micro nucleated cells indicates an

aneugenic and clastogenic activity, because according to Fenech (2002) micronucleus can be

derived from acentric fragments, which indicates clastogenic activity, or from entire

chromosomes, which indicates an aneugenic activity.

38

6. CONCLUSION AND RECOMMONDATIONS

6.1. Conclusion

Results obtained from this study showed that cytotoxicity, genotoxicity and chromosomal

aberrations were induced by the tannery effluent. These results were due to the presence of most

of the physicochemical parameters of the effluent above their discharge limit set by FEPA and

EEPA. The increase in the physicochemical properties of the tannery wastewater clearly

indicates that the effluent is a highly contaminated and toxic substance which has an adverse

effect on life and the environment. This study used the Allium cepa bioassay with different

toxicity endpoints (root growth, mitotic index, and occurrence of micronuclei, nuclear

abnormalities and chromosomal aberrations) to assess the potential of toxicity of tannery

wastewater and the bioassay revealed that the tannery effluent consistently contained Cyto-

genotoxic contaminations over the study period. The results of this study indicated that the

cytotoxic effect of Allium cepa depends on tannery effluent concentration, even the low doses

demonstrating a considerable rate of inhibition in root growth rate. Moreover, the destructive

effects of tannery effluent on the mitotic index of Allium cepa cells show that, it has a cytotoxic

effect on root tip cells. The genotocicity and structural chromosomal aberrations were also

observed in Allium cepa root cells, which were grown by tannery effluent. Aberrations can be

originated due to the development of the isolated chromosome that results from an unequal

distribution of genetic material due to the effect of the effluent components. However, their

induction is commonly used to detect genetic damages derived from exposure to mutagenic

chemicals. The abnormalities may be due to the presence of heavy metals in the effluent and it

has toxic effects on the genetic materials of living organism. It is thus concluded that the tannery

effluent used in this study has mutagenic components toxic to cells (life). In addition, the model

39

plant assay used in the current study (Allium cepa) test is an easy, fast and very sensitive assay to

detect environmental genotoxicity of chemicals. This assay is related to study the effect of

chemicals at the genetic level.

6.2. Recommendations

Based on the results obtained in this study, the following recommendations are forwarded:

According to this study, there is high potential toxicity risks of tannery wastewater

discharged directly from the Bahir Dar industry. Thus, the industry shall immediately

establish a waste treatment plant and the careless disposal of industrial wastes without

pretreatment should be discouraged.

The government body responsible for regulating the industries related to wastes they

release to the environment shall put the law into effect and shall have a regular

assessment of waste management by the industries.

The regulatory bodies shall create awareness of the effects of industrial effluents for the

general public as the toxicity of these effluents is in alarming condition.

Industries shall think of biological waste treatment methods such as plants which absorb

heavy metals from effluents until they establish the expensive waste treatment plants.

40

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48

8. APPENDIXS

A. Some representative onion images that were directly taken from lab

a. Root growth b. Root growth with tannery concentrations

B. Photos of chromosomal and nuclear aberrations taken from microscope

.

A. vagrant telophase b. bridge anaphase c. sticky chromosome d. binuculated cell

e. bridge f. sticky prophase g. bridge h. bridge

i. Chromosome break j. irregular metaphase k. bridge l. delayed anaphase