A STUDY ON THE

IMPACT OF PESTICIDES AND HERBICIDES

ON THE GROWTH AND REPRODUCTION OF
EARTHWORM IN KOTA REGION

A Thesis submitted by

ANAMIKA KHANDELWAL

for the award of

Ph.D. Degree
in the Faculty of Science

to the

UNIVERSITY OF KOTA, KOTA

Supervised by:

Dr. Anuradha Singh

Department of Zoology
Government College, Kota

2017
 UNIVERSITY OF KOTA, KOTA-324005
ALL RIGHTS RESERVED
 CANDIDATE’S DECLARATION

I hereby declare that the

1. Thesis entitled “A Study on the impact of Pesticides and Herbicides on

the Growth and Reproduction of Earthworm in Kota Region”
submitted by me is an original piece of research work, carried out by me
under the supervision of Dr. Anuradha Singh.

2. Matter presented in this thesis has not been submitted by me for the award
of any other degree of this or any other University.

Date:

Anamika Khandelwal
 SUPERVISOR’S CERTIFICATE

It is certified that the

1. Thesis entitled “A Study on the impact of Pesticides and Herbicides on

the Growth and Reproduction of Earthworm in Kota Region”
submitted by Anamika Khandelwal is an original piece of research work
carried out by the candidate under my supervision.

2. Literary presentation is satisfactory and the thesis is a form suitable for

publication.

3. Work evinces the capacity of the candidate for critical examination and
independent judgment.

4. Candidate has put in at least 200 days of attendance every year.

Date:

Dr. Anuradha Singh

Government College, Kota
Acknowledgements

I feel overwhelmed at this time of submission of my thesis. It was a long journey.

During studying for M.Sc. at Rohilkhand University Bareilly in 1994, I aspired to

pursue doctoral degree. On account of one or the other reason, I could not do so for
the next few years. After my marriage in Jaipur and settling at Kota due to my hus-

band’s job in RTU Kota, I started thinking over it again. In the meantime, I passed

SLET exam conducted by RPSC Ajmer, which made me eligible for Lecturership

in College without having Ph.D. degree. This again extended my dream of pursuing

research. Finally, I joined Ph.D. in July 2013. This became possible due to moti-
vation by my mother, Bina Khandelwal and my husband, Dr. Ajay Khunteta. Their

constant encouragement and undulating support has made my dream come true. Of

course, the unstinting support and co-operation of my daughter, Arunima was very

encouraging for me.

I would like to thank my supervisor Dr. Anuradha Singh, Sr. Lecturer in Zo-

ology Department, Government College Kota. Her constant encouragement and

keenness towards research made it possible for me. In addition to this, I am thank-
ful to Mr. Irshad (Head, Zoology deptt.), Dr. Prahlad Dube, Smt. Sandhya Singh,
Dr. Neeta Jain and all other faculty mambers for their critical and important sug-
gestions from time to time which not only guided me through the research work
but also helped me to enrich my research output. I also thank to supporting staff of
Zoology Department for their help during this period.
At this moment, I would like to give special thanks to Dr. C. A. M. Van Gestel

i
(Faculty of Earth and Life Sciences, Vrije Universiteit, Amsterdam, Netherlands)

and Dr. Juan Carlos Sanchez Hernandez (University of Castilla-La Mancha, Spain
) for their quick response via Email, whenever I contacted them regarding research
guidance. I would also like to express my sincere thanks to Dr. C. K. Mandal and Dr.
Ch. Satyanarayana of Zoological Survey of India, Kolkata (India) for species iden-
tification and Nanta agricultural farm, Kota (Office of Project Director, Crop, Govt.
of Raj.) for helping me in soil parameter measurement. Dr. B. S. Meena, Assis-

tant Professor (Agronomy), Agricultural University, Kota provided valuable inputs

regarding pesticides use in Kota region and Krishi Vigyan Kendra, Borkhera, Kota
provided earthworms Eisenia fetida (Lumbricidae) for experimentation. Thanks to

their valuable support and guidance.

With tearful eyes, I also remember my father Shrikant Khandelwal, father-in-

law Sh. S.N. Khunteta and mother-in-law Smt. Kamla Khunteta. They are no more

with me to share this moment of pride. Motivation of my brother Anurag and sister
Shalini was also very much valuable to me. All my family members provided me

much needed moral and unstinting support during this period.

Finally, I pray God to give me wisdom and strength to return some tiny useful

things to the society out of my research work.

Anamika Khandelwal

ii
List of Publications

Journals

1. Anamika Khandelwal Khunteta and Anuradha Singh. Avoidance behav-

ior of Eisenia fetida to Chlorpyrifos and 2, 4-D Ethyl ester in natural soil

from Kota (Rajasthan) in India. International Journal of Global Science

Research, vol. 3, issue 6, pp. 399–405, Oct. 2016, ISSN: 2348 − 8344
(http://www.ijgsr.com/web/admin/uploads/f2016051533p1.pdf).

2. Anamika Khandelwal Khunteta and Anuradha Singh. Study of Diversity of

Earthworm Species in Kota, (Rajasthan) India. Bioglobia, vol. 3 (2), pp. 25–

28, Dec. 2016, ISSN: 2349 − 5626 (http://www.bioglobia.in/Download.html).

3. Anamika Khandelwal Khunteta and Anuradha Singh. Individual and Com-

bined Effects of Triazophos and Pendimethalin, Chemicals used in Soybean

Crop, on the Growth and Reproduction of Eisenia fetida in Kota (India).
(Communicated to Elsevier’s Geoderma Regional).

Conference

1. Anamika Khandelwal Khunteta and Anuradha Singh. Effect of Triazophos

and Pendimethalin on Avoidance behavior of Eisenia fetida in Natural Soil
of Kota (Rajasthan). Proc. of ESW IV annual national conference on Impact
of Global Warming on Environment, Biodiversity and Ecotourism, Khajuraho,
India, 30-31 Jan. 2017 (http://godavariacademy.com/godavari/images/su.pdf).

iii
List of Abbreviations

AERC : Agro-Economic Research Centre

a.i. : Active ingredient

ARS : Agriculture Research Station

ERA : Environmental Risk Assessment

FICCI : Federation of Indian Chambers of Commerce and Industry

GDP : Gross Domestic Product

ha : Hectares (area)

ICAR : Indian Council of Agricultural Research

IFOAM : International Federation for Organic Agriculture Movements

MSL : Mean Sea Level

MT : Metric Tonnes
NPOF : National Project on Promotion of Organic Farming

NPOP : National Programme on Organic Production

NR : Net avoidance response (%)
NSDP : Net State Domestic Product
NSOP : National Standards for Organic Production
RAD : Recommended Agricultural Dose
TAS : Tropical Artificial Soil
Tha : Thousand Hectares
TP : Transformation Products

iv
Contents

Acknowledgements i

List of Publications iii

List of Abbreviations iv

List of Figures viii

List of Tables xi

1 Introduction 1

1.1 Indian agriculture at a glance . . . . . . . . . . . . . . . . . . . . . 1

1.2 Overview of Rajasthan state . . . . . . . . . . . . . . . . . . . . . 3

1.3 Agriculture scenario in Rajasthan . . . . . . . . . . . . . . . . . . . 4
1.4 Agro-climatic zones of Rajasthan . . . . . . . . . . . . . . . . . . . 5

1.5 Soil in Rajasthan . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.6 Kota region: An overview on agricultural perspective . . . . . . . . 10
1.7 Pesticides use and trends in India . . . . . . . . . . . . . . . . . . . 11
1.8 Earthworm: Farmer’s friend . . . . . . . . . . . . . . . . . . . . . 15
1.8.1 Ecological strategies of earthworms . . . . . . . . . . . . . 16
1.8.2 Factors affecting distribution of earthworms . . . . . . . . . 16
1.8.3 Importance of earthworms . . . . . . . . . . . . . . . . . . 17
1.9 Organic farming . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

v
 CONTENTS

2 Review of Literature 22
2.1 Study of avoidance behaviour in earthworms . . . . . . . . . . . . . 23

2.2 Study on the impact of chemicals on the growth and reproduction

of different earthworm species . . . . . . . . . . . . . . . . . . . . 27

2.3 Study on the impact of chemicals on the growth and reproduction

of earthworm Eisenia fetida together with other species . . . . . . . 38
2.4 Study on the impact of chemicals on the growth and reproduction
of earthworm Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . 42

3 Materials and Methods 56

3.1 Test soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

3.2 Test organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

3.3 Test chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

3.4 Identification of earthworm species in Kota . . . . . . . . . . . . . 58

3.5 Study of avoidance behaviour of Eisenia fetida . . . . . . . . . . . 61

3.6 Study on the impact of chemicals on the growth and reproduction

of Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

3.6.1 Earthworm growth and reproduction test . . . . . . . . . . . 67

4 Results 74
4.1 Identification of earthworm species in Kota . . . . . . . . . . . . . 74
4.2 Avoidance behaviour of Eisenia fetida . . . . . . . . . . . . . . . . 78
4.3 Impact of chemicals on the growth and reproduction of Eisenia fetida 84
4.3.1 Effect of Chlorpyrifos and 2,4-DE on growth and reproduc-
tion of Eisenia fetida . . . . . . . . . . . . . . . . . . . . . 84
4.3.2 Statistical analysis of effect of Chlorpyrifos and 2,4-DE . . 100
4.3.3 Effect of Triazophos and Pendimethalin on growth and re-
production of Eisenia fetida . . . . . . . . . . . . . . . . . 104
4.3.4 Statistical analysis of effect of Triazophos and Pendimethalin 120

vi
 CONTENTS

5 Discussion 124
5.1 Identification of earthworm species in Kota . . . . . . . . . . . . . 124

5.2 Avoidance behaviour of Eisenia fetida . . . . . . . . . . . . . . . . 125

5.3 Impact of chemicals on the growth and reproduction of Eisenia fetida127

6 Conclusion 133

Bibliography 136

Appendix 156

vii
List of Figures

1.1 Different agro-climatic zones of Rajasthan . . . . . . . . . . . . . . 7

1.2 Market share of different pesticides in India . . . . . . . . . . . . . 13

3.1 Kota region map showing search areas of earthworm . . . . . . . . 59

3.2 Digging of pit to search earthworms in Kota region . . . . . . . . . 60

3.3 Creating partition for avoidance test in earthworm . . . . . . . . . . 63

3.4 Top view of beaker after elimination of partition . . . . . . . . . . . 63

3.5 Placing of earthworm Eisenia fetida in the gap . . . . . . . . . . . . 64

3.6 Sun drying of raw cow dung . . . . . . . . . . . . . . . . . . . . . 70

3.7 Preparing manure in stages . . . . . . . . . . . . . . . . . . . . . . 70

3.8 Final form of manure for use . . . . . . . . . . . . . . . . . . . . . 71

3.9 Weighing of earthworms by electronic machine . . . . . . . . . . . 71
3.10 Counting of juveniles . . . . . . . . . . . . . . . . . . . . . . . . . 72

3.11 Juveniles after 56 days of exposure to chemicals (view 1) . . . . . . 72

3.12 Juveniles after 56 days of exposure to chemicals (view 2) . . . . . . 73

4.1 Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

4.2 Lampito mauritii . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
4.3 Perionyx excavatus . . . . . . . . . . . . . . . . . . . . . . . . . . 77
4.4 Avoidance behaviour of E. fetida in Chlorpyrifos with varying con-
centrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

viii
 LIST OF FIGURES

4.5 Avoidance behaviour of E. fetida in 2,4-DE with varying concen-

trations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

4.6 Avoidance behaviour of E. fetida in Triazophos with varying con-

centrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

4.7 Avoidance behaviour of E. fetida in Pendimethalin with varying

concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.8 Growth rate of E. fetida for different concentrations of Chlorpyrifos 90
4.9 Growth rate of E. fetida for different concentrations of 2,4-DE . . . 90

4.10 Growth rate of E. fetida for different combined concentration labels

of Chlorpyrifos and 2,4-DE . . . . . . . . . . . . . . . . . . . . . . 92
4.11 No. of juveniles produced per earthworm (E. fetida) for different

concentrations of Chlorpyrifos . . . . . . . . . . . . . . . . . . . . 92

4.12 No. of juveniles produced per earthworm (E. fetida) for different

concentrations of 2,4-DE . . . . . . . . . . . . . . . . . . . . . . . 95
4.13 No. of juveniles produced per earthworm (E. fetida) for different

combined concentration labels of Chlorpyrifos and 2,4-DE . . . . . 95

4.14 Earthworm swelling and shrinking due to the effect of Chlorpyrifos 96

4.15 Earthworm swelling due to the effect of 2,4-DE . . . . . . . . . . . 96

4.16 Earthworm cocoons . . . . . . . . . . . . . . . . . . . . . . . . . . 97

4.17 Earthworm cocoons showing emergence of juvenile (view 1) . . . . 97

4.18 Earthworm juveniles (view 1) . . . . . . . . . . . . . . . . . . . . . 98

4.19 Earthworm juveniles (view 2) . . . . . . . . . . . . . . . . . . . . . 99
4.20 Correlation between growth rate and no. of juveniles produced per
earthworm for different concentrations of Chlorpyrifos . . . . . . . 102
4.21 Correlation between growth rate and no. of juveniles produced per
earthworm for different concentrations of 2,4-DE . . . . . . . . . . 102

ix
 LIST OF FIGURES

4.22 Correlation between growth rate and no. of juveniles produced per
earthworm for different combined concentration labels of Chlor-

pyrifos and 2,4-DE . . . . . . . . . . . . . . . . . . . . . . . . . . 103

4.23 Growth rate of E. fetida for different concentrations of Triazophos . 110

4.24 Growth rate of E. fetida for different concentrations of Pendimethalin 110

4.25 Growth rate of E. fetida for different combined concentration labels
of Triazophos and Pendimethalin . . . . . . . . . . . . . . . . . . . 112
4.26 No. of juveniles produced per earthworm (E. fetida) for different

concentrations of Triazophos . . . . . . . . . . . . . . . . . . . . . 112

4.27 No. of juveniles produced per earthworm (E. fetida) for different
concentrations of Pendimethalin . . . . . . . . . . . . . . . . . . . 115

4.28 No. of juveniles produced per earthworm (E. fetida) for different

combined concentration labels of Triazophos and Pendimethalin . . 115

4.29 Earthworm swelling due to the effect of Triazophos . . . . . . . . . 116

4.30 Earthworm showing lesions due to the effect of Pendimethalin . . . 116

4.31 Earthworm cocoons showing emergence of juvenile (view 2) . . . . 117

4.32 Earthworm juveniles (view 3) . . . . . . . . . . . . . . . . . . . . . 118

4.33 Earthworm juveniles (view 4) . . . . . . . . . . . . . . . . . . . . . 119

4.34 Correlation between growth rate and no. of juveniles produced per
earthworm for different concentrations of Triazophos . . . . . . . . 122

4.35 Correlation between growth rate and no. of juveniles produced per
earthworm for different concentrations of Pendimethalin . . . . . . 122
4.36 Correlation between growth rate and no. of juveniles produced per
earthworm for different combined concentration labels of Triazophs
and Pendimethalin . . . . . . . . . . . . . . . . . . . . . . . . . . 123

x
List of Tables

1.1 Details of different agro-climatic zones in Rajasthan . . . . . . . . . 8

3.1 Combined concentration values of Chiorpyrifos and 2,4-DE

alongwith their labels . . . . . . . . . . . . . . . . . . . . . . . . . 69

3.2 Combined concentration values of Triazophos and Pendimethalin

alongwith their labels . . . . . . . . . . . . . . . . . . . . . . . . . 69

4.1 Avoidance (%) of Eisenia fetida for different concentrations of

Chlorpyrifos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.2 Mean and standard deviation (SD) of avoidance of Eisenia fetida

for different concentrations of Chlorpyrifos . . . . . . . . . . . . . 80

4.3 Avoidance (%) of Eisenia fetida for different concentrations of

2,4-DE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.4 Mean and standard deviation (SD) of avoidance of Eisenia fetida
for different concentrations of 2,4-DE . . . . . . . . . . . . . . . . 80
4.5 Avoidance (%) of Eisenia fetida for different concentrations of
Triazophos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.6 Mean and standard deviation (SD) of avoidance of Eisenia fetida
for different concentrations of Triazophos . . . . . . . . . . . . . . 82
4.7 Avoidance (%) of Eisenia fetida for different concentrations of
Pendimethalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

xi
 LIST OF TABLES

4.8 Mean and standard deviation (SD) of avoidance of Eisenia fetida

for different concentrations of Pendimethalin . . . . . . . . . . . . 82

4.9 Effect of different concentrations of Chlorpyrifos on biomass

growth of Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . . . 87

4.10 Effect of different concentrations of 2,4-DE on biomass growth of

Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4.11 Effect of different combined concentrations of Chlorpyrifos and
2,4-DE on biomass growth of Eisenia fetida . . . . . . . . . . . . . 89

4.12 Effect of different concentrations of Chlorpyrifos on reproduction

of Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4.13 Effect of different concentrations of 2,4-DE on reproduction of

Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

4.14 Effect of different combined concentrations of Chlorpyrifos and

2,4-DE on reproduction of Eisenia fetida . . . . . . . . . . . . . . . 94

4.15 Statistical analysis of biomass growth in Eisenia fetida due to

Chiorpyrifos and 2,4-DE . . . . . . . . . . . . . . . . . . . . . . . 101

4.16 Statistical analysis of reproduction in Eisenia fetida due to

Chlorpyrifos and 2,4-DE . . . . . . . . . . . . . . . . . . . . . . . 101

4.17 Effect of different concentrations of Triazophos on biomass growth

of Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

4.18 Effect of different concentrations of Pendimethalin on biomass

growth of Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . . . 108
4.19 Effect of different combined concentrations of Triazophos and
Pendimethalin on biomass growth of Eisenia fetida . . . . . . . . . 109
4.20 Effect of different concentrations of Triazophos on reproduction of
Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
4.21 Effect of different concentrations of Pendimethalin on reproduction
of Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

xii
 LIST OF TABLES

4.22 Effect of different combined concentrations of Triazophos and

Pendimethalin on reproduction of Eisenia fetida . . . . . . . . . . . 114

4.23 Statistical analysis of biomass growth in Eisenia fetida due to

Triazophos and Pendimethalin . . . . . . . . . . . . . . . . . . . . 121

4.24 Statistical analysis of reproduction in Eisenia fetida due to

Triazophos and Pendimethalin . . . . . . . . . . . . . . . . . . . . 121

xiii
Chapter 1

Introduction

1.1 Indian agriculture at a glance

India traditionally has an agricultural based economy and it is considered to be

the backbone of Indian economy. Agricultural sector employs approximately 55%

of the total workforce. The total share of agriculture and allied sectors (including

agriculture, livestock, forestry and fishery sub sectors) in terms of percentage of

gross domestic product (GDP) is 16.96% and 10% of export earnings. For the 12th

five year plan (2012-17), a growth target of 4% has been set for the agriculture
sector. Total food grain production in the year 2015-16 was 252 million tonnes.
As of today, India supports 18% of world’s population on 4.2% of world’s water
resources and 2.3% of global land. India’s per capita availability of resources is
about 4 to 6 times less as compared to world average. This will decrease further
due to increasing demographic pressure and consequent diversion of the land for
non-agricultural uses. The present cropping intensity of 136% has registered an
increase of only 25% since independence. Further, rain fed dry lands constitute
65% of the total net sown area. India’s arable land area of 159.7 million hectares
(394.6 million acres) is the second largest in the world, after the United States. Its
gross irrigated crop area of 82.6 million hectares (215.6 million acres) is the largest
 Chapter 1. Introduction

in the world (Agricultural Situation in India, 2015). India is among the top three
global producers of many crops, which includes wheat, rice, pulses, cotton, peanuts,

fruits and vegetables. In addition to growth in total output, agriculture in India has
shown an increase in average agricultural output per hectare in last 60 years. India’s

recent accomplishments in crop yields while being impressive, are still just 30% to
60% of the best crop yields achievable in the farms of developed as well as other
developing countries.
Apart from this, we also face many challenges as a second largest populated

country in the world. There is an unprecedented degradation of land, ground wa-

ter resource, and also fall in the rate of growth of total productivity factor. This
deceleration needs to be arrested and agricultural productivity has to be doubled to

meet growing demands of the population by 2050. The country recorded impres-

sive achievements in agriculture since the onset of green revolution in late sixties.

This enabled the country to overcome starvation, achieve self-sufficiency in food,

reduce poverty and bring economic transformation in millions of rural families. The

situation, however, started turning adverse for the sector around mid-nineties, with

slowdown in growth rate of output. Natural resource base of agriculture, like pure

water and fertile land, is shrinking and degrading, and is adversely affecting pro-

duction capacity. However, demand for agriculture is rising rapidly with increase in
population and per capita income and growing demand from industry sector. There

is, thus, an urgent need to identify severity of problem confronting agriculture sector
to restore its vitality and put it back on higher growth trajectory.
In addition to this, challenges associated with agriculture also includes the use
of fertilizers and pesticides. It is true that these chemicals enhance the growth but
the quest to achieve more has degraded our agriculture land quality severely and has
also created numerous health hazards. Organic farming provides a ray of hope in
this direction. Organic farming has fed India for centuries and it is a growing sector
in India due to public awareness regarding hazards of chemicals on human health.

2
 Chapter 1. Introduction

Organic production offers clean and green production methods without the use of
synthetic fertilizers and pesticides and it achieves a premium price in the market

place.

1.2 Overview of Rajasthan state

Rajasthan is the largest state of India constituting 10.4% of total geographical area
and 5.67% (6.86 crores) of total population of India (Census, 2011). State’s ru-
ral population is 75% of the total population residing in 7 divisions of its 33 dis-
tricts, which are further subdivided into 244 tehsils, 249 panchayat sammitees and

9,168 gram panchayats. Physiographically, the state can be divided into four ma-
jor regions, namely (i) the western desert with barren hills, rocky plains and sandy

plains; (ii) the Aravalli hills running south-west to north-east starting from Gujarat

and ending in Delhi; (iii) the eastern plains with rich alluvial soils; and (iv) the

south-eastern plateau. Mahi, Chambal and Banas are the three major rivers of the

state. Rajasthan is endowed with diverse soil and weather conditions comprising
of several agro-climatic zones that help the state to adopt a diversified cropping

pattern. The state is India’s largest producer of mustard, pearl millet (bajra), three

spices (coriander, cumin, and fenugreek), cluster beans, and isabgol. It is the second
largest producer of maize. The state has a substantial area under vegetable crops.
The state has a diverse weather conditions, warm and humid in south-eastern parts
to dry and cool in western parts of the state. About 65% population of the state is
dependent on agriculture and allied activities for their livelihood. The three major
canal irrigations, other than the vast area under arid and dry lands, offer great help
for agricultural development of the state. Agriculture in Rajasthan is primarily rain
fed, covering country’s 13.27% of available land. The diversity in climatic condi-
tions of the state creates potentiality to develop certain belts of horticultural crops.
Agriculture and allied sector plays an important role in state’s economy. Though

3
 Chapter 1. Introduction

its contribution in net state domestic product (NSDP) has fallen from about 35%
in 1990-91 to around 20% in 2015-16, agriculture still forms the backbone of the

state economy. Around two third of its population is still dependent on agricultural
activities for their livelihood. Thus, a higher priority to agriculture will achieve the

goals of reducing poverty and malnutrition as well as of inclusive growth. Though

agriculture forms the source of livelihood of the majority in the state, it is largely
dependent on rainfall. Only 34.5% of the net sown area is irrigated. Since the rain-
fall amount is very scanty and highly erratic, the expansion of irrigation provisions

and efficient water management are major challenging tasks for the policy makers.
The structural changes in Rajasthan agriculture have been in favour of growing of
oil seeds, pulses and horticultural crops.

1.3 Agriculture scenario in Rajasthan

Agriculture in Rajasthan is primarily rain-fed. As is well known, productivity of

crops in the state of Rajasthan varies greatly and largely depends on the behaviour
of the rainfall. A well-developed agriculture extension network has been created in

the state. The prospect of agriculture in the state largely depend on timely arrival of
monsoon. The major crops grown in different parts of Rajasthan are bajra, wheat,

jowar, maize, cotton, rapeseed and mustard, groundnut and horticultural crops. As

per the cropping pattern in the state, the crop groups such as total cereals (42%),
oil seeds (21%), pulses (18%) and fodder crops (15%) are major agriculture pro-
duce. Among the cereals, bajra (50.5%), wheat (27.9%), maize (10.5%) and jowar
(6.7%) are the major crops; while rapeseed and mustard (45.4%), taramira (21.7%),
soybean (14.0%), sesamum (10.0%) and groundnut (6.3%) are the major oil seeds
grown in the state. Among total pulses gram (37.5%), moth (33.5%) and moong
(22.1%) are the major crops. Agricultural pattern may broadly be subdivided be-
tween Rabi and Kharif seasons. Major Rabi produce includes rabi pulses, wheat,

4
 Chapter 1. Introduction

barley, gram, mustard, rapeseed and taramira; whereas Kharif crop mainly com-
prises kharif pulses, bajra, jowar, maize, sesamum, soybean and groundnut (AERC

Report, 2015). The normal rain fall of the state is 575.10 mm and the total water
requirement of major Kharif crops like bajra, maize and jowar is around 450 to 550

mm. It is not only the total amount of rainfall but its distribution which is also im-
portant for normal crop production. Sustainability in crop production depends very
much on productivity of crops along with the area sown for total production level.
Agriculture sector in Rajasthan has some major challenges such as network of

irrigation facilities, efficient water management due to scracity of water, low pro-
ductivity due to vastly different nature of soil strata. All these become more difficult
to attain due to its large geographic area which is also facing challenges associ-

ated with adverse climatic condition. Agro forestry programmes for the greening

of desert area, agricultural innovations, proper institutional mechanisms and other

modern reforms may be used to bring farm prosperity in the state.

1.4 Agro-climatic zones of Rajasthan

The entire country has been delineated into 126 agro-climatic zones by the Indian

Council of Agricultural Research (ICAR), out of which the Rajasthan state has been
divided into 10 agro-climatic zones as shown in Fig. 1.1 and Table 1.1. These
zones have been classified on the basis of agro-climatic parameters like rainfall,
temperature regime, topography, soil characteristics, cropping pattern and irrigation
availability.
The climate of Rajasthan state has varied contrasts and the presence of Aravallis
is the greatest influencing factor. The Aravallis play a significant role here, as on the
west of Aravallis the climate is arid having low rainfall, low humidity and extremes
of diurnal and annual temperature. To the east of Aravallis the climate is semi-arid
to sub-humid having lesser extremes of temperature, higher humidity and rainfall.

5
 Chapter 1. Introduction

The state can broadly be divided into Arid, Semi-arid, Sub-humid, humid and Very
humid regions, on the basis of rainfall intensities. Western Rajasthan comes in

the arid region with rainfall 10-20 cm. The region is characterized by low and
highly variable rainfall years creating inhospitable living condition to both human

and livestock population. Arid region covers Jodhpur, Bikaner, part of Ganganagar,
Jaisalmer and Barmer. Ganganagar, Hanumangarh, part of Jodhpur region having
rainfall of 20-40 cm belongs to Semi-arid region. Sub-humid region belongs to the
area having rain fall of 40-60 cm. Alwar, Jaipur, Ajmer, Jalore comes under the

Sub-humid region. Humid region with rainfall 60-80 cm comprises the districts of
Bharatpur, Dholpur, Sawai Madhopur, Bundi, Kota, Barmer and Rajsamand and
the north-eastern parts of Udaipur. Very Humid region with rain fall of 80-150 cm

consists of south-east Kota, Baran, Jhalawar, Banswara, south-west Udaipur and

adjacent areas of Mt. Abu. In Banswara, Chittaurgarh, Jhalawar, Baran and Kota

the rainfall varies between 70 cm to 100 cm. Kota comes under Zone-V which is
classified as Humid south Eastern Plain. This zone receives the highest rainfall in

the state ranging 70-100 cm. The plain is spread in the south eastern part of the

state covering Sawai Madhopur, Karauli, Jhalawar, Baran, Kota and Bundi districts.

The landscape is characterized by hill pediments and vast alluvial plain formed by

the rivers Chambal, Parbati, Parwan, Kalisindh and their tributaries. River, deep
gullies and ravines have been formed in the region due to these rivers. Land is very

productive due to the presence of fine textured alluvium deposited by the rivers
in this zone. The state witnesses great peculiarities in temperature. Winters are
very severe and temperature falls below freezing point at places like Ganganagar;
summers are intense and quite severe in region like the western Rajasthan. May
is generally the hottest month and generally January records the lowest minimum
temperature. Changes in the climate variables like temperature increase can affect
the hydrologic cycle and agriculture and allied sectors which exhibit high sensitivity
to climate stresses.

6
 Chapter 1. Introduction

Figure 1.1: Different agro-climatic zones of Rajasthan

7
 Table 1.1: Details of different agro-climatic zones in Rajasthan
Zone Area Districts Average Temp. Major crops Soil
covered rainfall range types
(cm) (oC) Kharif Rabi

IA Arid Barmer & part 20-37 8-40 Pearlmillet, Wheat, Desert soil and sand
western of Jodhpur Mothbean, Mustard, dunes aeolian soil,
plain Sesame Cumin coarse sand in
texture
some places
calcareous
IB Irrigated Sriganganagar, 10-35 4.7-42 Cotton, Wheat, Alluvial deposits
north Hanumangarh Clusterbean Mustard, calcareous, high
western Gram soluble salt &
plain exchangeable
sodium
IC Hyper arid Bikaner, 10-35 3-48 Pearlmillet, Wheat, Desert soil and sand
partial Jaisalmer, Mothbean, Mustard, dunes aeolian soil,
irrigated Churu Clusterbean Gram loamy coarse in
zone texture & calcareous
IIA Internal Nagaur, Sikar, 30-50 5.3-39.7 Pearlmillet, Mustard, Sandy loam, shallow
drainage Jhunjhunu, Clusterbean, Gram depth red soil in
dry zone part of Churu Pulses depressions
IIB Transitiona Jalore, Pali, 30-50 4.9-38 Pearlmillet, Wheat, Red desert soil in
l part of Sirohi, Clusterbean, Mustard Jodhpur, Jalore &
plain of Jodhpur Sesame Pali
Luni sierzems in Pali,
basin Sirohi
IIIA Semi arid Jaipur, Ajmer, 50-70 8.3-40.6 Pearlmillet, Wheat, Sierozens, eastern
eastern Dausa, Tonk Clusterbean, Mustard, part alluvial, west &
plains Sorghum Gram north west lithosols,
brown soil in foot
hills
IIIB Flood Alwar, 50-70 8.2-40 Pearlmillet, Wheat, Alluvial prone to
prone Dholpur, Clusterbean, Barley, water
eastern Bharatpur, Groundnut Mustard, logging,
plain Karoli, Gram alluvial
S. Madhopur calcareous in some
places
IVA Sub-humid Bhilwara, 50-90 8.1-38.6 Maize, Wheat, Soil is lithosols in
southern Sirohi, Pulses, Gram foot
plains Udaipur, Sorghum hills & alluvial in
Chittorgarh plain
IVB Humid Dungarpur, 50-110 7.2-39 Maize, Wheat, Predominantly
southern Udaipur, Paddy, Gram reddish
plains Banswara, Sorghum, medium texture,
Chittorgarh Black gram well
drained calcareous,
shallow on hills,
deep
soil in valleys
V Humid Kota, 65-100 10.6-42.6 Sorghum, Wheat, Black of alluvial
south Jhalawar, Soybean Mustard origin,
eastern Bundi, Baran clay loam,
plain ground water salinity
 Chapter 1. Introduction

1.5 Soil in Rajasthan

Extensive topography of Rajasthan includes rocky terrain, rolling sand dunes, wet-
lands, barren tracts or land filled with thorny scrubs, river-drained plains, plateaus,
ravines and wooded regions. The type of soils in Rajasthan are complex and highly
variable, reflecting a variety of different parent material and physiographic land
features. The soil in western region is light and coarse textured whereas the soil in
eastern parts is heavy and clayey in texture. The soils of the state have been cate-
gorized into five specific orders, viz. Aridisols, Alfisols, Entisols, Inceptisols and

Vertisols. All the soils have been identified into 22 soil series for particular charac-
teristic and problems. Due to scarcity of surface water in the state, it depends largely

on ground resources to a great extent. The ground water exploitation is very high in
the eastern as compared to the western region. The annual groundwater recharge is

relatively less in the western part of the state, largely owing to very low and erratic

nature of the rainfall, absence of surface water sources and high evapotranspiration.

The depth of water varies widely throughout the state and varies between 10 m to 25

m and 20 m to 80 m in eastern region to western region, respectively. It has been es-

timated that over 80% of the state has come under water level depletion zone during

the period 1984 to 2015.

The physiography of Kota region comprises vast area formed from the alluvium

brought down by Chambal and its tributaries passing through the residual hillocks
and gently sloping rocky plateau, which are barren with interspersed veneer of soils.
Moderately deep to very deep grayish brown, well drained, fine loamy/coarse loamy
either calcareous or non-calcareous soils are dominant feature of the soil scape
mainly on the plateau and intervening basin (NBSS, 1995).

9
 Chapter 1. Introduction

1.6 Kota region: An overview on agricultural per-

spective

Hadoti region, covering Kota, Bundi, Baran and Jhalawar districts, comes under
humid south eastern plain (Central Plateau and Hills Region). Regional headquarter

Kota is situated at Latitude of 25o 21′ N, Longitude of 75o 86′ E and having altitude
of 271 MSL.
Land use pattern of Kota district comprises total 521.3 thousand hectares (Tha)

of geographical area. Out of this, 269.1 Tha is cultivable area and 125.3 Tha is
forest area. This region comprises deep black clayey, deep brown clayey and deep

brown loamy soils having percentages of 42, 15 and 11 respectively. Out of the net
sown area of 269.1 Tha, 151.8 Tha of area sown more than once. Net irrigated area

in the region is 233.9 Tha. Sources of irrigation is canals, tanks and open / bore

wells. Rain fed area is 167.1 Tha. Most of the soil is irrigated by canals and wells.

Details of Rabi and Kharif crops in the region is as under:

• The Rabi crops are grown in winter season and are seeded in the months of
October and November. These crops are harvested in the months of March
and April and include barley, wheat, gram, pulses and oil seeds mainly. Rape

and mustard are the major oil seeds. Mustard, wheat and coriander are the

major crops with productivity of 175.2 kg/ha, 263.2 kg/ha and 40 kg/ha re-
spectively.

• The Kharif crops are summer crops and are sown in months of June and July.
Harvesting of these crops takes place in the months of September and Oc-
tober. Principal Kharif crops are bajra, pulses, jowar, maize, soybean and
ground nut. Soybean, paddy and maize are the major crops with productivity
of 1493 kg/ha, 3452 kg/ha and 1278 kg/ha respectively.

10
 Chapter 1. Introduction

1.7 Pesticides use and trends in India

India, second largest populated country of the world, currently supports nearly
17.84% of the world population having 2.4% land resources and 4% of water re-
sources. To meet the demands of growing population, the country needs to raise its
agricultural production to provide food as well as nutrition security. Good emerg-
ing trends and solutions for sustainable crop protection through use of time tested
agrochemicals, seed treatment, agronomy and bio-technological development have
raised the quantity and quality of agricultural production. It is important to note

that about 15-25% potential crop production is lost due to insect pests, weeds and
diseases. India today imports substantial quantities of pulses and oil seeds on a reg-

ular basis and sugar as well as other products. Such imports for longer term can
not be afforded by our nation. For ensuring farmer’s welfare, self reliance and in-

creasing the agricultural production is the need of the hour. Increasing pest attacks

in crops is one of the major challenges in enhancing the output in terms of quality

and quantity. At present, per hectare consumption of pesticides in India is amongst

the lowest in the world and stands at 0.6 kg/ha against 5-7 kg/ha in the UK and 13
kg/ha in China.

The total number of pests attacking major crops has increased significantly from
1940’s. For instance, the number of pests which are harmful for crops such as rice

has increased from 10 to 17, whereas for wheat pests increased from 2 to 19. The
increased damage to crops from pests and subsequent loss poses a serious threat
to food security and further underscores the importance of agrochemicals. Pests,
weeds and diseases are causing reduction in global crop output by approximately
25%. In such a scenario, agrochemicals have an increasing role to play in enhancing
productivity and crop protection post-harvest. Insecticides and herbicides are the
two major chemicals under the class of pesticides. Insecticides are the largest sub-
segment of agrochemicals with 60% market share, whereas herbicides with 16%
market share are the fastest growing segment in India (FICCI, 2016; Prasad et

11
 Chapter 1. Introduction

al., 2016). Traditionally, agrochemicals have been manufactured through chemical

synthesis but lately biochemical processes are also gaining popularity.

The agrochemicals are broadly classified into different categories as under:

1. Insecticides: Insecticides are those agrochemicals which protect the crops

either by killing insects or by preventing their attack. They control the popu-
lation of pest below a desired threshold level. On the basis of their mode of
action, further they are classified in two types:

a. Contact insecticides: These kill insects on direct contact. As these chemi-

cals leave no residual activity, they cause minimal damage to environment.

Fipronil, carbaryl, pyrethroids (esfenvalerate, bifethrin, cypermethrin, lamb-

dacyhalothin, cyfluthrin, permethrin, deltamethrin, tefluthrin or tralomethrin),

pyrethrins and spinosad are some of the examples.

b. Systemic insecticides: These are absorbed by the plant tissues and destroy in-

sects when they feed on the plant. Due to their long term residual activity, they

cause damage to environment and human health. Dimethoate, thiamethoxam,

imidacloprid, terbufos and dinotefuran are the exmples of such type of agro-
chemicals.

2. Fungicides: Fungicides are the agrochemicals which protect the crops from
the attack of fungi. Protectants and eradicates are the two popular types of
fungicide. Protectants prevent or inhibit fungal growth, while eradicates kill
the pests on application. In this way, fungicides improve productivity, reduce
blemishes on crop and improve storage life and quality of harvested crop.

3. Herbicides: Herbicides are also known as weedicides. These agrochemicals

kill undesirable plants. They can be of two types: selective and non-selective.
Selective herbicides kill specific plants, leaving the desired crop unharmed,
while non-selective herbicides are used for widespread clearance of ground.
Thus, non-selective herbicides are used to control weeds before crop planting.

12
 Chapter 1. Introduction

Figure 1.2: Market share of different pesticides in India

4. Bio-pesticides: Bio-pesticides are new age crop protection products which

are manufactured from natural substances like plants, animals, bacteria and
certain minerals. They are eco-friendly, easy to use and require lower dosage

amount for the same performance as compared to the chemical based pesti-

cides. Bio-pesticides have huge growth potential due to its non-toxic nature.

5. Others: Fumigants and rodenticides are the chemicals which protect the crops

from pest attacks during crop storage. Plant growth regulators help in con-
trolling or modifying the plant growth process and are usually used in cotton,
rice and fruits.

Though on one side, the use of pesticides in agricultural practices is very much
beneficial to increase productivity by protection of crop losses and vector disease
control, on the other side, their are numerous hazards associated with pesticides.
Some of the important hazards are as under:

• Direct impact on humans: Benefit of pesticides includes enhanced economic

13
 Chapter 1. Introduction

potential in terms of increased production of food and fiber. On the other

hand, disadvantages are in the form of serious health implications to man and

his environment. There is now overwhelming evidence that some of these

chemicals do pose a potential risk to humans and other life forms and un-

wanted side effects to the environment.

• Impact through food commodities: Pesticide residues in products of plant ori-

gin is tested to know the pesticide contamination in the food stuffs. It has been
found by a study that 13 pesticides (acephate, carbendazin, chlorothalonil,

chlopyriphos, DDT, diazinon, endosulfan, methamidophos, iprodione, met-

alaxyl, methidathion, thiabendazole, triazophos) were available in five com-

modities (mandarins, pears, bananas, beans and potatoes). This is just an
example to understand the gravity of the situation.

• Impact on environment: Pesticides can pollute soil, water, turf, and other

vegetation. In addition to killing insects or weeds, pesticides can be toxic to a

host of other organisms including birds, fish, beneficial insects, and non-target

plants. Insecticides are generally the most acutely toxic class of pesticides,

but herbicides can also pose risks to non-target organisms.

• Surface water contamination: Pesticides can contaminate surface water also

by reaching through runoff from treated plants and soil. Water contamination

may cause serious health hazards to human and aquatic organisms.

• Ground water contamination: Pesticides also pollute ground water in the long
run. This may damage our ground water quality during recharging of ground
in rainy season. According to a study, at least 143 different pesticides and 21
transformation products have been found in ground water.

• Soil contamination: The pesticides and their transformation products (TP) are
retained by soils to different degrees, depending on the interactions between

14
 Chapter 1. Introduction

soil and pesticide properties. The most influential soil characteristic is the
organic matter content. The larger the organic matter content, the greater the

adsorption of pesticides and TP’s.

• Effect on soil fertility (beneficial soil microorganisms): Heavy treatment of

soil with pesticides can decline the populations of beneficial soil microorgan-
isms also.

1.8 Earthworm: Farmer’s friend

Earthworms are terrestrial invertebrates belonging to the Phylum Annelida, Class

Chaetopoda, Order Oligochaeta. They have originated about 600 million years

ago during the pre-cambrian era (Piearce et al., 1990). Aristotle referred them

as “the intestines of earth and the restoring agents of soil fertility” (Shipley, 1970).
Earthworms with diverse habitat, improve soil fertility by changing the physical and

chemical composition of soil. Many scientists discussed the importance of earth-

worm in improving the soil fertility (Darwin, 1881; Edwards et al., 1995; Kale,

1998; Lalitha et al., 2000). Earthworms convert organic matter into soil humus
by ingesting soil, mixing of different soil components and then produce as surface

and sub surface castings (Jairajpuri, 1993). According to Shuster et al. (2000),
earthworms play a major role in the decomposition of organic matter through feed-

ing, aeration, fragmentation, turnover and dispersion. Presence of good population

of earthworms in soil indicates the presence of a large population of insects, spi-
ders, bacteria, viruses, fungi and other organisms and thus a healthy soil. Due to
this, earthworms are biological indicators of soil quality (Lachnicht and Hendrix,
2001).

15
 Chapter 1. Introduction

1.8.1 Ecological strategies of earthworms

Bouche (1977) and Lee (1985) categorised earthworms into three main ecological

groups. This classification is done on the basis of soil horizons in which the earth-
worms were commonly found i.e., litter, topsoil and sub soil. Classified three major

groups are the epigeics, anecics and endogeics. They may be defined as under :

• Epigeic: Species of earthworms which live on the soil surface are called
Epigeic. They are litter feeders, e.g. Allolobophora parva and Eisenia fetida.

• Anecic: Earthworms which live on topsoil are called Anecic and feed on the
leaf litter mixed with the soil. They predominantly form vertical burrows in

the soil, e.g. Lampito mauritii.

• Endogeic: These species of earthworms consume more soil than epigeic or

anecic species, deriving their nourishment from humus. They preferably form

horizontal burrows in soil, e.g. Metaphire posthuma.

1.8.2 Factors affecting distribution of earthworms

Physical and chemical characters of the soil, such as pH, moisture, organic matter,
temperature and soil texture affect the distribution of earthworms in soil (Edwards

and Bohlen, 1996).

1. As earthworms are sensitive to the hydrogen ion concentration, their distribu-

tion and abundance are very much affected by pH and factors that are related
to pH. (Staaf, 1987; Chalasani et al., 1998). Soils, having neutral pH, are
usually preferred by most species of earthworms.

2. Soil moisture plays an important role in the survival of earthworms, as their

bodies have 75-90% of water by weight (Grant, 1955). Soil moisture affects

16
 Chapter 1. Introduction

the number and biomass of earthworms (Wood, 1974). They can even sur-
vive in adverse moisture conditions, either by moving to a region with more

moisture (Valle et al., 1997) or by means of aestivation (Baker et al., 1992).

3. The distribution of earthworms is greatly influenced by the distribution of or-

ganic matter. Doube et al., 1997 reported that there is a strong positive corre-
lation between the organic matter content of the soil and earthworm numbers
and biomass. Earthworms are also recognised as soil managers because they
bring about physical, chemical and biological changes in the soil due to their

activities.

1.8.3 Importance of earthworms

• As a growth enhancer of plants: Earthworms prepare the ground in an excel-

lent manner for the growth of plants (Darwin, 1881). Many workers demon-

strated that earthworms have beneficial effects on soil as these effects increase
plant growth as well as yield of crops (Decaens et al., 1999; Lalitha et al.,

2000). Earthworms release auxins and cytokinins which are beneficial sub-

stances for plant growth (Krishnamoorthy and Vajranabhaiah, 1986).

• In organic farming : Burning of organic wastes originated from various sources

like domestic, agriculture and industrial has caused serious environmental

hazards and economic problems. This problem can be overcome by earth-

worms as they can process city refuse, household garbage, sewage and waste
from paper, food and wood industries. This composting process decreases the
time of stabilization of the waste and converts into an efficient bio-product,
i.e., vermicompost. Vermiculture and vermicompost associated with other
biological inputs have been found to be economically very productive. This
way it offers a solution to recycle and reuse organic agro wastes in ecofriendly
manner. By organic farming, we can eliminate the use of chemicals in the

17
 Chapter 1. Introduction

form of fertilizers/pesticides, convert waste into useful compost. This im-

proves soil, plant, animal and human health and creates an ecofriendly, sus-

tainable and economical bio-system models (Ansari and Ismail, 2001, 2001b).

• Vermicomposting: Vermicomposting is a process through which earthworms

and microorganisms biologically degrade and stabilize the organic waste into
vermicompost. The earthworms stimulate microbial activity, fragment the or-
ganic waste and induce rate of mineralization (Kale et al., 1982). All these
processes convert waste into humus-like substances with finer structure. By

adding vermicompost with clay soil, soil loosening takes place. This finally

improves the passage for the entry of air and hence improves soil porosity
(Lavelle and Spain, 2001). Vermicast improves water holding capacity of
the soil, as mucus associated with the cast is hydroscopic in nature. This also

prevents water logging of soil due to its better absorbing capacity. Nutrients,

beneficial for plants, are released slowly and steadily from the organic car-

bon present in vermicompost. Epigeic earthworms like Perionyx excavatus,

Eisenia fetida, Lumbricus rubellus and Eudrilus eugeniae are used for vermi-
composting but the local species like Perionyx excavatus has proved efficient

composting earthworms in tropical or subtropical conditions (Kale, 1998). In

this way, vermicompost is able to improve the physical, chemical and biolog-
ical properties of soil. It promotes growth of plants and has been found to
have a favourable influence on all crops.

• As a biomarker: A biomarker is defined as a “biochemical, cellular, physio-

logical or behavioural variations that can be measured in tissue or body fluid
samples, or at the level of whole organisms, to provide evidence of expo-
sure and/or effects from one or more contaminants” (Depledge, 1994). The
effects of contaminants occur more rapidly at lower levels, e.g. biochem-
ical, cellular, physiological in comparison to higher levels (e.g., ecological

18
 Chapter 1. Introduction

effects). This helps to indicate a more sensitive early warning of toxicolog-

ical effects within earthworm populations. Earthworm biomarkers represent

useful tools in soil monitoring and assessment as an early warning of adverse

ecological effects (Sanchez-Hernandez, 2006; Rodriguez-Castellanos &

Sanchez-Hernandez, 2007). As indicated by Sanchez-Hernandez (2006),

four types of approaches can be performed in soil pollution monitoring : 1)
biomarker analysis on native earthworm populations; 2) use of transplanted
organisms in in situ exposure bioassays; 3) exposure of a selected earthworm

population to the environmental medium (soil) in laboratory standardized

conditions; 4) simulated field studies.

Earthworm as biomarkers have become relevant for assessing the effects of

contaminants on soil organisms. Biomarker approach is used for soil pol-

lution monitoring. It is a relatively recent approach as compared to aquatic

environment monitoring. To develop this methodology to perfection, it is to

be further evaluated. First, it is necessary to identify and characterise ap-

propriate earthworm species for quick assessment of soil pollution. Second,

earthworm biomarkers studies have been mostly conducted for heavy metals.

It is necessary to study the exposure for a wider range of chemicals related

to soil pollution. Third, there is a need to increase the knowledge of biologi-

cal responses of earthworms to pollutants for the purpose of standardization.
This will help in detection of the pollutant induced stress syndrome in soil
organisms. Cytological, biochemical and transcriptomic parameters are also
used to assess the effect of contaminants by examining the effect on phys-
iological fluids, such as coelomic fluids and blood of earthworms. In this
regard, granulocyte morphometric alteration has been recently demonstrated
as a suitable general biomarker of contaminant’s effect. Finally, earthworm
biomarkers have been scarcely investigated under field conditions.

19
 Chapter 1. Introduction

1.9 Organic farming

The concept of organic farming includes the efficient use of local resources, soil
fertility management, recycling of nutrient to the possible extent, control of pests
and diseases by natural products. It can be a promising option for sustainable agri-
cultural development. Associated benefits of this are: (i) It provides greater yield
stability, particularly in tropical ecosystems, (ii) By using improved and the adapted
technology, higher yields and incomes are attainable in traditional farming systems,
(iii) It improves soil fertility which leads to long-term sustainability of farming sys-

tems, (iv) It reduces farmer’s dependence on external inputs, (v) By using organic
farming, restoration of degraded or abandoned land is possible and (vi) It strength-

ens the self-confidence as well as autonomy of farmers.

By organic farming, it is possible to produce safe and healthy food and fiber with

minimum or no adverse effects on the environment and resources. Organic culti-

vation follows different management approaches depending on climates, locations

and cropping systems.

Over the years, it has been scientifically proven that organic farming promises
environmental preservation, protection of variety and species, protection of soil,

purity of water and reduces the impact of agriculture on the atmosphere. Today’s
awareness regarding use of organically grown food and fiber has attracted the at-

tention of agricultural policy planners, promoters and producers all over the world.
Efforts were made by International Federation for Organic Agriculture Movements
(IFOAM) for promotion of organic farming has ensured the growing acceptance
of organic agricultural systems world over. Various countries, including India are
working seriously in this direction to develop and implement quality assurance sys-
tems to promote organic agriculture.
In India, ministry of commerce launched the “National Programme on Organic
Production” (NPOP) defining the National Standards for Organic Production (NSOP)
and the procedure for accreditation and certification in 2000. India now has 30

20
 Chapter 1. Introduction

accredited certification agencies for facilitating the certification to growers. For

area expansion and technology transfer, ministry of agriculture launched a Na-

tional project on Promotion of Organic Farming (NPOF). To augment the research

needs ICAR launched a Network Project on Organic Farming (NPOF-ICAR) un-

der Project Directorate of Farming System Research with 13 collaborating centers

across the country. As on March 2014, India has brought 4.72 million hectares
area under organic certification process, which includes 0.6 million hectares of cul-
tivated agricultural land and 4.12 million hectares of wild harvest collection area

in forests. Since 2004, many states embraced organic farming and drafted policies.
So far 11 states, namely Andhra Pradesh, Karnataka, Kerala, Uttarakhand, Maha-
rashtra, Madhya Pradesh, Tamil Nadu, Himachal Pradesh, Sikkim, Nagaland and

Mizoram have drafted the organic agriculture promotion policies. Sikkim became

the first state in the country by converting the entire state into organic.

21
Chapter 2

Review of Literature

Agrochemicals play a major role in fulfilling the ever growing demand of food by

increasing the productivity and protect the crops by controlling pests and weeds.

While their excessive use has resulted in increased yield, they have also resulted
in serious health complications to man and environment. Pesticides pollute water,

soil, and quality of other vegetation. These chemicals are found to be useful not

only in killing insects and weeds, but are toxic to host of other organisms which are

beneficial to crop and our ecosystem. Birds, fish, beneficial insects, and non-target

plants comes in this category. Insecticides are generally the most acutely toxic class
of pesticides, but the non-target organisms can be at a greater risk due to herbicides

also.

Earthworms play a pivotal role in farming through use of natural resources.

Earthworms are capable to recycle the organic waste and convert it into useful
organic manure which is very useful for agricultural application. Organic farm-
ing helps to provide many advantages like providing the alternative to fertiliz-
ers/pesticides, recycle and regenerate waste into useful manure, improve soil quality
and thus beneficial to plant, animal and human health.
Researchers studied the potential of earthworms for soil quality management
and enhancing its fertility. Following sections present the various key contributions.
 Chapter 2. Review of Literature

These research works were a motivation to explore further in this direction.

2.1 Study of avoidance behaviour in earthworms

Lukkari et al. (2005) assessed the toxicity of copper and zinc with standardised
earthworm acute toxicity and reproduction tests .They used avoidance test to find
out whether the earthworm Aporrectodea tuberculata avoids soils simultaneously
contaminated with Cu and Zn, and whether earlier exposure to metal-polluted soil
affects its avoidance response and also to compare the sensitivity of the earthworm
avoidance test to the standardised acute toxicity and reproduction tests. They ob-

served that A. tuberculata clearly avoided lower soil metal concentrations than those
that induced responses in the acute toxicity and reproduction tests. They found that

the standard species in the earthworm tests, Eisenia fetida, appeared to be more

tolerant to metals and it seemed to regulate the tissue metal concentrations more

strictly than A. tuberculata and concluded that the earthworms living in the metal

contaminated area were either better adapted or acclimatized to live in metal con-
taminated soil.

Zhou et al. (2007) assessed toxicity of chlorpyrifos-contaminated soil on earth-

worm with three different earthworm test methods. The test methods covered all
important ecological relevant endpoints (acute, chronic, and behavioural). They
found that at concentration of 78.91 mg/kg, chlorpyrifos caused significant toxic
effects in all test methods, but at lower test concentrations, only significant chronic
toxic effects could be observed. Results showed that chlorpyrifos had adverse ef-
fect on growth and fecundity in earthworm exposed to 5 mg/kg chlorpyrifos after
eight weeks. The avoidance response test, however, showed significant repellent
effects at concentration of 40 mg/kg chlorpyrifos. They concluded that for chlor-
pyrifos, concentration affecting avoidance response was far greater than growth and
fecundity, it seemed likely that earthworms were not able to escape from pesticide-

23
 Chapter 2. Review of Literature

contaminated soil into the clean soil in field and hence were exposed continuously
to elevated concentrations of pesticides.

Garcia et al. (2008) evaluated the avoidance behaviour of earthworms Eisenia

fetida for three pesticides (benomyl, carbendazim, lambda-cyhalothrin) in labora-

tory tests performed under temperate and tropical conditions. The results showed
that this test gives reproducible and reliable results. Toxicity values (NOEC, EC
50) are lower than those determined in 14 day-acute mortality tests and are approx-
imately in the same range such as those found in 56 day-chronic reproduction tests

with the same earthworm species, which were performed in parallel. Therefore, the
use of the earthworm avoidance tests is recommended as a screening tool for the
risk assessment of pesticides.

De Silva et al. (2009) studied comparative sensitivity of Eisenia andrei and Peri-

onyx excavatus in earthworm avoidance tests in the tropics, using chlorpyrifos and

carbofuran in artificial and natural soil. They exposed earthworms to concentrations

of 1-900 (chlorpyrifos) and 1-32 (carbofuran) mg a.i./kg dry soil in a two-chamber

system under tropical conditions (26 ± 2o C, 48h). They found no significant differ-

ence in the control tests comparing the two soils used; suggesting soil type did not

affect the distribution of the worms. Their results suggest a higher sensitivity of E.

andrei, with EC 50 for the effect on avoidance behaviour for both pesticides being
a factor of 2-3 lowers than for P. excavatus. They concluded that earthworm avoid-

ance tests with local species should therefore be used with caution when applied as a
tool for pesticide risk assessment in the tropics. Endpoints generated through avoid-
ance tests in their study are shown to be less sensitive than reproduction and more
sensitive than survival. They also concluded that Eisenia andrei was more sensitive
in avoidance test with two pesticides than P. excavatus under tropical conditions.
Marques et al. (2009) analysed the toxicity of formulated herbicides and their
active ingredients on natural soils by using earthworm avoidance behaviour. For
this, they used two herbicide active ingredients (a.i.) sulcotrione and penoxsulam

24
 Chapter 2. Review of Literature

and their respective commercial formulations–Mikado and Viper and earthworm

species was Eisenia andrei. Both herbicides induced avoidance behaviour on E.

andrei, stronger effects were denoted by penoxsulam and its respective formulated
product, Viper. Overall, avoidance tests provided a sensitive, valuable and feasible

response either to compare the habitat function of different standard and agricultural
natural soils or to test the effect of herbicides.
De Sousa and De Andrea (2011) studied the avoidance behaviour of Eisenia andrei
in three different agricultural soils treated with cypermethrin. Their experimental

result showed that after 48 h, there was no mortality, but the avoidance was clear
because all earthworms were found in the untreated section of each type of soil
(p < 0.05). They concluded that the different soil characteristics, cypermethrin

concentrations and formulation did not influenced the avoidance behaviour of the

earthworms.

Santos and Forrer (2011) observed avoidance behaviour of Eisenia fetida to car-
bofuran, chlorpyrifos, mancozeb and metamidophos in natural soils from the high-

lands of Colombia. Their observations showed that for the carbofuran and chlor-

pyrifos 100% avoidance was not reached and no significant avoidance behaviour

trend was found for mancozeb and methamidophos. However, for the case of car-

bofuran and methamidophos, differences of more than double in avoidance were

obtained.

Farrukh and Ali (2011) examined the effect of endosulfan on growth, reproduc-
tion and avoidance behaviour of earthworm Eisenia fetida. Their result showed
that growth was significantly affected at all three concentrations of pesticide used,
whereas earthworm of control group showed normal increase in growth. Repro-
duction seemed to be deliberately affected and avoidance test of 48 hours was also
found to be a sensitive parameter in evaluation of the toxic chemical.
Farruk and Ali (2011b) observed the effects of dichlorovos organophosphate on
growth, reproduction, and avoidance behaviour of earthworm Eisenia fetida. Their

25
 Chapter 2. Review of Literature

observation showed that all three doses of dichlorovos decreased the weight of
earthworms, whereas reproduction and avoidance behaviour which are sensitive pa-

rameters were found to be significantly affected.

Santos et al. (2012) assessed the effect of three commercial formulations contain-

ing the insecticides chlorpyrifos and endosulfan and the herbicide glyphosate to
non-target soil organisms. For this, they collected soil after spraying and dilution
series were prepared with untreated soil to determine the impact of the pesticides on
the avoidance behaviour and reproduction of the earthworm Eisenia andrei. Result

showed that a significant avoidance was observed at the recommended field dose in
case of endosulfan by earthworms (60%). In addition, both insecticides endosulfan
and chlorpyrifos affected the number of juveniles produced by the earthworms (EC

50 were below the recommended field dose). They concluded that glyphosate did

not seem to affect earthworms in the recommended field dose.

Alves et al. (2013) assessed the impact of the insecticides imidacloprid, fipronil,
thiametoxam, fungicides captan and carboxin plus thiram on the survival, reproduc-

tion, and behaviour of Eisenia andrei (Oligochaeta). All these chemicals are used

in the chemical treatment of crop seeds. Results showed that with the exception of

imidacloprid, none of the pesticides tested caused mortality in E. andrei in artificial

soils.
Morcillo et al. (2013) quantified the avoidance response of Lumbricus terrestris

in chlorpyrifos-spiked soils, depending on the pesticide concentration and expo-

sure duration. The effects of different chlorpyrifos concentrations were examined
in a standardised test (two-chamber system) with 0.6, 3 and 15 mg/kg chlorpyri-
fos. A modification of the test involved a pre-exposure step (24, 48 or 72 hrs)
in soils spiked with 15 mg/kg. In both protocols, earthworms were unable to
avoid the contaminated soils. They concluded that the avoidance behaviour test
for organophosphorus-contaminated soils could be supported by specific biomark-
ers to facilitate a better understanding of pesticide exposure and toxicity during this

26
 Chapter 2. Review of Literature

test.
Bucha et al. (2013) studied the toxicity of three pesticides, carbendazim, carbofu-

ran and glyphosate to Pontoscolex corethrurus (Muller, 1857) and Eisenia andrei
(Bouche, 1972) by using avoidance and mortality tests. They found that concentra-

tions applied in the field of these two pesticides have toxic effects on both species
and glyphosate showed no toxic effects for either species even at the highest con-
centration tested (47 mg a.i./kg), although earthworm species displayed avoidance
behaviour at this concentration. They observed that the sensitivity of P. corethrurus

appears to be similar to the standard species for the pesticides evaluated reinforcing
the notion that E. andrei is a good test species.
Acute and sub-acute effects of enrofloxacin on the earthworm species Eisenia fetida

in an artificial soil substrate were examined by Li et al. (2015). The results showed

that the LC 50 of enrofloxacin to earthworms was 11.01 gm/kg at day 14 , which

was far higher than the environmentally relevant concentration of at day 14 residues.
They observed that the reduction in the growth and reproduction of earthworms was

a dose dependent effect. Earthworms exposed to > 0.25 gm/kg of enrofloxacin

displayed avoidance response while the soil only spiked with > 2 gm/kg of en-

rofloxacin showed decreased habitat function. The respiration of earthworms was

inhibited when exposed to 2.0 gm/kg of enrofloxacin for more than 14 days or 1.0
gm/kg of enrofloxacin for more than 28 days. They concluded that enrofloxacin

could cause acute and sub-acute toxicity to earthworms (E. fetida) at relatively high
concentration.

2.2 Study on the impact of chemicals on the growth

and reproduction of different earthworm species

Various researchers reported the effect of pesticides on growth and reproduction

on different species of earthworms. In this section, detailed discussion is done on

27
 Chapter 2. Review of Literature

species other than Eisenia fetida.

Choo and Baker (1998) studied the influence of four commonly used pesticides

on the survival, growth, and reproduction of the earthworm Aporrectodea trape-

zoides (Lumbricidae) and found that endosulfan significantly reduced the weight of

juvenile Aporrectodea trapezoides within 5 weeks when applied to soil at normal

application rate in both the field and laboratory condition while fenamiphos did so
at normal application rate in the field only. Both fenamiphos and methiocarb re-
duced earthworm weight in the laboratory when applied at 10 times normal rate.

They also found that cocoon production in A. trapezoides was inhibited by endosul-
fan and fenamiphos at normal application rates and methiocarb at 10 times normal
rate.

Panda and Sahu (1999) investigated the decline and recovery of the growth and

reproduction of an earthworm Drawida willsi, for different doses of malathion to

soil. Study found that sharp decline in the growth of D. willsi (57% in 2.2 and
80% in 4.4 mg malathion per kg soil) was observed after 15 days. Study indicated

decline in growth and reproduction of D. willsi following application of a normal or

a double agricultural dose of malathion. However, the worms were able to resume

normal growth and reproduction after an interval of 105 days.

Maboeta et al. (1999) studied the effects of sublethal concentrations of lead nitrate
on growth and reproduction of earthworm Perionyx excavatus by exposing worms

in an organic substrate to lead nitrate-contaminated food over a period of 76 days.

Their results showed that growth was affected negatively by the presence of lead
while maturation rate and cocoon production was not affected. Lead accumulation
was of the same order of magnitude as for other previously studied species.
Panda et al. (1999b) studied the accumulation of zinc and its effects on the growth,
reproduction and life cycle of Drawida willsi (Oligochaeta), a dominant earthworm
in Indian crop fields. D. willsi did not revealed any significant changes in their mass
at any of the concentrations of Zn (50, 200 and 400 mg/kg) compared to in untreated

28
 Chapter 2. Review of Literature

soils. They found that concentrations in the exposed earthworms were significantly
increased, but they were able to regulate their body content of Zn within a range

of 116–125 mg/kg (dry wt) in 200–400 mg/kg Zn-treated soil. Reproduction was
significantly reduced when the Zn concentration in soil exceeded 200 mg/kg. The

drop in reproduction at elevated concentrations of Zn apparently resulted in a delay

in completion of the life cycle and a decline in the total population.
Booth et al. (2000) examined the effect of two organophosphates, chlorpyrifos and
diazinon in the earthworm Aporrectodea caliginosa and observed a reduction in

growth rate in all pesticide-treated worms.

Panda and Sahu (2000) assessed the recovery of population, biomass and repro-
duction of the earthworm Drawida willsi following the application of two recom-

mended agricultural doses of malathion (2.2 mg/kg single dose and 4.4 mg/kg

double dose) in a rice field agroecosystem for a period of 105 days. Observa-

tions showed that the average worm population and biomass were declined 12%
and 21%, respectively, in plots treated with single and double doses of malathion,

compared with control. The peak of worm population was observed after 60 days

in control and this peak value was shifted by 15 days (i.e. observed after 75 days)

in malathion-treated plots indicating delay in growth and maturation of the worms

following the application of malathion. Inhibition in the total number of cocoons

produced, and thus in the rate of reproduction, was also observed in malathion-

treated plots. One-way analysis of variance showed significant differences in the

population size and biomass of D. willsi up to 75 days, in rate of reproduction up to
90 days, and no difference thereafter. They concluded that under field conditions, D.
willsi worms took about 75–90 days after application of malathion to resume nor-
mal population, biomass and reproduction, and hence they suggested that a second
application of malathion in single and double agricultural doses should be avoided
before this time.
Panda and Sahu (2004) investigated the recovery of acetylcholine esterase (AChE)

29
 Chapter 2. Review of Literature

activity of Drawida willsi (Oligochaeta) by application of two recommended agri-

cultural (single and double) doses of butachlor (1.1 and 2.2 mg a.i./kg dry soil),

malathion (2.2 and 4.4 mg a.i./kg dry soil) and carbofuran (1.1 and 2.2 mg a.i./kg
dry soil) to the soil under laboratory conditions. A sharp decline in the AChE activ-

ity of D. willsi was observed up to 9 and 12 days following treatment of carbofuran

and malathion in both single and double doses, respectively, whereas very little in-
hibition was noticed in case of butachlor. They found that D. willsi worms took
45 and 75 days to resume normal AChE activity after exposure to both single and

double doses of malathion and carbofuran, respectively. They strongly suggested

that the time gap between the first and second application of malathion, irrespective
of single and double dosage, should be at least 90 days, whereas it should be at

least 105 days for carbofuran. Butachlor was found to be very toxic, suppressing

growth, sexual maturation and cocoon production of D. willsi at both single and

double doses. Researchers suggested that application of organochlorine pesticides

like butachlor should be avoided as far as possible to ensure maintenance of good

soil health.

Capowiez et al. (2005) tested lethal and sublethal effects of imidacloprid on two

earthworm species (Aporrectodea nocturna and Allolobophora icterica), which is

the major component of many widely used insecticides and is relatively persistent
in soils. Their result was consistent with previous findings obtained with other

earthworm species and natural soils, i.e. significant decreases in weight.

De Silva et al. (2009b) investigated the influence of temperature and soil type on
the toxicity of three pesticides to Eisenia andrei. They compared the toxicity of
chlorpyrifos, carbofuran and carbendazim to the earthworm Eisenia andrei at two
different temperatures reflecting temperate and tropical conditions. The toxicity of
the three pesticides in both conditions decreased in the order carbendazim > car-
bofuran > chlorpyrifos. For chlorpyrifos and carbofuran, but not for carbendazim,
survival was more sensitive at the higher temperature, probably due to increased

30
 Chapter 2. Review of Literature

earthworm activity. Sub-lethal effects (reproduction and growth) however, varied

inconsistently with temperature and soil types. They concluded that toxicity of pes-

ticides in tropics may not be predicted from data generated under temperate condi-
tions, even within the same species.

De Silva et al. (2010) assessed the toxicity of chlorpyrifos, carbofuran, mancozeb

and their formulations on survival, growth and reproduction of the tropical earth-
worm Perionyx excavates in standard artificial soil. They found that toxicity of the
three chemicals decreased in the order carbofuran > chlorpyrifos > mancozeb. In

general, formulations were more toxic than the active ingredients, but differences
in LC 50 and ECx values were significant only in two cases and notmore than a
factor of 2.0. This could mainly be due to masking of the effects of additives in

the soil. Comparison with available survival data revealed that P. excavatus is more

sensitive.

De Silva et al. (2010b) found that chlorpyrifos causes decreased organic matter
decomposition by suppressing earthworm and termite communities in tropical soil.

In their study, litterbag and earthworm field tests were performed simultaneously

at the same tropical field site sprayed with chlorpyrifos. Their experimental results

showed that the recommended dose of chlorpyrifos (0.6 kg a.i./ha) and two higher

doses (4.4, 8.8 kg a.i./ha) significantly decreased litter decomposition during the
first 3 months after application, which could be explained from lower earthworm

and termite abundances during this period. They observed species-specific effects
of chlorpyrifos on organism abundance and biomass, with termites being mostly
affected followed by the earthworm Perionyx excavatus; the earthworm Megascolex
species was least affected.
Capowiez et al. (2010) examined earthworm cast production as a new behavioural
biomarker for toxicity testing. They proposed a new and relatively simple ecotox-
icity test based on the estimation of cast production by Lumbricus terrestris under
laboratory conditions. Cast production was found to be linearly correlated to earth-

31
 Chapter 2. Review of Literature

worm biomass and to be greatly influenced by soil water content. Their experi-
mental result showed that azinphos-methyl had no effect on cast production at all

the concentrations tested. Significant decreases were observed at the normal appli-
cation rate for other pesticides with (imidacloprid, carbaryl, methomyl) or without

(ethyl-parathion and chlorpyrifos-ethyl) a clear concentration effect response. Cast

production is straight forward and rapidly measured and ecologically meaningful.
They thus believed it to be of great use as an endpoint in ecotoxicity testing.
Dittbrenner et al. (2010) studied the physiological and behavioural effects of im-

idacloprid on two ecologically relevant earthworm species (Lumbricus terrestris

and Aporrectodea caliginosa) commonly found in different agricultural soils. In
laboratory experiments they assessed sub-lethal effects (body mass change and cast

production) of imidacloprid. They observed a significant loss of body mass after

seven days in both species exposed to imidacloprid concentrations as low as 0.66

mg/kg dry soil. Cast production increased by 26.2% in A. caliginosa and by 28.1%
in L. terrestris at the lowest imidacloprid concentration tested (0.2 mg/kg dry soil),

but significantly decreased at higher concentrations (equal to and above 0.66 mg/kg

dry soil) in both earthworm species after the 7 days exposure experiment. They

concluded that the biomarkers used in this study, body mass change and changes

in cast production, may be of ecological relevance and have shown high sensitiv-
ity for imidacloprid exposure of earthworms. The measurement of changes in cast

production should be considered for inclusion in current standard tests.

Lister et al. (2011) studied a combined approach using mixture toxicity testing,
toxicokinetic studies and modelling to address the link between joint toxicity and
internal concentration. The study was conducted in Lumbricid earthworms (Lum-
bricus rubellus) with a binary mixture of a metal (nickel) and an organophosphate
insecticide (chlorpyrifos).
Dasgupta et al. (2012) studied the effects of carbaryl, chlorpyrifos and endosul-
fan on growth, reproduction and respiration of tropical epigeic earthworm, Perionyx

32
 Chapter 2. Review of Literature

excavatus (Perrier) under laboratory conditions. The experimental results showed

significant reduction in biomass, production and hatching of cocoon and produc-

tion of juveniles of the worms exposed to these insecticides. Endosulfan was found
most dangerous among the three insecticides followed by carbaryl and chlorpyri-

fos. Chlorpyrifos produced no change in respiration of the worms except at the

highest dose, while the worms showed an increase in evolution of CO2 at all doses
of carbaryl and endosulfan. Based on the recommended agricultural dose of each
insecticide, they concluded that application of endosulfan and carbaryl was poten-

tially dangerous to earthworms.

Bhattacharya and Sahu (2013) studied the effect of imidacloprid on mortality of
earthworm Drawida willsi, under ideal laboratory conditions to rice field soil. They

found that the 96 hrs LC 50 value for with their 95% confidence limit of juvenile,

immature and adult earthworm was 4.43, 7.96 and 12.45 mg a.i. imidacloprid /kg

dry soil respectively. Although the recommended dose of imidacloprid was lower
the 96 h LC 50 values of D. willsi for imidacloprid, but still it could affect the soil

biota by altering its vital rates and metabolism.

Suthar (2014) examined the toxicological impact of pesticide methyl parathion on

growth and reproduction performance in tropical earthworms: Metaphire posthuma

(endogeic), Lampito mauritii (anecic) and Allolobophora parva (epigeic). They

applied a total of three concentrations (a.i. g/kg dry test soil), 1.00, 1.125 and

2.25 of methyl parathion in test substrate over 60 days under laboratory conditions
and observed that methyl parathion caused significant mortality in all tested species
and the individual live weight loss was 27–37% in L. mauritii, 36–57.1% in M.
posthuma and 1.2–11.0% in A. parva in different test concentrations. The pesticide-
exposed worms produced less cocoons than control, but in L. mauritii, an unusual
reproduction (hormesis) was recorded. They suggested that the methyl parathion
had species-specific toxicity against tropical earthworms.
Murugan et al. (2014) assessed the stress of the monocrotophos (pesticide) and

33
 Chapter 2. Review of Literature

glyphosate (herbicide) on Eudrilus eugeniae. They found that the biomass of the
earthworm was decreased on using monocrotophos and observed that most earth-

worms showed their preference to be of neutral to slightly acidic when incorporated

into glyphosate, monocrotophos soil. They also observed that among the herbicides

and pesticides, the concentration of 0.44 ± 0.02 mg/kg of glyphosate accumulated

in the different regions of earthworms which were analysed during the study.
Sanchez-Hernandez et al. (2014) examined the integrated biomarker analysis of
chlorpyrifos metabolism and toxicity in the earthworm Aporrectodea caliginosa.

For this, they measured acetylcholinesterase (AChE), carboxylesterase (CbE), cy-

tochrome P450-dependent monooxygenase (CYP450), and glutathione S-transferase
(GST) activities in the body wall of earthworms.

Annapoorani (2014) studied the toxic effect of aluminium on reproduction and

survival of Eudrilus eugeniae (Kinberg) by exposing in moist leaf litter with alu-

minium. The morphological changes observed during the exposure of animals in

laboratory conditions. The reproduction and survival rate were higher in the control

than Al treatments. The activity of Al concentrations was found to influence the size

of the population and cocoon production and abnormal bulging of clitellar region

and over all emancipation of the size of the body.

Hattab et al. (2015) examined the stress response of earthworms (Eisenia andrei)
to exposure to a commonly used herbicide, 2,4 dichloro-phenoxy-aceticacid (2,4-

D). They exposed earthworms to three sublethal concentration of 2,4-D (3.5, 7, and
14 mg/kg) for 7 and 14 days. Results showed that exposure to 7 and 14 mg/kg
of 2,4-D significantly reduced both worm body weight and lysosomal membrane
stability. The latter is a sensitive stress biomarker in coelomocytes.
Singh and Singh (2015) exposed Eutyphoeus waltoni to different concentrations of
2,4-D (200, 300, 400, 450 mg/kg) in feed material (i.e., buffalo dung, wheat straw
and gram bran) and different concentrations of 2,4-D (150, 300, 350, 400 mg/kg)
in different types of soil (i.e., loamy soil, clay soil and sandy soil) from 24 h up to

34
 Chapter 2. Review of Literature

240 h. Result indicated that the toxic effect of herbicide 2,4-D on the earthworm
Eutyphoeus waltoni was both time and dose dependent. Maximum toxicity was

observed in the sandy soil, whereas minimum in the feed material of buffalo dung
with gram bran.

The toxicity of some selected widely used agricultural pesticides on earthworm

lumbricus terrestris were studied by Yuguda et al. (2015), using two different types
of bioassay, a 48 hours contact filter paper bioassay test and a 14 days soil bioassay
test. They also tested some selected heavy metals in the soil before and after the

experiment to determine the level of their presence in the selected pesticides. The
result of 48 hours contact filer paper test showed that pyrethroid, neonicotinoids and
organophosphates to be highly toxic to earthworm (lumbricus terrestris) with LC

50 values range from 0.000 ml–0.001 ml. Herbicides and fungicides were relatively

low toxic with LC 50 values ranges from 0.002 ml for butachlor and 0.006 ml for

mancozeb respectively. Their result for 14 days soil bioassay test revealed different
pattern of toxicity. Insecticides, pyrethroid and neonicotinoids, still showed high

level of toxicity under soil condition. Herbicides and fungicides do not pose a

serious threat to earthworm’s survival under soil condition. They concluded that

insecticides pyrethroid and neonicotinoids were highly toxic to earthworms in the

soil based on agricultural recommended dose/rate of application.

Govindarajan and Prabaharan (2015) determined the impact of monocrotophos

on the cocoon production of the earthworm Eudrilus eugeniae in laboratory con-

trolled soil. They observed that cocoon production was more sensitive than other
parameters such as growth, mortality, etc. They concluded that food played a major
role in reproduction potential (cocoon production).
Ahmed (2015) examined the influence of four pesticides (cyren, ridomil, triplen
and mamba) on Lumbricus terrestris earthworm for 4 weeks in the laboratory. He
observed that there were loss of weight in all cultures, signs and symptoms of toxic-
ity like, coiling, body swollen, sluggish movement and discharge of coelomic fluid.

35
 Chapter 2. Review of Literature

They concluded that among the four pesticides tested cyren was most toxic to earth-
worm Lumbricus terristris causing sever mortality, while triplen and mamba were

moderately toxic and ridomil was the least toxic pesticide.

Gaupp-Berghausen et al. (2015) evaluated the effect of glyphosate-based herbi-

cide on Lumbricus terrestris and Aporrectodea caliginosa. They observed that the
surface casting activity of vertically burrowing earthworms (L. terrestris) almost
ceased three weeks after herbicide application, while the activity of soil dwelling
earthworms (A. caliginosa) was not affected. Reproduction of the eathworms was

reduced by 56% within three months after herbicide application. They found that
herbicide application led to increased soil concentrations of nitrate by 1592% and
phosphate by 127%, pointing to potential risks for nutrient leaching into streams,

lakes, or groundwater aquifers.

Jeyanthi et al. (2016) studied the effect of pesticide (carbaryl) and metal (lead)

on the biochemical responses in three earthworm species, Eudrilus eugeniae, Peri-

onyx ceylanensis and Perionyx excavates. Their result showed that protein content

was slightly increased in E. eugeniae, P. ceylanensis when exposed to the lower

concentration of Pb (75 mg/kg). But decreased protein content was observed in P.

excavatus at the higher concentration of carbaryl (50 mg/kg), and with exposure to

300 mg/kg Pb in P. ceylanensis. They observed that due to exposure to carbaryl and
Pb the levels of antioxidant enzymes glutathione-S-transferase, glutathione perox-

idase, reduced glutathione, superoxide dismutase and catalase were changed. But
other antioxidant enzymes showed little variation at the time of exposure. They sug-
gested that both carbaryl and Pb induced the production of reactive oxygen species
by causing oxidative damage to cells.
Singh and Singh (2016) analyzed the lethal effect of different concentrations of
herbicide butachlor (2-chloro 2,6 diethyl N, butoxymethyl acetanilide) on the earth-
worm Eutyphoeus waltoni in different combination of feed materials i.e. buffalo
dung, wheat straw and gram bran and soils under the laboratory conditions. Their

36
 Chapter 2. Review of Literature

result showed that maximum toxicity was observed in the sandy soil whereas, min-
imum in combination of buffalo dung with gram bran.

Dominguez et al. (2016) studied the toxic effect of aminomethyl phosphonic acid
(AMPA) on the earthworm Eisenia andrei. Aminomethyl phosphonic acid is one of

glyphosate’s main metabolites which have been classified as persistent in soils, rais-
ing concern regarding the widespread use of glyphosate in agriculture and forestry.
They found that field-relevant concentrations of AMPA had no significant effects
on mortality in acute or chronic assays. Except at the highest concentration tested,

a significant biomass loss was observed compared to controls in the chronic as-
say. Their experimental results showed that the number of juveniles and cocoons
increased with higher concentrations of AMPA applied, but their mean weights

decreased. This mass loss indicates higher sensitivity of juveniles than adults to

AMPA. They suggested that earthworms coming from parents grown in contam-

inated soils may have reduced growth, limiting their beneficial roles in key soil
ecosystem functions.

Pelosi et al. (2016) examined the sublethal effects of epoxiconazole on the earth-

worm Aporrectodea icterica. In their study, they used different doses of a commer-

cial formulation of epoxiconazole, in a natural soil to evaluate their effect on earth-

worm mortality, uptake, weight gain, enzymatic activities (catalase and glutathione-
S-transferase), and energy resources (lipids and glycogens). Weight gains were 28,

19, and 13% of the initial weight after 28 days of exposure in the control and D1
and D10 (1 and 10 times the recommended dose) treatments, respectively. They
found that no difference was observed for catalase activity between the three treat-
ments, at 7, 14, or 28 days. The glutathion-S-transferase (GST) activity was two
times as high in D1 as in D0 at 14 days. At 28 days, glycogen concentration was
lower in D10 than in the D1 treatment. This study highlighted moderate sublethal
effects of the commercial formulation for earthworms. Considering that these ef-
fects were observed on a species found in cultivated fields, even at recommended

37
 Chapter 2. Review of Literature

rates, muchmore attention should be paid to this pesticide.

2.3 Study on the impact of chemicals on the growth

and reproduction of earthworm Eisenia fetida to-

gether with other species

Mahanthswamy and Patil (2003) studied on the toxicity of different pesticides on

four different species of earthworms Eudrillus eugeniae (Kinberg), Eisenia foetida

(Savigny), Perionyx excavatus (Stepenson), Lampito mauritii. Pesticides were ap-

plied by incorporation method to food material (mixture of soil, dried leaves and

farmyard manure). They observed that there was a variation in the toxicity of differ-

ent pesticides to different species of earthworms. Endosulfan 35 EC caused highest

mortality to all the species of earthworm. It caused cent percent mortality to L. mau-

ritii which was significantly higher than the remaining treatments. Dicofol caused

higher mortality next to endosulfan in all the species of earthworms except in E.

fetida. Carbendazim 50 WP caused maximum mortality to P. excavatus (40%) and

the lowest mortality to L. mauritii (10%). Trifluralin 48 EC caused maximum mor-

tality to E. eugeniae (52.50%) and lowest mortality to L. mauritii (22.5%).

Tripathi and Bhardwaj (2004) compared the composting potential, biomass growth

and biology of a non-native (Eisenia fetida) and an endemic (Lampito mauritii)

species of earthworm in the semiarid environment of Jodhpur district of Rajasthan
in India. They reared earthworm in a mixed bedding material comprising of biogas
slurry, cowdung, wheat straw, leaflitter, sawdust and kitchen waste. They observed
that percentage of organic carbon of the culture bedding material declined upto 105
days with E. fetida and 120 days with L. mauritii and percentage of nitrogen, phos-
phorous and potassium increased as a function of the vermicomposting period. In
contrast, C/N and C/P ratios decreased day by day. They found that both species

38
 Chapter 2. Review of Literature

were effective for decomposition and mineralization of mixed bedding in the semi-
arid environment. They assessed biomass growth and reproductive rates of E. fetida

and L. mauritii under controlled laboratory conditions using mixed bedding. Ex-
perimental results showed that cocoon production was higher for E. fetida than L.

mauritii and the net reproductive rate was 9 per month in the case of E. fetida and
1 per month for L. mauritii. Fertilized eggs of E. fetida and L. mauritii developed
into adults within 4 and 5.25 months, respectively. Their observations indicated that
the E. fetida may be a more efficient breeder than L. mauritii in the desert region of

Rajasthan.
The impact of organophosphate pesticides in orchards on earthworms in the West-
ern Cape, South Africa was observed by Reinecke and Reinecke (2007). Worm

densities were very low in orchards (22 per m2 ) compared to adjacent unculti-

vated fields (152 per m2 ) at a distance from the orchards. The possible effect of

organophosphate pesticides on the earthworms was investigated. Background soil

concentrations of chlorpyrifos prior to the start of the spraying season were low

(0.2–2.7 mg/kg) but persistent for up to 6 months after the last spraying event, and

the pesticide was, as a result of rainfall, transported to nontarget areas by runoff.

Background concentrations of azinphos methyl were higher than those of chlor-

pyrifos (1.6–9.8 mg/kg) but not detectable 2 weeks after the spraying event. Az-
inphos methyl was mostly transported by wind (spray drift) to adjacent areas. A

microcosm study indicated the effects of chlorpyrifos on earthworms as determined

by measuring biomass change and cholinesterase inhibition. It was concluded that
earthworms were affected detrimentally by the pesticides due to chronic (chlorpyri-
fos) and intermittent (azinphos methyl) exposure.
Reinecke and Reinecke (2007b) also worked on biomarker response and biomass
change of earthworms exposed to chlorpyrifos in microcosms. For this microcosms
were filled with soil from the same areas and earthworms of the species A. calig-
inosa were introduced. They treated microcosms with a series of concentrations

39
 Chapter 2. Review of Literature

of chlorpyrifos in the laboratory under controlled conditions. These concentra-

tions were chosen to fall within the background ranges found in the soils. They

determined biomass of the worms regularly for a period of 5 weeks and worms
in a state of estivation were noted. Earthworms were removed from the micro-

cosms for biomarker tests: for cholinesterase (ChE) inhibition assays every week
and for a neutral red retention determination 2 weeks after the exposures started.
They noted that most prominent biomass loss was in earthworms exposed to the
highest pesticide concentration of 8.0 mg/kg. Estivation was higher among earth-

worms exposed to higher exposure concentrations. Inhibition of ChE increased with

higher exposure concentrations and with time but there was no clear dose–related
response. They established a clear dose-related response with exposure concentra-

tion for the neutral red retention assay. A correlation between ChE inhibition and

biomass change existed directly after the second application of chlorpyrifos.

Tu et al. (2011) examined the effect of fungicides and insecticides on earthworm

behaviour in controlled environments and on the dynamics of earthworm commu-

nity in the field by using single application of insecticides sevin (carbaryl) and merit

(imidacloprid) at the manufacturer’s suggested dose. They observed that these in-

secticides significantly inhibited earthworm feeding activity for at least three weeks

without leading to any earthworm death. While fungicides did not show signifi-
cant toxicity to earthworms when applied only once, but their toxicities increased

as application frequency increased. T-methyl and sevin significantly reduced the

abundance and biomass of earthworms in the field, when applying six consecutive
weeks. Their suppressive effects lasting for at least 6 weeks after the chemical ap-
plication was terminated. They suggested that the surface activities of earthworms
in turfgrass systems may be managed through moderate application of pesticides at
peak periods of earthworm activities.
Dittbrenner et al. (2011) compared the sensitivity of Eisenia fetida to Aporrec-
todea caliginosa and Lumbricus terrestris after imidacloprid exposure. Results

40
 Chapter 2. Review of Literature

showed significant changes in body mass in E. fetida and A. caliginosa occurred

after exposure to imidacloprid concentrations as low as 0.2 (7 days) and 0.66 mg/kg

dry weight (14 days), significant body mass changes in L. terrestris observed to 2
and 4 mg/kg dry weight, for 7 and 14 days of exposure, respectively. They found

that significant cellular changes already occurred after 24 h exposure to the lowest
test concentrations in all species by histopathological examinations, but the degree
of detrimental effects as well as species-specific differences were dependent on the
monitor tissue. They concluded that E. fetida seemed to be more sensitive than L.

terrestris concerning cellular alterations.

Saxena et al. (2014) performed experiment to establish the use of Metaphire
posthuma as a sensitive test model for ecotoxicological studies. They carried out

acute toxicity testing of carbaryl, carbofuran, cypermethrin and fenvalerate on Eise-

nia fetida and Metaphire posthuma by using contact filter paper toxicity and soil

toxicity bioassays. They found among the tested chemicals, carbofuran was the
most toxic to both the earthworm species. On comparing the toxicity data of these

chemicals for both the earthworm species, they found M. posthuma was to be more

sensitive than E. fetida. Based on the acute toxicity data, the order of toxicity of

insecticides in both the test procedures was carbofuran > cypermethrin > carbaryl

> fenvalerate for M. posthuma where as for E. fetida it was carbofuran > carbaryl >
fenvalerate > cypermethrin. They observed some morphological changes in the or-

ganisms exposed to these chemicals which were more pronounced in M. posthuma

at lower concentrations than E. fetida in both the test procedures. They concluded
that the use of M. posthuma for ecotoxicity studies, being a more sensitive and
reliable model than E. fetida.

41
 Chapter 2. Review of Literature

2.4 Study on the impact of chemicals on the growth

and reproduction of earthworm Eisenia fetida

Helling et al. (2000) studied effects of the fungicide copper oxychloride on the
growth and reproduction of Eisenia fetida in the experiment lasting for 8 weeks.

They selected following life-history parameters: earthworm growth in consecutive

weeks, survival rate, maturation time, cocoon production, reproduction success, to-
tal number of hatchlings produced, and incubation time for their study. Earthworm

growth and cocoon production were significantly reduced at copper oxychloride

exposure concentrations of 8.92 mg/kg and higher. Reproduction success in the

8.92 mg/kg Cu substrate was highest. From an exposure concentration of 15.92

mg/kg Cu substrate and higher, there was a considerable impact of copper oxychlo-

ride on reproduction. They concluded that from a reduced reproduction success, a

reduced mean and maximum number of hatchlings per cocoon, and a longer incu-

bation time, indicating a strong effect of low copper oxychloride concentrations on

this earthworm species.

Vermeulen et al. (2001) determined the acute and sublethal effects of the fungi-

cide mancozeb on the earthworm Eisenia fetida in laboratory experiments. They

selected following life-history parameters for measurement: growth in consecutive

weeks over a 6-week period, survival rate, maturation time, cocoon production,
hatching success of cocoons, number of hatchlings per cocoon, and incubation time
of cocoons over another 4 weeks. Their results indicated that mancozeb had no
significant detrimental effect on either growth or reproduction of E. fetida at the
recommended dose (8 mg/kg) or at an estimated environmental concentration (44
mg/kg). The endings do not support the hypothesis that avoidance response to man-
cozeb could serve as an indication of toxicity.
Toxic effects of chlorpyrifos on morphology and acetylcholinesterase activity in the
earthworm, Eisenia fetida was studied by Rao et al. (2003). They carried out a

42
 Chapter 2. Review of Literature

48-h contact test for acute toxicity of chlorpyrifos in the earthworm, E. fetida, as
described by OECD guideline 207. The LC 50 of chlorpyrifos was 0.063 mg per m2 .

Scanning electron microscopic studies revealed the morphological abnormalities

in the worms. The study demonstrated a dose- and time-dependent exposure of

chlorpyrifos through skin results, morphological abnormalities, and inhibition of

AChE in the earthworm, E. fetida.
Xiao et al. (2004) studied single and joint effects of acetochlor and urea on earth-
worm Eisenia foetida populations in phaiozem. They found that acetochlor had an

enhanced toxicity from low concentration to high concentration. The mortality of

earthworms after a 6-day exposure was changed from 0 to 86.7%, and the weight
change rate ranged from 7.86 to 30.43%, when the concentration of acetochlor was

increased from 164 to 730 mg/kg. They observed that urea expressed its positive

and beneficial effects on earthworms when its concentration was lower than 500

mg/kg, but strongly toxic when the concentration of urea was higher than 1000
mg/kg. The mortality of earthworms exposed to urea reached 100% when its con-

centration was more than 1500 mg/kg. When the concentration of urea was lower

than 500 mg/kg, there were antagonistic effects between the two agrochemicals on

earthworms; when the concentration of urea was higher than 500 mg/kg, joint toxic

effects of acetochlor and excessive urea on earthworms were synergic. In any case,
excessive urea application is very harmful to the health of soil ecosystems.

Maboeta et al. (2004) studied the relationship between lysosomal biomarker and
organismal responses in an acute toxicity test with Eisenia foetida (Oligochaeta)
exposed to the fungicide copper oxychloride. Their aim was to establish whether a
lysosomal subcellular response, measured as neutral red retention times, could be
linked to the LC 50 and biomass changes. They found that changes in coelomo-
cyte membrane stability manifested earlier than effects on biomass and it may have
predictive value and may contribute information during acute toxicity tests, which
could be of greater ecological relevance than mortality data alone.

43
 Chapter 2. Review of Literature

Effects of perchlorate on earthworm (Eisenia fetida) survival and reproductive suc-

cess was examined by Landrum et al. (2006) using three types of tests: dermal

contact (filter paper), sand, and artificial soil. All studies utilized a range of per-
chlorate concentrations in order to simulate levels that are likely to occur in the

environment under different scenarios (typical soil levels vs. spill levels). Cocoon
production was highest in the control group, although overall cocoon production
appeared to be low. In contrast to the acute toxicity tests, perchlorate did affect
earthworm reproduction at environmentally relevant soil concentrations. In addi-

tion, preliminary data suggest that cocoons produced under perchlorate contamina-
tion did not hatch as well as cocoons produced in control soil despite incubation of
both sets of cocoons in clean soil or sand.

Xiao et al. (2006) showed the fate of herbicide acetochlor and its toxicity to Eise-

nia fetida under laboratory conditions. They studied the effect of the herbicide on

growth, reproduction, glutathione-S-transferases (GST) activity and cellulase activ-

ity of earthworms by taking 5 concentrations (5, 10, 20, 40 and 80 mg/kg soil). They

found that low concentrations had not significant effect on growth of E. fetida ex-

cept after 15 and 30 days of exposure. They observed that when concentration was

more than 20 mg/kg, growth rates and numbers of juveniles per cocoon decreased

significantly compared to the control in all treatments. However, cellulase activity

decreased significantly in all treatments. They concluded that growth, numbers of

juveniles per cocoon and cellulase activity can be regarded as sensitive parameters
to evaluate the toxicity of acetochlor on earthworms.
Yasmin and D’Souza (2007) investigated the effects of three different pesticides
(carbendazim, dimethoate, and glyphosate) and their mixture on the growth and
reproduction of the earthworm species, Eisenia fetida. Their results showed that the
pesticide treatment had a marked negative impact on the growth and reproduction
of earthworms. Carbendazim and dimethoate were found to cause greater harm to
the selected earthworm species than glyphosate.

44
 Chapter 2. Review of Literature

Shi et al. (2007) conducted laboratory tests to compare the effects of various con-
centrations of lindane and deltamethrin on mortality, growth, and cellulase activity

in earthworms (Eisenia fetida) cultured in artificial soil during typical acute (14
d) and subchronic (42 d) exposure periods. They observed that the toxicity order

for earthworm mortality from the 14-day exposure was lindane > deltamethrin,
with median lethal concentrations (LC 50) of 162.1 and 432.9 mg/kg, respectively.
Earthworms exposed to deltamethrin showed dose-dependent toxic effects on growth
and cellulose activity only from the acute exposures, whereas lindane’s effects on

these activities were seen correlated with both the acute and subchronic doses.
Rombke et al. (2007) investigated the effects of the fungicide benomyl on earth-
worms in laboratory tests under tropical and temperate conditions. They studied

whether the effects of the fungicide benomyl (chosen as a model substance) differ

between tropical and temperate regions and whether data generated under temper-

ate conditions can be used for the environmental risk assessment (ERA) in tropical
regions. The effect of benomyl on earthworms was evaluated in acute and chronic

laboratory tests modified for tropical conditions. These tests were performed at two

temperatures (20o C and 28o C) and with two strains (temperate and tropical) of the

compost worm Eisenia fetida. The fungicide was spiked in two natural and two

artificial soils. The results from the laboratory tests and a literature review showed
that the effects of benomyl were, on average, lower under tropical conditions (LC

50: 450–630 mg a.i./kg; EC 50: 0.8–12.9 mg a.i./kg) than under temperate condi-
tions (LC 50: 61–67 mg a.i./kg; EC 50: 1.0–1.6 mg a.i./kg) by a maximum factor
of 10.3 (acute tests) and 12.9 (chronic tests). They concluded that this result might
be caused by an increased degradation of benomyl, and/or its first metabolite car-
bendazim, at higher temperatures, but a different sensitivity of the two worm strains
cannot be ruled out. Despite the lower toxicity under tropical conditions and as-
suming comparable application rates, a preliminary assessment confirms the risk of
benomyl to soil invertebrates under both conditions.

45
 Chapter 2. Review of Literature

Reddy and Rao (2008) evaluated acute toxicity, morphological alterations and his-
tological effects of an organophosphorus insecticide, profenofos (PFF) to earth-

worm, Eisenia foetida by direct contact through a filter paper. They observed
that earthworms had body ruptures, bloody lesions, and internal excessive forma-

tion of glandular cell mass and disintegration of circular and longitudinal muscles,
which failed to regulate the internal coelomic pressure, leading to fragmentation
in earthworms. They assessed neurotoxic potentiality of PFF by measuring acetyl-
cholinesterase (AChE).

Rai and Bansiwal (2009) studied the impact of sublethal doses of an organophos-
phate pesticide- malathion on growth and reproduction of earthworm Eisenia foetida.
They analyzed the effects of malathion exposure for 35 days duration and recovery

for 35 days on growth and reproductive parameters of earthworm Eisenia foetida.

They found that the worms of control group gained more weight and produced more

numbers of cocoons and hatchlings in compare to malathion exposed worms. Their

study revealed that reduction in growth and cocoons productions were due to the

toxicity of malathion. It causes concentration dependent changes in growth and

reproduction.

Sarojini et al. (2009) studied effect of lignite fly ash on the growth and reproduc-

tion of earthworm Eisenia fetida. Supply of nutrients from fly ash with biosolids
may enhance their agricultural use. They assessed the growth and reproduction

of Eisenia fetida during vermicomposting of fly ash with cowdung and pressmud
in four different proportions and one control i.e., cow dung and pressmud alone.
The growth, cocoon and hatchlings production were observed at the interval of 15
days over a period of 60 days. The maximum worm growth and reproduction was
observed in bedding material alone.
Owojori et al. (2009) investgated the combined stress effects of salinity and copper
on the earthworm Eisenia fetida. They exposed Eisenia fetida in OECD artificial
soil spiked with a range of sub-lethal concentrations of NaCl and Cu singly and

46
 Chapter 2. Review of Literature

as mixtures. Mortality, weight change, and internal copper concentrations were

assessed in worms while Cu concentrations in the soil (total, di-ethylene-triamine-

penta acetic acid and CaCl2 extractable) were also determined. Their result showed
no worm mortality during this study in both individual and joint toxicity tests and

increased NaCl and Cu significantly affected the weight change and cocoon pro-
duction, as individual substances. In combination, the contaminants had mostly
additive effects on these worm parameters. They concluded that the effects of salin-
ity, resulting from increased NaCl, on the toxicity of copper to these earthworms

were mainly additive but also depended on the concentrations of both substances.
Correia and Moreira (2010) conducted laboratory tests to compare the effects of
various concentrations of glyphosate and 2,4-D on earthworms (Eisenia foetida)

cultured in argissol during 56 days of incubation. The effects on earthworm growth,

survival, and reproduction rates were verified for different exposure times. They

reported that there was no mortality in glyphosate treated soil samples, but showed
gradual and significant reduction in mean weight (50%) at all test concentrations.

For 2,4-D, 100% mortality was observed in soil treated with 500 and 1,000 mg/kg.

At 14 days, 30%-40% mortality levels were observed in all other concentrations.

They found no cocoons or juveniles in soil treated with either herbicide. They con-

cluded that glyphosate and 2,4-D demonstrated severe effects on the development
and reproduction of Eisenia foetida in laboratory tests in the range of test concen-

trations.
Gobi and Gunasekaran (2010) explored the effect of butachlor on the life history
parameters (biomass, clitellum development, and cocoon production) and the histo-
logical changes in the earthworm Eisenia fetida over 60 days. They took the dried
cow dung contaminated with 0.2575 mg/kg, 0.5150 mg/kg, and 2.5750 mg/kg of bu-
tachlor based on the LC 50 value, and a control was maintained. They found mean
earthworm biomass decreased with increasing herbicide concentration. Similarly,
cocoon production was also reduced by the increasing herbicide concentration. All

47
 Chapter 2. Review of Literature

earthworms in the exposed group were found to have glandular cell enlargement
and to be vacuolated.

Zhu et al. (2010) examined the bioaccumulation of penta-BDE (DE-71) in earth-

worms (Eisenia fetida) and the induced toxicities of penta-BDE (DE-71) on the

growth and reproduction of earthworms. They observed that all the major congeners
in DE-71 could be bioaccumulated in earthworms and the concentration found in
earthworms correlated to the spiked concentration in soil. DE-71 might inhibit the
growth and reproduction of cocoons and juveniles of earthworms. The toxicities

were dose dependent and increased with exposure time.

Zhou et al. (2011) assessed individual and combined toxic effects of cyperme-
thrin and chlorpyrifos on earthworm Eisenia fetida andrei for different responses

(acute, chronic, behavioural). Their results showed that the toxicity of the mixture

of cypermethrin and chlorpyrifos was significantly higher than either of these pes-

ticides individually, especially on the earthworms chronic responses. They found

that at a concentration of 5 mg/kg, the mixture caused significant reductions on the

growth and reproduction rates of earthworms, but did not cause any significant ef-

fect when the individual was tested. They concluded that the increase in toxicity of

the pesticide mixture means that the use of toxicity data obtained exclusively from

single-pesticide experiments may underestimate the ecological risk of pesticides

that actually present in the field.

Dasgupta et al. (2011) evaluated ecotoxicological risks of agricultural application

of six insecticides to Eisenia fetida by acute toxicity tests under laboratory condi-
tion following OECD guidelines. They found that the organochlorine insecticide
endosulfan (LC 50: 0.002 mg/kg) and the carbamate insecticides aldicarb (LC 50:
9.42 mg/kg) and carbaryl (LC 50: 4.81 mg/kg) were ecologically most dangerous
because LC 50 values of these insecticides were lower than the respective recom-
mended agricultural dose (RAD). Although E. fetida was found highly susceptible
to the pyrethroid insecticide cypermethrin (LC 50: 0.054 mg/kg), the value was

48
 Chapter 2. Review of Literature

higher than its RAD. The organophosphate insecticides chlorpyrifos (LC 50: 28.58
mg/kg), and monocrotophos (LC 50: 39.75 mg/kg) were found less toxic and eco-

logically safe because the LC 50 values were much higher than their respective
RAD.

Daam et al. (2011) compared the sensitivity of soil invertebrates to pesticides with
that of Eisenia fetida. They used the relative tolerance approach to enable compar-
ing toxicity thresholds obtained from the US-EPA ECOTOX database, for main pes-
ticidal types of action (insecticides, fungicides, herbicides, and other) and terrestrial

taxonomic groups separately. Their analysis reported lower and higher sensitivity
of collembolans to fungicides and insecticides, respectively as previously reported.
They found that arachnids and isopods were more sensitive to insecticides, and ne-

matodes to fungicides, as compared to E. fetida.

The effects of the insecticide lambda-cyhalothrin on the earthworm Eisenia fetida

under experimental conditions of tropical and temperate regions were studied by

Garcia et al. (2011). They performed their experiment with two strains (temperate

and tropical) of the compost worm Eisenia fetida and acute and chronic labora-

tory tests modified for tropical conditions, i.e at selected temperatures ((20o C and

(28o C). The insecticide was spiked in two natural soils, in OECD artificial soil and

a newly developed tropical artificial soil. They observed that the effects of lambda-
cyhalothrin did rarely vary in the same soil at tropical and temperate temperatures.

In tests with tropical soils and high temperature, effect values differed by up to a
factor of ten.
Wang et al. (2012) conducted a 48-h filter paper contact test to investigate compara-
tive toxicity of 45 pesticides, including insecticides, acaricides, fungicides, and her-
bicides, toward the epigeic earthworm Eisenia fetida. Results indicated that clothi-
anidin, fenpyroximate, and pyridaben were supertoxic to E. fetida, followed by car-
baryl, pyridaphenthion, azoxystrobin, cyproconazole, and picoxystrobin, while the
other pesticides ranged from being relatively nontoxic to very toxic to the worms.

49
 Chapter 2. Review of Literature

When experiment conducted in artificial soil for 14 days, clothianidin and picoxys-
trobin showed the highest intrinsic toxicity against E. fetida, followed by fenpy-

roximate. However, the herbicides fluoroglycofen, paraquat, and pyraflufen-ethyl

exhibited the lowest toxicities. In contrast, the other pesticides exhibited relatively

low toxicities.
Wu et al. (2012) examined biomarker responses of earthworms, Eisenia fetida,
exposured to phenanthrene and pyrene both singly and combined in microcosms.
For this, they considered following biomarker responses; growth inhibition, en-

zyme activity, malondialdehyde content, sperm count, neutral-red retention time

(NRRT) and annetocin and translationally controlled tumor protein gene transcrip-
tions. Their two-way ANOVA analysis showed that the combination of these two

compounds decreased growth, superoxide dismutase activities, NRRT and sperm

count synergistically, but increased the catalase activities and malondialdehyde con-

tent. A clear dose-related response with exposure concentration was established for
the NRRT. Their results demonstrated that earthworms were under physiological

stress at field dose of 0.5 (Phe) + 100 (Pyr) mg/kg soils. They stated that phenan-

threne and pyrene synergistically decreased sperm count and NRRT, but antagonis-

tically caused changes in antioxidant enzyme activities to disrupt the detoxification

functions and inhibit earthworm growth.

Neaman et al. (2012) explored the effects of lime and compost on earthworm

(Eisenia fetida) reproduction in copper and arsenic contaminated soils from the
Puchuncavi Valley, Chile to determine the effectiveness of lime and compost for
in situ immobilization of trace elements in the soils. Researchers concluded that
compost treatment was effective in improving the quality of soils of Puchuncavi
Valley, increasing earthworm reproduction.
Pal and Patidar (2013) studied the effect of insecticide malathion on cocoon pro-
duction in earthworm Eisenia foetida. They observed that the group of earthworms
that had been exposed to the high concentration of malathion, matured slowly and

50
 Chapter 2. Review of Literature

cocoon production in the resulting adults was reduced to 55.55% and 43.75% com-
pared to controls exposed for 15 days and 30 days respectively.

Chen et al. (2014) assessed the acute toxicity of butachlor, imidacloprid and chlor-
pyrifos with different modes of action on earthworm, Eisenia fetida. They also

compared ecotoxicities of these pesticides for earthworm E. fetida separately and in

combination in artificial soil and contact filter paper tests. They found that imida-
cloprid was the most toxic for E. fetida with LC 50 (lethal concentration 50) values
three orders magnitude lower than that of butachlor and chlorpyrifos in both tests.

They compared toxicity of the mixtures to that predicted by the concentration addi-
tion (CA) model and found the observed toxicities of all binary mixtures were less
than additive. They observed that the combined effects of the pesticides in contact

filter paper tests were not consistent with the results in artificial soil toxicity tests,

which may be associated with the interaction of soil salts with the pesticides.

Chen et al. (2014b) also studied combined toxicity of butachlor, atrazine and k-
cyhalothrin on the earthworm Eisenia fetida by combination index (CI)-isobologram

method in artificial soil and filter paper tests. The order of toxicity for the individ-

ual pesticides was ranked as atrazine > k-cyhalothrin > butachlor in both tests.

They found that synergism was observed in majority of the mixtures except for the

combination of butachlor plus k-cyhalothrin for most cases in artificial soil test.
This particular combination displayed opposite interaction in filter paper test. They

compared CI method with the classical models of concentration addition (CA) and
independent action (IA) and found that CI method could accurately predict the com-
bined toxicity and can serve as a useful tool in ecotoxicological risk assessment.
Jovana et al. (2014) assessd the toxic effects of three pesticides on the earthworm
Eisenia fetida (Savigny 1826) to artificial soil supplemented with different concen-
trations of the examined pesticides based on the recommended agricultural doses
(RAD) under laboratory conditions. Their commercial formulations were: galition
G-5 (insecticide: a.i. malathion and fenitrothion), terbis (herbicide: a.i. terbuthy-

51
 Chapter 2. Review of Literature

lazine), and gardene (limacide: a.i. metaldehyde). They chose mortality, biomass,
and growth inhibition as toxic endpoints. No death was recorded at the lowest con-

centration (1/4 RAD) of the insecticide or the limacide after 7 and 14-day exposures,
nor was it recorded at the highest concentration (4-RAD) of the insecticide or the

limacide after 7-day exposure. They found triazine herbicide terbis the most toxic
and ecologically dangerous to E. fetida, because it LC 50 value (1.26 mg per kg)
was very close to the respective RAD and the growth inhibition in all concentrations
was significantly positive. Although E. fetida was found susceptible to the galition,

due to the significant positive growth inhibition at the highest concentration, the
value of LC 50 was higher than its RAD. On the other hand, gardene was found
ecologically safe because the LC 50 value was higher than its RAD and weight was

not significantly changed.

Garcia-Torres et al. (2014) studied adult mortality, biomass, fecundity and via-

bility of cocoons in Eisenia fetida in response to glyphosate exposure in soil. They

found 71% mortality for E. fetida at the highest concentration of glyphosate (50,000

mg/kg), by day 7 of exposure. Although biomass of E. fetida, between the control

and 5,000 mg/kg dose at day 14, was not affected and only showed a significant

weight loss after 7 days of exposure to 50,000 mg/kg. They observed adverse

effects upon adult fecundity and cocoon viability at glyphosate concentrations of

5,000 mg/kg and above.

Santadino et al. (2014) observed sublethal effects of the widely used herbicide
glyphosate, on the earthworm (Eisenia fetida). They found that the control popula-
tion had a positive growth rate, both glyphosate treatments showed negative growth
rates.
Xu et al. (2014) studied the toxicity and bioaccumulation of ethofumesate enan-
tiomers in earthworm Eisenia fetida. They found a slight difference in toxicity to
earthworm between two enantiomers and indicated that the acute toxicity of etho-
fumesate enantiomers was enantioselective. Earthworm can uptake ethofumesate

52
 Chapter 2. Review of Literature

but the bioaccumulation curve did not reach the steady state. In the elimination
experiment, the concentrations of ethofumesate in earthworm declined following a

first-order decay model with a short half life of 1.8 day. The bioaccumulation and
elimination of ethofumesate in earthworm were both nonenantioselective.

Wang et al. (2015a) examined the combined toxicities of five insecticides (chlor-
pyrifos, avermectin, imidacloprid, cyhalothrin, and phoxim), two herbicides (atrazine
and butachlor), and a heavy metal (cadmium) using the acute toxicity test on the
earthworm, Eisenia fetida. They observed that imidacloprid exhibited the highest

acute toxicity toward the earthworm Eisenia fetida with a concentration of 2.75
mg/kg being lethal for 50% of the organisms. They also studied toxicological in-
teractions of these chemicals internary mixtures using the combination- index (CI)

equation method. They observed that twenty-one ternary mixtures exhibited vari-

ous interactive effects, in which 11 combinations showed synergistic effects, four

led to dual synergistic / additive behaviours, one exhibited an additive effect, and
five showed increasing antagonism within the entire range of effects. The CI method

was compared with the classical models of concentration addition and independent

action, and it was found that the CI method could accurately predict combined tox-

icity of the chemicals studied. The predicted synergism in the majority of the mix-

tures, especially at low-effect levels, might have implications in the real terrestrial
environment.

Wang et al. (2015b) applied the combination index(CI)-isobologram method which

allows computerized quantitation of synergism, additive effect and antagonism to
determine the nature of toxicological interactions of two pesticides λ-cyhalothrin,
imidacloprid, and heavy metal cadmium towards earthworm Eisenia fetida. They
found that, in an artificial soil test, λ-cyhalothrin and Cd combination was slightly
synergistic at low effect levels which turned into a slight antagonism above effect
level values of 0.6, while the binary mixtures containing imidacloprid exhibited
antagonism. The presence of imidacloprid in the ternary mixture also resulted in

53
 Chapter 2. Review of Literature

anantagonistic effect to the earthworms. This behaviour became more antagonistic

in the ternary mixture in filter paper tests.

Schnug et al. (2015) studied the effects of the insecticide esfenvalerate, the fungi-
cide picoxystrobin and the bactericide triclosan, applied individually and as a mix-

ture, on an earthworm (Eisenia fetida) community in the field. They observed that
effects on juvenile proportions were less pronounced and more variable than effects
on abundance. In general, effects were species-specific and chemical-specific as
well as temporal variations distinct. The mixture affected abundance and juvenile

proportions, but the latter only at high mixture concentrations.

Wang et al. (2015c) investigated the effects of five neonicotinoid insecticides on
reproduction, cellulase activity and the tissues of Eisenia fetida. The LC 50 of

imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid was 3.05, 2.69,

4.34, 0.93 and 2.68 mg/kg, respectively. These insecticides seriously affect the

reproduction of E. fetida, reducing the fecundity by 84.0%, 39.5%, 54.3%, 45.7%

and 39.5% at the sub-lethal concentrations of 2.0, 1.5, 0.80, 2.0 and 1.5 mg/kg,

respectively. Wang et al. (2015d) also studied the toxic effect of a neonicotinoid

insecticide guadipyr, in earthworm Eisenia fetida.

Yang et al. (2015) studied toxicological interactions of two pesticides chlorpyrifos

and atrazine and a heavy metal cadmium toward earthworm Eisenia fetida by arti-
ficial soil and filter paper acute toxicity tests. Their result showed that the binary

mixture of chlorpyrifos and atrazine was antagonistic toward E. fetida. The com-
bination of atrazine and Cd exhibited a slight degree of synergism throughout the
exposure range, while chlorpyrifos plus Cd combination led to dual antagonistic/
synergistic behaviour. The nature of binary combinations in filter paper displayed
opposite interaction to that in the artificial soil test, and the toxicity of ternary mix-
tures was not significantly synergistic than their binaries.
Shi et al. (2016) studied the survival, growth, activity of the biotransformation
system phase II enzyme glutathione-S-transferase (GST) and the oxidative defense

54
 Chapter 2. Review of Literature

enzyme catalase (CAT) of earthworms (Eisenia fetida) exposed to the contaminated

soils from a former DDT plant and reference soils, and compared with the corre-

sponding indicators in simulated soil-earthworm system, unpolluted natural soils

with spiked-in DDT series. Their results showed that mortality, growth inhibition

rates, GST and CST activities of earthworms exposed to the contaminated soils
were significantly higher than that in reference soils.
In addition to this, numerous review papers have been published in this area of
work. Frampton et al. (2006) reviewed and analysed effects of pesticides on soil

invertebrates in laboratory studies using species sensitivity distributions. Kale and

Karmegam (2010) presented the role of earthworms in tropics with emphasis on
Indian ecosystems. Bhadauria and Saxena (2010) studied role of earthworms in

soil fertility maintenance through the production of biogenic structures. Basic re-

search tools for earthworm has been compiled by Butt and Grigoropoulou (2010).

Yasmin and D’Souza (2010) presented a review on effects of pesticides on growth

and reproduction of earthworms. Weyers and Spokas (2011) studied the impact

of biochar on earthworm populations. Pelosi et al. (2013) published a review on

pesticides and earthworms.

55
Chapter 3

Materials and Methods

Earthworms are pivotal to achieve organic farming. They modify soil organic matter

both chemically and physically, mix leaf litter with the soil, facilitate the formation

and stabilization of soil aggregates and improve soil porosity. They are ideal test
organisms for soil risk assessment due to their high biomass in soil and frequently

observed sensitivity to relatively low concentrations of environmental toxicants.

3.1 Test soil

The natural soil was collected from an abandoned area in Kota (India), where no
prior agricultural activity was reported and was an area with no known history of
pesticides use. The physicochemical characterization of soil was provided by Nanta

agricultural farm (Office of Project Director, Crop) in the Kota district.

3.2 Test organisms

The earthworms Eisenia fetida (Lumbricidae), were obtained from Krishi Vigyan
Kend-ra, Borkhera, Kota. The animals were bred in cattle manure as food at tem-
perature ranging 20 − 30o C. For all tests, only adult worms with clitellum with a
fresh weight between 200 to 300 mg were used. All earthworms were fed accord-
 Chapter 3. Materials and Methods

ing to demand, usually once a week, with finely ground cattle manure free of any
chemical contamination. In addition, mass cultures of E. fetida were established

where the cultures were kept in room temperature at a light cycle of 16 hrs /8 hrs.
Finely ground cow dung, free of any chemical contamination, was used to feed the

earthworms with an interval of 7 to 10 days. Acclimatization of selected worms was

done in the selected soil 24 hrs before the onset of the experiment.

3.3 Test chemicals

Based on the data provided by the Agriculture Research Station (ARS), Umed Ganj,

Kota and by interaction with local farmers as well as shopkeepers, four chemicals
were selected to study their effect on avoidance, growth and reproduction of earth-

worms. The objective of the research work was to observe the effect of pesticides

used for one Rabi and Kharif season crop on earthworms. Wheat and soybean have

been selected as a Rabi and Kharif crop respectively for this study as these two are

main crops of the Kota region.

Chemicals used in Wheat crop:

• Chlorpyrifos (O,O-diethyl-O-3.5.6-trichloro-2-pyridyl phosphorothioate) is an

organophosphate insecticide. Chlorpyrifos was tested as Radar 20 EC (100

ml, Isagro (Asia) Agrochemical Pvt Ltd, Mumbai). It is used to kill insect
pests by disrupting their nervous system. Chlorpyrifos has an advantage over
other products in that it is effective against a wide range of plant-eating insect
pests.

• 2, 4-D Ethyl ester 38% EC (based on 86% w/w 2,4 dichloro-phenoxy-acetic

acid) is a selective herbicide effective against broad leaves weeds in sorgham,
maize, wheat, aquatic weeds etc. It was tested as Cut Off 38 (250 ml, Crystal
Crop Protection Pvt Ltd). It is a selective, systematic, post emergent herbicide

57
 Chapter 3. Materials and Methods

used mainly for control of annual/perennial broad leaved weeds in millets,

cereals, wheat, maize, rice, sugarcane and aquatic weeds etc. in accordance

with climatic conditions and as per advisory given by agriculture department.

Chemicals used in Soybean crop:

• Triazophos (O, O-diethyl O-(1- Phenyl -1 H -1,2,4-triazol -3 yl) phosphoroth-

ioate) is an organophosphates and it is a acetylcholinesterase (AChE) class of
inhibitor. Triazophos was tested as Trizocel 40% EC (250 ml, Excel Crop
Care Ltd). It is a broad-spectrum insecticide which acts upon insects through
contact and stomach action. It penetrates deeply into plant tissues. It is effec-

tive for control of stem borer, leaf folder, jassids, green leaf hopper, aphids

and bollworms in soybean.

• Pendimethalin (3, 4-Dimethyl-2,6-dinitro-N-pentan-3-yl-aniline) is an herbi-

cide of the dinitroaniline class. Pendimethalin was tested as Panida 30 EC

(250 ml, Rallis India Ltd). It is used to control annual grasses and certain
broadleaf weeds by inhibiting their cell division as well as cell elongation in

wheat, barley, corn, soybeans, rice, potato, legumes, fruits, vegetables, nuts

and other ornamental plants.

3.4 Identification of earthworm species in Kota

In the present work, earthworm species were collected from different areas around
Kota city and their species identified by renowned scientific organization of Gov-
ernment of India.
Earthworms were collected from six different sites on the outskirt areas of Kota
city as shown by blue and red rectangles in Figure 3.1. For collecting the earth-
worms, a pit was dug (four in each area at different places) by ploughing the area
(Figure 3.2). Earthworms were collected by hand picking and then transferred to
jars and preserved

58
 Figure 3.1: Kota region map showing search areas of earthworm

59
Chapter 3. Materials and Methods
 Chapter 3. Materials and Methods

Figure 3.2: Digging of pit to search earthworms in Kota region

60
 Chapter 3. Materials and Methods

in formaldehyde (formalin). Identification of the earthworms was done at Zoo-

logical Survey of India, Kolkata (India). Soil samples from these areas were also

collected and their physico-chemical parameters were obtained by sample test at

Nanta agricultural farm (Office of Project Director, Crop) in the Kota.

3.5 Study of avoidance behaviour of Eisenia fetida

The earthworm avoidance test was developed in USA (Yeardley et al., 1996) and
since then several studies have been conducted to observe the effects on earthworms
for various classes of chemicals.

Avoidance test is an easy and quick to perform. It is known to be sensitive

towards a wide range of chemicals. The principle of this test is that the earthworms

are simultaneously exposed to the soil sample spiked with the pesticide, and to the

control soil. The location of the earthworms is determined after the test period of

two days (ISO, 2008). The tendency of a species to avoid a certain study soil in

favour of the control soil (free of contaminants) is used as avoidance test to control
soil quality and the effects of certain chemicals on the behaviour of earthworm

species (ISO, 2011). These tests are based on the fact that chemicals in soil are

in different factions depending on the contamination level and soil type and can be
absorbed by earthworms. Earthworms can detect a wide range of contaminants due
to their chemoreceptors on their anterior segments and sensory tubercles located on
the surface body (Reinecke et al., 2002).
The avoidance assays with the earthworms were made based on the ISO guide-
line 17512-2 (ISO, 2011) and has been performed by two compartment method in a
container. Each plastic container (15.5 cm height and diameter 13 cm) was divided
into two equal sections with a plastic card (Figure 3.3), one-half of the container
received 250 gm (dry weight) of control soil and the other half 250 gm (dry weight)
of soil contaminated with the pesticide (Figure 3.4). All combinations of contami-

61
 Chapter 3. Materials and Methods

nated/ uncontaminated soil were tested, each one with four replicates. After placing
the soils into each container, the card divider was removed and 10 worms were

placed on the middle line (Figure 3.5). Afterwards, each container was covered
with a transparent lid perforated in order to allow aeration. The organisms were

incubated at (20 ± 2o C) with a photoperiod of 16 hrs : 8 hrs (light : dark) for 2

days. After the test period, the divider was put back to separate the control and test
soils, and the number of worms in both sections were counted.
The chosen eight concentrations tested were 0.316, 1, 3.162, 10, 31.62, 100,

316.22 and 1000 mg a.i./kg dry soil for all chemicals. For each replicate, the avoid-
ance response is calculated using

(C − T ) ∗ 100
NR = (3.1)
N

where, N R = net avoidance response (%), C = number of worms in control soil,

T = number of worms in pesticide-amended soil, and N = total number of worms

exposed.

62
 Chapter 3. Materials and Methods

Figure 3.3: Creating partition for avoidance test in earthworm

Figure 3.4: Top view of beaker after elimination of partition

63
 Chapter 3. Materials and Methods

Figure 3.5: Placing of earthworm Eisenia fetida in the gap

64
 Chapter 3. Materials and Methods

3.6 Study on the impact of chemicals on the growth

and reproduction of Eisenia fetida

The chosen agrochemicals for the present study were: Chlorpyrifos, 2,4-D Ethyl es-
ter, Triazophos and Pendimethalin. The concentrations used in our experimentation

were chosen based on recommended dose of these chemicals for Wheat and Soy-
bean crops in Kota, Rajasthan (India). These doses are 1.2 mg/kg for Chiorpyrifos,
0.75 mg/kg for 2,4-DE, 0.5 mg/kg for Triazophos and 1.5 mg/kg for Pendimethalin.

Doses are as per the recommendation of Department of Agriculture, Govt. of Ra-

jasthan, Kota division office. Dose calculations are as per the following:

• Chiorpyrifos dose calculation:

1. Recommended dose is 0.8 kg/hectare.

2. Assuming that the chemical would disperse into the top 5 cm soil (as

per literature), then volume of 1 hectare soil may be calculated as V =

9997.36 x 104 x 5 cm3 (1 hectare = 9997.36 x 104 cm2 ).

3. Test soil density calculation: 1 kg of dry powdered soil contained 750

ml of volume in a container, so density d of test soil is = 1000/750 =

1.33 gm/cm3 .

4. Now weight of soil in 1 hectare area (for 5 cm soil depth) is V.d =

9997.36 x 104 x 5 cm3 x 1.33 gm/cm3 = 6664824440 gm.

5. Recommended dose calculation in mg/kg, for dry soil, can now be ob-
tained by using dose of 0.8 kg/hectare. This comes out to be 1.2 mg/kg.

6. Five chosen concentrations are 1.2, 3.79, 12, 37.9 and 120 mg/kg dry
soil weight.

• 2,4-DE dose calculation:

1. Recommended dose is 0.5 kg/hectare.

65
 Chapter 3. Materials and Methods

2. Same soil is being used in all sets of experimentation, so soil density

under test and weight of 1 hectare soil are same as mentioned earlier.

3. Recommended dose calculation in mg/kg, for dry soil, can now be ob-
tained by using dose of 0.5 kg/hectare. This comes out to 0.75 mg/kg.

4. Five chosen concentrations are 0.75, 2.37, 7.5, 23.7 and 75 mg/kg dry
soil weight.

• Triazophos dose calculation:

1. Recommended dose 800 ml/hectare (v/w), now converted to (w/w) as

per formulation gives 0.320 kg/hectare.

2. Same soil is being used in all sets of experimentation, so soil density

under test and weight of 1 hectare soil are same as mentioned earlier.

3. Recommended dose calculation in mg/kg, for dry soil, can now be ob-

tained by using dose of 0.320 kg/hectare. This comes out to be 0.5

mg/kg.

4. Five chosen concentrations are 0.158, 0.50, 1.58, 5, and 15.8 mg/kg dry
soil weight.

• Pendimethalin dose calculation:

1. Recommended dose is 1 kg/hectare.

2. Same soil is being used in all sets of experimentation, so soil density

under test and weight of 1 hectare soil are same as mentioned earlier.

3. Recommended dose calculation in mg/kg, for dry soil, can now be ob-
tained by using dose of 1 kg/hectare. This comes out to be 1.5 mg/kg.

4. Five chosen concentrations are 0.474, 1.50, 4.74, 15, and 47.4 mg/kg
dry soil weight.

66
 Chapter 3. Materials and Methods

In our experimentation, five different concentrations were used with a multiplicative

factor of 3.167 times. This led to the scenario where every alternative concentration

is either 10 times or factored by 10. In our experimentation, one lower concentration

of Triazophosis and Pendimethalin is chosen from the recommended dose. This has

been done due to observed strong avoidance behaviour of earthworm at 10 mg/kg in

Triazophos and 31.6 mg/kg in Pendimethalin in our earlier experimentation. Strong
avoidance behaviour at certain concentration levels indicated regarding avoiding
very high dose of chemicals in our experimentation. In addition to this, study has

been conducted for observing the combined effect of Chiorpyrifos and 2,4-DE as
well as Triazophos and Pendimethalin . Combination values of five doses taken in
our experimentation were taken as per Tables 3.1 and 3.2.

3.6.1 Earthworm growth and reproduction test

Five sets of different concentrations were prepared to evaluate the growth of earth-

worms (in four weeks) and number of juveniles (after eight weeks) to test the ef-

ficacy of pesticides. For experimentation, raw cow dung was procured from dairy

farm and sun dried (Figures 3.6, 3.7 and 3.8). Test soil and cow dung powder were

taken in the ratio of 2:1. Dry weight of this mixture was 500 gms for each sample.
For experimentation, transparent plastic containers of 1 litre capacity were taken.

The soil was artificially contaminated by adding Chiorpyrifos, 2,4-DE and their
combination. In another set, Triazophos, Pendimethalin and their combination by
adding chosen concentrations of pesticides as stated earlier. In addition to this, one
set of experiment was conducted by taking control soil (no contamination). Earth-
worms were sorted out from the culture and acclimatized for 24 hrs in test soil. After
which they were washed with fresh water and left over the blotting paper for some
time before measuring their weight on electronic balance. Ten earthworms having
weight between 2 to 3 gms were selected (Figure 3.9) and left over on the soil sur-
face of each container. They immediately buried themselves into the soil. Close

67
 Chapter 3. Materials and Methods

watch was kept on moisture content of the soil by checking it weekly and maintain-
ing it at 50% by adding water, as required. Quantity of the water to be added was

decided by measuring the difference in the weight of the container as compared to

weight at the time of sampling. For growth rate observation, experiment lasted for

28 days. Adult earthworms were removed from the chemically treated soil as well
as from control soil after 28 days and their weight measured to observe the impact
on the biomass growth rate. Biomass growth rate is calculated by dividing average
weight of earthworms after 28 days of exposure with average weight of earthworms

at the begining of incubation.

For the study of effect on reproduction, cocoons were left in the soil for four
additional weeks. After the completion of the period, young worms present in the

soil were counted (Figures 3.10, 3.11 and 3.12). This count divided by ten provided

the parameter of number of juveniles produced per earthworm.

68
 Chapter 3. Materials and Methods

Table 3.1: Combined concentration values of Chiorpyrifos and 2,4-DE alongwith

their labels
1 2 3 4 5
Chiorpyrifos Chiorpyrifos Chiorpyrifos Chiorpyrifos Chiorpyrifos
1.2 mg/kg 3.79 mg/kg 12 mg/kg 37.9 mg/kg 120 mg/kg
+ + + + +
2,4-DE 2,4-DE 2,4-DE 2,4-DE 2,4-DE
0.75 mg/kg 2.37 mg/kg 7.5 mg/kg 23.7 mg/kg 75 mg/kg

Table 3.2: Combined concentration values of Triazophos and Pendimethalin along-

with their labels
1 2 3 4 5
Triazophos Triazophos Triazophos Triazophos Triazophos
0.158 mg/kg 0.5 mg/kg 1.58 mg/kg 5 mg/kg 15.8 mg/kg
+ + + + +
Pendimethalin Pendimethalin Pendimethalin Pendimethalin Pendimethalin
0.474 mg/kg 1.5 mg/kg 4.74 mg/kg 15 mg/kg 47.4 mg/kg

69
 Chapter 3. Materials and Methods

Figure 3.6: Sun drying of raw cow dung

Figure 3.7: Preparing manure in stages

70
 Chapter 3. Materials and Methods

Figure 3.8: Final form of manure for use

Figure 3.9: Weighing of earthworms by electronic machine

71
 Chapter 3. Materials and Methods

Figure 3.10: Counting of juveniles

Figure 3.11: Juveniles after 56 days of exposure to chemicals (view 1)

72
 Chapter 3. Materials and Methods

Figure 3.12: Juveniles after 56 days of exposure to chemicals (view 2)

73
Chapter 4

Results

4.1 Identification of earthworm species in Kota

Survey was conducted in six different areas of Kota for the presence of earthworms.

Of these, three areas showed presence of earthworms. These areas were Bundi road,
Rangpur road and Baran road areas (shown as red rectangle in Figure 3.1). Three

other areas Rawatbhata road, Jhawalwar road and Abhera areas (shown as blue rect-

angle) were predominantly marked by the absence of earthworm species. A total

number of three species of earthworms were identified belonging to two families

(Lumbricidae and Megascolecidae). The species identified under family Lumbri-
cidae is Eisenia fetida (Savigny, 1826). The number of species identified under
family Megascolecidae are Lampito mauritii (Kingberg, 1866) and Perionyx. All
three species are vermicomposting species. Eisenia fetida, as shown in Figure 4.1,
was found to be most abundant in Bundi road, Rangpur road and Baran road areas
around Kota city. Lampito mauritii (Figure 4.2) was found at Bundi road and Rang-
pur road area and Perionyx, as shown in Figure 4.3, was found only at Rangpur
road area. Earthworm identification report as received (by Email) from Zoological
Survey of India (Kolkata) is enclosed in Appendix.
 Chapter 4. Results

Figure 4.1: Eisenia fetida

75
 Chapter 4. Results

Figure 4.2: Lampito mauritii

76
 Chapter 4. Results

Figure 4.3: Perionyx excavatus

77
 Chapter 4. Results

Sample soil collected from survey areas contained the pH range 7.89 − 8.10, con-
ductivity in the range 0.320 − 0.475 m Mhos/cm. and organic carbon content be-

tween 0.42 − 0.60 %. Earthworm populations depend on both physical and chem-
ical properties of the soil; such as moisture, temperature, pH, salts, texture and

aeration as well as available food, and the ability of the species to reproduce and
disperse. These soil samples are represented as samples 1 to 3 in soil parameter test
report as received from Nanta agricultural farm (Office of Project Director, Crop)
and enclosed in Appendix.

4.2 Avoidance behaviour of Eisenia fetida

In the present study, it was found that there were no dead or missing worms in

the test for all the different concentrations of four chemicals used in experiments,

except at the highest concentration of Chlorpyrifos and Pendimethalin i.e. 1000 mg

a.i./kg of dry soil, where mortality was 100%. Therefore, this concentration was

excluded for the statistical analysis of avoidance behaviour.

When exposed to Chlorpyrifos, E. fetida showed a higher avoidance with higher

concentrations. Avoidance was seen 40% at very low concentration of 0.316 mg/kg.

55% avoidance (p < 0.05) was observed at 1 mg/kg. 75% avoidance was observed
at 3.16 mg/kg concentration and 60% avoidance was observed at 10 mg/kg. Earth-
worm showed more than 90% avoidance behaviour at highest three concentrations
(31.6, 100 and 316 mg/kg). Avoidance (%) of Eisenia fetida as well as mean and
standard deviation (SD) for different concentrations of Chlorpyrifos for four sam-
ples are given in Tables 4.1 and 4.2 respectively. Graphical representation of the
results of this experiment is shown in Figure 4.4. Experiment exhibited that avoid-
ance behaviour increased with higher concentration of Chlorpyrifos. The only vari-
ation was observed at 10 mg/kg concentration, where the avoidance found to be
little lower than previous concentration (3.16 mg/kg).

78
 Chapter 4. Results

For 2,4-DE chemical, the initial avoidance decreased with increase in concentra-
tion value upto 10 mg/kg and thereafter avoidance behaviour showed higher values

with higher concentration of the chemical. Avoidance was around 60% at 0.316
mg/kg and 1 mg/kg. Avoidance was seen 20% at 3.16 mg/kg. Minimum avoidance

of 5% was shown at 10 mg/kg. 25% avoidance was reflected at the concentration

of 31.6 mg/kg. The result of the experiment showed gradual increase for the re-
maining higher three concentrations and varied between 55% to 90% progressively.
Avoidance (%) of Eisenia fetida as well as mean and standard deviation (SD) for

different concentrations of 2,4-DE for four samples are given in Tables 4.3 and 4.4
respectively. Graphical representation of the results of this experiment is shown in
Figure 4.5.

For Triazophos, earthworm showed strong attraction behaviour 85% at 0.316

mg/kg of dry soil. Avoidance was minimum 10% at 1 mg/kg concentration. Avoid-

ance was 70% at 3.16 mg/kg. 85% avoidance was shown at 10 mg/kg. Avoidance
was 95% at concentrations of 100 mg/kg and 316 mg/kg. 100% avoidance was ob-

served in earthworms at the highest concentration of 1000 mg/kg. Avoidance (%)

of Eisenia fetida as well as mean and standard deviation (SD) for different concen-

trations of Triazophos for four samples are given in Tables 4.5 and 4.6 respectively.

Graphical representation of the results of this experiment is shown in Figure 4.6.

When exposed to Pendimethalin, E. fetida showed attraction response of 15%

and 10% at the very low concentrations of 0.316 mg/kg and 1 mg/kg respectively.
30% and 50% avoidance was observed in earthworms at concentration values of
3.16 mg/kg and 10 mg/kg respectively. Earthworm showed 70% avoidance be-
haviour at concentrations of 31.6 and 100 mg/kg. 80% avoidance was showed at
concentration 316 mg/kg of dry soil. Avoidance (%) of Eisenia fetida as well as
mean and standard deviation (SD) for different concentrations of Pendimethalin for
four samples are given in Tables 4.7 and 4.8 respectively. Graphical representation
of the results of this experiment is shown in Figure 4.7.

79
 Chapter 4. Results

Table 4.1: Avoidance (%) of Eisenia fetida for different concentrations of Chlor-
pyrifos

Chemical Replica 0.316 1 3.16 10 31.6 100 316 1000

name no. mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
Chlor- Sample 1 40 60 80 60 100 100 100 died
pyrifos Sample 2 40 60 80 80 80 100 100 died
Sample 3 60 60 60 40 80 100 80 died
Sample 4 20 40 80 60 100 80 100 died

Table 4.2: Mean and standard deviation (SD) of avoidance of Eisenia fetida for
different concentrations of Chlorpyrifos

Chemical Statistical 0.316 1 3.16 10 31.6 100 316 1000

name parameter mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
Chlor- Mean 40 55 75 60 90 95 95 –
pyrifos SD 16.33 10 10 16.33 11.55 10 10 –

Table 4.3: Avoidance (%) of Eisenia fetida for different concentrations of 2,4-DE

Chemical Replica 0.316 1 3.16 10 31.6 100 316 1000

name no. mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
2,4-DE Sample 1 60 60 20 00 20 60 60 80
Sample 2 60 60 00 20 40 40 80 100
Sample 3 80 40 40 00 20 60 60 80
Sample 4 40 60 20 00 20 60 60 100

Table 4.4: Mean and standard deviation (SD) of avoidance of Eisenia fetida for
different concentrations of 2,4-DE

Chemical Statistical 0.316 1 3.16 10 31.6 100 316 1000

name parameter mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
2,4-DE Mean 60 55 20 5 25 55 65 90
SD 16.33 10 16.33 10 10 10 10 11.55

80
 Chapter 4. Results

Figure 4.4: Avoidance behaviour of E. fetida in Chlorpyrifos with varying concen-

trations

Figure 4.5: Avoidance behaviour of E. fetida in 2,4-DE with varying concentrations

81
 Chapter 4. Results

Table 4.5: Avoidance (%) of Eisenia fetida for different concentrations of Tria-
zophos

Chemical Replica 0.316 1 3.16 10 31.6 100 316 1000

name no. mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
Tria- Sample 1 -80 00 80 100 100 100 100 100
zophos Sample 2 -80 20 60 80 100 80 100 100
Sample 3 -80 20 60 80 80 100 100 100
Sample 4 -100 00 60 80 80 80 80 100

Table 4.6: Mean and standard deviation (SD) of avoidance of Eisenia fetida for
different concentrations of Triazophos

Chemical Statistical 0.316 1 3.16 10 31.6 100 316 1000

name parameter mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
Tria- Mean -85 10 65 85 90 90 95 100
zophos SD 10 11.55 10 10 11.55 11.55 10 00

Table 4.7: Avoidance (%) of Eisenia fetida for different concentrations of

Pendimethalin
Chemical Replica 0.316 1 3.16 10 31.6 100 316 1000
name no. mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
Pendi- Sample 1 -20 00 40 40 60 60 80 died
methalin Sample 2 -20 -20 20 60 60 80 80 died
Sample 3 00 -20 20 40 80 60 60 died
Sample 4 -20 00 40 60 80 80 80 died

Table 4.8: Mean and standard deviation (SD) of avoidance of Eisenia fetida for
different concentrations of Pendimethalin
Chemical Statistical 0.316 1 3.16 10 31.6 100 316 1000
name parameter mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
Pendi- Mean -15 -10 30 50 70 70 75 –
methalin SD 10 11.55 11.55 11.55 11.55 11.55 10 –

82
 Chapter 4. Results

Figure 4.6: Avoidance behaviour of E. fetida in Triazophos with varying concentra-

tions

Figure 4.7: Avoidance behaviour of E. fetida in Pendimethalin with varying con-

centrations

83
 Chapter 4. Results

4.3 Impact of chemicals on the growth and reproduc-

tion of Eisenia fetida

The natural soil for the purpose of experimentation was collected from an aban-
doned area in Kota with no history of agricultural activity and hence no known

history of pesticides use. Sample soil of our experiments contained the pH 8.33,
conductivity 0.291 m Mhos/cm and organic carbon content is 0.03%. Soil color
was brown. This natural soil is represented as sample 4 in soil parameter test re-

port as received from Nanta agricultural farm (Office of Project Director, Crop) and
enclosed in Appendix.

4.3.1 Effect of Chlorpyrifos and 2,4-DE on growth and repro-

duction of Eisenia fetida

• Effect on growth

Chlorpyrifos caused decline in growth rates of the treated earthworms com-

pared to the growth rates of the control earthworms. After 28 days of expo-

sure, growth rate of the control earthworms was found 138% ± 19.1% while

growth reduced to 122.9% ± 9.8% in treated soil with concentration of 1.2

mg/kg. Growth rates were 80.4%±6.47%, 64.38% ±3.19%, 51.18% ±8.78%

and 13.8%±7.35% at the other concentration values of 3.79 mg/kg, 12 mg/kg,
37.9 mg/kg and 120 mg/kg respectively. The results of five samples for five
different concentrations of Chlorpyrifos on biomass growth of Eisenia fetida
together with biomass growth in control soil are shown in Table 4.9. Graphi-
cal results of effect on growth of Eisenia fetida due to Chlorpyrifos is shown
in Figure 4.8.

2,4 DE produced a sharp decline in the growth rates as compared to control.

This is found to be 89.54% ± 3.39%, 78.5% ± 7.83% and 78.66% ± 14.12%

84
 Chapter 4. Results

for concentration values of 0.75 mg/kg, 2.37 mg/kg and 7.5 mg/kg respec-
tively. Similarly, growth rates were 44.32% ± 5.47% and 26.36% ± 1.91%

for concentration values of 23.7 mg/kg and 75 mg/kg respectively. Table 4.10
shows the result of five samples for five different concentrations of 2,4-DE

on biomass growth of Eisenia fetida together with biomass growth in control

soil. Graphical results of effect on growth of Eisenia fetida due 2,4-DE is
shown in Figure 4.9.

Combined effects of these two chemicals have also been studied in our exper-

iments and their combined concentrations are reflected in Table 3.1. Growth
rate for combination no. 1 was found 80%±4.73%, while for combination no.
2, 3, 4 and 5 growth rates were 62.4%±3.12%, 43.6%±2.99%, 33.4%±3.35%

and 10.2% ± 1.84% respectively. Results are shown in Table 4.11. Graphical

results of effect on growth of Eisenia fetida due to Chlorpyrifos plus 2,4-DE

chemicals are shown in Figure 4.10.

• Effect on reproduction

The effect of Chlorpyrifos on reproduction was observed and found to be

drastically reduced as compared to control soil. After 56 days of exposure,

number of juveniles produced per earthworm in control soil was found 22.82±
2.72. In treated soil, number of juveniles reduced to 13.2 ± 0.69 with concen-

tration of 1.2 mg/kg Chlorpyrifos. Number of juveniles produced per earth-

worm were 6.52 ± 1.022, 4.7 ± 0.412, 3.86 ± 0.919 and 1.08 ± 0.104 at the
other concentration values of 3.79 mg/kg, 12 mg/kg, 37.9 mg/kg and 120
mg/kg respectively. The results of five samples for five different concentra-
tions of Chlorpyrifos on reproduction of Eisenia fetida together with repro-
duction in control soil are shown in Table 4.12. Graphical results of effect on
reproduction of Eisenia fetida due to Chlorpyrifos is shown in Figure 4.11.

2,4-DE also caused decline in number of juveniles produced per earthworm.

85
 Chapter 4. Results

This is found to be 8.96 ± 1.08, 11.41 ± 1.71, 8.82 ± 1.38, 7.96 ± 2.13 and
6.22 ± 0.222 for concentration values of 0.75 mg/kg, 2.37 mg/kg, 7.5 mg/kg,

23.7 mg/kg and 75 mg/kg respectively. Table 4.13 show the results of five
samples for five different concentrations of 2,4-DE on reproduction of Eisenia

fetida together with reproduction in control soil. Graphical results of effect

on reproduction of Eisenia fetida due to 2,4-DE is shown in Figure 4.12.

Combined effects of Chlorpyrifos and 2,4-DE on reproduction have also been

studied in our experiments. Number of juveniles produced per earthworm in

combination no. 1 was found 8.76 ± 0.845, while for combination no. 2, 3, 4

and 5, number of juveniles produced per earthworm were 7.7 ± 0.978, 5.04 ±
0.347, 3.9 ± 0.362 and 1.9 ± 0.439 respectively. Results show that combined

concentrations caused severe negative impact on growth and reproduction.

Results are shown in Table 4.14. Graphical results of effect on reproduction

of Eisenia fetida due to Chlorpyrifos plus 2,4-DE chemicals are shown in

Figure 4.13.

The test chemicals, Chlorpyrifos and 2,4-DE not only caused a decline in
the growth rate and number of juveniles reduced, but at higher concentra-

tions they also caused swelling on the earthworms body surface. The effect
of Chlorpyrifos is shown in Figure 4.14 and the effect of 2,4-DE is shown
in Figure 4.15. The cocoons of the earthworm and the emergence of the
earthworms from the cocoon are depicted in Figure 4.16 and Figure 4.17 re-
spectively. Figures 4.18 and 4.19 show the earthworm juveniles.

86
 Chapter 4. Results

Table 4.9: Effect of different concentrations of Chlorpyrifos on biomass growth of

Eisenia fetida

Chemical Replica Biomass Mean Standard Std. Lower

name and no. growth growth deviation error of and upper
concentration (%) (AvG) (SD) mean (SE) 95% CI mean
Control soil Sample 1 120 138.8 15.42 6.895 119.7
Sample 2 157 and
Sample 3 152 157.9
Sample 4 130
Sample 5 135
Chlorpyrifos Sample 1 112.9 122.9 7.877 3.523 113.1
1.2 Sample 2 126.5 and
mg/kg Sample 3 134 132.7
Sample 4 120
Sample 5 121
Chlorpyrifos Sample 1 74.6 80.4 6.063 2.711 72.87
3.79 Sample 2 85.5 and
mg/kg Sample 3 87.9 87.93
Sample 4 75
Sample 5 79
Chlorpyrifos Sample 1 64.8 64.38 2.566 1.147 61.19
12 Sample 2 68 and
mg/kg Sample 3 61.1 67.57
Sample 4 63
Sample 5 65
Chlorpyrifos Sample 1 39 51.18 7.072 3.163 42.4
37.9 Sample 2 57 and
mg/kg Sample 3 54.9 59.96
Sample 4 53
Sample 5 52
Chlorpyrifos Sample 1 5.7 13.8 5.92 2.647 6.449
120 Sample 2 18.3 and
mg/kg Sample 3 20 21.15
Sample 4 15
Sample 5 10

87
 Chapter 4. Results

Table 4.10: Effect of different concentrations of 2,4-DE on biomass growth of Eise-

nia fetida

Chemical Replica Biomass Mean Standard Std. Lower

name and no. growth growth deviation error of and upper
concentration (%) (AvG) (SD) mean (SE) 95% CI mean
Control soil Sample 1 120 138.8 15.42 6.895 119.7
Sample 2 157 and
Sample 3 152 157.9
Sample 4 130
Sample 5 135
2,4-DE Sample 1 93.5 89.54 2.734 1.223 86.15
0.75 Sample 2 90.3 and
mg/kg Sample 3 86.9 92.93
Sample 4 87
Sample 5 90
2,4-DE Sample 1 68 78.5 6.305 2.82 70.67
2.37 Sample 2 79 and
mg/kg Sample 3 80.5 86.33
Sample 4 85
Sample 5 80
2,4-DE Sample 1 98.9 78.66 11.37 5.085 64.54
7.5 Sample 2 75.2 and
mg/kg Sample 3 72.2 92.78
Sample 4 74
Sample 5 73
2,4-DE Sample 1 38.1 44.32 4.375 1.956 38.89
23.7 Sample 2 47.5 and
mg/kg Sample 3 49 49.75
Sample 4 45
Sample 5 42
2,4-DE Sample 1 25 26.36 1.533 0.6856 24.46
75 Sample 2 25.1 and
mg/kg Sample 3 28.7 28.27
Sample 4 26
Sample 5 27

88
 Chapter 4. Results

Table 4.11: Effect of different combined concentrations of Chlorpyrifos and 2,4-DE

on biomass growth of Eisenia fetida

Chemical Replica Biomass Mean Standard Std. Lower

name and no. growth growth deviation error of and upper
concentration (%) (AvG) (SD) mean (SE) 95% CI mean
Control soil Sample 1 120 138.8 15.42 6.895 119.7
Sample 2 157 and
Sample 3 152 157.9
Sample 4 130
Sample 5 135
Chlorpyrifos Sample 1 85 80 3.808 1.703 75.27
1.2 mg/kg Sample 2 80 and
+ Sample 3 75 84.73
2,4-DE Sample 4 82
0.75 mg/kg Sample 5 78
Chlorpyrifos Sample 1 65 62.4 2.51 1.122 59.28
3.79 mg/kg Sample 2 60 and
+ Sample 3 62 65.52
2,4-DE Sample 4 60
2.37 mg/kg Sample 5 65
Chlorpyrifos Sample 1 45 43.6 2.408 1.077 40.61
12 mg/kg Sample 2 42 and
+ Sample 3 47 46.59
2,4-DE Sample 4 41
7.5 mg/kg Sample 5 43
Chlorpyrifos Sample 1 35 33.4 2.702 1.208 30.05
37.9 mg/kg Sample 2 30 and
+ Sample 3 32 36.75
2,4-DE Sample 4 37
23.7 mg/kg Sample 5 33
Chlorpyrifos Sample 1 10 10.2 1.483 0.6633 8.358
120 mg/kg Sample 2 8 and
+ Sample 3 12 12.04
2,4-DE Sample 4 11
75 mg/kg Sample 5 10

89
 Chapter 4. Results

Figure 4.8: Growth rate of E. fetida for different concentrations of Chlorpyrifos

Figure 4.9: Growth rate of E. fetida for different concentrations of 2,4-DE

90
 Chapter 4. Results

Table 4.12: Effect of different concentrations of Chlorpyrifos on reproduction of

Eisenia fetida

Chemical Replica No. of Mean Standard Std. Lower

name and no. juveniles/ no. of juv. deviation error of and upper
concentration earthworm (AvJ) (SD) mean (SE) 95% CI mean
Control soil Sample 1 22.5 22.82 2.995 1.34 20.10
Sample 2 25 and
Sample 3 18.4 25.54
Sample 4 19
Sample 5 24.2
Chlorpyrifos Sample 1 12.6 13.2 0.5523 0.247 12.51
1.2 Sample 2 12.9 and
mg/kg Sample 3 13 13.89
Sample 4 14
Sample 5 13.5
Chlorpyrifos Sample 1 5.5 6.52 0.8228 0.368 5.498
3.79 Sample 2 7.6 and
mg/kg Sample 3 6.5 7.542
Sample 4 7
Sample 5 6
Chlorpyrifos Sample 1 4.3 4.7 0.3317 0.1483 4.288
12 Sample 2 4.8 and
mg/kg Sample 3 4.4 5.112
Sample 4 5
Sample 5 5
Chlorpyrifos Sample 1 3.8 3.86 0.7403 0.3311 2.941
37.9 Sample 2 5 and
mg/kg Sample 3 4 4.779
Sample 4 3
Sample 5 3.5
Chlorpyrifos Sample 1 1.1 1.08 0.0836 0.0374 0.976
120 Sample 2 1 and
mg/kg Sample 3 1.2 1.184
Sample 4 1
Sample 5 1.1

91
 Chapter 4. Results

Figure 4.10: Growth rate of E. fetida for different combined concentration labels of
Chlorpyrifos and 2,4-DE

Figure 4.11: No. of juveniles produced per earthworm (E. fetida) for different
concentrations of Chlorpyrifos

92
 Chapter 4. Results

Table 4.13: Effect of different concentrations of 2,4-DE on reproduction of Eisenia

fetida

Chemical Replica No. of Mean Standard Std. Lower

name and no. juveniles/ no. of juv. deviation error of and upper
concentration earthworm (AvJ) (SD) mean (SE) 95% CI mean
Control soil Sample 1 22.5 22.82 2.995 1.34 20.10
Sample 2 25 and
Sample 3 18.4 25.54
Sample 4 19
Sample 5 24.2
2,4-DE Sample 1 8.4 8.96 0.8735 0.3906 7.875
0.75 Sample 2 7.8 and
mg/kg Sample 3 9.1 10.04
Sample 4 9.5
Sample 5 10
2,4-DE Sample 1 11.9 11.14 1.374 0.6145 9.434
2.37 Sample 2 12 and
mg/kg Sample 3 8.8 12.85
Sample 4 11
Sample 5 12
2,4-DE Sample 1 7.1 8.82 1.112 0.4974 7.439
7.5 Sample 2 10 and
mg/kg Sample 3 8.5 10.2
Sample 4 9.5
Sample 5 9
2,4-DE Sample 1 6 7.96 1.718 0.7685 5.826
23.7 Sample 2 8.9 and
mg/kg Sample 3 10.4 10.09
Sample 4 7
Sample 5 7.5
2,4-DE Sample 1 6.5 6.22 0.1789 0.08 5.998
75 Sample 2 6.2 and
mg/kg Sample 3 6.2 6.442
Sample 4 6
Sample 5 6.2

93
 Chapter 4. Results

Table 4.14: Effect of different combined concentrations of Chlorpyrifos and 2,4-DE

on reproduction of Eisenia fetida

Chemical Replica No. of Mean Standard Std. Lower

name and no. juveniles/ no. of juv. deviation error of and upper
concentration earthworm (AvJ) (SD) mean (SE) 95% CI mean
Control soil Sample 1 22.5 22.82 2.995 1.34 20.10
Sample 2 25 and
Sample 3 18.4 25.54
Sample 4 19
Sample 5 24.2
Chlorpyrifos Sample 1 9.8 8.76 0.6804 0.3043 7.915
1.2 mg/kg Sample 2 9 and
+ Sample 3 8.5 9.605
2,4-DE Sample 4 8
0.75 mg/kg Sample 5 8.5
Chlorpyrifos Sample 1 9 7.7 0.7874 0.3521 6.722
3.79 mg/kg Sample 2 7.5 and
+ Sample 3 7.8 8.678
2,4-DE Sample 4 7
2.37 mg/kg Sample 5 7.2
Chlorpyrifos Sample 1 4.8 5.04 0.2793 0.1249 4.693
12 mg/kg Sample 2 5.3 and
+ Sample 3 5.3 5.387
2,4-DE Sample 4 5.1
7.5 mg/kg Sample 5 4.7
Chlorpyrifos Sample 1 4 3.9 0.2915 0.1304 3.538
37.9 mg/kg Sample 2 4.1 and
+ Sample 3 4.2 4.262
2,4-DE Sample 4 3.7
23.7 mg/kg Sample 5 3.5
Chlorpyrifos Sample 1 2.4 1.9 0.3536 0.1581 1.461
120 mg/kg Sample 2 2.1 and
+ Sample 3 1.5 2.339
2,4-DE Sample 4 1.7
75 mg/kg Sample 5 1.8

94
 Chapter 4. Results

Figure 4.12: No. of juveniles produced per earthworm (E. fetida) for different
concentrations of 2,4-DE

Figure 4.13: No. of juveniles produced per earthworm (E. fetida) for different
combined concentration labels of Chlorpyrifos and 2,4-DE

95
 Chapter 4. Results

Figure 4.14: Earthworm swelling and shrinking due to the effect of Chlorpyrifos

Figure 4.15: Earthworm swelling due to the effect of 2,4-DE

96
 Chapter 4. Results

Figure 4.16: Earthworm cocoons

Figure 4.17: Earthworm cocoons showing emergence of juvenile (view 1)

97
 Chapter 4. Results

Figure 4.18: Earthworm juveniles (view 1)

98
 Chapter 4. Results

Figure 4.19: Earthworm juveniles (view 2)

99
 Chapter 4. Results

4.3.2 Statistical analysis of effect of Chlorpyrifos and 2,4-DE

The statistical data of growth rate under influence of Chlorpyrifos, 2,4-DE and their

combined concentrations are presented in Table 4.15. Data shows that growth rate is
not significant in Chlorpyrifos for 95% confidence interval (CI) of mean. Whereas

for 2,4-DE and combined concentrations, results are significant for 95% CI of mean.
Correlation value for these chemicals lies in the range of -0.995 to -0.8579. This
ascertains the adverse effect on growth rate.
The statistical data showing the number of juveniles produced per earthworm

under influence of Chlorpyrifos, 2,4-DE and their combined concentrations is given

in Table 4.16. Results show that effect on number of juveniles produced per earth-
worm under influence of Chlorpyrifos and 2,4-DE is not significant with 95% CI

of mean. Whereas for combined concentrations, result is significant with 95% CI

of mean. Correlation values between number of juveniles produced per earthworm

and concentration lies in the range of -0.9920 to -0.7240. This ascertains the ad-
verse effect on number of juveniles produced per earthworm due to the effect of

chemicals.

Most of the P (two-tailed) values are closer to zero as reflected in Tables 4.15

and 4.16. This result ascertains negative effect of chemicals over important life
history parameters of earthworm. While Rsquared values are nearer to one. This
shows that data is closer to the fitted regression line around its mean.
The combined plot showing correlation between growth rate of earthworms and
number of juveniles produced per earthworm for different concentrations of Chlor-
pyrifos, 2,4-DE and their combined concentrations are shown in Figures 4.20, 4.21
and 4.22.

100
 Chapter 4. Results

Table 4.15: Statistical analysis of biomass growth in Eisenia fetida due to Chior-
pyrifos and 2,4-DE

Chemical Correlation 95% R P Significant

name Concentration v/s CI squared (two- (alpha=0.05)
Biomass growth (%) tailed) Confidence 95%
Chlorpyrifos -0.8579 -0.9905 to 0.7361 0.0629 No
0.1001
2,4-DE -0.9205 -0.9948 to 0.8473 0.0266 Yes
-0.2035
Chlorpyrifos -0.995 -0.9997 to 0.9901 0.0004 Yes
and 2,4-DE -0.9233

Table 4.16: Statistical analysis of reproduction in Eisenia fetida due to Chlorpyrifos

and 2,4-DE

Chemical Correlation 95% R P Significant

name Concentration v/s CI squared (two- (alpha=0.05)
Juvenile/earthworm tailed) Confidence 95%
Chlorpyrifos -0.7240 -0.9802 to 0.5241 0.1667 No
0.4382
2,4-DE -0.8488 -0.9898 to 0.7204 0.0690 No
0.1333
Chlorpyrifos -0.992 -0.9995 to 0.9841 0.0009 Yes
and 2,4-DE -0.8795

101
 Chapter 4. Results

Figure 4.20: Correlation between growth rate and no. of juveniles produced per
earthworm for different concentrations of Chlorpyrifos

Figure 4.21: Correlation between growth rate and no. of juveniles produced per
earthworm for different concentrations of 2,4-DE

102
 Chapter 4. Results

Figure 4.22: Correlation between growth rate and no. of juveniles produced per
earthworm for different combined concentration labels of Chlorpyrifos and 2,4-DE

103
 Chapter 4. Results

4.3.3 Effect of Triazophos and Pendimethalin on growth and re-

production of Eisenia fetida

• Effect on growth

Triazophos caused decline in growth rates of the treated earthworms com-

pared to the growth rates of the control earthworms. After 28 days of ex-
posure, growth rate of the control earthworms was found 153% ± 20.9%
while in treated soil with concentration of 0.158 mg/kg growth reduced to
97.94% ± 4.04%. At the other concentration values of 0.50 mg/kg, 1.58

mg/kg, 5 mg/kg and 15.8 mg/kg, growth rates were 93.6% ± 12.6%, 69.6% ±

4.53%, 40% ± 4.73% and 13.6% ± 7.58% respectively. The results of five

samples for five different concentrations of Triazophos on biomass growth of

Eisenia fetida together with biomass growth in control soil are shown in Ta-

ble 4.17. Graphical results of the effect on growth of Eisenia fetida exposed

to Triazophos is shown in Figure 4.23.

Pendimethalin also produced a sharp decline in the growth rates. The growth

rate at concentration values of 0.474 mg/kg, 1.5 mg/kg, 4.74 mg/kg, 15 mg/kg
and 47.4 mg/kg is found to be 38.88% ± 11.45%, 25.48% ± 2.29%, 20.54% ±
4.70%, 19.68% ± 6.35% and 10.2% ± 4.99% respectively. Triazophos caused

lesser decline in growth rate as compared to Pendimethalin. The result of five

samples for five different concentrations of Pendimethalin on growth of Eise-

nia fetida together with growth in control soil are shown in Table 4.18. Graph-
ical results of the effect on growth of Eisenia fetida due to Pendimethalin is
shown in Figure 4.24.

Combined effects of these two chemicals have also been studied in our exper-
iments and their combined concentrations are given in Table 3.2. Growth rate
for combination no. 1 was found 106.3% ± 4.1%, while for combination no.
2, 3, 4 and 5 growth rates were 69.96% ± 4.97%, 57% ± 4.56%, 40% ± 4.73%

104
 Chapter 4. Results

and 11% ± 1.96% respectively. Tabular results are shown in Table 4.19.
Graphical results of effect on growth of Eisenia fetida due to Triazophos plus

Pendimethalin chemicals are shown in Figure 4.25.

• Effect on reproduction

The effect on reproduction has been found to be drastically reduced as com-

pared to control soil. After 56 days of exposure, number of juveniles produced
per earthworm in control soil was found 20.2 ± 3.21, while in soil treated
with concentration of 0.158 mg/kg Triazophos, number of juveniles reduced

to 8.68 ± 1.41. At the other concentration values of 0.50 mg/kg, 1.58 mg/kg,

5 mg/kg and 15.8 mg/kg, number of juveniles produced per earthworm were

7.5 ± 0.47, 5.9 ± 0.8, 4.04 ± 0.45 and 2.1 ± 0.53 respectively. The results

for five different concentrations (five samples) of Triazophos on reproduction

of Eisenia fetida together with reproduction in control soil are shown in Ta-

ble 4.20. Graphical results of effect on reproduction of Eisenia fetida due to

Triazophos is shown in Figure 4.26.

Pendimethalin also caused decline in number of juveniles produced per earth-

worm. For concentration values of 0.474 mg/kg, 1.5 mg/kg, 4.74 mg/kg, 15
mg/kg and 47.4 mg/kg, this is found to be 5.84 ± 0.69, 4.1 ± 0.36, 3.28 ±
0.87, 2.82 ± 1.23 and 1.38 ± 0.78 respectively. Triazophos caused lesser
decline in juvenile numbers as compared to Pendimethalin. The results for

five different concentrations (five samples) of Pendimethalin on reproduction

of Eisenia fetida together with reproduction in control soil is shown in Ta-
ble 4.21. Graphical results of effect on reproduction of Eisenia fetida due to
Pendimethalin is shown in Figure 4.27.

Combined effect of Triazophos and Pendimethalin on reproduction have also

been studied in our experiments. Number of juveniles produced per earth-
worm in combination no. 1 was found 7.26 ± 2.11, while for combina-

105
 Chapter 4. Results

tion no. 2, 3, 4 and 5, number of juveniles produced per earthworm were

6.2 ± 0.71, 3.32 ± 0.62, 2.5 ± 0.34 and 1.7 ± 0.5 respectively. Results show

that combined concentrations caused severe negative impact on growth and

reproduction. Result of the combined effects of these two chemicals on repro-

duction are shown in Table 4.22. Graphical results of effect on reproduction

of Eisenia fetida due to Triazophos plus Pendimethalin chemicals are shown
in Figure 4.28.

Figure 4.29 shows swelling on the body surface of earthworm when exposed

to higher concentration of Triazophos and Figure 4.30 shows lesions on earth-

worm body due to the effect of higher concentration of Pendimethalin. Fig-
ure 4.31 shows emergence of juvenile. Figures 4.32 and 4.33 show juveniles

of earthworms during counting process done after 56 days.

106
 Chapter 4. Results

Table 4.17: Effect of different concentrations of Triazophos on biomass growth of

Eisenia fetida

Chemical Replica Biomass Mean Standard Std. Lower

name and no. growth growth deviation error of and upper
concentration (%) (AvG) (SD) mean (SE) 95% CI mean
Control soil Sample 1 161 153.3 16.81 7.52 132.4
Sample 2 130.9 and
Sample 3 140 174.2
Sample 4 166.6
Sample 5 168
Triazophos Sample 1 101 97.94 3.251 1.454 93.9
0.158 Sample 2 100.7 and
mg/kg Sample 3 99 102
Sample 4 95
Sample 5 94
Triazophos Sample 1 93 93.6 10.14 4.534 81.01
0.5 Sample 2 111 and
mg/kg Sample 3 90 106.2
Sample 4 85
Sample 5 89
Triazophos Sample 1 65 69.6 3.647 1.631 65.07
1.58 Sample 2 68 and
mg/kg Sample 3 70 74.13
Sample 4 75
Sample 5 70
Triazophos Sample 1 45 40 3.808 1.703 35.27
5 Sample 2 42 and
mg/kg Sample 3 40 44.73
Sample 4 38
Sample 5 35
Triazophos Sample 1 10 13.6 6.107 2.731 6.017
15.8 Sample 2 15 and
mg/kg Sample 3 18 21.18
Sample 4 20
Sample 5 5

107
 Chapter 4. Results

Table 4.18: Effect of different concentrations of Pendimethalin on biomass growth

of Eisenia fetida

Chemical Replica Biomass Mean Standard Std. Lower

name and no. growth growth deviation error of and upper
concentration (%) (AvG) (SD) mean (SE) 95% CI mean
Control soil Sample 1 161 153.3 16.81 7.52 132.4
Sample 2 130.9 and
Sample 3 140 174.2
Sample 4 166.6
Sample 5 168
Pendimethalin Sample 1 31 38.88 9.218 4.122 27.43
0.474 Sample 2 53 and
mg/kg Sample 3 33.7 50.33
Sample 4 43.4
Sample 5 33.3
Pendimethalin Sample 1 27.4 25.48 1.843 0.824 23.19
1.5 Sample 2 24 and
mg/kg Sample 3 24.8 27.77
Sample 4 27.5
Sample 5 23.7
Pendimethalin Sample 1 19.2 20.54 3.787 1.694 15.84
4.74 Sample 2 19.5 and
mg/kg Sample 3 27.2 25.24
Sample 4 17.7
Sample 5 19.1
Pendimethalin Sample 1 28.5 19.68 5.117 2.289 13.33
15 Sample 2 19.6 and
mg/kg Sample 3 16 26.03
Sample 4 17.7
Sample 5 16.6
Pendimethalin Sample 1 9.1 10.2 3.874 1.732 5.39
47.4 Sample 2 16.5 and
mg/kg Sample 3 6 15.01
Sample 4 9
Sample 5 10.4

108
 Chapter 4. Results

Table 4.19: Effect of different combined concentrations of Triazophos and

Pendimethalin on biomass growth of Eisenia fetida

Chemical Replica Biomass Mean Standard Std. Lower

name and no. growth growth deviation error of and upper
concentration (%) (AvG) (SD) mean (SE) 95% CI mean
Control soil Sample 1 161 153.3 16.81 7.52 132.4
Sample 2 130.9 and
Sample 3 140 174.2
Sample 4 166.6
Sample 5 168
Triazophos Sample 1 108 106.3 3.324 1.487 102.2
0.158 mg/kg Sample 2 108.6 and
+ Sample 3 108.8 110.4
Pendimethalin Sample 4 105
0.474 mg/kg Sample 5 101
Triazophos Sample 1 65.7 69.94 3.757 1.68 65.27
0.5 mg/kg Sample 2 70 and
+ Sample 3 75 74.61
Pendimethalin Sample 4 72
1.5 mg/kg Sample 5 67
Triazophos Sample 1 55 57 3.674 1.643 52.44
1.58 mg/kg Sample 2 52 and
+ Sample 3 57 61.56
Pendimethalin Sample 4 61
4.74 mg/kg Sample 5 60
Triazophos Sample 1 45 40 3.808 1.703 35.27
5 mg/kg Sample 2 42 and
+ Sample 3 40 44.73
Pendimethalin Sample 4 38
15 mg/kg Sample 5 35
Triazophos Sample 1 10 11 1.581 0.7071 9.037
15.8 mg/kg Sample 2 12 and
+ Sample 3 13 12.96
Pendimethalin Sample 4 11
47.4 mg/kg Sample 5 9

109
 Chapter 4. Results

Figure 4.23: Growth rate of E. fetida for different concentrations of Triazophos

Figure 4.24: Growth rate of E. fetida for different concentrations of Pendimethalin

110
 Chapter 4. Results

Table 4.20: Effect of different concentrations of Triazophos on reproduction of

Eisenia fetida

Chemical Replica No. of Mean Standard Std. Lower

name and no. juveniles/ no. of juv. deviation error of and upper
concentration earthworm (AvJ) (SD) mean (SE) 95% CI mean
Control soil Sample 1 20.1 20.2 2.586 1.156 16.99
Sample 2 21.2 and
Sample 3 16 23.41
Sample 4 23.4
Sample 5 20.7
Triazophos Sample 1 7.1 8.68 1.139 0.5093 7.266
0.158 Sample 2 9.3 and
mg/kg Sample 3 9 10.09
Sample 4 10
Sample 5 8
Triazophos Sample 1 7.5 7.5 0.3808 0.1703 7.027
0.5 Sample 2 8 and
mg/kg Sample 3 7 7.973
Sample 4 7.3
Sample 5 7.7
Triazophos Sample 1 5.5 5.9 0.6519 0.2915 5.091
1.58 Sample 2 5 and
mg/kg Sample 3 6.5 6.709
Sample 4 6
Sample 5 6.5
Triazophos Sample 1 4 4.04 0.3647 0.1631 3.587
5 Sample 2 4.2 and
mg/kg Sample 3 4.5 4.493
Sample 4 4
Sample 5 3.5
Triazophos Sample 1 2 2.1 0.4301 0.1924 1.566
15.8 Sample 2 2.2 and
mg/kg Sample 3 2.1 2.634
Sample 4 1.5
Sample 5 2.7

111
 Chapter 4. Results

Figure 4.25: Growth rate of E. fetida for different combined concentration labels of
Triazophos and Pendimethalin

Figure 4.26: No. of juveniles produced per earthworm (E. fetida) for different
concentrations of Triazophos

112
 Chapter 4. Results

Table 4.21: Effect of different concentrations of Pendimethalin on reproduction of

Eisenia fetida

Chemical Replica No. of Mean Standard Std. Lower

name and no. juveniles/ no. of juv. deviation error of and upper
concentration earthworm (AvJ) (SD) mean (SE) 95% CI mean
Control soil Sample 1 20.1 20.2 2.586 1.156 16.99
Sample 2 21.2 and
Sample 3 16 23.41
Sample 4 23.4
Sample 5 20.7
Pendimethalin Sample 1 6.5 5.84 0.555 0.2482 5.151
0.474 Sample 2 5 and
mg/kg Sample 3 5.7 6.529
Sample 4 6.1
Sample 5 5.9
Pendimethalin Sample 1 4.2 4.1 0.2915 0.1304 3.738
1.5 Sample 2 4.5 and
mg/kg Sample 3 4 4.462
Sample 4 3.7
Sample 5 4.1
Pendimethalin Sample 1 2.4 3.28 0.6979 0.3121 2.413
4.74 Sample 2 2.7 and
mg/kg Sample 3 4 4.147
Sample 4 3.5
Sample 5 3.8
Pendimethalin Sample 1 1.3 2.82 0.9834 0.4398 1.599
15 Sample 2 3 and
mg/kg Sample 3 2.5 4.041
Sample 4 3.8
Sample 5 3.5
Pendimethalin Sample 1 1.9 1.38 0.6301 0.2818 0.5977
47.4 Sample 2 1 and
mg/kg Sample 3 0.5 2.162
Sample 4 2
Sample 5 1.5

113
 Chapter 4. Results

Table 4.22: Effect of different combined concentrations of Triazophos and

Pendimethalin on reproduction of Eisenia fetida

Chemical Replica No. of Mean Standard Std. Lower

name and no. juveniles/ no. of juv. deviation error of and upper
concentration earthworm (AvJ) (SD) mean (SE) 95% CI mean
Control soil Sample 1 20.1 20.2 2.586 1.156 16.99
Sample 2 21.2 and
Sample 3 16 23.41
Sample 4 23.4
Sample 5 20.7
Triazophos Sample 1 8.7 7.26 1.704 0.762 5.144
0.158 mg/kg Sample 2 8.6 and
+ Sample 3 4.5 9.376
Pendimethalin Sample 4 7
0.474 mg/kg Sample 5 7.5
Triazophos Sample 1 7 6.2 0.5701 0.255 5.492
0.5 mg/kg Sample 2 6 and
+ Sample 3 6.5 6.908
Pendimethalin Sample 4 5.5
1.5 mg/kg Sample 5 6
Triazophos Sample 1 3.8 3.32 0.497 0.2223 2.703
1.58 mg/kg Sample 2 3.9 and
+ Sample 3 2.8 3.937
Pendimethalin Sample 4 3
4.74 mg/kg Sample 5 3.1
Triazophos Sample 1 2.5 2.5 0.2739 0.1225 2.16
5 mg/kg Sample 2 2.4 and
+ Sample 3 2.7 2.84
Pendimethalin Sample 4 2.8
15 mg/kg Sample 5 2.1
Triazophos Sample 1 1.7 1.7 0.4062 0.1817 1.196
15.8 mg/kg Sample 2 2.3 and
+ Sample 3 1.8 2.204
Pendimethalin Sample 4 1.5
47.4 mg/kg Sample 5 1.2

114
 Chapter 4. Results

Figure 4.27: No. of juveniles produced per earthworm (E. fetida) for different
concentrations of Pendimethalin

Figure 4.28: No. of juveniles produced per earthworm (E. fetida) for different
combined concentration labels of Triazophos and Pendimethalin

115
 Chapter 4. Results

Figure 4.29: Earthworm swelling due to the effect of Triazophos

Figure 4.30: Earthworm showing lesions due to the effect of Pendimethalin

116
 Chapter 4. Results

Figure 4.31: Earthworm cocoons showing emergence of juvenile (view 2)

117
 Chapter 4. Results

Figure 4.32: Earthworm juveniles (view 3)

118
 Chapter 4. Results

Figure 4.33: Earthworm juveniles (view 4)

119
 Chapter 4. Results

4.3.4 Statistical analysis of effect of Triazophos and Pendimethalin

The statistical data of growth rate under influence of Triazophos, Pendimethalin

and their combined concentration are presented in Table 4.23. Data shows that
effect on growth rate is significant in Triazophos and combined concentrations for

95% confidence interval (CI) of mean. Whereas for Pendimethalin, results are not
significant for 95% CI of mean. Correlation value for these chemicals lies in the
range of -0.986 to -0.7947. This again ascertains the adverse effect on growth rate.
The statistical data showing the number of juveniles produced per earthworm

under influence of Triazophos, Pendimethalin and their combined concentrations

is given in Table 4.24. Results shows that effect on number of juveniles produced
per earthworm under influence of Pendimethalin is not significant with 95% CI of

mean. Whereas for Triazophos and combined concentrations, results are significant

with 95% CI of mean. Correlation values between number of juveniles produced

per earthworm and concentration lies in the range of -0.9714 to -0.8403. This as-
certains the adverse effect on number of juveniles produced per earthworm in our

experiments.

Most of the P (two-tailed) values are closer to zero as reflected in Tables 4.23

and 4.24. This result ascertains negative effect of chemicals over important life
history parameters of earthworm. While Rsquared values are nearer to one. This
shows that data is closer to the fitted regression line around its mean.
The combined plot showing correlation between growth rate of earthworms
and number of juveniles produced per earthworm for different concentrations of
Triazophos, Pendimethalin and their combined concentrations are shown in Fig-
ures 4.34, 4.35 and 4.36.

120
 Chapter 4. Results

Table 4.23: Statistical analysis of biomass growth in Eisenia fetida due to Tria-
zophos and Pendimethalin

Chemical Correlation 95% R P Significant

name Concentration v/s CI squared (two- (alpha=0.05)
Biomass growth (%) tailed) Confidence 95%
Triazophos -0.9172 -0.9946 to 0.8413 0.0282 Yes
-0.1832
Pendimethalin -0.7947 -0.9858 to 0.6316 0.1082 No
0.2930
Triazophos -0.986 -0.9991 to 0.9722 0.0020 Yes
and Pendimethalin -0.7977

Table 4.24: Statistical analysis of reproduction in Eisenia fetida due to Triazophos

and Pendimethalin
Chemical Correlation 95% R P Significant
name Concentration v/s CI squared (two- (alpha=0.05)
Juvenile/earthworm tailed) Confidence 95%
Triazophos -0.9005 -0.9935 to 0.8109 0.0371 Yes
-0.0887
Pendimethalin -0.8403 -0.9892 to 0.7061 0.0747 No
0.1622
Triazophos -0.9714 -0.9982 to 0.9435 0.0058 Yes
and Pendimethalin -0.6229

121
 Chapter 4. Results

Figure 4.34: Correlation between growth rate and no. of juveniles produced per
earthworm for different concentrations of Triazophos

Figure 4.35: Correlation between growth rate and no. of juveniles produced per
earthworm for different concentrations of Pendimethalin

122
 Chapter 4. Results

Figure 4.36: Correlation between growth rate and no. of juveniles produced
per earthworm for different combined concentration labels of Triazophs and
Pendimethalin

123
Chapter 5

Discussion

5.1 Identification of earthworm species in Kota

Survey of the areas revealed that earthworms were found only in the agricultural

areas of Kota i.e., Bundi road, Rangpur road and Baran road. As Rawatbhata road,
Jhawalwar road and Abhera area constitute the rocky terrain and also because of

closed proximity of Abhera to the Thermal plant, no earthworms were found. Eise-

nia fetida was found to be the prominent species in all the agricultural areas around

Kota.
Eisenia fetida also known as the red wiggler, brandling worm, dung worm, or
the tiger worm, is found extensively not only in the ground but also in various
habitats around the world. This is a common worm associated with garbage and
waste and used also as a fishing bait. It is also probably the most widely used worm
for vermicomposting. Eisenia fetida is the standard test organism used in terrestrial
ecotoxicology, because it can easily be bred on a variety of organic wastes with short
generation times (ISO 1998, OECD 1984 and 2004). Although, earthworm species
vary in their tolerance, reports have shown a decline in the earthworm populations
in response to large amounts of organic chemical deposition (Bayer and Foy 1982).
The anecic earthworm, i.e. Lampito mauritii, is commonly found in Indian
 Chapter 5. Discussion

soils, used as an efficient tool for organic waste reduction (Tripathi and Bharad-
waj 2004). The composting efficiency and biology of Lampito mauritii, is well

documented in literature. Several authors have reported the vermicomposting po-

tential of Lampito mauritii by using a variety of organic wastes (Manivannan 2005,

Suthar and Singh 2008). It can withstand wide range of temperature, soil moisture
and various other physical factors (Kale 1988).
Perionyx excavatus (Perrier, 1872), is a beautiful worm with an iridescent blue
or violet sheen to its skin clearly visible under bright light. This species is mainly

found in tropical regions, especially in Asia (Blakemore et al., 2006) and is also
present in Europe and North America (Edwards et al., 1998). Although primarily a
compost worm, it is commonly found in the top soil layer (0-15 cm) at temperatures

ranging between 20.8o C and 28.8o C and pH of 6.4-7.4 as discussed by Bhattachar-

jee and Chaudhuri (2002). Hallatt et al. (1990), Joshi and Dabral (2008) and

Reinecke and Hallatt (1989) studied the life cycle and biology of this species ex-
tensively. This species makes excellent fishing bait. Like all tropical worm species,

this species has a very poor tolerance for low temperatures, fluctuations in their

environment or disruption to the system.

5.2 Avoidance behaviour of Eisenia fetida

It has been well established that earthworms are suitable biomarkers for the assess-
ment of soil quality. The receptors present on their body are extremely sensitive
to the changes in pH, temperature and chemicals. Hence, avoidance behaviour
of earthworms for the two chemicals used for wheat crop and the two chemicals
for soybean crop were conducted. Results show that at the lowest concentration
of Triazophos and lower two concentrations of Pendimethalin, attraction towards
the chemicals was noticed but the earthworms exhibited a very strong avoidance
at higher concentration of all four chemicals. Avoidance response was higher in

125
 Chapter 5. Discussion

Chlorpyrifos and Triazophos in comparison to 2,4-DE and Pendimethalin for higher

concentration values. The results show a direct link between the concentrations of

chemicals and avoidance behaviour. In general, there is a gradual increase in avoid-

ance behaviour with increase in dose concentration. Numerous research papers

have presented study on avoidance behaviour of Eisenia fetida for Chlorpyrifos.

Whereas, avoidance behaviour of Eisenia fetida for 2,4-DE chemical has not been
attempted much by researchers.
For the organophosphate Chlorpyrifos, Eisenia fetida proved to be a potentially

suitable species to assess the soil contamination. In our study, Chlorpyrifos showed
increased avoidance response with higher contaminant levels. Garcia-santos et
al. (2011) reported that Eisenia fetida showed increased avoidance response with

higher contaminant levels of Chlorpyrifos. Comparable trends of avoidance be-

haviour was reported at higher levels of concentrations of Chlorpyrifos using Eise-

nia fetida (Zhou et al., 2007) and by De Silva et al. (2009) using Eisenia andrei
in a two compartment system for natural tropical soil from Sri Lanka. De Silva et

al. (2009) observed that Perionyx excavatus was significantly attracted by Chlor-

pyrifos at the lowest three concentrations (1-10 mg a.i./ kg dry soil), whereas such

an effect was not seen for Eisenia andrei. When neglecting this negative avoidance

behaviour and focusing on the avoidance of Chlorpyrifos in OECD (1984) artificial

and natural soil, Eisenia andrei was 2.5 and 2.0 times more sensitive, respectively,

than Perionyx Excavatus.

In case of 2,4-DE, we found that Eisenia fetida showed decrement in avoid-
ance behaviour as the concentration of 2,4-DE increases upto 10 mg/kg. Avoidance
behaviour at this concentration was only 5%. Beyond this concentration, the avoid-
ance behaviour increased as the concentration of 2,4-DE increased and observed
value was 90% at 1000 mg/kg concentration. It seems that the concentration of
2,4-DE at around 10 mg/kg is favourable to Eisenia fetida as comparison to other
concentration in our experimentation.

126
 Chapter 5. Discussion

Study on avoidance behaviour of Eisenia fetida for Triazophos and Pendimethalin

is not available in literature. This may be due to heavy use of these chemicals

in our study area as compared to other areas and other countries. At lowest con-
centration of Triazophos and Pendimethalin, attraction behaviour was observed in

Eisenia fetida. This type of attraction behaviour in earthworms was also observed
by other researchers. Li et al. (2015) reported that at concentration of 0.1 mg/kg
of enrofloxacin, Eisenia fetida showed attraction behaviour. Garcia et al. (2008)
observed that tropical Eisenia fetida in TAS soil and in LUFA soil indicate a sig-

nificant avoidance behaviour of earthworms at concentrations > 1.0 mg a.i./kg for

fungicide carbendazim. While in artificial OECD (1984) soil, a significant attrac-
tion was observed at the lowest concentration (1.0 mg a.i./kg). De Silva and Gestel

(2009) found same type of attraction behaviour with Perionyx excavatus at the low-

est three concentrations (110 mg/kg dry soil) by Chlorpyrifos. Eisenia andrei also

showed same attraction behaviour with carbofuran (Bucha et al. 2013). Marques
et al. (2009) found that Eisenia andrei showed attraction behaviour at concentra-

tion of 31.7 mg/kg of formulated herbicide Mikado (a.i. is sulcotrione). Alves et al.

(2013) observed that more worms were found in the polluted compartments of thi-

ametoxam, fungicides captan and carboxin plus thiram, at the lowest concentrations

than in the control compartments. However, TAS treated with higher concentrations
of these three pesticides was avoided by the worms. They also reported the same

type of strong attraction by Eisenia andrei with fipronil, carboxin plus thiram, cap-
tan and thiametoxam chemicals. All these findings support our present study.

5.3 Impact of chemicals on the growth and reproduc-

tion of Eisenia fetida

The sensitivity of earthworm to chemicals is well established, hence the experiments

were conducted to study the effect of most commonly used pesticides and herbicides

127
 Chapter 5. Discussion

for wheat and soybean crop on the growth and reproduction of Eisenia fetida. Since,
meagre work is available on 2,4-DE, Triazophos and Pendimethalin, the results of

the present study are discussed in light of the Eisenia fetida affected with other
chemicals.

Statistical analysis of Chlorpyrifos showed that it’s effect on growth rate and re-
production of Eisenia fetida is not significant for 95% confidence interval of mean,
but Chlorpyrifos showed negative impact on growth rate and reproduction of Eise-
nia fetida. This type of finding were also discussed by other scientists in their

research work. Zhou et al. (2007) observed that Chlorpyrifos has negative effect
on growth and reproduction of Eisenia fetida. They also found that except 5 mg/kg
Chlorpyrifos, in all concentrations (10, 20, 40 and 60 mg/kg) growth deffered from

control, as the concentration increased growth was decreased. Effect of Chlorpyri-

fos was also negative on reproduction, with significant decrease in reproduction at

the lowest concentration. Similar type of behaviour was also reflected from our
study. Zhou et al. (2011) also reported that Chlorpyrifos siginificantly affected

the growth rate and reproduction of earthworm Eisenia fetida andrei. Reproductive

rates in all concentrations were found lower than the control group. These find-

ing confirms the results of present investigation. Booth et al. (2000) also assessed

the effect of Chlorpyrifos on Aporrectodea caliginosa and observed that earthworm

weight was greatly reduced after four weeks of exposure of Chlorpyrifos. They

also reported that cocoon production and hatching success were also reduced sig-
nificantly by pesticide exposure which support the present findings. Santos et al.
(2012) investigated the effect of Chlorpyrifos on Eisenia andrei which found similar
type of observation as we found in present study with Eisenia fetida that there was
gradual decrease in number of juveniles produced as the concentration of Chlor-
pyrifos in soil increased.
Statistical analysis of 2,4-DE showed that for Eisenia fetida, it’s effect on growth
rate is significant for 95% confidence interval of mean but it is not significant for

128
 Chapter 5. Discussion

reproduction. In case of 2,4-DE, we found in our study that at concentrations 0.75,

2.37 and 7.5 mg/kg, growth rate was almost similar. Such type of behaviour is

also seen in case of reproduction where at concentrations values of 0.75 and 7.5
mg/kg, number of juveniles per earthworms produced were almost similar (approx.

9) while at 2.37 mg/kg it was 11.14. This seems that 2.37 mg/kg concentration of
2,4-DE is more favorable as compared to recommended agriculture dose, though
even this concentration has negative impact on growth and reproduction of Eisenia
fetida as compared to control. Correia and Moreira (2010) observed similar type

of reduction in growth rate of Eisenia fetida as compared to control due to 2,4-D.

They observed no cocoon or juveniles in any experiment using soil containing 2,4-
D with concentrations of 10, 100, 500 and 1000 mg/kg. These findings are not in

an ageement with the present study. In our experiment, we observed juveniles in

all concentartions under study. This is due very high concentrations ( 100, 500 and

1000 mg/kg) used by the researchers. No juveniles were found even at concentration
of 10 mg/kg in their experimentation. This is in contrast to our results. Hattab

et al. (2015) studied the effect of 2,4-D on Eisenia andrei and observed that at

concentration of 7 and 14 mg/kg 2,4-D significantly reduced the body weight of

Eisenia andrei after exposure for 7 and 14 days. This result shows similarity with

present study in which 2,4-DE also reduced the body weight of Eisenia fetida after
exposure to the chemical.

Results showed that effect of 2,4-DE on growth of earthworms is more as com-

pared to Chlorpyrifos for initial two lower concentrations and their combined effect
is more severe as compared to their individual effect. Chlorpyrifos, 2,4-DE and their
combined concentrations show gradual fall in growth rate of biomass with increase
in level of concentration. In case of study on reproduction, Chlorpyrifos was found
to be more toxic than 2,4-D except at the lower concentration. In case of 2,4-DE,
lowest concentration (0.75 mg/kg) shows more negative impact than next concen-
tration i.e. 2.37 mg/kg. Further increase in concentration of 2,4-DE decreases the

129
 Chapter 5. Discussion

number of juveniles produced per earthworm.

While studying the combined effect of Chlorpyrifos and 2,4-DE, it has been

observed that their effect on growth rate and reproduction of Eisenia fetida is sig-
nificant for 95% confidence interval of mean. Analysis of these results reflects that

used insecticide and herbicide (Chlorpyrifos and 2,4-DE) showed a negative impact
on growth rate and reproduction. Effect of their combined doses is more toxic in
comparison to Chlorpyrifos and 2,4-DE individually. Overall, all these chemicals
give negative values of correlation coefficient with respect to different values of

chemical concentrations.
In the present study, combined effect of Chlorpyrifos and 2,4-DE was found
more toxic than their individual effect on growth and reproduction of Eisenia fetida.

This type of synergistic effects are supported by finding of Yasmin and D’Souza

(2007). They found that the combined effect of carbendazim, glyphosate and dimeth-

oate was more toxic in comparison to their individual effect on the growth and re-
production of Eisenia fetida. This type of effect also confirmed by Zhou et al.

(2011). In their experiment, they found that combined effect of Chlorpyrifos and

cypermethrin was more harmful than their individual effect on the growth and re-

production of Eisenia fetida. Yang et al. (2015) found that mixture of Chlorpyrifos

and atrazine was antagonistic towards Eisenia fetida.

Statistical analysis of Triazophos showed that it’s effect on growth rate and re-

production of Eisenia fetida is significant for 95% confidence interval of mean. On

the other hand, effect of Pendimethalin on growth rate and reproduction of Eise-
nia fetida is not significant. In the study of combined effect of Triazophos and
Pendimethalin, it has been observed that their effect on growth rate and reproduc-
tion of Eisenia fetida is significant. Analysis of these results reflects that used in-
secticide and herbicide (Triazophos and Pendimethalin) show a negative impact on
growth rate and reproduction. Effect of Pendimethalin is more toxic in compari-
son to Triazophos and their combined doses. Overall, all these chemicals mostly

130
 Chapter 5. Discussion

give negative values of correlation coefficient with respect to different values of

chemical concentrations. In our study, we observed that same level of concen-

tration of Pendimethalin (when used as a sole chemical) shows more toxicity as

compared to its combined dose with Triazophos. It might be due to masking effect

of Triazophos over Pendimethalin. At Pendimethalin dose levels of 4.74, 15 and

47.4 mg/kg, growth rate was reduced significantly. On the other hand, Triazophos
caused gradual fall in growth rate. Almost similar behaviour is observed with com-
bined concentrations. Experiment on the study of number of juveniles produced per

earthworm shows a sharp decline in soil treated with Triazophos and Pendimethalin
as compared to control soil but there was no clear dose-response relationship be-
tween the substrate concentrations and the mean number of juveniles produced per

earthworm.

The effect of these chemicals on earthworm species has not been discussed by

other researchers in literature but they have discussed similar negative effect due to
others chemicals on Eisenia fetida. Vermulen et al. (2001) found that mancozeb

had no significant negative effect on either growth or reproduction of Eisenia fetida

at recommended dose (8 mg/kg) or at an estimated environmental concentration

(44 mg/kg), which is not in an agreement with present work. Rai and Bansiwal

(2009) found that malathion also showed negative effect on growth and reproduc-
tion of Eisenia fetida, as we observed in our present study. Similar observations

were reported by Gobi and Gunasekaran (2010) while studying effect of herbi-
side (butachlor) on Eisenia fetida. Biomass and cocoon production were decreased
with increased herbicide concentration, which supports the present finding.
In case of studying the combined effect of Triazophos and Pendimethalin, we
found that combined effect was more toxic in comparison to individual effect of
Triazophos. On the other hand, combined effect of Triazophos and Pendimethalin
was observed to be less toxic in comparison to individual effect of Pendimethalin.
This type of antagonistic effect might be due to masking effect of Triazophos on

131
 Chapter 5. Discussion

Pendimethalin. Many scientists studied the combined effect of different chemicals

on toxicity of Eisenia fetida. Chen et al. (2014b) showed that combined toxicity

of butachlor, atrazine and k-cyhalothrin on Eisenia fetida in which majority of mix-

tures except for combination of butachlor plus k-cyhalothrin showed synergism in

artificial soil test. Wang et al. (2015) also studied combined effect of insecticides,
herbicides and cadmium on Eisenia fetida in ternary combinations. They found that
eleven combinations showed synergistic effects, four led to dual synergistic / addi-
tive behaviours and other five showed increasing antagonism within the entire range

of effects.
In the present study, the synergistic effect was observed in case of combined
effect of Chlorpyrifos and 2,4-DE, while antagonistic effect was observed in case

of combined effect of Triazophos and Pendimethalin, which is in an agreement with

the above mentioned works.

132
Chapter 6

Conclusion

Kota situated on the bank of river Chambal presents a diverse topographical fea-

tures. Some areas, mainly along the bank of the river are agriculturally very pro-

ductive and produce two seasons of crops; the Rabi and the Kharif. Whereas, other
areas present a rocky terrain and have a flourishing stone mining business.

Earthworm species were abundantly found in the agricultural areas whereas

their absence in rocky terrain and area in the vicinity of Thermal station was quite

marked. Three species of earthworms were identified and all the species are ver-

micomposting. Of these, one species Eisenia fetida was predominantly found in

all agriculturally inclined areas followed by Lampito mauritii and Perionyx was the

least abundant. Presence of earthworms is beneficial to agro-ecosystem as earth-

worms are very efficient biomarkers to assess the soil quality and any contamination
thereof.
Interactions with the local farmers in the survey areas and shopkeepers selling
agricultural products revealed that the farmers are using pesticides excessively to
enhance crop production. Chemicals used for Rabi crop (wheat) were Chlorpyrifos
and 2,4-DE as insecticide and herbicide respectively. For Kharif crop (soybean),
the chemicals used in practice are Triazophos and Pendimethalin as insecticide and
herbicide respectively. The excessive use of chemicals is not only detrimental to
 Chapter 6. Conclusion

the soil quality but also affects the diversity of earthworms and if discretion in use
of these pesticides and concrete implementation are not enforced, the species of

earthworms may soon be extinct.

Avoidance behaviour help us to determine the range of doses to be used for the

study of the effect of chemicals on the growth and reproduction of earthworms. It

can be concluded from the results that the concentration of Chlorpyrifos and avoid-
ance behaviour are directly proportional. Increase in concentration resulted in in-
creased avoidance behaviour. However, for 2,4-DE, avoidance decreases with in-

crease in concentration value upto 10 mg/kg and thereafter the avoidance behaviour
increases with the increase in the concentration of the chemical.
In case of Triazophos, the attraction was found to be highest at lowest concentra-

tion (0.316 mg/kg). Beyond this concentration, the avoidance behaviour increased

with the increased concentration of Triazophos. For Pendimethalin, it was seen that

the lower concentration exhibited attraction behaviour but as the concentration in-
creased, there was a gradual increase in the avoidance behaviour. The avoidance

behaviour was higher for Triazophos as compared to Pendimethalin.

Study was conducted to know the impact of chemicals on growth and reproduc-

tion of Eisenia fetida by taking five different concentrations. Chlorpyrifos caused

decline in the growth of earthworm as compared to the control. Similarly, reproduc-

tion also reduced drastically. 2,4-DE produced a sharp decline in the growth rate as

compared to the control and also decline in the reproduction was observed. Com-
bined effect of these chemicals caused a severe negative impact both on the growth
and reproduction of earthworms.
Triazophos caused a drastic decline both in the growth and reproduction of
earthworms. Similarly, Pendimethalin also caused a sharp decline both in the growth
and reproduction of earthworms as compared to controls. The combined concentra-
tions of these chemicals resulted in severe negative impact on both the growth and
reproduction of earthworms.

134
 Chapter 6. Conclusion

Normally, the effect of the combined concentrations of chemicals was found to

be more toxic as compared to their individual effect due to synergistic effect but in

the case of Triazophos and Pendimethalin, the combined effect was less toxic to the
individual effect of Pendimethalin. It may be concluded that it is due to the masking

effect of Triazophos over Pendimethalin.

Conclusively, the toxicity of used chemicals for growth was in the order: Pendi-
methalin > 2,4-DE > Triazophos > Chlorpyrifos. Similarly, toxicity of the used
chemicals for reproduction was in the order: Pendimethalin > Triazophos > 2,4-

DE > Chlorpyrifos.
It has been established by the present study that the growth and reproduction
parameters of earthworms exposed to agrochemicals are very efficient bioindicators

of soil contamination. Study on the impact of all the four chemicals used on the

above mentioned parameters showed a negative impact even at the recommended

doses. Indications are that a long term exposure of earthworms to these chemicals
will adversely affect their populations resulting in the low abundance of earthworm

in the soil.

The present study was conducted on one of three species found in the study area.

Additional research with the other species of the region is recommended. Further

research on the chemical nature, mode of action and means of degradation of pesti-
cides in soil is required so that the harm to the soil microorganisms as well as higher

ups in the food chain can be contained and minimized. Choosing organic farming
would be a very good method of achieving this goal. Implementation of the policies
drafted by NPOF-ICAR as adopted by Sikkim would be a step towards protecting
the earthworm populations, maintaining soil nutrients and leading to second green
revolution in the country.

135
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Appendix
 GOVERNMENT OF INDIA
ZOOLOGICAL SURVEY OF INDIA
Telegram: “Zoology” Kolkata Zoological Survey of India,
Phone: 033-22861608, General Non-Chordata Section,
Fax: 033-22861610 27-J.L.Nehru Road,
E-mail: [email protected] Kolkata-700016.

F.No. GNC/I&A/2014-15/936 Date: 21.10.2014

To
Dr. Anuradha Singh
Research Supervisor
Deptt. of Zoology,
Govt. College
Kota, Rajasthan

Sub: Earthworm Identification report

Ref: Your letter, dated10.09.2014

Dear Dr. Anuradha,

With reference to the above mentioned request received from you by us on
01.10.2014 by hand, the specimen sent by you is examined and identified by group expert
Dr. C.K.Mandal (e-mail: [email protected]). The identification report is as follows.

Phylum : Annelida
Class : Clitellata
Order : Haplotaxida
Family : Megascolecidae
1. LampitomauritiiKingberg, 1866. 3 ex.
2. Perionyxsp. 1 ex.

Family : Lumbricidae

3. Eiseniafetida(Savigny, 1826). 4 ex.

It is expected that the identification is duly acknowledged in your reports and publications.
All the best for all your endeavours.
Yours faithfully
-sd-
(Dr. Ch. Satyanarayana)
Officer- in- charge,
General Non-ChordataSecion.
Zoological Survey of India
Kolkata: 700016