ABSTRACT

This research work focuses on determining the impact of dichlorvos and chlorpyrifos on the microbial diversity of agricultural soil. The soil samples were obtained from an agricultural soil in Michael Okpara University of Agriculture, Umudike. Serial dilutions of the samples resulted in 22 isolates; 10 bacteria, 12 fungi. 5 each of both the bacterial and fungal isolates were used for this study and were identified as Proteus species, Bacillus species, Klebsiella species, Pseudomonas species, Actinomycetes species and Fusarium species, Penicillium species, Aspergillus species, Rhizopus species, Mucor species. The impact of the two pesticides was studied by employing susceptibility test using the disc diffusion method. Pure cultures of the isolates were subjected to 5 different concentrations of the pesticides; 100µg/ml, 50µg/ml, 25µg/ml, 12.5µg/ml, 6.25µg/ml with 0µg/ml as the control concentration. The inhibition zone diameter of each of the isolates was obtained in millimeter. The result showed that the bacterial isolates were more susceptible to the two pesticides than the fungal isolates with the tolerance level order for dichlorvos being Klebsiella species> Actinomycetes species> Proteus species> Bacillus species >Pseudomonas species and that of chlorpyrifos being Bacillus species < Klebsiella species and Proteus species <Actinomycetes species< Pseudomonas species. The order of maximum effect of the two pesticides on the isolates was 100µg/ml > 50µg/ml > 25µg/ml > 12.5µg/ml > 6.25µg/ml. Also the lowest concentration of the pesticides that inhibited the growth of the isolates was determined. This work revealed that the use of pesticides at higher concentrations could be detrimental to soil microorganisms especially soil bacteria which play essential role in bioremediation and maintenance of soil fertility. 

TABLE OF CONTENTS

Title page . . . . . . . . . . i

Certification . . . . . . . . . . ii

Dedication . . . . . . . . . . iii

Acknowledgement . . . . . . . . . iv

Table of contents . . . . . . . . . v

List of tables . . . . . . . . . . ix

Abstract . . . . . . . . . . x

CHAPTER ONE: INTRODUCTION

1.1 Aims and Objectives . . . . . . . 3 

CHAPTER TWO: LITERATURE REVIEW

2.1 Pesticides . . . . . . . . . 4

2.2.0 Classification of Pesticides . . . . . . . 5

2.2.1 Organophosphate Insecticide . . . . . . . 5 

2.2.2 Carbamate Insecticides . . . . . . . 6

2.2.3 Pyrethroids . . . . . . . . . 6

2.2.4 Neonicotinoids . . . . . . . . 7 

2.2.5 Biopesticides . . . . . . . . . 7

2.2.5.1 Microbial Pesticides . . . . . . . . 8

2.2.5.2 Plant-Incorporated-Protectants (PIPS) . . . . . 8

2.2.5.3 Biochemical Pesticides . . . . . . . 9

2.3 Uses . . . . . . . . . . 9

2.4 Health Effects . . . . . . . . 10

2.5 Environmental Effect . . . . . . . 11

2.6 Chlorpyrifos . . . . . . . . . 12

Toxicological Effects . . . . . . . . 14

Effects from Exposure during Pregnancy, . . . . . 14

 Infancy, And Childhood

2.6.1.2 Effects from Exposure during Adulthood . . . . . 15

2.6.1.3acute Toxicity . . . . . . . . . 15

2.6.1.4 Environmental Fate . . . . . . . . 17

2.6.1.5 Breakdown in Soil . . . . . . . . 17

2.6.2 Breakdown in Water . . . . . . . . 17

2.7.0 Dichlorovinyl Dimethyl Phosphate . . . . . . 18

Mechanism of Action . . . . . . . . 19

2.7.2 Regulation . . . . . . . . . 20

2.8.0 Soil Microbial Diversity . . . . . . . 20

2.8.1 Actinomycetes . . . . . . . . . 21

2.8.2 Bacteria . . . . . . . . . 22

2.8.3 Fungi . . . . . . . . . . 22

CHAPTER THREE: MATERIAL AND METHODS

3.1 Collection of Pesticides . . . . . . . 24

3.2 Sampling . . . . . . . . . 24

3.2.1 Selection of Sampling Area . . . . . . . 24

3.2.2 Sampling Procedure . . . . . . . . .24

3.2.3 Soil Sample Preparation . . . . . . . 24

3.3 Media Used . . . . . . . . . 25

3.4 Sterilization . . . . . . . . . 25

3.5 Paper Disc Preparation . . . . . . . 25

3.6 Microbiological Procedures . . . . . . . 25

3.6.1 Isolation of Microorganisms from Soil . . . . . 25

3.7      Identification of Fungal Isolates . . . . . . 26

3.7.1   Lactophenol Cotton Blue Staining . . . . . . 26

3.8      Identification of Bacterial Isolates . . . . . 27

3.8.1    Gram Staining . . . . . . . . 27

3.8.2 Catalase Test . . . . . . . . 28

3.8.3 Citrate Test . . . . . . . . . 28

Indole Test . . . . . . . . . 28

Methyl Red Test . . . . . . . . 29

3.8.6 Voges Proskauer Test . . . . . . . 29

3.8.7 Sugar Test . . . . . . . . . 29

3.9 Pesticide Tolerance . . . . `. . . . 30

3.9.1 Agar Disc Diffusion . . . . . . . . 30

3.9.2 Minimum Inhibitory Concentration . . . . . . 30

CHAPTER FOUR: RESULTS 

4.1 Results . . . . . . . . . . 31

CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATION

Discussion . . . . . . . . . . 40

5.2 Conclusion . . . . . . . . . 40

5.3 Recommendation . . . . . . . . . 40

REFERENCES

LIST OF TABLES

Table Title

Table 1: Plate Count of the Isolates . . . . . . 32

Table 2: Preliminary Identification of Bacterial Isolates . . . 33

Table 3: Biochemical Tests Result . . . . . . 34

Table 4: Morphology and Microscopy of Fungal Isolates . . . 35

Table 5a: Inhibition Zone Diameter of dichlorvos (Mm) . . . 36

Table 5b: Inhibition Zone Diameter of Chlorpyrifos (Mm) . . . 37

Table 6: Minimum Inhibitory Concentration (MIC) results of the . . 38

Pesticides