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
2.6.1 Toxicological Effects . . . . . . . . 14
2.6.1.1 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
2.7.1 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
3.8.4 Indole Test . . . . . . . . . 28
3.8.5 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
5.1 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
CHAPTER ONE
1.0 INTRODUCTION
Soil is the most important agricultural resource next to water. It is important to study the possible effects of specific practices on soil properties. Agricultural practices now rely heavily on the use of pesticides for ridding the plants of pests. Application of pesticides on agricultural crop results in adverse effect on the properties of the soil as well as the microbial activities of beneficial bacteria (Rehman and Motoyama, 2000). Wide spread use of these pesticides over the years has resulted in problems caused by their interaction with biological systems in the environment (Kanekar et al., 2004). When pesticides are applied, the possibility exists that these chemicals may exert certain effects on non-target organisms, including soil microorganisms (Wardle and Parkinson, 1990; Simon-Sylvester and Fournier, 1979). Excessive and persistent use of pesticides results in deterioration of the environment and also the quality of soils, ground water, surface waters as well as the air, is compromised by pesticide contamination (Surekha et al., 2008). The microbial biomass plays an important role in the soil ecosystems where they fulfill a critical role in nutrient cycling and decomposition (De Lorenzo et al., 2001). The side effects of pesticides on the soil microflora were studied by several authors (Anderson, 1978; Duah-Yentumi and Johnson, 1986; Wardle and Parkinson, 1990; Perucci et al., 2000). Pesticides may affect the microbial population by controlling the survival and reproduction of individual species. On the other hand several microbes were reported to degrade some pesticides (Hata et al., 1986; Topp et al., 2000; De Lorenzo et al., 2001; Morgan and Watkinson, 1989). The important microflora, beneficial for growth of plants includes nitrogen fixing bacteria and phosphate-solubilizing bacteria, present in the rhizosphere of the plants. Since the fertility of soil depends upon number and type of organism present in the soil, studies on effect of pesticide application on soil were carried out (Sarnaik et al., 2006). Approximately less than 0.1% of applied pesticide, reaches the target pests, living bulk to effect the environment (Malinowaski, 2000). Microorganisms are frequently the major and sometimes the only means by which the chemicals are eliminated from a variety of ecosystems. Jones (1956) and Fletcher (1960) have shown the effect of different pesticide on type, number and activities. They explain that herbicide and insecticides can destroy soil microorganism or suppress their activities if applied at excessive rate but when applied at recommended rate these chemicals reach soil concentration of more than 2 or 3 part per million (ppm). Chlorpyrifos is an outstanding example of pesticide and probably the most commonly used (Sayali et al., 2012). It is used around the world to control pest insects in agricultural, residential, and commercial settings, although its use in certain residential applications has been restricted in several countries. According to the Dow Chemical Company, chlorpyrifos is registered for use in nearly 100 countries and is applied to approximately 8.5 million crop acres each year. The crops with the most intense chlorpyrifos use are cotton, corn, almonds, and fruit trees including oranges, bananas and apples. Chlorpyrifos is the common name for the chemical 0, 0-diethyl 0-(3, 5, 6-trichlor-2-pyridinyl) – phosphorothioate (Sayali et al., 2012). The Chemical Abstracts Service number is 2921-88-2 (Sayali et al., 2012). Dichlorvos or DDVP is an organophosphate, widely used as an insecticide to control household pests, in public health, and protecting stored product from insects. The compound has been commercially available since 1961 and has become controversial because of its prevalence in urban waterways and the fact that its toxicity extends well beyond insects It is effective against mushroom flies, aphids, spider mites, caterpillars, thrips, and whiteflies in greenhouse, outdoor fruit, and vegetable crops. Dichlorvos does not seem to bind to soil. This means it can move through soil fairly rapidly. The breakdown in soil is less rapid than in air or water. Dichlorvos breakdown is most rapid in moist soil that contained 200 parts of dichlorvos per million parts of soil (200ppm), 30% of dichlorvos remained in the soil after 3 days. Dichlorvos remains for longer periods in dry, acidic soil. Certain bacteria and other microorganisms in the soil can also breakdown dichlorvos. However, some pesticides are obtained from plant and minerals, while few others are obtained by the mixed culture of microorganisms. Insecticides like pyrethin, cruelties and nicotine are extracted from plant. Also toxins produced by Bacillus thuringensis are active ingredient against moth and butterfly larvae. The toxins are sometime called miracle gene. In many areas of the world locally available plant materials are widely used to protect stored products against damage by insects’ infestation (Golob and Webley 1980; Singh et al., 2003). Botanical products are environmentally safe, less hazardous, economical and easily available and can be used as an alternative to chemical pesticide (Talukder et al., 1990).
1.1 Aim and Objectives
i. To isolate, characterize and identify microbial diversity from agricultural soil.
ii. To determine the effect of pesticide Dichlorvos (DDVP) and Chlorpyrifos on the microbial diversity of agricultural soil.
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