**2. Materials and methods**

## **2.1. Laboratory experiment**

quantities. The use of pesticides has become an integral part of the modern agricultural system. It is estimated that 4 million tons of pesticides have been applied to world crops annually for pest control [1]. The residual pesticides may become the contamination sources and pose a serious threat to the soil and groundwater environment through the rainfall infiltration process. Some pesticides act on biochemical processes that are common to many animals, plants and microorganisms, and thus are a greater hazard to non-target organisms. Imidaclo‐ prid is a systemic chloronicotinyl insecticide and is used for soils, seeds and foliar applications for the control of sucking insects, including rice hoppers, aphids, thrips, whiteflies, termites, turf insects, soil insects and some beetles [2]. The active chemical works by interfering with the transmission of stimuli in the insect's nervous system [3]. Imidacloprid is a Category II acute toxicant, and thus, is classified as a General Use Pesticide. Imidacloprid is hazardous to

In humans it is linked to reproductive and mutagenic effects and is considered neurotoxic. Reproductive toxicity testing also showed that imidacloprid is an agonist to the acetylcholine receptors that regulates the endocrine system in the brain [4]. The over accumulation of this pesticide in environment requires higher awareness about this pesticide. Imidacloprid is reported to have different impacts on soil bacterial community and also cluster analyzing clearly showed that imidacloprid has significant negative impact on soil bacterial diversity in highly polluted farms and soil microbial balance has been gradually upset by the application

Oxidative stress is a misbalance between reactive oxygen species (ROS) generation and detoxification, resulting in increased levels of enzyme activity. ROS are of increasing interest in environmental toxicity as they may provide insights to toxicity mechanisms and may identify novel biomarkers. ROS can modify and inactivate proteins in a variety of ways. It is commonly recognized that *Escherichia coli* is the most suitable model system for the investiga‐ tion of the cell response to oxidative stress. When organisms or cells are exposed to low levels of certain harmful physical or chemical agents, the organisms acquire an induced tolerance against the adverse effects. The effect of hydrogen peroxide on the activity of Sox RS and Oxy R regulon enzymes in different strains of *Escherichia coli* has been studied. Exposure to acetamiprid in *Escherichia coli*, *Pseudomonas* and *Bacillus subtilis* resulted in synthesis of stress

The term "genomics" was first used by Winkler to describe the haploid set of chromosomes and the genes associated with them. Genomics includes many scientific disciplines [6]. Toxicogenomics is the subdiscipline combining the fields of genomics and toxicology [7]. It has also been described as the study of genes and their products important in adaptive responses to chemical-derived exposures. The toxicogenomic approach provides opportuni‐ ties to improve understanding of the molecular mechanisms underlying toxic responses to environmental contaminants [8]. Therefore, the present investigation was carried out to study the effect of 10–7 to 10–3 molar concentrations of imidacloprid for a period of 24, 48, 72 and 96 h on three antioxidant enzymes superoxide dismutase, catalase and peroxidase in *Bacillus*

the insects, especially honeybees, and also to fish, birds and algae.

of more pesticide.

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enzymes [5].

The experiment was carried out during the summer of 2011 at the laboratory of Department of Biotechnology and Microbiology, Karanatak University, Dharwad, Karnataka. The soil samples were collected from cotton fields around Hubli city. These fields did not have a history of imidacloprid applications for the past 5 years. Soil was collected at a depth of 15 cm and samples were passed through a sieve of 2 mm to remove stones and plant debris. One gram of soil was mixed with 9 ml of sterilized water and mixed by shaking for even distribution of soil in water. And 1 ml of solution from this test tube was then added to another test tube with 9 ml sterilized water. This gives a dilution of 10–2 and in the same pattern dilutions up to 10–7 were prepared. And 100 μl of solution from 10–6 dilution was spread on nutrient plates containing different concentration (125, 250, 500 and 1,000 ppm) of imidacloprid. These plates were incubated at 37°C for 48 h. After incubation, bacterial colonies were counted using colony counter and results were expressed as the number of bacteria in 1 g of soil [9].

### **2.2. Field experiment**

Imidacloprid was applied to experimental field at recommended rates and at 1.5× rates on two plots on same field in replicates, the plot without application served as control. Soil samples were taken on 7, 14, 21 and 28th day of application. About 1 g of sample was suspended in 9 ml of sterilized water. Serial dilutions were done as mentioned earlier. Then, 100 μl of solution from 10–6 dilution was spread-plated on nutrient agar plates. These plates were incubated at 37°C for 48 h. After incubation, colonies of bacteria were counted using colony counter and results were expressed as the number of bacteria in per gram of soil [9].

## **2.3. Preparation of stock solution of imidacloprid**

The stock solution of one molar imidacloprid was prepared and further diluted to give 10–3, 10–4, 10–5, 10–6 and 10–7 molar. Soil isolate was isolated and identified from soil as described in our previous publication. The bacterium was maintained at 4°C on nutrient agar and subcultured every fortnight. The medium used for toxicity testing was an optimized medium (dextrose – 0.65 g /l: yeast extract – 1.05 g /l; K HPO –0.30 g/l; NaCl – 0.25 g /l) [9].

## **2.4. Preparation of inoculum**

Pre-inoculum was prepared by inoculating a loop full of bacteria from the overnight incubated nutrient agar slant cultures on a 100 ml sterilized optimized growth medium and incubated for 24 h at 37°C under static conditions [9].

#### **2.5. Identification of bacterial isolate**

Imidacloprid tolerant colonies isolated and identified morphological, cultural and biochemical characters and 16S rDNA identification as described in our previous publication. The pure culture was grown on nutrient agar medium [10].

#### **2.6. Experimental procedures**

Five millilitres of the pre-inoculum was inoculated to 250 ml Erlenmeyer's flask containing 100 ml of sterilized optimized growth medium amended with different molar concentrations of imidacloprid. The flasks were incubated at 37°C for 96 h under shaking conditions at 120 rpm on a rotary shaker. At regular intervals, sample was taken out from each flask aseptically for analysis [10].

#### **2.7. Extraction of enzymes**

The cells were centrifuged at 8,000 rpm for 3 min and the pellet was dissolved in 0.2 ml of lysis buffer (50 mM tris-cl and 10 mM lysozyme). The tubes were incubated at 37°C for 10 min and centrifuged at 10,000 rpm for 10 min. Supernatant was used as the source of enzyme.

#### **2.8. Estimation of stress enzyme activity**

The activity of SOD, catalase and peroxidase were assayed using the supernatant from centrifugation (15,000 rpm) for 12 min at 4°C homogenate by standard methods [11–12].

#### **2.9. Isolation of genomic DNA**

The genomic DNA was isolated from soil isolate SP-03 by CTAB method. The DNA stock samples were quantified using Nanodrop spectrophotometer at 260 and 280 nm using the convention that one absorbance unit at 260 nm wavelength equals 50 μg DNA per ml. Quality and purity of DNA were checked by agarose gel electrophoresis. The DNA was used further for PCR. And 250 μl of the isolated genomic DNA was taken and treated with 1 μl of RNase enzyme and incubated at 37°C in a water bath for 30 min and further incubated at 60°C for 10 min in the water bath and used as a template with PCR mix [13].


**Table 1.** Composition of PCR mix


**Table 2.** Primers of stress enzymes

**2.5. Identification of bacterial isolate**

**2.6. Experimental procedures**

**2.7. Extraction of enzymes**

**2.8. Estimation of stress enzyme activity**

**2.9. Isolation of genomic DNA**

**Table 1.** Composition of PCR mix

for analysis [10].

278 Insecticides Resistance

culture was grown on nutrient agar medium [10].

Imidacloprid tolerant colonies isolated and identified morphological, cultural and biochemical characters and 16S rDNA identification as described in our previous publication. The pure

Five millilitres of the pre-inoculum was inoculated to 250 ml Erlenmeyer's flask containing 100 ml of sterilized optimized growth medium amended with different molar concentrations of imidacloprid. The flasks were incubated at 37°C for 96 h under shaking conditions at 120 rpm on a rotary shaker. At regular intervals, sample was taken out from each flask aseptically

The cells were centrifuged at 8,000 rpm for 3 min and the pellet was dissolved in 0.2 ml of lysis buffer (50 mM tris-cl and 10 mM lysozyme). The tubes were incubated at 37°C for 10 min and

The activity of SOD, catalase and peroxidase were assayed using the supernatant from centrifugation (15,000 rpm) for 12 min at 4°C homogenate by standard methods [11–12].

The genomic DNA was isolated from soil isolate SP-03 by CTAB method. The DNA stock samples were quantified using Nanodrop spectrophotometer at 260 and 280 nm using the convention that one absorbance unit at 260 nm wavelength equals 50 μg DNA per ml. Quality and purity of DNA were checked by agarose gel electrophoresis. The DNA was used further for PCR. And 250 μl of the isolated genomic DNA was taken and treated with 1 μl of RNase enzyme and incubated at 37°C in a water bath for 30 min and further incubated at 60°C for 10

**Contents Volume (µl)**

1.0 0.5 2.0 1 + 1 0.4 0.3 3.8

min in the water bath and used as a template with PCR mix [13].

10× Taq Assay buffer MgCl2 Template Primers (Forward + Reverse) (10 pM) dNTP mix Taq DNA polymerase HPLC grade water

centrifuged at 10,000 rpm for 10 min. Supernatant was used as the source of enzyme.


**Table 3.** PCR Conditions

The annealing temperatures for catalase peroxidase and for superoxide dismutase were 46°C.

#### **2.10. Statistical analysis**

Statistic significance between the control and the experimental data were subjected to analysis of variance (ANOVA) followed by post-hoc Dunnett's test (*P* ≤ 0.05) [9].
