Akoijan

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Biodegradation of imidacloprid and its metabolism in sandy

loam soil by Bacillus aerophilus

Journal: International Journal of Environmental Analytical Chemistry

Manuscript ID: GEAC-2014-0119

Manuscript Type: Original Paper

Date Submitted by the Author: 06-Apr-2014

Complete List of Authors: AKOIJAM, ROMILA; PUNJAB AGRICULTURAL UNIVERSITY, ENTOMOLOGY

Keywords: Imidacloprid, Metabolites, Bacillus, Soil, Biodegradation

URL: http://mc.manuscriptcentral.com/geac

International Journal of Environmental Analytical Chemistry

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Biodegradation of imidacloprid and its metabolism in sandy loam soil by Bacillus aerophilus 1

Romila Akoijama and Balwinder Singh

b 2

aPesticide Residue Analysis Laboratory, Department of Entomology, Punjab Agricultural 3

University, Ludhiana-141004, Punjab, India 4

Phone : +91-8427479475; E-mail: [email protected] 5

6

bPesticide Residue Analysis Laboratory, Department of Entomology, Punjab Agricultural 7

University, Ludhiana-141004, Punjab, India 8

Phone : +91-9814746304; E-mail: [email protected] 9

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______________________________ 19

*Address correspondence to Romila Akoijam, Pesticide Residue Analysis Laboratory, Department 20

of Entomology, Punjab Agricultural University, Ludhiana-141004, Punjab, India; 21

Phone : +91-8427479475; Fax: +91161-2412359; E-mail: [email protected] 22

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Abstract 24

Soil samples were fortified with imidacloprid @ 50, 100 and 150 mg kg-1

along with 45x107 cfu 25

of Bacillus aerophilus. Each treatment was replicated thrice and from each fortified (insecticide + 26

microbes) sample, 15 g soil sample was taken at 0, 7, 15, 30, 45, 60, 90 and 120 days after 27

application. The parent compound, imidacloprid was found to be more persistent. The residues of 28

metabolites, 6-chloronicotinic acid, olefine, urea and 5-hydroxy were observed at 60 days of 29

application whereas nitroguanidine and nitrosimine were not observed at 30 and 45 days, 30

respectively when imidacloprid applied @ 50 mg kg-1

. Among metabolites, urea and olefine were 31

found to be the maximum, 5-hydroxy, 6-chloronicotinic acid, nitrosimine and nitroguanidine were 32

also observed in all the treatments. Total imidacloprid residues did not follow the first order 33

kinetics for its application @ 50, 100 and 150 mg kg-1

in sandy loam soil amended with B. 34

aerophilus. The half-life values for 50, 100 and 150 mg kg-1

were worked out to be 14.33, 15.05 35

and 18.81 days, respectively. 36

Keywords: Imidacloprid; Metabolites; Bacillus; Soil; Biodegradation 37

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1. Introduction 47

Imidacloprid is a systemic chloronicotinyl insecticide belongs to the family of neonicotinoids, 48

which is widely used for the control of sucking insects [1]. The mode of action of imidacloprid is 49

brought about by its binding to the nicotinergic acetylcholine receptor and interfering with the 50

transmission of stimuli in the insect nervous system [2-4]. Although imidacloprid has greater 51

advantages of high insecticidal activity as well as low mammalian toxicity, it is extremely toxic to 52

beneficial predatory ground beetles, spiders, honeybees, parasitoid wasps, birds and aquatic 53

animals even at low concentrations [5-8]. Moreover, imidacloprid can be easily released into 54

diverse environments and persists for a long time. In soils, this compound can persist for 48-190 55

days [9]. In the absence of light, the longest half-life of imidacloprid in soil was 997 days in 56

laboratory studies [10]. Imidacloprid can also be taken up by crops and thus enter the food chain 57

which may result in harm to aquatic organisms and humans [11-13]. The majority of toxicity 58

studies have been focused on the parent compound, imidacloprid. The metabolites of imidacloprid 59

viz. olefin and nitrosimine have greater insecticidal activity than the parent compound [13] while 60

the guanidine metabolite does not possess insecticidal properties, but has a higher mammalian 61

toxicity than the parent compound [14]. 62

Different studies revealed that imidacloprid can be removed by natural processes in natural 63

environments, such as hydrolysis, photodegradation and biodegradation [15-18]. Biodegradation is 64

the process of using biological agents to clean up contaminants because of its easily operation, 65

high applicability, low cost and complete destruction of the contaminants from the environment 66

[19]. Ge et al. [20] reported a bacterium, Stenotrophomonas maltophilia was isolated from the 67

soils which was found to degrade imidacloprid. A bacterium belonging to the genus Leifsonia was 68

isolated from soil that was capable of degrading imidacloprid in agricultural soils [21]. Gopal et al. 69

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[15] also reported that Burkholderia cepacia degraded 69% of imidacloprid within 20 days when 70

50 g ml-1

of imidacloprid was applied in agricultural soils. Another bacterial strains, SP-01 71

(Brevundimonas sp. MJ 15) and BC-1 (Ochrobactrum anthropic) were found to degrade 72

imidacloprid [22-23]. 73

There are several advantages of biodegradation, which may be employed in areas which 74

cannot be reached easily without excavation. Biodegradation process has its natural ability of 75

microorganisms to degrade wasteful organic compounds. With the integration of proper utilization 76

of natural or modified microbial abilities with suitable engineering designs to make available for 77

their optimum growth environments, biodegradation can be successful in the field. So, 78

understanding the nature of microbial communities and their response to the natural environment 79

and contaminants is very important for developing sustainable environment. Expanding the 80

knowledge of the genetics of the microbes to increase capabilities for pollutants degradation and 81

conducting research on new biodegradation techniques, which are cost effective that afford 82

potential for significant advances. In laboratory condition, B. aerophilus was found to be the 83

highest capability among other organisms to degrade imidacloprid, showing reduction per cent of 84

42.85 after 15 days of incubation [24]. Therefore, the present study was undertaken to study the 85

biodegradation of imidacloprid on sandy loam soil by the use of Bacillus aerophilus. 86

2. Materials and Methods 87

2.1 Chemicals and reagents 88

The technical grade analytical standards of imidacloprid (99.9 %) and its metabolites like 89

nitrosimine (90.6 %), olefin (97.9 %), urea (99.4 %), 6-chloronicotinic acid (98.8 %), 5-hydroxy 90

(96.8 %) and nitroguanidine (99.0 %) were supplied by M/s Bayer Crop Science India Ltd., 91

Mumbai, India. Imidacloprid (Confidor 17.8 SL) formulation used for fortification was also 92

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obtained from M/s Bayer CropScience India Ltd., Mumbai, India. Sodium chloride, activated 93

anhydrous MgSO4 and solvents like HPLC grade acetonitrile and water were obtained from E. 94

Merck (India) Limited, Mumbai, India. Sodium sulfate anhydrous was from S D. Fine Chemicals, 95

Mumbai. Primary Secondary Amine (PSA) sorbent and activated graphitic carbon black (GCB 400 96

mesh) were obtained from Agilent Technologies Pvt. Ltd., Bangalore. All the solvents used were 97

of laboratory grade. All common solvents were redistilled in all glass apparatus before use. The 98

suitability of the solvents and other chemicals was ensured by running reagent blanks before actual 99

analysis. 100

The bacteriological media, Luria broth (LB) was used for the growth of imidacloprid 101

degrading bacteria (Bacillus aerophilus). The composition of Luria broth was - trypton 20.0 g, 102

yeast-extract 1.5 g, NaCl 1.5 g, distilled water 1.0 L, pH 7.0. The pH of the each medium was 103

adjusted and all the media were sterilized by autoclaving (121C, 15 psi of steam, 20 min) before 104

use. 105

2.2 Instrumentation 106

Analysis of imidacloprid and its metabolites was carried out on high performance liquid 107

chromatograph (Model DGU-2045) equipped with reverse phase (RP) C18 column and photo 108

diode array (PDA) detector, dual pump was supplied by M/S Shimadzu Corporation, Kyoto, Japan. 109

The HPLC column, a Luna 5m C18 column (250 x 4.6 mm size, 5.200.30 m particle size, 110

2.200.30 (90:10) particle distribution, 9515 A pore diameter, 43040 m2 g-1

surface area, < 111

55.0 ppm metal content, 19.000.70 % total carbon and 3.250.50 moles m-2 surface coverage) 112

was obtained from M/S Spincotech Pvt. Ltd. Chennai, India. The sample injector was equipped 113

with a 20 L loop. Acetonitrile and water (30:70) were used as mobile phase @ 0.3 mL min-1

. For 114

instrument control, data acquisition and processing, LC Solution software was used. 115

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2.3 Collection of soil samples 116

Samples of sandy loam soil were collected from Entomological Farm of Punjab Agricultural 117

University, Ludhiana. A composite sample (10 kg) was collected, consisting of several cores from 118

0-15 cm deep soil. The samples were packed in gunny bags and brought to the laboratory for 119

further analysis. Each sample was mixed well and sieved, and extraneous matter, including stones 120

or pebbles were removed and were dried under shade. 121

2.4 Physico-chemical properties of soils 122

The physical characteristics viz. nature of soil, organic matter, pH and electrical conductivity were 123

ascertained before initiating the experiment. The physico-chemical properties of sandy loam soil 124

used in this study were organic carbon = 0.153 %; pH = 8.32; sand = 76.0 %; silt = 16.0 % clay = 125

8.0 % and electrical conductivity = 0.44 ds m-1

. 126

2.5 Inoculum preparation in LB broth 127

The bacterial culture (Bacillus aerophilus) was streaked on LB agar plates. After 24 hours of 128

incubation at 37 OC, single colony of the bacterial cultures was picked up and inoculated in 3 ml 129

of LB and ampicillin media. Again after 24 hours of incubation, 500 l of media was inoculated in 130

50 ml of LB and ampicillin broth, which was used for the experiment. 131

2.6 Imidacloprid degradation in soil amended with B. aerophilus 132

To study the biodegradation and metabolism of imidacloprid, sandy loam soil was autoclaved to 133

destroy the microbes responsible for degradation of pesticides. Sterile sandy loam soil samples 500 134

g were fortified using three doses of imidacloprid @ 50, 100 and 150 mg kg-1

along with 135

inoculated cultures (45x107 cfu) of B. aerophilus. Each treatment was replicated thrice. From 136

each fortified (insecticide + bacterial cultures) sample, 30 g soil sample was taken, filled in plastic 137

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cups and covered with aluminum foil. The cups were moistened with water at 7 days interval to 138

maintain 70 % moisture content throughout the experiment. The cups containing soil samples with 139

insecticide and bacterial cultures as well as control soil samples were incubated at 25 2 C. 140

Fortified soil samples along with control samples were withdrawn at 0, 7, 15, 30, 45, 60, 90 and 141

120 days of time interval for the analysis after inoculation with bacterium, B. aerophilus. 142

2.7 Residue analysis 143

2.7.1 Extraction and cleanup 144

A representative 15 g sample of soil was taken into a 50 ml centrifuge tube and 30 ml of 145

acetonitrile was dispensed into all centrifuge tubes. The samples were well shaken and then 146

homogenized @ 15,000 rpm for 3 min using a Heidolph homogenizer. Ten gram sodium chloride 147

was added to each sample and shaken vigorously by rotospin for 5 minutes. The samples were 148

centrifuged using a laboratory centrifuge for 3 min @ 2,500 rpm. From each tube, 15 ml of the top 149

organic layer was decanted into another 50 ml centrifuge tube containing 10 g of activated sodium 150

sulfate. It was then shaken using a rotospin for 2 min. Six ml of the sample extract was transferred 151

to centrifuge tube containing primary secondary amine (PSA) sorbent (0.15 g), activated 152

anhydrous magnesium sulfate (0.90 g) and graphitic carbon black (0.05 g). The tube was tightly 153

capped and vortexed for 30 seconds. The tubes were centrifuged using a laboratory centrifuge for 1 154

minute @ 2,500 rpm. 4 ml of the top extract was transferred into a test tube and concentrated to 2 155

ml with rotary evaporator under 35oC for further quantification by HPLC. 156

2.7.2 Estimation by HPLC 157

Analysis of imidacloprid and its metabolites was carried out by HPLC equipped with photo diode 158

array (PDA) detector. Before use, the column was primed with several injections of standard 159

solution of imidacloprid and its metabolites till a consistent response was obtained. An injection 160

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volume of 20 l was used in all the experiments. Under these operating conditions, the retention 161

time of 6-chloronicotinic acid, nitroguanidine, olefin, nitrosimine, urea, 5-hydroxy and 162

imidacloprid were found to be 4.93, 7.91, 9.12, 11.32, 13.82, 15.45 and 22.47 min, respectively. 163

The compounds in the sample were identified and quantified by comparison of the retention times 164

and peak heights of the sample chromatograms with that of standards run under identical operating 165

conditions. 166

3. Results and Discussion 167

3.1 Efficiency of the method for estimation of imidacloprid and its metabolites 168

In the present investigations, recovery experiments were carried out at different levels to establish 169

the reliability and validity of analytical method and to know the efficiency of extraction and clean 170

up procedures. Soil samples were spiked at levels of 0.01, 0.05, 0.10, 0.25, 0.50 and 0.10 mg kg-1

. 171

These were extracted, cleaned up and analyzed following the method already described. The 172

control samples and reagent blanks were also processed in the same way so as to find out the 173

interferences, if any, due to the substrate and reagents, respectively. The mean per cent recoveries 174

of imidacloprid and its metabolites like 6-chloronicotinic acid (6-CNA), nitroguanidine (NTG), 175

olefine, nitrosimine, urea and 5-hydroxy were found to be 81.20 to 99.14 % (Table 1). The average 176

recovery values were found to be more than 80 %; therefore, the results have been presented as 177

such without applying any correction factor. 178

3.2 Limit of detection (LOD) and limit of quantification (LOQ) 179

Half-scale deflection was obtained for 1.0 ng imidacloprid which could be easily identified from 180

the baseline. A 15 g of soil samples was extracted, cleaned up and final volume made to 2 ml, 20 181

l of sample (equivalent to 20 mg soil) when injected did not produce any background 182

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interference. Thus, the limit of quantification (LOQ) was found to be 0.01 mg kg-1

and limit of 183

detection (LOD) being 0.003 mg kg-1

. 184

3.3 Biodegradation and metabolism of imidacloprid in sandy loam soil amended with Bacillus 185

aerophilus 186

Sandy loam soil samples were fortified with imidacloprid alone @ 50, 100 and 150 mg kg-1

for 187

control samples and the other soil samples fortified with the same doses along with 45x107 cfu 188

Bacillus aerophilus microbe cells. From each control and fortified (insecticide + B. aerophilus) 189

samples, 30 g soil sample was taken and filled in plastic cup. The samples of sandy loam soil were 190

analyzed at 0, 7, 15, 30, 45, 60, 90 and 120 days after application. When imidacloprid was applied 191

@ 50 mg kg-1

, the residue of imidacloprid was found to be 47.01 mg kg-1

at 0 day. In control 192

samples, the residue of total imidacloprid was reduced to 40.30 mg kg-1

at 7 days and further 193

degraded to 1.68 mg kg-1

at 120 days of application whereas in soil amended with B. aerophilus, 194

the total residue was degraded to 32.07 mg kg-1

at 7 days and it was further reduced to 2.06 mg kg-

195

1 at 60 days of imidacloprid application and the residues were not detected at 90 days of 196

application.. All the metabolites, 6- chloronicotinic acid, nitroguanidine, olefine, nitrosimine, urea 197

and 5-hydroxy were found to be detected at 7 days of application in both the conditions. Among 198

metabolites, urea and olefine were found to be the maximum, 5-hydroxy, 6-chloronicotinic acid, 199

nitrosimine and nitroguanidine were also observed in soils amended with bacteria (Table 2). 200

Following application of imidacloprid @ 100 mg kg-1

, the residues were not detected at 90 201

days in soil amended with bacteria. The residues of metabolites, nitroguanidine and 5-hydroxy in 202

soil inoculated with bacteria were found to be higher as compared to control samples. The same 203

trends of metabolites were observed as found in 50 mg kg-1

of application. The per cent reduction 204

values in control samples were observed to be 13.65, 31.39, 49.59, 69.05, 82.15, 87.27 and 93.21 205

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% at 7, 15, 30, 45, 60, 90 and 120 days, respectively but it was found to be higher in soil 206

inoculated with bacteria and were observed as 19.71, 41.00, 62.79, 85.01, 94.03, 100 %, 207

respectively at 7, 15, 30, 45, 60 and 90 days (Table 3). 208

With the increase of concentration of insecticides applied, the residue obtained was also 209

increased. The residue of imidacloprid at 0 day was found to be 129.81 mg kg-1

following 210

imidacloprid application @ 150 mg kg-1

. The total residue of imidacloprid and its metabolites was 211

found to be degraded to 7.10 mg kg-1

at 120 days and the metabolites such as 6-chloronicotinic 212

acid, olefine and urea were found to be observed. In samples of soil amended with bacteria, the 213

total residues of imidacloprid and its metabolites were not recorded at 120 days but at 90 days of 214

application, the parent compound, imidacloprid and the metabolites viz. 6-chloronicotinic acid, 215

olefine, urea and 5-hydroxy were found to be observed. As with the other studies, the total residues 216

of imidacloprid on soil declined gradually with time. The same trend of per cent reduction was 217

recorded in both the conditions. From the above findings, the parent compound, imidacloprid and 218

was found to be more persistent (Table 4). 219

Hu et al. [23] studied the bioremediation of imidacloprid using an indigenous imidacloprid 220

degrading bacterial strain, BCL-1 (Ochrobactrum anthropic) following application of imidacloprid 221

@ 100 mg kg-1

and amended with a concentration of 1.0 x 106 cfu g-1

of O. anthropic. The 222

degradation rate was approximately 67.67 % within 48 hours with optimum pH of 8 and 30C. 223

Anhalt et al. [21] also studied biodegradation of imidacloprid by an isolated microbe. The microbe, 224

Leifsonia strain PC-21, obtained from the enrichment cultures, degraded 37 to 58 % of 25 mg L 1 225

imidacloprid in tryptic soy broth containing 1 gL 1 succinate and D-glucose at 27C incubation 226

over a period of three weeks. The metabolites produced were identified as imidacloprid-guanidine 227

and imidacloprid-urea. The enrichment media without microorganisms had no loss of 228

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imidacloprid. A strain named NJ2, Stenotrophomonas maltophilia was isolated from the soils and 229

found to degrade imidacloprid with the formation of 5-hydroxy metabolite [20]. Another bacterial 230

strain, SP-01, Brevundimonas sp. MJ 15 was found to degrade imidacloprid in minimal salt 231

medium and tryptic soya broth containing 10 -3 molar imidacloprid by 38 and 69 %, respectively 232

[22]. 233

3.4 Degradation dynamics of total imidacloprid residues in sandy loam soil amended with B. 234

aerophilus 235

The degradation kinetics of the imidacloprid and its metabolites in control sandy loam soil and 236

sandy loam soil amended with B. aerophilus were determined by plotting residue concentration 237

against time, and the maximum squares of correlation coefficients found were used to determine 238

the equations of best fit curves. Confirmation of the first order kinetics was further made 239

graphically from the linearity of the plots of logC against time (C= residues 100). Total 240

imidacloprid residues for control soil samples followed first order kinetics with correlation co-241

efficient of 0.996 and 0.999 for its application @ 50 and 150 mg kg-1

but in case of control 242

imidacloprid fortification @ 100 mg kg-1

, the total residues of imidacloprid and its metabolites did 243

not follow the first order kinetics with correlation co-efficient of 0.982. The half-life (T1/2) values 244

of imidacloprid calculated as per Hoskins [25] were worked out to be 25.08 days for 50 mg kg-1

245

and 30.10 days for both 100 and 150 mg kg-1

. Total imidacloprid residues did not follow the first 246

order kinetics with correlation co-efficient of 0.989, 0.984 and 0.988 for its application @ 50, 100 247

and 150 mg kg-1

in sandy loam soil amended with B. aerophilus. The corresponding half-life 248

values for were found to be 14.33, 15.05 and 18.81 days, repectively (Figure 1). 249

4. Conclusions 250

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Biodegradation is a natural process by using biological agents to clean up contaminants from the 251

environment. With the use of B. aerophilus, the reduction percentage of imidacloprid in sandy 252

loam soil was found to be higher in all the three doses (50, 100 and 150 mg kg-1

) as compared to 253

that of the control samples. The metabolites, urea and olefine were found to be the maximum, 5-254

hydroxy, 6-chloronicotinic acid, nitrosimine and nitroguanidine were also observed in the soils 255

amended with B. aerophilus. 256

Acknowledgements 257

The authors are thankful to the Professor and Head, Department of Entomology, PAU, Ludhiana 258

for providing the necessary research facilities. 259

References 260

[1] W. Leicht, Pestic. Outlook 4, 17 (1993). 261

[2] D. Bai, S.C. Lummis, W. Leicht, H. Breer, and D.B. Sattelle. Pestic. Sci. 33, 197 (1999) 262

[3] J.A. Gervais, B. Luukinen, K. Buhl, and D. Stone. Retrieved 12 April (2012). 263

[4] E.E. Oliveira, S. Schleicher, A. Buschges, J. Schmidt, P. Kloppenburg, and V.L. Salgado. 264

Insect Biochem. Mol. Biol. 41, 872 (2011). 265

[5] J.E. Cresswell. Ecotoxicol. 20, 149 (2011). 266

[6] A. Elbert, B. Becker, J. Hartwig, and C. Erdelen. Pflanzenschutz Nachrichten Bayer. 267

44,113 (1991). 268

[7] M.Y. Liu, J. Lanford, and J.E. Casida. Pestic. Biochem. Physiol. 46, 200 (1993). 269

[8] R. Schmuck, R. Schoning, A. Stork, and O. Schramel. Pest Manag. Sci. 57, 225 (2001). 270

[9] S. Baskaran, R.S. Kookana, and R. Naidu. J Chromatogr A. 787, 271 (1997). 271

[10] M. Fossen. 2006;Accessed on 9th June; (2011). 272

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[11] H. Chopade, D. Eigenberg, E. Solon, P. Strzemienski, J. Hostetler, and T. McNamara. Res. 273

Appl. Vet. Med. 11, E1 (2010). 274

[12] U. Kapoor, M.K. Srivastava, and L.P. Srivastava. Food Chem. Toxicol. 49, 3086 (2011). 275

[13] R. Nauen, K. Tietjen, K. Wagner, and A. Elbert. Pestic. Sci. 52, 53 (1998). 276

[14] M. Tomizawa, and J.E. Casida. Br. J. Pharmacol. 127, 115 (1999). 277

[15] M. Gopal, D. Dutta, S.K. Jha, S. Kalra, S. Bandyopadhyay, and S.K. Das. Pestic. Res. 23, 36 278

(2011). 279

[16] V. Kitsiou, N. Filippidis, D. Mantzavinos, and I. Poulios. Appl. Catal. B Environ. 86, 27 280

(2009). 281

[17] J. Tang, X. Huang, X. Huang, L. Xiang, and Q. Wang. Environ. Earth Sci. 66, 441 (2012). 282

[18] C.A. Zaror, C. Segura, H. Mansilla, M.A. Mondaca, and P. Gonzalez. Water Pract. Technol. 283

1, 1 (2009). 284

[19] A. Kumar, B.S. Bisht, V.D. Joshi, and T. Dhewa. Intl. J. Environ. Sci. 1, 1079 (2011). 285

[20] F. Ge, Y.J. Dai, and T. Chen. Wei Sheng Wu Xue Bao. (Abstract) 46, 557 (2006) 286

[21] J.C. Anhalt, T.B. Moorman, and W.C. Koskinen. J. Environ. Sci. Heal. B. 42, 509 (2007). 287

[22] A.A. Shetti, and B.B. Kaliwal. Intl. J. Curr. Res. 4, 100 (2012). 288

[23] G. Hu, Y. Zhao, B. Liu, F. Song, and M. You. J. Microbiol. Biotechnol. 23, 1617 (2013). 289

[24] S. Sharma. Ph.D. diss., Punjab Agricultural University, Ludhiana, India, (2012). 290

[25] W.M. Hoskins. Proc. Nat. Acad. Sci. 9, 163 (1961). 291

292

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FIGURE CAPTION: 295

Figure 1. Semi-logarithm graph showing dissipation kinetics of total imidacloprid residues in 296

sandy loam soils fortified @ (a) 50, (b) 100 and (c) 150 mg kg-1 and amended with Bacillus 297

aerophilus 298

299

300

301

302

303

304

305

306

307

308

309

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(a) 313

314

(b) 315

316

y = -0.012x + 3.7001

R = 0.9965

y = -0.0215x + 3.6648

R = 0.9896

0

0.5

1

1.5

2

2.5

3

3.5

4

0 20 40 60 80 100 120 140

Log (residues X 100) mg/kg

Days after treatment

Control Bacillus aerophilus

y = -0.01x + 3.9045

R = 0.9821

y = -0.0203x + 3.9852

R = 0.9842

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 20 40 60 80 100 120 140




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For Peer Review O

nly

317

(c) 318

Figure 1. Semi-logarithm graph showing dissipation kinetics of total imidacloprid residues in 319

sandy loam soils fortified @ (a) 50, (b) 100 and (c) 150 mg kg-1

and amended with Bacillus 320

aerophilus 321

y = -0.0106x + 4.1167

R = 0.9995

y = -0.0163x + 4.1061

R = 0.9886

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 20 40 60 80 100 120 140




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For Peer Review Only

Table 1 322

Recovery (%) of imidacloprid and its metabolites from fortified samples of sandy loam soil (n=3) 323

Level of

fortification

(mg kg-1)

Imidacloprid

Metabolites

6-

chloronicotinic

Acid

Nitroguanidine Olefine Nitrosimine Urea 5-hydroxy

0.01 a83.271.81 81.792.90 85.622.11 85.160.79 87.621.79 85.970.86 93.761.41

0.05 82.221.34 89.462.16 87.511.20 83.120.45 93.262.00 81.200.98 96.650.76

0.10 86.342.08 91.030.87 81.440.59 84.201.20 98.703.82 90.461.76 89.161.32

0.25 85.380.93 85.172.01 80.880.74 92.990.91 83.020.86 97.432.45 90.960.98

0.50 99.141.87 98.621.67 90.531.78 99.002.02 90.061.97 91.810.64 90.511.08

1.00 93.011.13 91.080.78 91.770.92 98.331.32 83.002.68 92.722.14 97.911.72

324

aMean Standard Deviation of three replicate determinations 325

326

327

328

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Table 2 329

Residues of imidacloprid and its metabolites (mg kg -1) in sterilized sandy loam soil fortified @ 50 mg kg

-1 (control) and sterilized 330

sandy loam soil fortified with the same dose amended with Bacillus aerophilus 331

Treatment DAT Imidacloprid Metabolites Percent

reduction 6-

chloronicotinic

Acid Nitroguanidine Olefine Nitrosimine Urea

5-

hydroxy

Total

residues

Control

(without B.

aerophilus)

0 a47.012.03

bBDL BDL BDL BDL BDL BDL 47.012.03

7 39.671.09 0.080.02 0.010.00 0.210.04 0.050.02 0.230.05 0.050.01 40.301.18 14.27

15 32.240.53 0.100.03 BDL 0.170.06 0.040.02 0.180.09 0.040.00 32.770.92 30.29

30 24.181.33 0.090.01 BDL 0.110.02 0.010.00 0.120.03 BDL 24.511.37 47.86

45 14.871.90 0.060.02 BDL 0.050.02 BDL 0.080.02 BDL 15.061.98 67.96

60 10.200.72 0.040.01 BDL 0.020.01 BDL 0.030.01 BDL 10.290.85 78.11

90 4.490.33 0.010.01 BDL BDL BDL BDL BDL 4.500.35 90.42

120 1.680.10 BDL BDL BDL BDL BDL BDL 1.680.10 96.42

0 47.012.03 BDL BDL BDL BDL BDL BDL 47.012.03

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332

aMean Standard deviation,

bBDL= Below determination limit of 0.01 mg kg

-1. aMean of three replications 333

334

335

336

337

338

339

Soil

amended

with B.

aerophilus

7 31.673.19 0.050.03 0.010.00 0.110.06 0.030.01 0.140.07 0.060.01 32.074.21 31.70

15 19.922.98 0.070.04 0.020.00 0.060.04 0.020.01 0.090.05 0.040.01 20.223.17 56.98

30 11.511.14 0.060.02 BDL 0.040.02 0.010.00 0.050.02 0.030.00 11.702.10 75.11

45 5.830.86 0.030.01 BDL 0.020.01 BDL 0.030.02 0.020.01 5.931.04 87.38

60 2.020.22 0.010.00 BDL 0.010.00 BDL 0.010.00 0.010.00 2.060.27 95.61

90 BDL BDL BDL BDL BDL BDL BDL BDL 100


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Table 3 340





reduction 6-

chloronicotinic


5-

hydroxy

Total

residues

Control

(without B.

aerophilus)

0 a85.843.21


7 73.014.58 0.130.07 0.020.00 0.380.10 0.070.02 0.420.16 0.090.03 74.124.67 13.65

15 57.981.86 0.170.09 BDL 0.310.07 0.050.01 0.330.10 0.050.01 58.892.05 31.39

30 42.572.09 0.150.06 BDL 0.250.08 0.020.00 0.260.07 0.020.00 43.272.31 49.59

45 26.121.64 0.110.04 BDL 0.130.05 BDL 0.200.05 BDL 26.561.95 69.05

60 15.030.90 0.080.02 BDL 0.070.01 BDL 0.140.04 BDL 15.321.32 82.15

90 10.800.57 0.020.02 BDL 0.030.02 BDL 0.070.02 BDL 10.920.64 87.27

120 5.810.19 BDL BDL BDL BDL 0.010.02 BDL 5.820.21 93.21

0 85.843.21 BDL BDL BDL BDL BDL BDL 85.843.21

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343




345

346

347

348

349

350

351

352

Soil

amended

with B.

aerophilus

7 68.213.06 0.080.04 0.030.01 0.200.08 0.050.02 0.240.10 0.110.01 68.923.38 19.71

15 50.032.21 0.120.07 0.020.00 0.170.09 0.030.01 0.190.08 0.080.02 50.642.26 41.00

30 31.531.05 0.100.02 BDL 0.110.05 0.020.00 0.120.05 0.060.01 31.941.86 62.79

45 12.611.11 0.080.03 BDL 0.050.02 BDL 0.080.04 0.040.00 12.861.23 85.01

60 5.030.55 0.030.01 BDL 0.020.02 BDL 0.020.02 0.020.01 5.120.65 94.03



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Table 4 353





reduction 6-

chloronicotinic


5-

hydroxy

Total

residues

Control

(without B.

aerophilus)

0 a129.814.40


7 110.573.85 0.190.06 0.030.01 0.570.13 0.120.03 0.650.12 0.130.05 111.264.01 14.29

15 89.632.69 0.250.08 0.010.00 0.410.15 0.090.02 0.510.17 0.070.03 90.972.78 29.92

30 65.121.52 0.200.05 BDL 0.290.09 0.040.02 0.380.09 0.040.01 66.071.69 46.02

45 44.801.63 0.170.07 BDL 0.180.05 0.020.00 0.210.07 BDL 45.381.72 65.04

60 30.010.57 0.110.04 BDL 0.080.03 BDL 0.110.04 BDL 30.310.63 76.65

90 14.300.81 0.070.02 BDL 0.050.02 BDL 0.070.03 BDL 14.490.92 88.83

120 7.020.26 0.030.01 BDL 0.020.01 BDL 0.030.00 BDL 7.100.35 94.53

Soil 0 129.814.40 BDL BDL BDL BDL BDL BDL 129.814.40

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amended

with B.

aerophilus

7 96.334.87 0.140.07 0.030.01 0.30 0.12 0.080.04 0.340.15 0.160.03 97.384.91 24.98

15 75.012.71 0.190.05 0.020.01 0.200.04 0.050.02 0.270.04 0.120.02 75.862.89 41.56

30 46.321.69 0.150.04 0.010.00 0.130.08 0.030.00 0.170.08 0.070.01 46.881.72 63.88

45 22.951.71 0.090.02 BDL 0.070.04 BDL 0.100.05 0.050.00 23.261.96 82.08

60 10.510.62 0.050.02 BDL 0.040.02 BDL 0.050.02 0.030.01 10.680.94 91.77

90 5.011.84 0.020.01 BDL 0.010.01 BDL 0.010.02 0.010.00 5.060.05 96.10


356

357




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