Isolation of the Novel Chiral Insecticide Paichongding (IPP) Degrading

Subscriber access provided by EPFL | Scientific Information and Libraries

Article

Isolation of the Novel Chiral Insecticide Paichongding Degrading Strains and Biodegradation Pathway of RR/SS-IPP, SR/RS-IPP in Aqueous System Jing Wang, Jie Chen, Wenjuan Zhu, Jiangtao Ma, Yan Rong, and Zhiqiang Cai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02862 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 17, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

Journal of Agricultural and Food Chemistry

1

Isolation of the Novel Chiral Insecticide Paichongding Degrading

2

Strains and Biodegradation Pathway of RR/SS-IPP, SR/RS-IPP in

3

Aqueous System

4 5

Jing Wang, Jie Chen, Wenjuan Zhu, Jiangtao Ma, Yan Rong, Zhiqiang Cai*

6

Lab. of Applied Microbiology and Biotechnology, School of Pharmaceutical

7

Engineering & Life Science, Changzhou University, Changzhou 213164, China

8 9

ABSTRACT:

10

Chiral insecticide Paichongding (IPP) is a member of cis-nitromethylene

11

neonicotinoid insecticides used in China. Paichongding was the promising

12

replacement for imidacloprid due to its higher activity against imidacloprid-resistant

13

insects. Two pairs of enantiomers: RR/SS-IPP and SR/RS-IPP, were separated by

14

preparative high-performance liquid chromatography and employed in aqueous

15

system to investigate their biodegradation process. In this study, the strains

16

G1-13/G1-14 and G2-19 with effective paichongding-degrading capability were

17

isolated from agricultural soils. G1-14 was mutated from G1-13 by UV light exposed.

18

Sequence alignment of 16S rRNA proved these three strains belonged to the family of

19

Sphingobacterium. Degradation rate of RR/SS-IPP by Sphingobacterium sp. G1-13

20

and G1-14 reached 13% and 30% within 6 and 4 days, respectively. And the

21

degradation rate of SR/RS-IPP by Sphingobacterium sp. G2-19 could reach 35%

22

within 5 days. Degradation intermediates (I1-I6) of enantiomers were detected and 1

ACS Paragon Plus Environment


23

two possible biodegradation pathways were proposed based on the identification of

24

metabolites.

25 26

KEY WORDS: Paichongding, Sphingobacterium; aqueous system; Degradation

27

intermediates; Biodegradation pathways

28

2


Page 2 of 28

Page 3 of 28


29

■ INTRODUCTION

30

Many chiral pesticides have been introduced into the commercial market and applied

31

in agriculture.1 Generally, a chiral pesticide contains several pairs of enantiomers,

32

which proved the same physical and chemical properties and shown different

33

activities in bioprocess. Recently, enantioselectivity or stereoselectivity of chiral

34

pesticide has attracted increasing attention, especially with regards to environmental

35

protection and human healthy.2-3

36

Over the past three decades, the discovery of neonicotinoids was considered as a

37

milestone in pesticide research. Neonicotinoids insecticides have been one of the

38

world’s largest selling insecticides (24% of the total world market) in more than 120

39

countries, owing to the advantages of low mammalian toxicity, broad insecticidal

40

scope and high potency when compared to many other classes of insecticides, such as

41

organochlorines, organophosphates and methylcarbamates.1-6 Paichongding (IPP,

42

1-((6-chloropydidin-3-yl) methyl) -7- methyl- 8-nitro- 5-propoxy- 1, 2, 3, 5, 6, 7-

43

hexahydroimidazo[1, 2-α-]-pyri-dine)) is a novel cis-nitromethylene neonicotinoid

44

insecticide developed by Jiangsu Kwin Co. Ltd. and East China University of Science

45

and Technology. In the case of growing resistance to imidacloprid, the novel

46

insecticides are likely to have a great future development. Paichongding has shown

47

relatively low mammalian toxicity and used for control a broad spectrum of sucking

48

and biting insects, such as cowpea aphids (Aphis cracivora) and fabricius (Nephotettix

49

bipunctatus).6 In addition, the insecticidal activity of IPP was much higher than that of

50

imidacloprid against imidacloprid-resistant populations of the brown planthopper 3



51

(Nilaparvata lugens), an economically important insect pest of rich crops in many

52

parts.1,7-8 Since 2009, the use of IPP was estimated to soon reach about 1000 tons or

53

3.3 million hectares. 9-11 IPP has two chiral centers and consists of two couples of

54

enantiomers. One pairs were 5R, 7R-paichongding (RR-IPP) and 5S, 7S-

55

paichongding (SS-IPP), and another pairs were 5S, 7R-paichongding (SR-IPP) and 5R,

56

7S-paichongding (RS-IPP) (Figure 1).9 Previous investigation of IPP was focused on

57

its photodegradation in aqueous solution, microbial degradation, bound residues

58

formation and isomer-specific transformation in soil.7-13 The fate of characteristics and

59

biodegradation of enantiomers RR/SS-IPP and SR/RS-IPP in aqueous solution have

60

been unknown.

61

In this study, two pairs of enantiomers were separated by preparative

62

high-performance liquid chromatography (prep-HPLC) and then they were used in

63

aqueous solution degradation studies. Sphingobacterium sp. G1-13 was isolated from

64

soil and Sphingobacterium sp. G1-14 mutageniced by UV light exposed from

65

Sphingobacterium sp. G1-13. Mutagenic strain Sphingobacterium sp. G1-14 with a

66

higher RR/SS-IPP degrading activity compared to non-mutagenic Sphingobacterium

67

sp. G1-13. Bacterial strain Sphingobacterium sp. G2-19 could degradation SR/RS-IPP

68

with efficient degrading activity isolated from soil. Liquid chromatography-tandem

69

high resolution mass spectrometry (LC-MS/MS) was used to identify the degradation

70

intermediates of enantiomers RR/SS-IPP and SR/RS-IPP in aqueous system.

71

■ MATERIALS AND METHODS

72

Sampling Sites and Preparation of Stereoisomers RR/SS-IPP and SR/RS-IPP. 4


Page 4 of 28

Page 5 of 28


73

Soil samples were collected at a depth of 5-10 cm in sterile 20 mL sealed bag from

74

different stations in Jiangsu Province, China. Then they were all stored at 4 °C in the

75

dark until apply to isolate the RR/SS-IPP and SR/RS-IPP degrading bacteria.14-15

76

The stereoisomers of IPP were separated by preparative high-performance liquid

77

chromatography (prep-HPLC) Shimadzu with a C18 column (5 µm, 20 × 250 mm,

78

particle size, Shimadzu, Japan). Prep-HPLC condition was as follows: the mobile

79

phase was 40% acetonitrile and 60% pure water for 30 minutes at 354 nm for 30 °C

80

and flow rate was 5.0 mL·min-1.9

81

RR/SS-IPP and SR/RS-IPP were determined with HPLC (Agilent 1260), 354 nm,

82

Agilent C18 column (3.5 µm, 4.6 × 250 mm) and 30 °C. Acetonitrile (HPLC grade)

83

and pure water (HPLC grade) was 40:60 and the flow rate was 1.0 mL·min-1 for 10

84

minutes. The combined fractions containing the pure stereoisomers were then

85

concentrated to near dryness on a vacuumed rotary evaporator.9

86

Isolation of RR/SS-IPP and SR/RS-IPP Degrading Bacteria by Enrichment

87

Culture Method. LB medium, mineral salt medium (MSM), and agar plate/slant

88

medium were used in this research to isolate RR/SS-IPP and SR/RS-IPP degrading

89

bacteria (seeing supporting Table S1). RR/SS-IPP and SR/RS-IPP were prepared as a

90

stock solution (1000 mg·L−1) and filter sterilized. It’s concentration in media was

91

varied as required.

92

Enrichment cultures were performed in 250 mL erlenmeyer flasks containing 100

93

mL LB medium and 1 g soil sample. These flasks were incubated for 2 days at 30 °C in

94

a rotary shaker operating at 120 r·min-1. Then, 1 mL of the inoculum was transferred to 5



95

fresh MSM for subsequent subcultures. After a series of five further subcultures, 100

96

µL inoculum from the flask was streaked on MSM with 1% (V/V) IPP stereoisomers as

97

the carbon source in solid medium, and phenotypically different colonies were isolated.

98

All the obtained colonies were streaked for purity. Isolated strains were preserved at

99

4 °C on LB agar slants and at −80 °C in LB broth supplemented with 20% sterilized

100

glycerol.

101

UV Mutagenesis of RR/SS-IPP Degrading Bacteria. MSM media was used for

102

maintenance and propagation of RR/SS-IPP degrading bacteria. An UV survival curve

103

of degrading bacteria was defined by liquid culture (1 mL) was exposed to UV for 0,

104

15, 30, 45, 60, 75, 90, 105, 120 s using UV light (254 nm) and the distance of

105

exposure at 30 cm.16-17 Mutant strains were streaked on MSM plate and incubated for

106

36 h at 30 °C to get single colony. In addition, all the survival strains were streaked

107

for purity. Isolation and screening of mutant strains were according to the isolation

108

methods of G1-13/G2-19.

109

Identification of RR/SS-IPP and SR/RS-IPP Degrading Bacterial Strains. The

110

morphological characterization of the bacterial strains was performed by Gram staining.

111

Cell morphology was examined with a light microscope after Gram staining and a Cold

112

field emission scanning electron microscope (Hitachi. Japan).

113

A comparative 16S rRNA gene sequence analysis of RR/SS-IPP and SR/RS-IPP

114

degrading bacterial strains was performed according to the previous report,5

115

sequencing of the gene fragment was fulfilled by Shanghai Sangon Biological

116

Engineering Technology and Services Co. Ltd. (seeing supporting Table S2). A blast 6


Page 6 of 28

Page 7 of 28


117

search of 16S rRNA nucleotide sequences was performed by National Center for

118

Biotechnology Information (NCBI) sequence database. The sequences were analyzed

119

using Molecular Evolutionary Genetic Analysis (MEGA v.5.1).18

120

Degradation of RR/SS-IPP and SR/RS-IPP in Aqueous System by Degrading

121

Bacterial Strains. The biodegradation studies were conducted in a series of 250 mL

122

sterile glass flasks. 10 mL of inoculum was transferred aseptically to each glass flask

123

containing 100 mL MSM supplemented with varying amounts of IPP stereoisomers as

124

the only carbon (RR/SS-IPP and SR/RS-IPP solution of 50 mg·L−1) added as

125

requirement from the stock solution (10000 mg·L−1, filter sterilized beforehand). The

126

pH of the broth was measured with a Precision pH meter (Sartorius Group. Germany).

127

Control test was conducted under the same manner as glass flask without bacteria.

128

Extraction and Analysis of RR/SS-IPP and SR/RS-IPP Degradation Products.

129

The cultivation was centrifuged under 8000 r·min-1 for 5 min and the supernatant was

130

transferred to a 250 mL separating funnel. The supernatant was extracted with

131

dichloromethane three times,5, 15 the organic phases through a rotary evaporator at

132

40 °C until nearly dry, and then they were volume to 10 mL with acetonitrile.

133

RR/SS-IPP and SR/RS-IPP residual amount and biodegradation intermediates were

134

monitored by high-performance liquid chromatography (HPLC, Agilent 1260, Agilent

135

Company, USA). An ultraviolet detector was operated at 354 nm, with an Agilent C18

136

(3.5 µm, 4.6 × 250 mm) and the column temperature at 30 °C. Acetonitrile (HPLC

137

grade) and pure water (HPLC grade) were at a gradient elution (min/% acetonitrile:

7



138

0/3, 7/3, 9/35, 18/35, 20/85, 35/85, 37/100, 45/100, 47/15, 55/15) at a flow rate of 1.0

139

mL·min-1.

140

A Dionex U3000 HPLC system coupled with Bruker maXis 4G ion trap mass

141

spectrometer with an electrospray ionization source (ESI) was used for LC-MS/MS

142

analysis. The separate conditions were in accordance with those used for HPLC

143

analysis. The ion source temperature was controlled at 250 °C, and the capillary

144

voltage was -4.5 kV. The analysis mode of ionization was electrospray ionization (ESI,

145

positive). The operation conditions were as follows: collision energy, 10.0 eV; ISCID

146

energy and ion energy, 5.0 eV; dry gas, 6 L·min-1; dry temperature, 180 °C; gas

147

pressure, 1.5 bar. The continuous full scanning from m/z 50 to 500 Da was performed

148

in positive ion mode.15, 18-22

149 150 151

■ RESULTS AND DISCUSSION Isolation and Characterization of RR/SS-IPP and SR/RS-IPP Degrading

152

Bacteria. Two pairs of IPP stereoisomers: RR/SS-IPP and SR/RS-IPP, were achieved

153

when C18 column (5 µm particle size, 20 × 250 mm, Shimadzu, Japan) was used under

154

the optimized condition (Figure 2).

155

After several weeks of enrichment followed by isolation, the bacterial colonies of 50

156

different morphological types were isolated from the soil samples collected from

157

different sites in Jiangsu Province, China. In particular, Two potential strains (named as

158

G1-13 and G2-19, respectively.) were eventually selected for further study.

159

A graph of survived rates of mutated strains obtained according to different times of 8


Page 8 of 28

Page 9 of 28


160

UV exposure shown the times of 90 and 105 seconds with UV exposure (under the

161

given condition) was efficient for 10-15% survival rate (Figure 3). Obtained

162

Surviving efficient mutant strain was named as G1-14.

163

Morphological characterization by Gram staining showed G1-14 more large than

164

G1-13 in the same time of streak culture (seeing supporting Table S3 and Figure S1).

165

Gram stain and microscopic examination showed that G1-13, G1-14 and G2-19 were

166

no-spore and Gram-negative. Strain G1-13 grew rapid after inoculation 4 days and

167

mutant strain G1-14 grew rapid after 3 days. Then, these strains grew slowly and

168

RR/SS-IPP concentration also decreased by about 13% and 30% (Figure 4a),

169

respectively. G2-19 grew rapidly after inoculation within 3 days during SR/RS-IPP

170

concentration decreased by about 35% (Figure 4b). At the end of incubation period

171

(seeing supporting Table S4), the effect degradation rates of RR/SS-IPP and

172

SR/RS-IPP reached 30% and 35%, respectively. Photolysis was negligible because all

173

the studies were carried out in the dark. Therefore a weak decrease of RR/SS-IPP and

174

SR/RS-IPP in all blank control groups without bacterial strains might result from

175

hydrolysis. The results suggested that the degradation of SR/RS-IPP was easier than

176

RR/SS-IPP in aqueous solution.5 The degradation rate of SR/RS-IPP by G2-19 was

177

much higher than RR/SS-IPP by G1-13/G1-14 in aqueous solution. Although the

178

reason for the above conclusion was still unclear, the result agreed with previous

179

research that SR/RS-IPP was easier degrading than RR/SS-IPP in soils reported by

180

Cai et el. and Fu et el.7,19-22

181

Bacterial strains were classified according to the best match of their 16S rRNA 9



182

sequences with the GenBank database.19 Sequences of these three strains were

183

deposited in the GenBank database at the National Center for Biotechnology

184

Information (NCBI), USA. Isolation bacteria strains G1-13/G1-14 had the highest

185

similarity values of the sequences closely related to Sphingobacterium sp. 7 SY-2016

186

(100% identity), and G2-19 had the best match of the sequences to Sphingobacterium

187

sp. IN42 (100% identity). The phylogenetic tree of these bacterial strains was drawn

188

based on the 16S rRNA by Clustal and Neighbor-Joining algorithms of MEGA5.1

189

(Figure 5).

190

Identified RR/SS-IPP and SR/RS-IPP Degradation Intermediates.

191

Stereoisomers of RR/SS-IPP and SR/RS-IPP had retention time of 14.30 min and

192

14.75 min, respectively. The protonated ion [M+H+] and the fragmentation pattern

193

from MS/MS2 spectral comparison with mass spectral data of degradation

194

intermediates indicating that these two stereoisomers were matching with elemental

195

formula of C17H23ClN4O3 (Figure 6a), with m/z 367.1535, and m/z 367.1538,

196

including daughter ions of 306.1358 (100), 137.1084 (44), and 306.1365 (100),

197

137.1082 (14), respectively.

198

Intermediates (I2, I3, I5, I6) were observed and analyzed by strains

199

Sphingobacterium sp. G1- 13/ G1-14 degrading RR/SS-IPP in aqueous solution.

200

Intermediate I2 with m/z 301.1399 (M+H, Figure 6c) matched elemental formula

201

C13H19ClN4O2, I2 was identified as 8-Amino-1-(6-chloro-pyridin-3-ylmethyl)-

202

octahydro-imidazo [1, 2-a]-pyridine-5, 7-diol, including daughter ions of 301.1399

203

(100), 142.1600 (45) and 217.1038 (10) according to the structure of I2. Intermediate 10


Page 10 of 28

Page 11 of 28


204

I2 was tentatively determined to be hydroxylated derivative of RR/SS-IPP and it was

205

corresponding to M6 according to the previous reports by Cai et al..4, 15, 22 Thus I2

206

monitored and detected not only from IPP-degrading produces in soil but also could

207

be detected from degradation intermediates of RR/ SS-IPP in aqueous system.

208

Intermediate I3 had a retention time of 29.05 min, containing daughter ions of

209

282.2780 (100) and 142.1602 (14). The protonated molecular ion clearly showed

210

there was a mass unit loss of 17 matched to -OH was likely a result of the

211

hydroxyl-elimination reaction and hydroxylation of methyl on I2. Intermediates I3,

212

with m/z 282.2781 (Figure 6d), matched elemental formula C13H19ClN4O and

213

identified as 8-Amino-1-(6-chloro-pyridin-3-ylmethyl)-octahydro-imidazo[1,2-a]

214

pyridin-7-ol analyzed by the calculated mass and fragment pattern.

215

Intermediate I5, with m/z 142.1591 (M+H, Figure 6f). Which likely corresponded

216

to the broken of C-N bond between 2-Chloro-5-ethyl-pyridine and 8-amino-

217

octahydro-imidazo [1, 2-a] pyridin-7-ol of I3, and then hydroxyl was eliminated from

218

tetrahydropyridine to form I5. The I5 was identified as octahydro-imidazo [1, 2-a]

219

pyridin-8-ylamine, and matched formula of C7H15N3 based on the protonated ion and

220

the fragmentation pattern.

221

Intermediate I6, with m/z 226.9502 (M+H, Figure 6f ), matched formula of

222

C10H17N3O3, which was tentatively determined to be generated from the broken of

223

C-N bond between 2-Chloro-5-ethyl-pyridine and 8-amino-octahydro-imidazo [1, 2-a]

224

pyridin-7-ol of RR/SS-IPP compound, and then methylene was eliminated from

225

tetrahydropyridine ring to form I6. Intermediate I6 was identified as 8-Nitro-511



226

propoxy-1, 2, 3, 5, 6, 7- hexahydro-imidazo [1, 2-a] pyridine.

227

Four intermediates (I1-I4) were analyzed and identified from degradation products

228

of SR/RS-IPP by LC-MS and LC-MS/MS in aqueous system by Sphingobacterium sp.

229

G2-19. I2 and I3 were mentioned as above. The molecular ion of intermediate I1

230

wasn’t the daughter ions of SR/RS-IPP, and the loss of 42 mass units corresponding to

231

C3H7 was likely a result of de-propyl derivative of SR/RS-IPP. The metabolite I1, with

232

the m/z 325.1052 (M+H, Figure 6b), matched formula of C14H17ClN4O3, which was

233

identified as 1-(6-Chloro-pyridin-3-ylmethyl)-7-methyl-8-nitro-1, 2, 3, 5, 6, 7-

234

hexahydro-imidazo [1, 2-a] pyridin-5-ol and it was unobserved in several metabolites

235

of RR/SS-IPP in aqueous system.

236

Intermediate I4, with m/z 276.0884 (M+H, Figure 6e), was matched elemental

237

formula of C14H14ClN3O, I4 was positively identified as the derivative of elimination

238

of amino and then hydroxy was oxidized to a carbonyl on tetrahydropyridine ring of

239

I2 based on the elemental formula fit corresponded to previous reported by Cai et al.,

240

Fu et al. and zhao et al. .5, 7, 13 Thus I4 was determined as 1-(6-Chloro-pyridin-3-

241

ylmethyl)-7-methyl-2, 3-dihydro-1H-imidazo [1, 2-a] pyridin-5-one.

242

Aqueous solution degradation products of RR/SS-IPP and SR/RS-IPP were

243

observed and preliminarily identified by HPLC and LC-MS/MS (seeing supporting

244

Table S5). All degradation intermediates of SR/RS-IPP were found compared to the

245

products of RR/SS-IPP except I2 and I4 in aqueous solution. The results clearly

246

demonstrated that the degradation products of RR/SS-IPP and SR/RS- IPP in aqueous

247

system less than microbial degradation products of IPP in soils. Possible aqueous 12


Page 12 of 28

Page 13 of 28


248

solution degradation of RR/SS-IPP and SR/RS-IPP lacked the effective of soil

249

microbial community on the degradation process. In addition, I2 and I6 only

250

monitored among of degradation intermediates in aqueous system.

251

RR/SS-IPP and SR/RS-IPP Biodegradation Pathway in Aqueous System. In

252

this study, three strains G1-13, G1-14 and G2-19 were identified as Sphingobacterium

253

isolated from agricultural soil long-term administration for insecticides by enrichment

254

culture method. Sphingobacterium sp.G1-13, G1-14 and G2-19 were used to degrade

255

RR/SS-IPP and SR/RS-IPP with high degrading activity. Biodegradation pathway in

256

aqueous solution of RR/SS-IPP and SR/RS-IPP were proposed based on the identified

257

degradation intermediates (Figure 7). The results demonstrated that the

258

tetrahydropyridine ring of RR/SS-IPP and SR/RS-IPP structure was very unstable and

259

easily broken under fermentation condition. The microbial transformation of

260

RR/SS-IPP and SR/RS-IPP revealed two modification patterns of the

261

tetrahydropyridine ring and the chloropydidine ring on the RR/SS-IPP and SR/RS-IPP

262

parent compounds: (a) elimination of propyl group, nitro, or hydroxyl; (b) conversion

263

of methyl group to a hydeoxyl; (c) reduction of nitro group to an amino; (d) oxidation

264

of tetrahydropyridine ring; (e) fracture of methylene.

265

The degradation pathway of RR/SS-IPP in aqueous system by Sphingobacterium

266

sp.G1-13/ G1-14 as follows (Figure 7a): the propyl group was eliminated from

267

tetrahydropyridine ring on RR/SS-IPP and then hydroxyl was generated, meanwhile,

268

nitro group was reduced to an amino group, methyl was converted to hydroxyl, C=C

269

was hydrogenation to produce C-C at the tetrahydropyridine ring to form intermediate 13



270

I2; Subsequently, hydroxyl was eliminated at the tetrahydropyridine ring on I2 to

271

form I3; then, the broken of C-N bond between 2-Chloro-5-ethyl-pyridine and

272

8-amino-octahydro-imidazo[1, 2-a]pyridin-7-ol, and hydroxyl was eliminated from

273

tetrahydropyridine ring on I3 to create I5; In addition, another metabolic intermediate

274

I6 was generated through broken of C-N bond between 2-Chloro-5-ethyl-pyridine and

275

8-amino-octahydro-imidazo[1, 2-a] pyridin-7-ol, and methyl was eliminated at

276

RR/SS-IPP parent compound.

277

The biodegradation pathway of SR/RS-IPP by Sphingobacterium sp. G2-19 in

278

aqueous solution was found (Figure 7b): the propyl group was eliminated through

279

broken of C-O bond and hydroxyl to form I1. Subsequently, the nitro group was

280

reverted to amino, methyl was conversed to hydroxyl and C=C was hydrogenation to

281

produce C-C at the tetrahydropyridine ring of I1 to form I2; then, hydroxyl was

282

eliminated at the tetrahydropyridine ring on I2 to produce I3; moreover, I4 was

283

probable produced by C-O was oxidized to C=O, C-C was transformed to C=C and a

284

nitro was cancellation at the tetrahydropyridine ring of I1; in addition, I4 was also

285

possible generated through the propyl group was eliminated by broken of C-O bond

286

and hydroxyl, C-O was oxidized to C=O, C-C was transformed to C=C and a nitro

287

was cancellation at the tetrahydropyridine ring on SR/RS-IPP parent compound.

288

Basis on the analysis of biodegradation products and pathways of RR/SS-IPP and

289

RS/SR-IPP, the distance of degradation pathways between two pairs of stereoisomers

290

was shown in Figure 7. The biodegradation of SR/RS-IPP mainly occurred on the

291

tetrahydropyridine ring rather than on the chloropydidine ring. However, in the 14


Page 14 of 28

Page 15 of 28


292

degradation pathway of RR/SS-IPP, the broken of C-N bond between 2-Chloro-5-

293

methyl-pyridine and 8-amino-octahydro-imidazo [1, 2-a] pyridin-7-ol, and cleavage

294

of the chloropydidine ring was detected. Moreover, the degradation products of

295

SR/RS-IPP were striking different from RR/SS-IPP. The results may be caused by the

296

isomer’s different spatial conformation.

297

In this study, biodegradation of novel chiral insecticide, RR/SS-IPP and RS/SR-IPP,

298

in aqueous solution had been studied. Three degradation strains were isolated from

299

agriculture soil and identified as Sphingobacterium. The degradation of RR/SS-IPP

300

and SR/RS-IPP in aqueous solution over time then reached a balanced state after 4

301

and 5 days, respectively. During the logarithmic phase and the stabilization period of

302

the strains, degradation rates correlated with the strains grown. And the degradation

303

rate of RR/SS-IPP and SR/RS-IPP reached 30% and 35%, respectively. Photolysis

304

was negligible because all the studies were carried out in the dark. Therefore a weak

305

decrease of RR/SS-IPP and SR/RS-IPP in all blank control groups without bacteria

306

strains might result from hydrolysis. The result of this research was shown that

307

SR/RS-IPP easier degrading than RR/SS-IPP in aqueous solution.5 In addition,

308

biodegradation products of enantiomers had been identified, and a distinct of

309

degradation pathways between two pairs of stereoisomers was also proposed. The

310

main biodegradation of SR/RS-IPP occurred on the tetrahydropyridine ring, and

311

microbial degradation of RR/SS-IPP not only on the tetrahydropyridine, also on the

312

chloropydidine ring. The study of RR/SS-IPP and SR/RS-IPP degrading strains and

313

pathway in solution system will improve the understanding of their transformations 15



314

and fate in biological systems, and reduce the amount of pesticides released into the

315

environment.

316 317

318

Corresponding author.

319

*(Z. Cai) Phone: 86-519-86330160. Fax: 86-519-86330160. Email:

320

[email protected].

321

Funding

322

The research was financially funded by the grants “National Natural Science Foundation of China”

323

(Project No. 11275033) and “Natural Science Foundation of Jiangsu Province, China” (Project No.

324

BK20151185).

325

Note

326

The authors declare no competing financial interest.

AUTHOR INFORMATION

327 328

REFERENCE

329

(1) Wang, H.; Yang, Z.; Liu, R.; Fu, Q.; Zhang, S.; Cai, Z.; Li, J.; Zhao, X.

330

Stereoselective uptake and distribution of the chiral neonicotinoid insecticide,

331

Paichongding, in Chinese pak choi (Brassica campestris ssp. chinenesis) . J. Hazard.

332

Mater. 2013, 262, 862-869.

333 334 335

(2) Liu, W.; Ye, J.; Jin, M. Enantioselective phytoeffects of chiral pesticides. J. Agric. Food Chem. 2009, 57, 2087-2095. (3) Ye, J.; Zhao, M.; Liu, J.; Liu, W. Enantioselectivity in environmental risk 16


Page 16 of 28

Page 17 of 28


336 337

assessment of modern chiral pesticides, Environ. Pollut. 2010, 58, 2371-2383. (4) Cai, Z.; Ma, J.; Wang, J.; Rong, Y.; Chen, J.; Li, S.; Zhang, W.; Zhao, X. Aerobic

338

biodegradation kinetics and pathway of the novel cis-nitromethylene neonicotinoid

339

insecticide Paichongding in yellow loam and Huangshi soils.[J]. Appl. Soil Ecol. 2016,

340

98, 150-158.

341

(5) Zhang, M.; Zhao, H.; Yang, X.; Dong, A.; Zhang, H.; Wang, J.; Liu, G.; Zhai, X.

342

A simple and sensitive electrochemical sensor for new neonicotinoid insecticide

343

Paichongding in grain samples based on β-cyclodextrin-graphene modofied glassy

344

carbon electrode. Sens. Actuators. B. 2016, 229, 190-199.

345

(6) Lou, Y.; Xu, Y.; Chai, Z.; Shao, X.; Zhao, G.; Li, Z. Organocatalytic Michael

346

addition of 2-nitromethylene imidazolidines to α,β-unsaturated aldehydes: concise

347

synthesis of chiral insecticide Paichongding. Tetrahedron lett. 2015, 71, 6651-6658.

348

(7) Fu, Q.; Wang, Y.; Zhang, J.; Zhang, H.; Bai, C.; Li, J.; Wang, W.; Wang, H.; Ye,

349

Q.; Li, Z. Soil Microbial Effects on the Stereoselective Mineralization, Extractable

350

Residue,Bound Residue, and Metabolism of a Novel Chiral Cis Neonicotinoid,

351

Paichongding. J. Agric. Food Chem. 2013, 61, 7689-7695.

352

(8) Xu, X.; Bao, H.; Shao, X.; Zhang, Y.; Yao, X.; Liu, Z.; Li, Z. Pharmacological

353

characterization of cis-nitromethylene neonicotinoids in relation to imidacloprid

354

binding sites in the brown planthopper, Nilaparvata lugens. Insect Mol. Biol. 2010, 19,

355

1-8.

356 357

(9) Li, J.; Zhang, J.; Li, C.; Wang, W.; Yang, Z.; Wang, H.; Gan, J.; Ye, Q.; Xu, X.; Li, Z. Stereoisomeric Isolation and Stereoselective Fate of Insecticide Paichongding in 17



358

Flooded Paddy Soils. Environ. Sci. Technol. 2013, 47, 12768-12774.

359

(10) Shao, X.; Lee, P.; Liu, Z.; Xu, X.; Li, Z.; Qian, X. cis-Configuration: a new

360

tactic/rationale for neonicotinoid molecular design. J. Agric. Food Chem. 2011, 59,

361

2943-2949.

362 363

(11) Shao, X.; Ye, Z.; Bao, H.; Li, Z.; Xu, X.; Li, Z.; Qian, X. Advanced research on cis-Neonicotinoids. Chimia. 2011, 65, 957-960.

364

(12) Fu, Q.; Zhang, J.; Xu, X.; Wang, H.; Wang, W.; Ye, Q.; Li, Z. Diastereoselective

365

metabolism of a novel cis-nitromethylene neonicotinoid paichongding in aerobic soils.

366

Environ. Sci. Technol. 2013, 47, 10389-10396.

367

(13) Zhao, X.; Shao, X.; Zou, Z.; Xu, X. Photodegradation of novel nitromethylene

368

neonicotinoids with tetrahydropyridine-fixed cisconfiguration in aqueous solution. J.

369

Agric. Food Chem. 2010, 58, 2746-2754.

370

(14) Mohanram, R.; Jagtap, C.; Kumar, P. Isolation, screening, and

371

characterization of surfaceactive agent-producing, oil-degrading marine

372

bacteria of Mumbai Harbor. Mar. Pollut. Bull. 2016, 1-8.

373

(15) Cai, Z.; Zhang, W.; Li, S.; Ma, J.; Wang, J.; Zhao, X. Microbial

374

Degradation Mechanism and Pathway of the Novel Insecticide Paichongding

375

by a Newly Isolated Sphingobacterium sp.P1 3 from Soil. J. Agric. Food Chem.

376

2015, 63, 3823-3829.

377

(16) Taloria, D.; Samanta, S.; Das, S.; Pututunda, C. Increase in Bioethanol

378

Production by Random UV Mutagenesis of S.cerevisiae and by Addition of Zinc Ions

379

in the Alcohol Production Media. APCBEE Procedia, 2012, 2, 43-49. 18


Page 18 of 28

Page 19 of 28


380

(17) Beacham, T.; Mora Macia, V.; Rooks, P.; White, D.; Ali, S. Altered lipid

381

accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced

382

mutagenesis. Biotechnol. Rep. 2015, 7, 87-94.

383

(18) Cai, Z.; Wang, H.; Shi, S.; Wang, W.; Chen, Q.; Zhao, X.; Ye,Q. Aerobic

384

biodegradation kinetics and pathway of the novel herbicide ZJ0273 in soils. Eur. J.

385

Soil Sci. 2013. 64, 37-46.

386

(19) Seo, J.; Keum, Y.; Harada, R.; Li, Q. Isolation and Characterization of Bacteria

387

Capable of Degrading Polycyclic Aromatic Hydrocarbons (PAHs) and

388

Organophosphorus Pesticides from PAH-Contaminated Soil in Hilo, Hawaii. J. Agric.

389

Food Chem. 2007, 55, 5383-5389.

390 391 392

(20) Bollag, J.; Myers, C.; Minard, R. Biological and chemical interactions of pesticides with soil organic matter. Environ. Sci. Technol. 1992, 123-124, 205-217. (21) Cai, Z. Q.; Chen, Q. L.; Wang, H. Y.; He, Y., Wang, W.; Zhao, X.; Ye, Q.

393

Degradation of the novel herbicide ZJ0273 by Amycolatopsis sp. M3-1 isolated from

394

soil. Appl. Microbiol. Biotechnol. 2012, 96, 1371-1379.

395

(22) Cai, Z.; Wang, J.; Zhu, X.; Cai, J.; Yang, G. Anaerobic degradation pathway of

396

the novel chiral insecticide paichongding and its impact on bacterial communities in

397

soils. J. Agric. Food Chem. 2015, 63, 7151-7160.

398

19



399

FIGURE CAPTIONS

400

Figure 1. Stereochemical structures of IPP stereoisomers with two chiral centers (R or

401

S mark chiral centers).

402

Figure 2. (a) The stereoisomers of IPP were separated by prep-HPLC; (b) The HPLC

403

chromatogram of RR/SS-IPP; (c) The HPLC chromatogram of SR/RS- IPP;

404

Figure 3. Survival rate of mutated strain when exposed by UV light.

405

Figure 4. Degradation efficiency of RR/SS-IPP with G1-13/G1-14 (a) and SR/RS-IPP

406

with G2-19 (b).

407

Figure 5. Phylogenetic tree of strains G1-13, G1-14 and G2-19 based on the 16S

408

rRNA by Clustal and Neighbor-Joining algorithms of MEGA 5.1.

409

Figure 6. Mass spectra of metabolites (I1-I6) formed during RR/SS-IPP and

410

SR/RS-IPP biodegradation in aqueous systems. (a) IPP; (b) I1; (c) I2; (d) I3; (e) I4; (f)

411

I5, I6;

412

Figure 7. Proposed degradation pathways of RR/SS-IPP and SR/RS-IPP in aqueous

413

systems: (a) RR/SS-IPP biodegradation by Sphingobacterium sp. G1-13/ G1-14; and

414

(b) SR/RS-IPP biodegradation by Sphingobacterium sp.G2-19.

415

20


Page 20 of 28

Page 21 of 28


416

417 418 419 420 421

Figure 1. Stereochemical structures of IPP stereoisomers with two chiral centers (R or S mark chiral centers).

21



422

423 424

425 426

427 428 429 430 431 432

Figure 2. (a) The stereoisomers of IPP were separated by prep-HPLC; (b) The HPLC chromatogram of RR/SS-IPP; (c) The HPLC chromatogram of SR/RSIPP;

22


Page 22 of 28

Page 23 of 28


433

434 435 436 437

Figure 3. Survival rate of mutated strain when exposed by UV light.

23



438

439 440

441 442 443 444

Figure 4. Degradation efficiency of RR/SS-IPP with G1-13/G1-14 (a) and RS/SR-IPP with G2-19 (b).

24


Page 24 of 28

Page 25 of 28


445

446 447 448 449 450

Figure 5. Phylogenetic tree of strains G1-13, G1-14 and G2-19 based on the 16S rRNA by Clustal and Neighbor-Joining algorithms of MEGA 5.1.

25



451

452

453 454 455 456 457

Figure 6. Mass spectra of metabolites (I1-I6) formed during RR/SS-IPP and SR/RS-IPP biodegradation in aqueous systems. (a) IPP; (b) I1; (c) I2; (d) I3; (e) I4; (f) I5, I6; 26


Page 26 of 28

Page 27 of 28


458 459

460 461 462 463 464 465

Figure 7. Proposed degradation pathways of RR/SS-IPP and SR/RS-IPP in aqueous systems: (a) RR/SS-IPP biodegradation by Sphingobacterium sp. G1-13/ G1-14; and (b) SR/RS-IPP biodegradation by Sphingobacterium sp. G2-19.

27



*TOC graphic


Page 28 of 28

Isolation of the Novel Chiral Insecticide Paichongding (IPP) Degrading

Recommend Documents