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Aerobic versus Anaerobic Microbial Degradation of Clothianidin Under Simulated California Rice Field Conditions Rebecca Anne Mulligan, Patrick L. Tomco, Megan W. Howard, Tabitha S. Schempp, Davis J. Stewart, Phillip M. Stacey, David B. Ball, and Ronald S. Tjeerdema J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02055 • Publication Date (Web): 08 Aug 2016 Downloaded from http://pubs.acs.org on August 30, 2016

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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.

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Journal of Agricultural and Food Chemistry

ACS Paragon Plus Environment


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Aerobic versus Anaerobic Microbial Degradation of Clothianidin Under Simulated California Rice Field Conditions Rebecca A. Mulligan, Patrick L. Tomco*, Megan W. Howard**, Tabitha Schempp***, Davis J. Stewart ****, Phillip M. Stacey****, David B. Ball**, and Ronald S. Tjeerdema Department

of

Environmental

Toxicology,

College

of

Agricultural

and

Environmental Sciences, University of California, One Shields Avenue, Davis California 95616-8588 *Department of Chemistry, University of Alaska, Anchorage, 3211 Providence Drive, Anchorage, AK 99508 **Department of Biological Sciences, University of Alaska, Anchorage, 3211 Providence Drive, Anchorage, AK 99508. Current Address: Battelle Memorial Institute, 505 King Ave, Columbus, OH 43201 ***Department of Chemistry and Biochemistry, California State University, Chico, 400 West First Street, California 95929-0210

1

Abstract. Microbial degradation of clothianidin was characterized under aerobic

2

and anaerobic California rice field conditions. Rate constants (k) and half-lives

3

(DT50) were determined for aerobic and anaerobic microcosms, and an enrichment

4

experiment was performed at varying nutrient conditions and pesticide

5

concentrations. Temperature effects on anaerobic degradation rates were

6

determined at 22 ± 2oC and 35 ± 2oC. Microbial growth was assessed in the

7

presence of varying pesticide concentrations and distinct colonies were isolated and

8

identified. Slow aerobic degradation was observed but anaerobic degradation


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9

occurred rapidly at both 25 and 35oC. Transformation rates and DT50 values in

10

flooded soil at 35 ± 2oC (k= -7.16 x 10-2 ± 3.08 x 10-3 d-1, DT50 =9.7 d) were

11

significantly faster than in 25 ± 2oC microcosms (k= -2.45 x 10-2 ± 1.59 x 10-3 d-1,

12

DT50 = 28.3 d). At the field scale, biodegradation of clothianidin will vary with

13

extent of oxygenation.

14 15

Introduction. Clothianidin, a synthetic neonicotinoid insecticide, is registered for

16

pre-flood (to field soil) and post-flood (to field water) application to protect

17

California rice fields against the rice water weevil (RWW), Lissoroptrus oryzophilus.

18

1-3

19

nervous system of insects and is of interest to California rice culture as an

20

alternative to the broad-spectrum pyrethroids currently in use. 2-4

It acts as a potent agonist at post-synaptic cholinergic receptors within the

21 22

Researchers have suggested that the environmental fate of neonicotinoid

23

insecticides in soil is primarily controlled by microbial metabolism, however no

24

bacteria have been found to posses a complete set of genes required for their

25

complete mineralization.

26

neonicotinoids have been characterized previously, though little is known about the

27

ability of clothianidin to undergo similar degradation.5,

28

investigation we address microbial degradation under both flooded (anaerobic) and

29

non-flooded (aerobic) soil conditions.

5-8

Soil microbial degradation processes involving other

30


7-12

Therefore, in this


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31

In California rice culture, flooded conditions are established prior to planting and

32

maintained throughout the growing season. The abundance and variability of

33

microbial communities in flooded soils is complex and heavily influenced by

34

environmental conditions.13, 14 Once flooded, aerobic microbes rapidly metabolize

35

oxygen stores in surface soils while anaerobes reduce alternative electron acceptors

36

in anoxic bulk soil.13, 15 Although transformation processes in a flooded field are

37

predominately anaerobic, oxic-anoxic regions may occur within the soil-water

38

interface, within the rhizosphere soil and near the root system, allowing for the

39

possibility of both aerobic and anaerobic processes, respectively.

40

Furthermore, the rate of microbial transformation of a pesticide is dependent upon

41

the degree to which it is accessible to microbes and is therefore influenced by

42

temperature-dependent partitioning between soil and water.18-21

13, 16, 17

43 44

In this report we describe the results of a laboratory incubation study conducted

45

under representative California rice field conditions for the purpose of calculating

46

biodegradation rates, assessing microbial growth dynamics and identifying colonies

47

capable of growing in the presence of the insecticide. Flooded and non-flooded

48

microcosms were constructed based on OECD guideline 307 and sample extracts

49

were

50

transformation products.22 Throughout the growing season, California air and soil

51

temperatures are subject to daily temperature fluctuations ranging between 20-36

52

oC

53

representative flooded soil were determined at both 22 ± 2 oC and 35 ± 2oC. An

monitored

for

disappearance

of

clothianidin

and

appearance

of

and 14-26 oC, respectively.23 Thus, transformation rates of clothianidin in


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54

enrichment experiment was conducted using varying nutrient conditions and

55

dosages to determine optimal biodegradation conditions and assess which

56

microorganisms can survive in the presence of the pesticide with no added carbon

57

source.

58 59

Materials and Methods

60

Soil and reagents. Aerobic and anaerobic microbial degradation rates were

61

evaluated for soil collected from the UC Rice Experiment Station (Biggs, CA) in

62

January 2015 and April 2015, respectively. The field had no history of clothianidin

63

application. Several kilograms of soil were collected from the 0-10 cm layer, air-

64

dried and ground to pass through a 2-mm sieve; soil was stored at -23oC and used

65

within 8 weeks. Both physical-chemical properties and organic carbon content were

66

determined by the UCD Analytical Laboratory; the methods can be found on their

67

website (http://analab.ucdavis.edu). The soil, an Esquon Neerdobe clay, was

68

taxonomically classified as a fine, smectic, thermic Xeric Epiaquerts containing

69

14.5% sand, 1.0% organic carbon, 33.0% silt and 52.5% clay; moisture content and

70

pH were 27.8% and 5.1, respectively.

71 72

Chemicals.

Analytical grade clothianidin (Fluka, 99.9% purity), and clothianidin-

73

d3 (Fluka, ≥ 97.0% purity), and reagent grade nitroguanidine (Aldrich, contains ≥

74

25.0% water) and methyl nitroguanidine (Aldrich, contains ≥ 22.0% water), KH PO

75

MgSO ●7 H O, MnSO ●7H O, and glucose were purchased from Sigma Aldrich (St.

76

Louis, MO, USA).

2

4

2

4

4,

2

K HPO and FeSO ●7 H O were purchased from J.T. Baker Inc. 2

4

4

2



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77

(Phillipsburg, NJ), while ammonium monohydrogen phosphate ((NH ) HPO ) was from

78

EMD Chemicals Inc. (Gibbstown, NJ). The pancreatic digest of gelatin and beef extract

79

were both purchased from BD Biosciences (San Jose, CA). Formic acid (puriss. p.a.,

80

~98%), deionized water (≤4.3μS cm-1), methanol, acetone and water were

81

purchased from Sigma-Aldrich. Synthesis of the degradation product N- (2-chloro-1,

82

3-thiazole-5-ylmethyl)-N-methylurea (TZMU; ≥ 95.0% purity) was described in

83

Mulligan et al. 2015. 24

4 2

4

84 85

Synthesis of TZNG. A homogenous solution of 2-chlorothiazol-5-yl methanaminium

86

chloride (1.92g, 10.4mmol), 100 ml of ethanol and diisopropylethylamine (3.61ml,

87

20.7mmol) was prepared in a round bottom flask equipped with a stir bar. (E)-

88

methyl N'-nitrocarbamimidothioate (1.89g, 10.4mmol) was added and the solution

89

was stirred at 22 oC for 16 h. Upon completion, 100 mL of water was added to the

90

reaction

91

nitroguanidine (TZNG) was extracted from the aqueous suspension with 30 mL of

92

ethyl acetate. The process was repeated three times; the combined organic extracts

93

were dried over magnesium sulfate and concentrated in vacuo to yield a brown

94

solid (1.01g, 41.2%). Identity (≥95.0% purity) was confirmed via NMR using a

95

Varian 300VX high-resolution magnet (Palo Alto, CA): 1H (300 MHz, DMSO-d6),

96

δ8.08 (s, 2H), δ7.6o (s, 1H), δ4.51 (s, 2H) ppm;

97

δ150.4, δ140.1, δ138.3, δ36.8 ppm.

flask.

Thiazolylnitroguanidine

(N-(2-Chlorothiazol-5-ylmethyl)-N’-

13C

(125 MHz, DMSO-d6) δ158.7,

98


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99

Whole Soil Microcosm Experiment. Aerobic microcosms were prepared using 20

100

g of soil in 50 mL amber glass jars (uncapped) and covered with foil. Soil moisture

101

content was adjusted and maintained to 40% water-holding capacity. Controls were

102

triple autoclaved prior to the addition of 20 μg clothianidin (in 1 mL water) at the

103

start of the experiment. Samples (n=3) and controls (n=2) were placed on a rotary

104

shaker (100 rpm) at 25 ± 2oC, and sacrificial microcosms were sampled over 0, 4, 6,

105

11, 21, 28, 35, 42, 53, and 59 days.

106 107

Anaerobic microcosms were constructed with a 5-cm soil layer (40 g) and a flood

108

depth of 4 cm (79 mL) in Nalgene plastic screw-top bottles (125 mL). Controls were

109

prepared using sterile water and autoclaved soil. Samples (n=4) and controls (n=3)

110

were flushed with N2 gas, wrapped with Parafilm, placed in a light protected rotary

111

shaker (100 rpm) and incubated at 22 ± 2oC or 35 ± 2oC. The relative redox potential

112

of flooded soil (as Ehs) and pH values were measured throughout the experiment

113

using a calibrated ORP/pH meter (Hanna Instruments; Woonsocket, RI). To ensure

114

anaerobic conditions were achieved prior to the addition of clothianidin, soil redox

115

potential was monitored daily in triplicate microcosm bottles. At t=0 days, 40μg of

116

clothianidin (in 1 mL water) was added to each jar, corresponding to approximately

117

three times the maximum yearly application rate (0.2 lb a.i. acre-1), and destructive

118

sampling of microcosms occurred over 0, 1, 3, 8, 11, 15, 18, 22, 27 and 30 days.

119 120

Soil and water phases of anaerobic microcosms were separated by centrifugation

121

(100 x RCF) for 10 min, and supernatant was transferred to a 10-mL volumetric



122

flask containing clothianidin-D3 (1 μg in 200 μL methanol). A 1-mL aliquot of the

123

mixture was passed through a 13 mm Acrodisc syringe tip filter with a 0.2 μm

124

polypropylene hydrophilic filter (PTFE; Pall, Port Washington, NY) and stored in

125

amber HPLC vials at 4 oC until analysis. Each soil sample (anaerobic and aerobic)

126

was suspended in 40 mL of acetone and placed in a sonication bath for 4 h. Soil-

127

acetone slurries were separated by centrifugation (100 x RCF) for 10 min. This

128

process was repeated four times, combined acetone fractions were transferred to a

129

50-mL amber PTFE screw-top centrifuge tube and acetone was evaporated under N2

130

gas. Residues were reconstituted in 10 mL of methanol with 1 μg of clothianidin-D3,

131

and samples were filtered and stored prior to analysis by LC-MS/MS.

132 133

LC-MS/MS conditions. Soil and water samples were analyzed using an Agilent

134

(Santa Clara, CA) model 1220 HPLC with an 1220 series autosampler coupled to a

135

model 6420 triple quadrupole mass spectrometer equipped with an atmospheric

136

pressure chemical ionization source (APCI) and controlled by Mass Hunter (version

137

B.06.00). An Xterra C18 column (3.5 μm particle size, 4.6 x 250 mm i.d.; Waters,

138

Milford, MA) with gradient elution (0.4 mL min-1, 37oC) and a 5-μL injection volume

139

was used. The solvent gradient profile was as follows: Solvent A, water (0.1% formic

140

acid); solvent B, methanol (0.1% formic acid); 0 min., 90% A: 10% B; 0 – 14 min.,

141

2.5% A: 97.5% B; 15.1 min., 90% A, 10% B.

142 143

Detection and quantification of clothianidin and degradation products were

144

performed in positive ion mode, using multiple reaction monitoring (MRM) with


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145

protonated molecular ions (M-H)+ as precursor ions. The retention time, MRM

146

quantification and qualifier transitions dwell time, fragmentor voltage, collision

147

energy and collision cell voltages for each compound are summarized in Table 1.

148

APCI source parameters were as follows: gas temperature, 325oC; APCI heater

149

temperature, 350oC, curtain gas flow, 4 L min-1; nebulizer, 20 psi; and capillary

150

voltage, 4500 V. Internal standard calibration curves using clothianidin-D3 were

151

constructed for clothianidin, MNG, NG and TZNG-37 were linear in the range of

152

0.007 to 2 ug mL-1 (R2= 0.999, residuals less than 10%).

153 154

2.6 Enrichment Microcosms. Microbial enrichment cultures were performed on

155

thawed soil; control microcosms containing sterile soil (samples autoclaved four

156

sequential cycles at 121 °C, 15 psi for 30 min, cooling between cycles). Sub-samples

157

of sterile soil were used for control microcosms.

158 159

Aerobic microcosms were prepared in 250 mL Erlenmeyer flasks containing 125 mL

160

minimal media (MM): 2.5 g K2HPO4, 2.5 g KH2PO4, 1.0 g (NH4)2HPO4, 2.0 g MgSO4●7

161

H2O, 0.01 g FeSO4●7 H2O and 0.007 g MnSO4●7H2O per liter), minimal media plus

162

glucose (MMG: MM recipe with additional 0.1% w/v glucose per liter), or nutrient

163

broth (NB: 5.0 g pancreatic digest of gelatin, 3.0 g beef extract, pH 7.0 per

164

liter). Four microcosms per media were created, each adjusted to 0.05 µg/mL, 0.5

165

µg/mL, 5 µg/mL, or 50 µg/mL clothianidin (using a 500 µg/mL clothianidin solution

166

in MM). Thawed soil samples (2.5 g) were added to each microcosm, thoroughly

167

triturated, capped (not sealed), and maintained on a shaker at 150 rpm at room-



168

temperature (RT). Control microcosms of each media were created, and included

169

0.5 µg/mL clothianidin and 2.5g sterile soil, and were treated identically to

170

experimental microcosms.

171 172

Enrichment microcosms were sampled at day 0, 2, 4, 7, 14, and 28. Aqueous

173

samples were filtered through a 0.22 µm MillixGP syringe filters (EMD Millipore,

174

Temecula, Ca.) and stored at -20°C until analysis. Detection and quantification of

175

clothianidin was performed in positive ion mode LC-MS/MS as described above.

176 177

Bacterial Enumeration and Isolation: Enrichment microcosms were sampled in

178

triplicate daily from 0-7 d, then at 14 and 28 days. Turbidity (optical density, OD)

179

was measured as absorbance at 600 nm using an Evolution 201 Spectrophotometer

180

(Fisher Scientific, Pittsburgh, PA). Samples were clarified via centrifugation prior to

181

absorbance readings. Microbial load was determined using the standard plate count

182

enumerated in duplicate on nutrient agar (5.0 g pancreatic digest of gelatin, 3.0 g

183

beef extract, 15 g agar per liter) after 48 hour incubation at RT. Colonies on

184

enumeration plates were counted and microbial load was calculated as CFU/g soil.

185 186

Bacteria capable of growth in the presence of differing concentrations of

187

clothianidin were isolated from enumeration plates. Enumeration plates were

188

screened for colonies that were visually distinct from one another, and were sub-

189

cultured onto nutrient agar. As colony growth became visually present, colonies

190

were sub-cultured from each microcosm onto fresh nutrient agar plate and


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incubated for two days prior to DNA extraction. DNA was extracted from colonies

192

using a modified PowerSoil DNA Isolation Kit (MoBio, Carlsbad, CA) as previously

193

described.25 Genomic DNA was PCR amplified using 16S ribosomal RNA primers 27F

194

(5’- AGAGTTTGATCMTGGCTCAG-3’) and 1492 (5’- GGTTACCTTGTTACGACTT-3’), as

195

described previously.26 Reactions were conducted in 20 mL volumes, using

196

DreamTaq Green PCR 2X mastermix (Thermo Fisher, Grand Island, NY). PCR cycling

197

conditions were: 95°C for 5’, followed by 35 cycles of 95°C for 30”, 57°C for 30” and

198

72°C for 2’. Amplicons were visually inspected using agarose gel electrophoresis,

199

and amplicons were purified using the Agencourt AMPure XP - PCR Purification

200

(Beckman Coulter, Indianapolis, IN) at a ratio of AMPure XP to sample of 0.6X.

201

Sequencing was performed on an ABI 3730xl sequencer using the 27F primer for

202

sequence initiation. Amplification, purification and sequencing were performed at

203

the DNA Services Facility at the University of Illinois at Chicago.

204 205

Microbial Phylogeny. Identification of phylogenetic neighbors was initially carried

206

out by the BLASTN program against the database containing type strains with

207

validly published prokaryotic names and representatives of uncultured phylotypes27,

208

28.

209

calculation of pairwise sequence similarity using global alignment algorithm, which

210

was implemented at the EzTaxon server (http://www.ezbiocloud.net/eztaxon).28, 29

211

Identification of phylogenetic neighbors was initially carried out by the BLAST and

212

megaBLAST programs against the database of type strains with validly published

213

prokaryotic names.27, 28, 30 The top thirty sequences with the highest scores were

The top 30 sequences with the highest scores were then selected for the



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214

then selected for the calculation of pairwise sequence similarity using global

215

alignment

216

(http://eztaxon.ezbiocloud.net/).28

algorithm,

which

was

implemented

at

the

EzTaxon

server

217 218

Data Analysis. Data were log-transformed and rate constants were calculated

219

based on pseudo first-order kinetics using Equation 1:

ln = −

220

(1)

221

where k is the first-order rate constant, Co is the initial amount of clothianidin

222

applied (mg kgsoil-1), and Ct is the amount recovered (mg kgsoil-1) at time t (min). The

223

half-life (DT50) was calculated from the absolute value of the first-order rate

224

constant k using Equation 2: DT =

225

(2)

226

Data were analyzed with JMP software package version 10.0 (SAS Institute Inc., Cary,

227

NC). First-order rate constants (k), standard error (SE) and R2 values were obtained

228

from a linear regression analysis of the semi-log transformed degradation data.

229

Discrete differences between anaerobic temperature treatments, controls and non-

230

sterile controls were determined by calculating the p-values associated with one-

231

way analysis of variance (ANOVA) and post hoc comparison (Tukey HSD test, α =

232

0.05); significance was determined at a α=0.05 confidence level.

233 234

Results and Discussion


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235

Redox potential and pH of flooded rice field soil. Redox potential (Ehs) was

236

monitored throughout a 14-day incubation period and determined for each sampled

237

flask (Figure 1). No significant differences were observed between Ehs values for

238

the 25 and 35oC temperature treatments. Average Ehs values of flooded microcosms

239

after a 14 day pre-incubation period ranged between -169 ± 17 mV and -146 ± 12

240

mV for 25 and 35oC, respectively – values comparable to soil redox potentials

241

observed in flooded rice fields (-123 ≤ Ehs ≤ -217 mV).16, 23 The pH of flooded soils

242

was 6.5 for the 25 and 35oC microcosms – values also comparable to flooded rice

243

fields.

244 245

Aerobic degradation kinetics. The rate of aerobic degradation (k), standard error,

246

R2, and DT50 are summarized in Table 2. The percent recovery from aerobic

247

microcosms remained consistent throughout the experiment; the average mass of

248

clothianidin recovered at t=0 d was 80± 1.5% and 79± 0.5% compared to 74± 1.0%

249

and 76± 0.6% after 59 d for non-sterile samples and sterile controls, respectively.

250

No significant differences were observed between percent recovery values in non-

251

sterile samples and sterile controls at 25oC indicating that clothianidin remained

252

persistent under aerobic conditions throughout the 59 d incubation period (Figure

253

2).

254 255

Anaerobic degradation kinetics. Degradation data for clothianidin under

256

anaerobic conditions were log-transformed and fit to the first-order kinetic model

257

using Equation 1. Plots for 25 and 35oC treatments were linear with R2 values of



258

0.862 and 0.925, respectively; corresponding degradation rate constants (k),

259

standard errors and DT50 values are presented in Table 2.

260 261

The percent recovery of clothianidin from sterile controls and non-sterile samples

262

for 25 and 35oC treatments over time are presented in Figure 3. Overall, the average

263

mass of pesticide recovered from sterile anaerobic controls was 84.0 ± 4% and

264

102.0 ± 12% for 25 and 35oC, respectively, indicating that loss of clothianidin in

265

non-sterile samples can be attributed to biodegradation.

266 267

Temperature effects on anaerobic degradation. Microbial degradation of

268

clothianidin occurred rapidly under flooded conditions at both 25 and 35oC. A

269

continuous degradation pattern (without a lag period) was observed for both

270

temperatures. After 30 days at 25oC, less than 46.0 ± 2% of the initial mass of

271

clothianidin remained compared to 14.9 ± 4% after 24 days at 35oC. ANOVA

272

demonstrated significant differences between degradation rates and half-lives for

273

the 25oC (-2.45 x 10-2 ± 1.59 x 10-3 d-1, DT50 = 28.3 d) and 35oC (-7.16 x 10-2 ± 3.08 x

274

10-3 d-1, DT50 =9.7 d) treatments (P< 0.05). The temperature sensitivity of soil

275

microbial degradation rates is well characterized and commonly expressed in terms

276

of the Q10 relationship.

277

degradation at elevated temperature may be attributed to the Q10 relationship, the

278

increased rate observed at 35oC might also be a consequence of increased

279

availability of clothianidin in the soil solution.33 It is generally accepted that only

280

unbound residues are accessible to microorganisms; higher temperatures may

31, 32

Although it is possible that the observed increase in


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281

significantly increase the bioavailability of pesticides by promoting the release

282

(desorption) of bound pesticides residues into the soil solution (via enhanced

283

solubility).33-38 Mulligan et al. 2014 previously demonstrated that the overall

284

sorption capacity for clothianidin is low and desorption (hysteresis) will decrease at

285

elevated temperatures.39 Overall, these findings suggest that temperature will be a

286

primary factor controlling the rate of biodegradation of clothianidin in flooded rice

287

fields.

288

experienced in the Sacramento Valley during the summer growing season could

289

stimulate accelerated anaerobic degradation of the insecticide in rice fields.

It is therefore likely that the elevated temperatures (i.e. 32 to 40oC)

290 291

Transformation products. Reports generated by the registrant list TZMU, TZNG,

292

methyl-nitroguanidine and nitroguanidine as potential microbial transformation

293

products under aerobic conditions.40-42 Although soil extracts and aqueous samples

294

(for each time point) from anaerobic microcosms were analyzed using LC-MS/MS,

295

no transformation products were observed (instrumental limit of detection ≥ 10

296

ppb), and no anaerobic soil transformation data have been published to our

297

knowledge.40-42

298 299

Degradation in enrichment microcosms. At an initial concentration of 0.05

300

µg/mL, which corresponds to a 0.1X application rate, clothianidin degraded fastest

301

when incubated in nutrient broth (NB), a general purpose bacterial culture media

302

(Figure 4). The pseudo-first order degradation rate constant (+/- SE) was k =

303

0.01196 +/- 0.00471 d-1, compared to k = 0.00838 +/- 0.00561 d-1 in minimal media



304

(MM) conditions and k = 0.00448 +/- 0.0023 d-1 in minimal media supplemented

305

with 0.1% glucose (MMG). At 1X application rate (0.5 µg/mL), significant

306

degradation was also noted with k = 0.00647 +/- 0.00412 d-1 for NB, k = 0.00597 +/-

307

0.00219 d-1 for MM, and k = 0.01455 +/- 0.00268 d-1 when incubated in MMG media.

308

For the 10X application rate (5.0 µg/mL), only soil incubated in NB revealed

309

significant degradation with k = 0.00681 +/- 0.00198 d-1. For the 100X application

310

rate (50 µg/mL), no degradation in any enrichment microcosm was observed.

311

Control microcosms at 0.1X did not show degradation, except those incubated in

312

MM, a media with no additional carbon source added, which revealed k = 0.00575

313

+/- 0.00327 d-1.

314 315 316

Microbial Growth in enrichment microcosms Microbial growth in the enriched microcosms revealed a classical substrate response

317

dynamic when incubated under aerobic conditions in varying nutrient conditions.

318

Heterotrophic bacterial counts revealed typical microbial growth curves, consisting of a

319

brief period of rapid growth followed by a tapering period as the microbial community

320

adjusted to their respective nutrient conditions, or depleted available nutrients (Figure 5).

321

Microbial load was generally highest when incubated in higher nutrient conditions (NB),

322

with maximum microbial load observed at 2 days post-inoculation (heterotrophic counts

323

varied from 2.75-6.62 x109 CFU/g soil). Minimal nutrient conditions, consisting of media

324

with no added carbon source (MM), produced the lowest microbial load, with maxima at

325

4 days post-inoculation (heterotrophic counts varied from 5.50 x104 - 2.66 x105 CFU/g

326

soil). Sterilized samples incubated in the presence of 0.1x clothianidin did not show

327

microbial growth under minimal nutrient conditions (MM, MMG). The only sterilized


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328

samples to reveal growth were incubated in nutrient rich media (NB); microbial growth

329

in NB was observed on 2, 7, and 14 d post-inoculation.

330 331

Clothianidin biodegradation was found to be sensitive to both nutrient conditions

332

and initial concentration in enriched microcosms. Clothianidin was readily

333

biodegraded in microcosms supplemented with 0.1X and 1X insecticide application

334

rates; with pseudo-first order half-lives varying from 48-155 d depending on

335

nutrient conditions. At higher application rates (10X and 100X), the insecticide was

336

not readily biodegraded; only nutrient-rich conditions (NB) supplemented with 10X

337

insecticide showed biodegradation (an appreciable half-life of 102 d). Higher

338

application rates of clothianidin in NB did not show any biodegradation, potentially

339

due to toxic effects of the compound on bacteria at high concentrations, as

340

previously observed in microcosm studies with other pesticides.43

341 342

Clothianidin was depredated when soil was incubated under aerobic low nutrient

343

conditions (MM).

344

clothianidin application rates, with t1/2 = 83 d. Clothianidin degradation occurred

345

fastest when incubated in MMG media supplemented with 1X insecticide application

346

rate, with t1/2 = 48 d.

Degradation rates were fastest in MM enriched with 0.1X

347 348

MM and MMG microcosms contained either no supplemented carbon (MM) or only a

349

small amount of supplemented labile carbon (MMG), other than carbon already

350

present in the soil sample. Our enriched microcosm system exhibited a specific



Page 18 of 31

351

range of nutrient availability and pesticide concentration that allowed for

352

biodegradation. The decreased clothianidin half-life under low nutrient conditions

353

(MM, MMG) suggested that microbial populations could be degrading clothianidin

354

via metabolism using clothianidin as a carbon source, directly, co-metabolically or

355

as a side reaction of other metabolic processes.

356 357

In order to identify a subset of bacteria able to survive typical (0.1X and 1X) and

358

higher (10X, 100X) clothianidin application rates in soil, a subset of bacterial

359

colonies were isolated from enumeration plates and identified via 16S ribosomal

360

DNA sequencing. Forty-seven distinct colonies were identified on enumeration

361

plates; organisms were isolated and DNA sequence was used to identify bacterial

362

species to the genus level.

363

predominantly Gram negative bacteria and one member of the Gram positive

364

Bacillus genus. Gram negative bacteria were identified to the genus (Pseudomonas,

365

Pedobacter

366

Enterobacteriaceae and Oxalbacteraceae).

and

Sequencing (Supplemental Table S1) identified

Flavobacterium)

and

family

level

(Chryseobacterium,

367 368

The distribution of soil microbiota in agricultural soil is highly variable, even within

369

cm- to meter-scales and can vary seasonally and inter-annually based on

370

management practices and soil type.44, 45 The community composition of rice field

371

soil in particular is sensitive to flooding practices with a lower redox potential when

372

flooded; this can further complicate the microbial composition and affect substrate

373

decomposition rates.46, 47 Overall, these data indicate that aerobic degradation of


Page 19 of 31


374

clothianidin is possible given appropriate nutrient conditions but is expected to be

375

highly variable at field scale. Supplemental experiments with isolated bacteria in the

376

absence of soil particulates should be conducted to determine details of biotic

377

clothianidin degradation.

378 379

Conclusions. The persistence of pesticide residues released into the Sacramento

380

River Basin is a major concern to California farmers, researchers, and regulators.

381

Clothianidin is highly toxic towards pollinators and sediment dwelling organisms;

382

detailed and regionally specific dissipation and degradation data are necessary to

383

address concerns over the safety and suitability of its use in California rice culture.

384

In this study, clothianidin underwent rapid microbial transformation in flooded

385

microcosms and enrichment experiments demonstrated aerobic degradation is

386

possible given appropriate nutrient concentrations. The rate of anaerobic

387

degradation was higher at 35oC, compared to 25oC. These data indicate that the rate

388

of microbial transformation in rice fields will be faster after permanent flood is

389

established and air temperatures have reached elevated values characteristic of the

390

Sacramento Valley during peak growing season.

391



Page 20 of 31

ACKNOWLEGEMENTS. We thank M. J. Hengel, M.R. Yoder, C. Rering, K. Williams, Z. Redman, K. Trunnelle, M. Maier, P. Kuzmicky and D. Tjeerdema for assistance. The research was supported by the California Rice Research Board (Award RP-5) and the Donald G. Crosby Endowment in Environmental Chemistry.

Table 1: Compound names, structures and MRM parameters for clothianidin and its degradation products.

Name

nitroguanidine

Structure

Mass Transition (m/z) 105 59.1

Retention Fragmentor Collision Cell Time Voltage Energy Acclerator (min) (V) (V) (V)

8.67

66

105 75.2

methyl nitroguanidine

119 73.1

207 175

9.42

66

236 155.1

16.9

94

253.04 172.1

18

94

17

4

12

1

15

18.3

40

253.04 132

14

4

18

250 169.1

clothianidin

4

25

236 132.1

clothianidin-d3

17 1

207 86.1

thiazolnitroguanidine

4

5

119 89.1

thiazolmethylurea

9

14 18.3

250 132


40

4 18

Page 21 of 31


Table 2: Comparison of aerobic and anaerobic degradation rates with standard error values, R2 values, and corresponding half-lives. Temperature (oC) Conditions

Type

R2

DT 50 (day)

Significant Differencea

Samples

Aerobic

25

-2.24 x 10-04 ± -7.48 x 10 -04

0.0007

> 187

A

Samples

Anaerobic

25

-2.45 x 10-02 ± 1.59 x 10 -03

0.8622

28.3

B

Samples

Anaerobic

35

-7.81 x 10-02 ± 2.91 x 10 -03

0.9246

9.7

C

Same letter indicates significant difference (Tukey HSD test, α = 0.05).

400

300

200

Soil Redox Poten al (mV)

a

k (day-1 )

100

0 0

5

10

15

20

25

30

-100

-200

-300

Time (days) post flood


35

40

45

50


Page 22 of 31

Figure 1: Average relative redox potential profiles for flooded soil microcosms at 35oC (

) and 25oC (

) over the course of the experiment. Error bars represent

± SE (n≥ 3).

100%

95%

Recovery (% Clothianidin Applied)

90%

85%

80%

75%

70%

65%

60%

55%

50% 0

10

20

30

40

50

60

70

Time (day)

Figure 2: Percent recovery of clothianidin (expressed as percent of clothianidin applied) for non-sterilized soil ( ) and autoclaved controls ( ) under aerobic conditions. Data points represent mean values. Error bars represent ± SE (controls, n≥ 2; samples, n≥ 3).


Page 23 of 31


A1. Recovery (% Clothianidin Applied)

120%

100%

80%

60%

40%

20%

0% 0

5

10

15

20

25

30

35

Time (day)

A2.

0.5

0.0

ln C/Co

-0.5

-1.0

-1.5

-2.0

-2.5 0

5

10

15

20

25

me (day)


30

35


Page 24 of 31

B1. Recovery (% Clothianidin Applied)

140%

120%

100%

80%

60%

40%

20%

0% 0

5

10

15

20

25

30

Time (day)

B2.

0.5

0.0

ln C/Co

-0.5

-1.0

-1.5

-2.0

-2.5 0

5

10

15

20

25

30

me (day)

Figure 3: Percent applied mass recovered (A1 and B1) and pseudo-first order anaerobic degradation kinetics (A2 and B2) of clothianidin for biologically active soil ( ) and autoclaved controls ( ) for 25 oC (A1 and A2) and 35 oC (B1 and B2) temperature treatments. Data points represent mean values ± SE (controls, n≥ 3; samples, n≥ 4)


Page 25 of 31


Degradation rate, k (d-1)

0.02 0.018 0.016 0.014 0.012

MM

0.01 0.008

MMG

0.006

NB

0.004 0.002 0 0.1X sterile

0.1X

1X

10X

100X

Enrichment Level Figure 4. Pseudo-first order degradation rates of clothianidin, in k (d ) (+/- 1 SE) for three enrichment media (MM = minimal media, MMG = minimal media + glucose, NB = nutrient broth) at varying application rates. -1



Figure 5. indicated by nutrient growth MMG, NB). conducted with - 100x Data are means day 0, n=2

Page 26 of 31

Microbial load, as plate counts under conditions (MM, Incubations were clothianidin at 0.1x concentrations. +/- 1 SE (n=3 for afterwards).


Page 27 of 31


Supplemental Table S1. Identification of heterotrophic bacteria capable of growth in the presence of clothianidin. Bacterial identification and match percentage obtained from EzTaxon server.28



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