Evaluation of Diuron Tolerance and Biotransformation by Fungi from a

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Evaluation of diuron tolerance and biotransformation by fungi from a sugarcane plantation sandy-loam soil Bruna Perissini Lopes, Tássia Chiachio Egea, Diego Alves Monteiro, Ana Cláudia Vici, Danilo Grunig Humberto Da Silva, Daniela Correa de Oliveira Lisboa, Eduardo Alves de Almeida, John Robert Parsons, Roberto da Silva, and Eleni Gomes J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03247 • Publication Date (Web): 21 Nov 2016 Downloaded from http://pubs.acs.org on December 1, 2016

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Journal of Agricultural and Food Chemistry

1

Evaluation of diuron tolerance and biotransformation by fungi from a

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sugarcane plantation sandy-loam soil

3 4

Bruna Perissini-Lopes1*, Tássia Chiachio Egea1, Diego Alves Monteiro1,

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Ana Cláudia Vici2, Danilo Grünig Humberto Da Silva1, Daniela Correa de

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Oliveira Lisboa1; Eduardo Alves de Almeida1, John Robert Parsons3, Roberto

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Da Silva1, Eleni Gomes1.

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1

Universidade Estadual Paulista Julio de Mesquita Filho - Campus de São José

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do Rio Preto Cristóvão Colombo, 2265, Jardim Nazareth, São José do Rio

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Preto, SP, BR 15054-000.

12 13 14

2

Universidade de São Paulo Faculdade de Medicina de Ribeirão Preto

Bandeirantes, 3900, Monte Alegre, Ribeirão Preto, SP, BR 14040-900

15 16

3

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Nieuwe Achtergracht 199, 1018 WV Amsterdam, NL 1018 WV, +31205256580

University of Amsterdam, Institute for Biodiversity and Ecosystem Dynamics,

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*Corresponding author: Bruna Perissini-Lopes ([email protected]),

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Address: Universidade Estadual Paulista, Brazil. Phone #: +55-17-996070620

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1

ACS Paragon Plus Environment


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Abstract

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Microorganisms capable of degrading herbicides are essential to minimize

24

the amount of chemical compounds that may leach into other environments.

25

This work aimed to study the potential of sandy-loam soil fungi to tolerate the

26

herbicide Herburon® (50% diuron) and to degrade the active ingredient diuron.

27

Verticillium sp. F04, Trichoderma virens F28 and Cunninghamella elegans B06

28

showed the highest growth in the presence of the herbicide. The evaluation of

29

biotransformation showed that Aspergillus brasiliensis G08, Aspergillus sp. G25

30

and Cunninghamella elegans B06 had the greatest potential to degrade diuron.

31

Statistical analysis demonstrated that glucose positively influences the potential

32

of the microorganism to degrade diuron, indicating a cometabolic process. Due

33

to metabolites founded by diuron biotransformation, it is indicated that the fungi

34

are relevant in reducing the herbicide concentration in runoff, minimizing the

35

environmental impact on surrounding ecosystems.

36 37

Keywords: Fungi soil, biotransformation, diuron, Plackett-Burman, Central

38

Composite Rotational Design, HPLC, LC/MS/MS.

39 40

2


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1. Introduction

42 43

According to Ministry of Agriculture, Livestock and Food Supply, Brazil is the

44

largest producer of sugarcane and the first in the world in the production of

45

sugar and ethanol, accounting for more than half the sugar traded in the world.1

46

The cultivation of sugarcane can suffer from weed infestation, causing problems

47

that raise the cost of production. Several herbicides are used to eliminate

48

weeds from sugarcane crops,2 but their effects are not limited only to the

49

application site. Once in the environment, they can disperse contaminating

50

groundwater, surface water, and soil, causing alterations in the structure and

51

function of the local microbiota, and may compromise the health and resilience

52

of non-target organisms.3,4

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Diuron [3-(3,4-dichlorophenyl-1,1-dimethylurea)] is one of the most important

54

herbicides applied in sugarcane crops with pre- and post-emergent application,

55

often in combinations with other herbicides such as hexazinone, ametrine and

56

tebuthiuron.5 Diuron is used extensively in the state of São Paulo and this state

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contributes about 53% of the Brazilian sugarcane production,1 and the impact of

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diuron in this area is understudied. It is difficult to make generalizations about

59

the behavior of herbicides in soil, since the relation herbicide-soil-biota is very

60

peculiar. Therefore specific studies in a particular region may help complete

61

understanding of the impact of these compounds in the environment.

62

Furthermore, it is important to study the metabolites formed from the

63

degradation of herbicides, because many compounds have degradation

64

products that can be more toxic to the environment, affecting non-target

3



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organisms,3-5 and evaluate the degradation of these metabolites in order to

66

understand the real herbicide impact in the region.

67

Diuron has low solubility in water, high organic carbon-water partition

68

coefficient Koc (499 cm3 g-1) and low Henry’s Law value Kh (5.1 x 10-5 Pa m3 mol

69

L-1), resulting in strong adsorption to soil organic particles.5 Thus, in more

70

organic rich and clayey soils, the persistence of diuron in the environment is

71

greater, since the retained molecules in the soil become less available to plants

72

and microorganisms.6 In contrast, application to soils with low organic matter

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content, such as loam-sandy and sandy, can result in more leaching of diuron to

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the aquatic environment.7

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The chloroaromatic structure of diuron is responsible for its recalcitrance and

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toxic potential in the environment and it is classified as extremely harmful to

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aquatic ecosystems, affecting organisms such as seagrasses, mangroves,

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corals, benthic microalgae and fish.8-12 Furthermore, diuron is also classified as

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endocrine disrupting, i.e. a chemical which can interfere with the natural

80

function of the endocrine system of animal species, including humans, and

81

causing carcinogenic effects in rats.13,14

82

Microorganisms are involved in biodegradation process of a wide range of

83

herbicides.15-19

84

chlorophenols,

85

agrochemicals and textile dyes, have been shown to be degraded by different

86

fungal species.20-22 Fungi play an important role in cycling of xenobiotics in the

87

environment, due to their ability to increase the soil respiratory activity and

88

secrete different enzymes.22 The mycelial growth allows the fungal hyphae go

Several

xenobiotics,

polycyclic

aromatic

such

as

aliphatic

hydrocarbons,

hydrocarbons, chloroaromatics,

4


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89

through air gaps and pores in the soil, reaching contaminated sites previously

90

inaccessible by other microorganisms.23,24

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Even though many studies have been carried out involving diuron

92

degradation,20,25-28 the complexity of this process still require a lot of research to

93

fully understand the environmental fate and risks of this herbicide and its main

94

metabolites. Due to the relevance of fungi in contribute to herbicides

95

biodegradation, this work aimed to study the potential of these microorganisms

96

from an herbicide-treated sandy-loam soil to tolerate high concentrations of the

97

herbicide Herburon® (50% diuron) and to degrade its active ingredient. We also

98

report the potential of compounds in the culture medium to increase the rates of

99

diuron degradation by fungi, thereby reducing its concentration in runoff and

100

minimizing the environmental impact on surrounding ecosystems.

101 102

2. Materials and Methods

103 104

2.1 Microorganisms

105

The fourteen fungi strains used in this study were previously isolated from

106

sugarcane plantation soil subjected to frequent application of diuron and

107

characterized by Egea et al.29 (Table 1). The soil used for fungal isolation is

108

located in the tropical zone of the northwest of São Paulo state, Brazil. The

109

fungi were maintained on Potato Dextrose Agar (PDA) and mineral oil at 5 ºC.

110 111

2.2 Chemicals

112

The commercial herbicide Herburon® (composed by 500 g L-1 of the active

113

ingredient diuron [3-(3,4-dichlorophenyl-1,1-dimethylurea)] and 678 g L-1 of inert 5



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ingredients), was obtained from Milenia Agrociências S/A – Londrina, Brazil.

115

Analytical-grade diuron (98%), DCPMU [1-(3,4-dichlorophenyl)-3-methylurea]

116

(99%), DCPU [1-(3,4-dichlorophenyl)urea] (99%) and DCA (3,4-dichloroaniline)

117

(98%) were purchased from Sigma Aldrich Brazil. HPLC and LC-MS grade

118

solvents were obtained from Sigma Aldrich. Potato Dextrose Agar (PDA) was

119

purchased from Himedia – Curitiba, Brazil.

120 121

2.3 Evaluation of herbicide effects on fungal growth

122

Previous studies suggest that fungi are tolerant to herbicide concentrations

123

higher than those occurring in the field.30 Thus, tolerance experiments were

124

conducted in duplicate by cultivating fungi on Petri dishes containing PDA

125

medium and commercial diuron (Herburon®). Herburon® concentrations were

126

2.5, 10 and 20 g L-1, corresponding to 1.25, 5 and 10 g L-1 of active ingredient,

127

respectively.

128

concentrations may represent the accumulation of diuron from previous

129

harvests, current crop and the application of this herbicide in combination with

130

others, due to the intense sugarcane production and low rotation culture in the

131

region.

Diuron-free

media

were

used

as

controls.

These

high

132

The center of each Petri dish plate was inoculated with a 12 mm diameter

133

agar disc containing fungal mycelia from pre-cultivated fungi and incubated at

134

28 °C for 144 h. After 6 days of incubation, the diameters of the fungal colonies

135

were measured and the growth inhibition was calculated compared to the

136

control, according Equation 1.

137

C = (Ch.100)/Cc (Equation 1)

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C = growth (%); Ch = diameter of colony in presence of herbicide; Cc = diameter of

139

control

140 141

2.4 Evaluation of diuron biotransformation in liquid medium

142

The inocula were obtained by growing fungal mycelia in 100-mL sloping

143

Erlenmeyers containing 30 mL of solid medium (PDA). After 120 h of incubation

144

at 28 ºC, 30 mL of a sterile solution of Tween 80 (0.01%) were added to obtain

145

a mycelial suspension. Biotransformation experiments were performed in 125-

146

mL Erlenmeyers, inoculated with 107 spores in 30 mL of minimal medium (pH

147

5.0; g L-1):

148

MgSO4.7H2O; 4.0 peptone; 0.5 yeast extract; 5 mL micronutrients (g L-1: 22.0

149

ZnSO4.7H2O; 11.0 de H3BO3; 5.0 MnCl2.4H2O; 5.0 FeSO4.7H2O; 1.6

150

CoCl2.5H2O; 1.6 CuSO4.5H2O; 1.1 (NH4)Mo7O24.4H2O; 50 EDTA).

5.0 glucose; 1.4 (NH4)2SO4; 2.0 KH2PO4, 0.3 CaCl2; 0.2

151

The biotransformation experiments were performed in duplicate, adding 10

152

mg L-1 of diuron. The flasks were incubated at 28 °C and 160 rpm, and after

153

seven days, the samples were filtered under vacuum, centrifuged at 10000 x g

154

at 5 ºC for 10 min. The supernatant was filtered with non-sterile syringe filters

155

(0.22 µm) and analyzed by HPLC to evaluate the herbicide transformation and

156

formation of metabolites. The biomass was dried at 105 °C for 24 h31 for

157

evaluation of fungal growth.

158

The spontaneous transformation of diuron was accounted with an abiotic

159

control, and was subtracted from the diuron degradation rates obtained with the

160

fungi. A biotic control was performed by cultivating the fungus in the same

161

medium using glucose as a sole carbon source, but without diuron, to verify the

162

biomass produced and the presence of signals on analytical analysis. 7



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163 164

2.5 Analytical methods

165

HPLC

166

Culture

samples

were

analyzed

by

High

Performance

Liquid

167

Chromatography (HPLC) on a Perkin Elmer Model Flexar instrument. The

168

injection volume was 10 µL using an Agilent Zorbax Eclipse Plus C18 (250 x 4.6

169

mm

170

water/acetonitrile (40/60; v/v) as mobile phase at a flow rate of 1.0 mL min-1 at

171

40 °C. The UV detector was set at 240 nm and run time was 9 min. Calibration

172

curves were determined at concentrations from 0.5 to 50 mg L-1 for diuron and

173

0.05 to 10 mg L-1 for the metabolites. The regression coefficients were greater

174

than 0.999 for all curves.

x

5µm

–

PN:

959990-902)

column and isocratic

elution

with

175 176

SPE extraction

177

Before mass spectrometer analysis, the samples were prepared by solid

178

phase extraction (SPE) method according to Gatidou et al.32 The samples were

179

extracted using Oasis HLB (60 mg) cartridges in accordance with the

180

manufacturer’s instruction (Waters). Ten mL of the culture medium, centrifuged

181

and filtered (0.22 µm filter), were applied to cartridges in a manifold (Supelco -

182

12 samples) coupled to a vacuum pump (Marconi) at a flux of 6 mL min-1. The

183

cartridges were conditioned with 3 mL of methanol and 3 mL of ultra-pure water

184

and were eluted with 1 mL of methanol.

185 186

LC/MS/MS

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A 500-MS ion-trap mass spectrometer (Agilent Technologies), coupled to

188

electrospray ion source (ESI) and Turbo DDS software was used to confirm

189

diuron metabolites. The binary mobile phase consisted of water with 0.2%

190

formic acid (A) and methanol with 0.2% formic acid (B); gradient curve, 20% B

191

at 0 min, 90% B at 14 min, 90% B at 15 min, 20% B at 16 min, 20% at 18 min,

192

with a flow rate of 0.5 mL min-1. The ESI ionization was operated in positive ion

193

mode. The MS/MS were performed in Turbo DDS survey scan mode to detect

194

daughter ions, using trigger thresholds of 40,000 (ion counts), at radio

195

frequency of 682 khz. The nebulizer gas (N2) pressure was 35 psi, drying gas

196

(N2) pressure 10 psi, drying gas (N2) temperature 350 ºC and trap damping gas

197

(He) flow 0.8 mL min-1. The parameters which were optimized by directly

198

injecting are listed on Table 2.

199 200

2.6 Effect of media components on diuron biotransformation

201

Factorial design was used to evaluate the influence of glucose, nitrogen

202

[(NH4)2SO4] and phosphorus (KH2PO4) concentrations, and pH on diuron

203

transformation. A Plackett-Burman design (PBD) was screened in eight

204

experimental runs and four central point replications. For analysis of the effects

205

of the variables a confidence level of 90% was considered. The basal medium

206

used was as described above but omitting the yeast extract.

207

A second step in the optimization of the culture media was the experimental

208

design and response surface of the selected variables in PBD. In this analysis

209

the variables used were glucose and nitrogen [(NH4)2SO4] concentrations, and

210

pH. A factorial design Central Composite Rotational Design (CCRD) was used

211

when the factorial design analyzing indicated the curvature. When this did not 9



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happen, the first order analysis – Full Factorial Design (FFD) – was performed.

213

The lowest (-1), highest (+1) and external (±1.68) levels of each factor are listed

214

in Table 3. Three central point repetitions were added to verify the

215

reproducibility of the analysis. For the analysis of the variable effect, we

216

considered a confidence level of 95%. A polynomial equation was constructed

217

for each response surface analysis. An ANOVA analysis with student's t-test

218

was used to verify the statistical significance of the regression coefficients. The

219

Statistica v.12.0 statistical software was used for analyzing the experimental

220

data.

221 222

3. Results and Discussion

223 224

3.1 Diuron tolerance and transformation by fungal strains

225

Petri dish experiments have been used in several studies to assess the

226

tolerance of xenobiotics by fungi and the results depend on the fungal strain,

227

the type of compound and the concentration of each contaminant in the culture

228

media.33,34 In this study, 12 of the 14 fungi showed decreasing growth when

229

exposed to increasing diuron concentrations (Figure 1). In contrast, Verticillium

230

sp. F04 and Fusarium sp. B26 showed a increase growth when the herbicide

231

concentration raised up from 2.4 to 10 g L-1. Once the concentration reached 20

232

g L-1, the mycelial growth for both fungi revealed a slight increase compared to

233

the growth observed in 10 g L-1. This result suggests that Verticillium sp. F04

234

and Fusarium sp. B26 may be using the herbicide as a carbon source, thus

235

these fungi are expected to show high rates of diuron transformation.

236

Aspergillus sp. G25, Absidia cylindrospora F27, Mucor hiemalis G23 were the 10


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most affected by diuron, while Trichoderma virens F28, Trichoderma harzianum

238

G15, Cunninghamella elegans B06, Verticillium sp. F04 and Fusarium sp. F13

239

showed growth higher than 70%. Trichoderma and Fusarium strains were

240

reported as tolerant genera to different herbicides and other compounds such

241

as oil and naphthalene.30,35

242

Diuron transformation ranged from 12 to 45% (Table 1). Despite the results

243

of the Petri dish tests, Verticillium sp. F04 and Fusarium sp. B26 did not

244

respond as expected, showing only 20 and 33% herbicide biotransformation,

245

respectively. A. brasiliensis G08, Aspergillus sp. G25 and C. elegans B06

246

showed the highest biotransformation. Vroumsia et al.36 noted the potential for

247

diuron degradation by several genera and observed that Cunninghamella sp.

248

(54%) and Aspergillus sp. (45%) showed highest degradation rates. The genera

249

Cunninghamella and Aspergillus were also reported by Tixier et al.37 as able to

250

degrade 100% and 75% of diuron, respectively, in five days.

251

It is known that for microorganisms to be capable of degrading herbicide,

252

first they must be tolerant to the compound. However, the results indicated that

253

there is no relation between the tolerance to an herbicide and the high ability of

254

the fungi to degrade it. Thus, this test is not reliable for predicting fungal

255

potential of diuron transformation, since the fungus that showed highest growth

256

rate was not the best in the biotransformation tests (Table 1). The fungal sandy-

257

loam soils studied showed tolerance to high concentrations of the herbicide

258

Herburon® (50% diuron) and potential to biotransform diuron into different

259

metabolites by cometabolic processes. Based on these results, the fungi A.

260

brasiliensis G08, Aspergillus sp. G25 and C. elegans B06 were selected for

11



261

statistical

analysis

262

transformation.

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of the effects of medium composition on diuron

263 264 265

3.2 Optimization of diuron biotransformation by A. brasiliensis G08, Aspergillus sp. G25 and C. elegans B06

266

Experimental design is commonly used to increase biocompound

267

production and this approach proved to be an interesting tool in this work.

268

Moreover, this tool has been increasingly used to optimize degradation by

269

microorganisms such as lindane by Pleurotus ostreatus,38 naphthalene by

270

Pseudomonas putida S239 and petroleum hydrocarbons by Aspergillus

271

vesicolor.40

272

The PBD is a qualitative experimental design type used to screen the

273

effects of medium components in order to optimize the yields of bioproducts or

274

biodegradation.41 The results showed wide variations as may be observed in

275

the Pareto chart (Figure 2). Phosphorus concentrations showed no significant

276

effect on any results. None of the variables showed effects on the

277

transformation of diuron by the fungus A. brasiliensis G08 under the conditions

278

assayed. On the other hand, glucose, nitrogen and pH affected diuron

279

transformation by Aspergillus sp. G25 and C. elegans B06. For this reason,

280

glucose, nitrogen and pH were selected for a FFD to evaluate the effects of

281

medium composition on diuron transformation.

282

The values for each variable for FFD and CCRD (Table 3) were changed as

283

indicated by the effect calculated in the PBD (negative or positive effect), as

284

described in Pareto chart (Figure 2). Thus, on FFD and CCRD, the pH values

285

used were lowered – pH had a negative effect on PBD – and the glucose and 12


Page 13 of 32


286

nitrogen concentrations were increased, due to the positive effect on the PBD.

287

Table 3 shows the results for the 17 experimental conditions used in the diuron

288

biotransformation assay, which in bold is found the better results for each fungi.

289

The experimental design revealed the transformation rates for the three fungi

290

were all superior to those achieved previously in this work. These design

291

increased the fungi biotransformation potential by 2.2, 1.5 and 1.8 times for A.

292

brasiliensis G08, Aspergillus sp. G25 and C. elegans B06, respectively. A first

293

order analysis was performed for A. brasiliensis G08 (Equation 2), while for

294

Aspergillus sp. G25 (Equation 3) and C. elegans B06 (Equation 4) CCRD was

295

carried out, for the reason that a significant curvature was indicated by the

296

second order analysis.

297 Equation 2 (G08)

298

% = 59.95 + 14.36 − 4.31

299

% = 40.14 + 14,02 − 5,63 − 6,65 + 4,94 ² Equation 3 (G25)

300

% = 73.16 + 12.18 + 1.99 − 7.51

301

Dt is Diuron transformation, in percentage, and G, N and pH are glucose, (NH4)2SO4

302

and pH variables, respectively.

Equation 4 (B06),

303 304

According to the significance of the equations, glucose had a positive effect

305

on diuron transformation for the three fungi. Higher fungal biomass production

306

was also observed under high glucose concentrations. This fact suggest that

307

fungi are not consuming diuron as a carbon source, and the transformation of

308

diuron is occurring by a cometabolic process. Previous studies revealed that

309

fungi of the genera Aspergillus and Cunninghamella are able to carry out the

310

degradation

of

a

variety

of

xenobiotic

compounds

by

cometabolic 13



Page 14 of 32

311

processes.42,43 It has been reported that addition of extra substrate as extra

312

carbon source to the system is effective in enhancing the cometabolic

313

degradation of persistent organic pollutants.44

314

The assay showed a negative effect of (NH4)2SO4 on the activity of A.

315

brasiliensis G08, suggesting that lower concentrations of this nutrient would be

316

more suitable for diuron transformation by this fungus. The pH effect clearly

317

depends on the fungal species. The CCRD assay showed a negative effect of

318

increasing pH on diuron transformation by Aspergillus sp. G25 but a positive

319

effect for C. elegans B06. It has been reported previously that Aspergillus

320

versicolor degraded higher concentrations of hydrocarbons at pH 7 and 8.40

321

Similarly, the biodegradation of phenol by Aspergillus niger showed that there

322

was maximum degradation at neutral pH due to maximum utilization of phenol

323

as carbon source.45

324

The ANOVA analysis of diuron transformation showed coefficients of

325

variation of 0.96, 0.97 and 0.98 for A. brasiliensis G08 and Aspergillus sp. G25

326

and C. elegans B06, respectively, and Fcalc values were higher than Flisted in all

327

cases (Table 4). This method generates surface graphs between the significant

328

variables (Figure 3). The analysis of these results led to the determination of an

329

ideal culture medium for diuron transformation by each fungus separately. In

330

spite of the fact that this work shows an increase in diuron degradation potential

331

for only 3 fungi, the results revealed that the presence of carbon sources which

332

are easily assimilable by microorganisms in the soil can be beneficial in

333

reducing the concentration of herbicides.

334 335

3.3 Identification of metabolites of diuron 14


Page 15 of 32


336

Degradation studies should not only focus on the disappearance of the

337

primary contaminant, but also in the formation of potentially accumulating and

338

toxic intermediate products in order to define the actual environmental impact of

339

a pollutant. HPLC analysis revealed that all the fungi isolated were able to

340

transform the diuron to at least the first metabolite, DCPMU. For A. brasiliensis

341

G08, Aspergillus sp. G25 and C. elegans B06, the metabolites DCPMU and

342

DCPU were identified by HPLC by comparison with analytical standards and

343

confirmed by LC-MS analysis. DCA was only detected by HPLC for Aspergillus

344

sp. G25, but this metabolite was detected by LC-MS for the other fungi. The first

345

reactions in the diuron biodegradation pathway therefore involve demethylation

346

and removal of the urea group, resulting in DCA (Figure 4). This pathway has

347

also been observed by other authors in the biodegradation of diuron by fungi

348

and bacteria.37,46-49

349

Low concentrations of DCA found in this work (Table 5) led to question

350

whether this metabolite was accumulating and inhibiting the biodegradation

351

pathway, or was consumed quickly, continuing the process. For this reason, the

352

fungi were grown in the presence of 10 mg L-1 of the three metabolites,

353

separately, under the optimized conditions described above. High DCA

354

biotransformation rates for A. brasiliensis G08 and C. elegans B06 revealed the

355

fungi were able to modify this compound, continuing the diuron degradation

356

process. However, the formation of DCA as metabolite from diuron may inhibit

357

the degradation of DCPMU and DCPU due to the higher susceptibility of DCA to

358

be biotransformed by fungi.

359

Many studies confirmed the toxicity of DCA to various organisms, including

360

mammals.5 Thus soil fungi are extremely important for the decrease of diuron 15



Page 16 of 32

361

metabolites concentrations in the environment, contributing to the reduction of

362

effects on environments in the surroundings of herbicides application sites.

363

The analysis of DCA samples by LC-MS showed the presence of the

364

metabolite 3,4-dichloroacetanilide (DCAC) (Figure 4) for A. brasiliensis G08 and

365

C. elegans B06. This metabolite corresponds to the acetylation of the DCA

366

molecule, where the N-acetyltransferase enzyme catalyzes the transfer of the

367

acetyl group present in Acetyl-coenzyme (acetyl-CoA) to the amino group in

368

aniline.50 Although the acetylation process is most commonly described for

369

bacteria,47 Ellegaard-Jensen et al.28 reported the potential of Aspergillus niger,

370

Beauveria bassiana, Cunninghamella elegans and Mortierella isabelina in

371

acetylate DCA molecule to DCAC. This pathway has been reported as a

372

mechanism used by fungi to reduce the toxicity of DCA, which has high

373

importance, since this biotransformation results in a metabolite that has been

374

shown to be less toxic to Vibrio fisheri than DCA.47,31,50 The identification of

375

metabolites formed by diuron biotransformation, including 3,4-dichloroaniline

376

and the degradation of this potentially accumulating product show that fungi are

377

relevant in reducing diuron concentrations in soil and runoff, minimizing the

378

environmental impact on surrounding ecosystems.

379 380

Abbreviations

381

Diuron – 3-(3,4-dichlorophenyl-1,1´-dimethylurea)

382

PDA – Potato Dextrose Agar

383

Herburon® – 50% of diuron as active ingredient

384

DCPMU – 1-(3,4-dichlorophenyl)-3-methylurea

385

DCPU – 1-(3,4-dichlorophenyl)urea 16


Page 17 of 32


386

DCA – 3,4-dichloroaniline

387

DCAC – 3,4-dichloroacetanilide

388

HPLC – High Performance Liquid Chromatography

389

SPE – Solid phase extraction SPE

390

MS – Mass spectrometer

391

PBD – Plackett-Burman Design

392

CCRD – Central Composite Rotational Design

393

FFD – Full Factorial Design

394 395

Funding

396

This work was supported by FAPESP (Process Number 2011/50885-3,

397

2011/01577-4, 2011/22387-9), and CAPES.

398 399

Acknowledgment

400

We are grateful to “Usina Açucareira Virgulino de Oliveira S/A” for permission to

401

collect soil samples.

402 403 404 405

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Page 25 of 32


Figure 1: Percentage of fungal growth in the presence of three concentrations of Herburon® in comparison with the control media growth.

25



Page 26 of 32

Figure 2: Pareto graph for Plackett-Burman analysis. (A) A. brasiliensis G08 (R2 = 0.15); (B) Aspergillus sp. G25 (R2 = 0.74); (C) C. elegans B06 (R2 = 0.90). p = significance level at 10% (or 0.1).

Figure 3: Surface response with significant variables for diuron transformation. First order model for (A) A. brasiliensis G08 and second order response for (B) Aspergillus sp. G25 and (C) C. elegans B06. The variation from green to brown indicates increased diuron degradation rate.

26


Page 27 of 32


Figure 4: Proposed pathway for the biotransformation degradation of diuron by A. brasiliensis G08; Aspergillus sp. G25 and C. elegans B06. Solid line: metabolites from diuron; Dotted line: metabolite from DCA.

27



Page 28 of 32

Table 1: Fungi growth in the presence of Herburon® and diuron transformation rate of the fungi studied. 1 2 Fungal strain % growth % degradation Absidia cylindrospora F27 28 19 Aspergillus brasiliensis G08 52 38 Aspergillus sp. G25 27 45 Cunninghamella elegans B06 72 43 Cunninghamella sp. F19 40 31 Fusarium sp. B19 69 18 Fusarium sp. B26 68 33 Fusarium sp. F13 71 29 Mucor hiemalis G23 28 35 Paecilomyces sp. P07 49 20 Trichoderma harzianum G15 72 12 Trichoderma sp. G19 49 30 Trichoderma virens F28 73 27 Verticillium sp. F04 107 20 1 Growth in 20 g L-1 of Herburon® 2 -1 Degradation of 10 mg L of diuron

Table 2: LC/MS/MS parameters optimized by directly injecting. Parameter Diuron DCPMU DCPU DCA Spray Voltages (V) 5708 5546 5700 5700 Capillary voltages (V) 62.8 47.3 100 100 Radio Frequency (%) 77 75 70 70 190-240 150-225 100-210 100-210 Scan Range (m/z) 233 219 205 162 [M+H+] (m/z) 187 and 107 162 and 203 162 and 188 127 Fragments (m/z)

28


Page 29 of 32


Table 3: Diuron biotransformation assay performed by experimental design under 17 conditions.

Runs

Variables Glucose

pH

ab

(%) (NH4)2SO4

Diuron degradation (%) G08

G25

1 0.10 (-1) 3.00 (-1) 0.30 (-1) 44.2 29.1 2 0.40 (+1) 3.00 (-1) 0.30 (-1) 84.6 59.7 3 0.10 (-1) 4.00 (+1) 0.30 (-1) 54.5 22.7 4 0.40 (+1) 4.00 (+1) 0.30 (-1) 71.6 50.8 5 0.10 (-1) 3.00 (-1) 0.60 (+1) 40.2 30.1 6 0.40 (+1) 3.00 (-1) 0.60 (+1) 69.4 57.6 7 0.10 (-1) 4.00 (+1) 0.60 (+1) 41.3 17.5 8 0.40 (+1) 4.00 (+1) 0.60 (+1) 69.5 51.8 9 0 (-1.68) 3.50 (0) 0.45 (0) 13.0 0.2 10 0.50 (+1.68) 3.50 (0) 0.45 (0) 57.2 42.5 11 0.25 (0) 2.70 (-1.68) 0.45 (0) 79.4 67.0 12 0.25 (0) 4.30 (+1.68) 0.45 (0) 60.7 41.3 13 0.25 (0) 3.50 (0) 0.198 (-1.68) 56.2 42.5 62.7 41.0 14 0.25 (0) 3.50 (0) 0.702 (+1.68) 15 0.25 (0) 3.50 (0) 0.45 (0) 61.0 45.1 16 0.25 (0) 3.50 (0) 0.45 (0) 61.0 40.18 17 0.25 (0) 3.50 (0) 0.45 (0) 62.2 36.99 a The real value of the variables (w/v). b Encoded values in parenthesis. Maximum degradation and growth of each fungus is shown in bold.

Biomass (mg)

B06

G08

G25

B06

49.5 75.7 58.1 76.5 50.3 75.1 60.0 81.3 28.6 73.6 71.3 72.3 67.4 75.9 73.5 73.8 72.4

10.2 26.0 12.8 18.0 9.8 16.3 7.9 22.3 6.5 20.8 16.4 15.5 16.6 11.9 13.2 14.9 15.0

4.9 11.1 4.4 11.8 2.7 14.7 4.6 1.3 13.9 13.2 16.7 13.3 11.0 11.3 15.8 14.2 12.3

6.5 10.6 2.2 19.5 5.5 16.6 4.9 17.1 3.7 20.8 11.2 10.4 13.5 14.0 11.7 11.5 12.8

29



Page 30 of 32

Table 4: ANOVA for full factorial and composite central rotational design (reduced model). Aspergillus brasiliensis G08

Source of

Aspergillus sp. G25

Cunninghamella elegans B06

variation SSa

DFb

MSc

Fcalcd/Flistede

Model (R) 974.8 2 487.4 54.09/ 4.46 Residual (r) 72.1 8 9.01 Total (T) 1046.8 10 104.68 2 R = 0.96, 0.97 and 0.98 for G08, G25 and B06, respectively. a Sum Square b Degree of freedom c Mean square d Fcalc= MSR/MSr e Flisted with confidence level of the 95%

SSa

DFb

MSc

Fcalcd/Flistede

SSa

DFb

MSc

Fcalcd/Flistede

4228.7 106.1 4334.8

4 12 16

1057.17 8.84 270.92

119.59/3.26

2851 61.1 2912.1

3 13 16

950.33 4.70 182.01

202.19/ 3.41

30


Page 31 of 32


Table 5: Fungi biotransformation percentage of 10 mg L-1 of diuron, DCPMU, DCPU and DCA, and the concentrations of diuron metabolites produced by fungi. Diuron DCPMU DCPU DCA -1 -1 -1 Fungal strains % mg L from % mg L from % mg L from Diuron Diuron Diuron 85 2.19 65 4.3 41 Nd A. brasiliensis G08 Aspergillus sp. G25 67 5.5 53 0.27 63 0.2 C. elegans B06 81 5.0 73 3.9 56 Nd Nd: not detected

31


% 96 30 98


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TOC Graphic

32


Evaluation of Diuron Tolerance and Biotransformation by Fungi from a

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