The Mode of Action of AdjuvantsâRelevance of Physicochemical

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Agricultural and Environmental Chemistry

The mode of action of adjuvants – Relevance of physicochemical properties for effects on the foliar application, cuticular permeability and greenhouse performance of Pinoxaden Katja Arand, Elisabeth Asmus, Christian Popp, Daniel Schneider, and Markus Riederer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01102 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 22, 2018

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Journal of Agricultural and Food Chemistry

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The mode of action of adjuvants – Relevance of physicochemical properties for

2

effects on the foliar application, cuticular permeability and greenhouse

3

performance of Pinoxaden

4 5 6

Katja Aranda§, Elisabeth Asmusa§#, Christian Poppb, Daniel Schneiderb and Markus

7

Riederera*

8 9

a

University of Würzburg, Julius von Sachs Institute of Biosciences, Julius-von-Sachs-

10

Platz 3, Würzburg, D-97082, Germany

11

b

12

Münchwilen, CH-4333, Switzerland

Syngenta

Crop

Protection,

Global

Formulation

Technology,

Breitenloh

5,

13 14 15

1 ACS Paragon Plus Environment


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ABSTRACT

17

We comprehensively studied the complexity of mode of action of adjuvants by

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uncoupling the parameters contributing to the spray process during foliar application

19

of agrochemicals. The ethoxylated sorbitan esters Tween 20 and Tween 80 improved

20

the efficiency of Pinoxaden (PXD) in controlling grass-weed species in greenhouse

21

experiments by aiding retention, having humectant properties, maintaining the

22

bioavailability and increasing the cuticular penetration of PXD. The non-ethoxylated

23

sorbitan esters Span 20 and Span 80 showed minimal effects on retention, droplet

24

hydration or cuticular penetration, resulting in reduced PXD effects in the

25

greenhouse. Tris(2-ethylhexyl)phosphate (TEHP) does not contribute much to

26

retention and spreading but strongly enhances the diffusion of PXD across isolated

27

P. laurocerasus cuticular membranes. As TEHP was most efficient in controlling the

28

growth of grass-weed species, we propose, that the direct effect of penetration aids

29

on cuticular permeation plays a key role in the efficiency of foliar applied

30

agrochemicals.

31 32 33 34 35 36 37 38

KEYWORDS: Non-ionic surfactants, Polysorbates, Cuticular Penetration, Foliar

39

Application, Mode of Action

40 2 ACS Paragon Plus Environment

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INTRODUCTION

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Foliar spray application of agrochemicals is standard practice to protect crop plants

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against various pests and diseases and to control weeds. The spray process is very

44

complex, and several factors such as droplet formation, droplet retention, the wetting

45

and spreading process on the leaf surface but also spray deposit formation and

46

hydration followed by the permeation of the active ingredient (AI) through the plant

47

cuticle are closely connected.1,2 The cuticular membrane is an extracellular

48

biopolymer layer, mostly composed of hydroxyl modified and esterified fatty acids.

49

The cutin matrix is impregnated with very long chain aliphatics with several functional

50

modifications and/or cyclic triterpenoids, the so-called cuticular waxes.3 Foliar-applied

51

AIs have to permeate the lipophilic layer by the physical process of diffusion4 to reach

52

the underlying plant tissue.5 In many species, very complex three-dimensional wax

53

crystalloids structures on the surface of the cuticle strongly contribute to the

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hydrophobic surface properties and make them extremely difficult to wet.6 Thus,

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spray retention would be dramatically reduced. As the plant cuticle is physically and

56

chemically designed to protect the plant against water loss and other biotic and

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abiotic environmental factors,7 the overall effect of spray droplets are generally

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challenged by this barrier. Therefore, adjuvants are used to optimize the

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physicochemical characteristics of the spray liquid to improve the biological

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performance of the AI. This can be achieved through spray liquid modifying

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properties8-11 like pH adjustment, foaming activity, retention,1 wetting and spreading

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behavior12 or humectancy.13-15 Penetration aids impact the bioavailability of the AI in

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the spray deposit16,17 or modify the cuticular transport properties.13,18-24 Considering

64

the various processes during spray application, it is evident that adjuvants can either



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impact individual or several steps of the application procedure. Thus, one adjuvant

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can have multifunctional properties, which make the elucidation of the mode of action

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of adjuvants very complex. Therefore, the aim of this work was to investigate the

68

individual

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physicochemical and structural properties of the adjuvant solutions, their effect on

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cuticular penetration of the AI Pinoxaden and its efficacy on weeds. Besides the

71

commercially

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ethylhexyl)phosphate (TEHP) two commonly used polysorbates (Tween 20 and

73

Tween 80) were selected. The corresponding non-ethoxylated sorbitan esters Span

74

20 and Span 80 were also included in this study because they share the basic

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structure with the Tweens but without polyethoxylation. From this point of view, this

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study will systematically contribute to a fundamental understanding of the distinct

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mode of actions of adjuvants in foliar applied formulations.

functions

used

of

adjuvants

built-in

from

adjuvant

different

for

perspectives,

Pinoxaden

products

including

tris(2-

78

79

MATERIALS AND METHODS

80

Active ingredients

81

The post-emergence graminicide Pinoxaden (PXD) (8-(2,6-Diethyl-p-tolyl)-1,2,4,5-

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tetrahydro-7-oxo-7H-pyrazolo[1,2-d][1,4,5]oxadiazepin-9-yl-2,2-dimethylpropionat)

83

(log KO/W 3.2) was used in form of the commercial non-adjuvanted EC100 formulation

84

Axial 100 EC (100 g l-1 PXD, Syngenta Crop Protection AG, Switzerland) containing

85

the safener cloquintocet-mexyl.

86 87

Adjuvants

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The non-ionic surfactants sorbitan monolaurate (Span® 20, CAS: 1338-39-2) and

89

sorbitan monooleate (Span® 80, CAS: 1338-43-8) and their polyethoxylated 4 ACS Paragon Plus Environment

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derivatives polyoxyethylene sorbitan monolaurate (Tween® 20, CAS: 9005-64-5) and

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polyoxyethylene sorbitan monooleate (Tween® 80, CAS: 9005-65-6) (Figure 1) were

92

obtained from Croda (Nettetal, Germany).

93

The lipophilic adjuvant tris(2-ethylhexyl)phosphate (TEHP) is known to have

94

substantial accelerating effects on the uptake of PXD25 and was therefore used as a

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positive control in cuticular penetration and greenhouse experiments. TEHP was

96

emulsified with polyvinyl alcohol (TEHP EW400; 400 g l-1) (Syngenta Crop Protection

97

AG, Switzerland) which has no accelerating effects on cuticular penetration itself.

98

Isodecyl alcohol ethoxylate (Trend 90; 900 g l-1) (DuPont de Nemours, La Défense

99

Cedex, France) was used as a positive control adjuvant in retention experiments,

100

because of its pronounced surface tension lowering properties.

101

If not stated otherwise, all samples were used as aqueous solutions or emulsions

102

with an adjuvant concentration of 0.1% (w/v) which is in the typical range for the use

103

of agricultural formulation products.

104



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Plant material

106

For the retention experiments and the contact angle measurements, greenhouse-

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grown winter wheat plants (Triticum aestivum cv. Arina), grown under 14-h light

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period with 18/17 °C day/night temperature and 70% relative humidity (RH), were

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used in growth state BBCH 12 (2-leaf-stadium).

110

For greenhouse experiments winter wheat (Triticum aestivum cv. Horatio) and the

111

five grass-weed species wild oat (Avena fatua), Italian rye-grass (Lolium multiflorum),

112

green foxtail (Setaria viridis), awned canary-grass (Phalaris paradoxa) and black

113

grass (Alopecurus myosuroides) were used in growth state BBCH 12 (2-leaf-

114

stadium). Plants were sown together in bio troughs in soil and grown in a greenhouse

115

with a 16h light period and 20/17 °C day/night temperature at around 65% RH.

116 117

Cuticular membranes

118

Cuticular membranes (CM) were obtained from the upper, astomatous surfaces of

119

fully expanded leaves of Prunus laurocerasus cv. Herbergii plants growing in the

120

Botanical Garden of the University of Würzburg. Enzymatic isolation was carried out

121

as described previously.26

122 123

Static surface tension

124

The static surface tension (SST) of aqueous surfactant solutions was determined

125

with the drop shape analyzer DSA100S (Krüss GmbH, Hamburg, Germany) using

126

the pendant drop method.

127


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Dynamic surface tension

129

The dynamic surface tension (DST) of surfactant solutions was measured using the

130

bubble pressure tensiometer BP100 (Krüss GmbH, Hamburg, Germany).

131 132

Retention and leaf coverage

133

The retention tests were carried out in a spray cabin equipped with a variable speed

134

track sprayer at the Global Formulation Technology Centre, Syngenta (Münchwilen,

135

Switzerland). Ten Plants were cut at the bottom and fixed vertically and well-spaced

136

in the spray cabin to prevent spraying shadows. Surfactant solutions were blended

137

with Helios SC 500 (Novartis, Basel, Switzerland) to obtain a concentration of 0.1%

138

of the fluorescent ingredient Tinopal OB CO (2,5-thiophenediylbis(5-tert-butyl-1,3-

139

benzoxazole) and sprayed 50 cm above the plants with a Teejet XR11003VP flat fan

140

nozzle with 2 bars and 8 km h-1. After a 10 min drying period, leaf surfaces were fixed

141

on a red colored surface, illuminated with UV light and photographed on both sides.

142

From the respective pseudo color images, the leaf surface area, as well as the leaf

143

coverage (%), was determined with the software FluorSoft v0.1 (Syngenta Crop

144

Protection, Switzerland). The plants were washed with 8 ml of acetonitrile for 30 s.

145

The solution was filtered into UV glass tubes, and the concentration of Tinopal was

146

measured with the Fluorimeter 96 (Novartis, Basel, Switzerland) with two technical

147

replicates. The excitation light was 375 nm, and the emission light was 435 nm.

148

Retention was calculated as ng Tinopal per leaf surface area.

149 150

Contact angle measurement

151

Contact angle (CA) measurements were performed with the optical contact angle

152

measuring device OCA 15 plus (DataPhysics Instruments GmbH, Filderstadt,



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Germany) including a drop shape analysis (DSA) software. The second leaf of wheat

154

plants was fixed with double-sided adhesive tape on glass slides. A 3 µl droplet of

155

water or the surfactant solutions, each containing 0.1% (w/v) of the strawberry-red

156

azo dye Sanolin Ponceau 4RC 82 (Clariant, Muttenz, Switzerland), was placed on

157

the adaxial leaf surface. Droplet spreading was recorded with 1 fps during 1 minute,

158

and the contact angles were measured at frame 1, 30 and 60. After drying, the

159

droplet spread area of the red colored residue was determined using a microscope

160

(Leica DMR, Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) with the

161

software AxioVision Rel. 4.8 (Carl Zeiss Microscopy GmbH, Jena, Germany).

162

Measurements of at least 10 replicates were carried out for each treatment.

163 164

Simulation of foliar penetration experiments

165

The experimental set-up ‘simulation of foliar uptake/penetration’ (SOFU/SOFP)27 was

166

used to investigate the cuticular penetration of PXD. Cuticular membranes were

167

mounted on chambers made of stainless steel, with the physiological outer side

168

facing towards the atmosphere. A 5 µl droplet of a mixture of Axial 100 EC in water (2

169

g l-1 PXD) without or with adjuvant (4 g l-1) was applied to the outer surface. After

170

droplet drying, the chambers were inverted, and 1 ml of deionized water was added

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as receiver solution. The chambers were placed in closed plastic cups over a

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glycerol-water mixture (79.7:20.3, w/w) at 25°C to achieve 50% RH.28 At

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approximately 24 h time intervals, 10 µl aliquots were sampled from the receiver

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solution and PXD concentrations were quantified by UPLC-MS according to Asmus

175

et al. (2016).29 Plotting the amount of penetrated PXD as a function of time, the

176

resulting slope of the linear section represents the flow rate in µg s-1.

177


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Greenhouse experiments

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Greenhouse experiments were conducted with five different concentrations of

180

Axial EC100 with application rates of 1.875, 3.75, 7.5, 15 and 30 g PXD ha-1 either in

181

water as a negative control, with 0.1% surfactants or with 0.2% TEHP as a positive

182

control. All treatment combinations were sprayed on a selection of six different

183

monocot species in a spray cabin with a Teejet XR11002VP flat fan nozzle at 2 bars

184

and 3.3 km h-1 that resulted in an actual spray quantity of 200 l ha-1 at the plant

185

intercept point. Three replicates were made. The experiment was fully randomized.

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Plants were further grown in a greenhouse as described previously. The assessment

187

was conducted 14 days after application (DAA). Expected symptoms of plants

188

treated with an ACCase inhibiting herbicide are yellowish chloroses at the new

189

growing meristems. The herbicide damage in percent including also the loss of

190

biomass due to inhibited growth and tillering was estimated visually in comparison to

191

non-treated plants.

192 193

RESULTS

194

Surface tension

195

The dynamic surface tension (DST) was measured between 10 ms and 30 s surface

196

age and can be interpreted as a function of time. The static surface tension (SST)

197

was investigated independently at the equilibrium state. The DST and SST of water

198

were constant at around 72 mN m-1 (Figure 1). All adjuvant solutions, except Trend

199

90, showed high initial DST values of about 65 to 72 mN m- 1 but did decrease the

200

surface tension during 30 s with different kinetics (Figure 1). The DST for Span 80

201

stayed consistently high at 72 mN m-1 up to 20 s and then decreased rapidly to a low

202

SST value of 29.3 mN m-1. The DST for Span 20 moderately decreased over time 9 ACS Paragon Plus Environment


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starting from 200 ms and resulted in a final SST value of 48 mN m-1. At young

204

surface ages, the DST for TEHP followed the same kinetic as for Span 20, but from

205

about 1 s, the DST decreased much faster until an SST of 32 mN m-1 was reached.

206

The DST of Tween 20 and Tween 80 decreased linearly with time to ultimately reach

207

SST values of 46 mN m-1 (Tween 80) and 38 mN m-1 (Tween 20). The initial value for

208

Trend 90 was comparably low at about 47 mN m-1, and SST was reached at

209

27.8 mN m-1.

210 211

Contact angle and spread area

212

The initial contact angle of water droplets on the adaxial surface of wheat leaves was

213

about 152° and did not change within 60 s after droplet application (Table 1). The

214

dried-down droplet residue covered an area of about 0.6 mm². With all surfactants,

215

the initial contact angles were about 10° to 20° lower than for water. For Tween 20

216

and Tween 80, contact angles remained constant over the first 60 s resulting in a

217

spread area of about 2 mm² (Table 1). A slight decrease of the CA was observed for

218

both Spans but was more distinct for Span 20 (Table 1). After drying, the residue of

219

Span 20 covered a surface area of about 60 mm², while the coverage of the Span 80

220

residue was nearly tenfold smaller (~6 mm²) (Figure S1).

221 222

Retention and leaf coverage

223

In spray experiments, the maximum retention was obtained with Trend 90 (0.95 ng

224

mm-²) which also caused the highest leaf coverage (~14 %) followed by both Tweens

225

which showed less retention (Tween 20: 0.8 ng mm², Tween 80: 0.6 ng mm²) and

226

achieved a leaf coverage of about 5.3% (Table 1). The retention for both Spans was

227

similar to water (~0.2 ng mm²), but the resulting leaf coverage was about 2 to 4 times


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higher compared to water (~1 %) (Table 1). The retention and leaf coverage values

229

for TEHP were in the medium range (Table 1).

230 231


232

The diffusion of PXD across isolated P. laurocerasus cuticles was studied from a

233

rehydrated deposit containing 10 µg PXD at 50% RH. Without additional adjuvants,

234

the flux rate of PXD was about 0.1×10- 6 µg s-1 (Figure 2). With Span 20 or Span 80,

235

the PXD flow was comparable to the non-adjuvanted control (Figure 2). By adding

236

Tween 20 or Tween 80, the PXD flow was significantly enhanced to 4.7×10- 6 µg s-1

237

or 2.9×10- 6 µg s-1, respectively (Figure 2). With TEHP, the cuticular penetration of

238

PXD (24×10- 6 µg s-1) was increased significantly by more than two orders of

239

magnitude (Figure 2).

240 241


242

After spraying, typical ACCase symptoms25,30 were visible at the meristematic

243

younger tissue. New leaf tissue turned slowly yellow (chlorotic), or brown leaf spots

244

became visible (necrotic) (Figure 3). The assessment of the grass-weed species

245

Phalaris paradoxa was not conducted, because of the contamination with a

246

considerable amount of a rogue species. Differences were observed due to adjuvant

247

treatment, PXD concentration level or between the plant species (Figure 3, Figure

248

4). No damage on Triticum aestivum was detected (Figure 3) due to the presence of

249

the crop safener cloquintocet-mexyl. Comparing the weed plant species, most

250

damages for all treatments were found for Setaria viridis (SETVI) (Figure 4 A). With

251

increasing PXD concentration, weed damage increased for all plant species and all

252

adjuvant treatments. The overall ranking of adjuvants relating to weed damage was



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almost similar for all plant species. The negative control (no adjuvant) resulted in

254

minimum damage. Span 20 and Span 80 showed only small differences between

255

them but still more damage than the non-adjuvanted control (Figure 4). Tween 20

256

and Tween 80 resulted in higher damage rates than the Spans used in this study.

257

The maximum weed damage was always observed for TEHP whereby 80% damage

258

rates were already reached with medium PXD concentrations of 7.5 g PXD ha-1

259

(Figure 4).

260 261

DISCUSSION

262

Surface active action

263

The retention process of a droplet on the target plant surface is a crucial prerequisite

264

for successful spray application of plant protection agents1,12,31 especially for

265

superhydrophobic plant surfaces which are difficult to wet.32,33 During foliar spray

266

application, droplets are in flight for only about 50-250 ms1,31 and an effective

267

reduction of the surface tension during this critical time frame favors successful

268

droplet adhesion upon impact on the leaf surface.12,34,35 Surface tension

269

measurements showed that all surfactants could effectively reduce the surface

270

tension over time, but in most cases, SST was not even reached after 30 s surface

271

age (Figure 1). The surface tension at a surface age of about 100 ms (94 ms, value

272

depends on the measurement procedure of maximum bubble pressure method) was

273

used to test whether the selected surfactants aid droplet retention on wheat leaves in

274

track sprayer experiments through a reduction in surface activity.

275

For all adjuvants tested except for Trend 90, droplet retention has increased with

276

decreasing surface tension at 94 ms surface age (Figure 5) and amongst them best

277

retention was achieved with Tween 20. This correlation was also previously shown 12 ACS Paragon Plus Environment

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for several other surfactants on maize plants1 which are also very difficult to wet.

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Nevertheless, at 94 ms surface age, Span 20, Span 80 and TEHP showed a surface

280

tension close to water (Figure 1) and thus, retention on wheat leaves was not

281

significantly improved with these adjuvants (Figure 5).

282

Trend 90 was shown to have potent surface tension lowering properties within the

283

relevant time frame of the spray droplets being in flight (Figure 1). For this reason, it

284

is used as a retention aid, too. However, in our experiments, the retention of Trend

285

90 was comparable to Tween 20 and was lower than would be expected from the

286

correlation obtained with all other adjuvants. One reason might be the risk of droplet

287

shattering36 that tended to increase with decreasing surface tension, especially for

288

large, fast-moving droplets.37 In our track sprayer experiments with well-spaced

289

plants, they would be easily lost to the ground. Under field conditions with much

290

higher leaf area index, small decelerated satellite droplets produced upon the first

291

impact would eventually adhere to neighboring leaves on secondary impact due to

292

their low surface tension. This would improve overall retention.

293

The retention process involves short-term dynamics1,31 and therefore can be

294

correlated to the surface tension at very young surface ages. In contrast to that, the

295

spreading process, which is initiated after successful retention, is less time-bound

296

and can also be affected by adjuvants which reduce the surface tension at surface

297

ages far beyond 250 ms. The wetting or spreading properties of the spray droplets

298

can be characterized by the contact angle between the leaf surface and the droplet.38

299

A very high contact angle for water, as measured for greenhouse-grown wheat

300

leaves

301

surface.33,39 The water droplet forms a sphere on top of the wax crystalloids33

302

resulting in a minimal physical contact area between the droplet and the leaf surface

(151°, Table

1)

indicating

a

superhydrophobic,

very

difficult-to-wet



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(Figure S1). For both Tweens and Spans and also TEHP, the initial contact angles

304

were reduced by about 13°-22° only during the first minute upon droplet application

305

(Table 1), but after complete drying, enormous differences in the spread area were

306

detected (Table 1, Figure S1). The contact angle for Span 20 droplets decreased

307

steadily during the first 60 s (and even further until dry-down, not measured) and the

308

final spread area was a hundredfold bigger compared to a pure water droplet residue

309

(Table 1). In contrast to that, the initial contact angle for Tween 20 was lowest within

310

the sorbitan esters tested and only surpassed by Trend 90. This is also reflected in

311

the highest retention effects of Tween 20 (except for Trend 90) within spray

312

experiments (Table 1, Figure 5). Further spreading during the first minute was not

313

detected and the resulting spread area (1.9 mm2) was only about 3-times higher than

314

for pure water. Under controlled laboratory conditions with comparatively high droplet

315

volumes, Span 20 is a very potent spreading agent on extremely hard-to-wet

316

surfaces, while Tween 20 rather acts as a retention aid. In spray experiments, the

317

leaf coverage for Span 20 was similar to Tween 20 (about 5%) even though retention

318

was lower for Span 20. Since the spray droplets are very tiny, they will have been

319

evaporated before the spreading behavior of Span 20 comes into significant effect.

320

Additionally, the total droplet deposition on the leaf surface would not be sufficient

321

with Span 20 due to its poor retention properties.

322

Trend 90 showed by far the lowest initial contact angle, which also decreased over

323

time, resulting in a ca. 30-fold enhanced spread area compared to water. The

324

combination of good retention and spreading properties led to the highest leaf

325

coverage in track spray experiments (Table 1). Nevertheless, the physical value of

326

surface tension alone cannot explain why Span 20 produces a much bigger spread

327

area than Trend 90 when applied as a single droplet. Possible influences of


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interfacial properties40-42 or the size, velocity, and viscosity of the droplets34 were not

329

elucidated in this study.

330 331


332

After droplet adhesion and spreading on the leaf surface, AI uptake into the leaf is

333

initiated. Thereby, cuticular waxes act as a potential barrier to the permeation of

334

organic and inorganic solutes.43,44 The rate of AI permeation is considerably

335

determined by their solubility characteristics related to the physicochemical

336

properties of the cuticle, which are indicated by the octanol/water (KO/W) or

337

cuticle/water (KC/W) partition coefficient.5 The permeation of hydrophilic solutes (log

338

KO/W < 1) takes place via hydrated sites in the cuticle and is therefore influenced by

339

the actual hydration status of the cuticle.45 Cuticular permeation of nonpolar, lipophilic

340

solutes (log KO/W > 1) is favored by factors impairing the resistance of cuticular

341

waxes, e.g. by reducing their crystallinity.43 Thereby; low EO surfactants are known

342

to readily penetrate the cuticle enhancing the lipophilic pathway20,46,47 whereas higher

343

EO surfactants rather increase the permeability for hydrophilic solutes.14,19,48

344

In cuticular penetration experiments with P. laurocerasus, the addition of TEHP

345

significantly enhanced the flow of PXD. Muehlebach et al.25 stated that TEHP might

346

act in the spray deposit by “overcoming active ingredient chemical stability or

347

crystallization issues” thus, maintaining the driving force. On the other hand, it is very

348

likely that the lipophilic adjuvant enters the cuticle and change the structure or the

349

composition of the cuticular pathway,19 thus increasing the mobility of PXD.

350

With Span 20 and Span 80, the PXD diffusion across isolated P. laurocerasus

351

cuticles at 50% RH was not affected significantly, indicating that Spans neither

352

improve the bioavailability of PXD in the spray deposit nor increase its mobility in the 15 ACS Paragon Plus Environment


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353

rate limiting barrier of the cuticle. In contrast, Tween 20 and Tween 80 enhanced the

354

PXD flow by factors of about 40 and 25, respectively. Tweens are highly diverse and

355

complex mixtures of different molecules varying in the degree of ethoxylation and

356

also in the fatty acid chain length distribution.49,50 Therefore, the potential of the low

357

EO fraction to act within the cuticle20,46,47 might also be a contributing factor for

358

mobilization of PXD. Conversely, it was stated that Tween 20 is known to have poor

359

foliar uptake properties and the activation action is due to humectant properties.47

360

Recently, Asmus et al.15 published a comprehensive data set about the humectant

361

activity (nws) of several surfactants at different humidities, showing that the humectant

362

activity increases with increasing ethoxylation. For the sorbitan esters used in our

363

study, the PXD diffusion across P. laurocerasus cuticles at 50% RH rises with

364

increasing humectant activity and respectively with increasing EO content. These

365

results indicate the enhanced uptake of PXD with Tweens in contrast to Spans is

366

also promoted by their humectant property, preventing the AI from crystal

367

precipitation and thus improving the bioavailability.14

368 369


370

PXD is a graminicide for the control of wide-spread annual grass-weed species in

371

mainly wheat, and barley.25,51 Since the cuticular membrane of grass species cannot

372

be isolated for further uptake experiments, cuticular membranes of the model plant P.

373

laurocerasus were used to elucidate the underlying mechanisms concerning cuticular

374

penetration. By using a plant species without epicuticular wax crystalloids, we also

375

minimized the effect of wetting and spreading, and the contact area with the droplet

376

was comparable for all adjuvants. To take a step forward, greenhouse experiments

377

were performed with target grass-weeds, to determine whether our findings from 16 ACS Paragon Plus Environment

Page 17 of 40


378

model systems can be applied to living plants. To minimize retention effects, we used

379

an 02 flat fan nozzle producing a narrow droplet spectrum, so the droplets generated

380

adhere to the leaf surface more easily. Furthermore, plants were grown tightly

381

together resulting in a much bigger leaf area index compared to the retention

382

experiment, where single plants were used. Droplets which bounce off at first impact

383

will be captured at an adjacent leaf instead. PXD is usually applied at application

384

rates of 15-60 g AI ha-1, depending on the target weed species.30,51 To detect

385

differences in the adjuvant response of PXD, we used PXD rates far below the

386

recommended field rate.

387

The mode of action of PXD is the inhibition of the fatty acid biosynthesis which

388

impacts the formation of biomembranes (HRAC-class A).30 The physiological

389

symptoms like plant leaf chlorosis at the growing meristems followed by browning

390

become visible in the grass-weed species within 1-3 weeks after treatment.25 At the

391

time of assessment (14 DAT) considerable differences in the absolute plant damage,

392

depending on plant species, adjuvants and PXD rates were observed (Figure 4). To

393

quantify the adjuvant effects, we calculated the slope of the initial linear part of the

394

dose-response relationship (Figure 4) for each adjuvant. This slope can be used as

395

a proxy to determine the effect of the adjuvant on the biological performance of PXD.

396

Even though the different weed species showed different absolute responses, the

397

same ranking of adjuvants was observed across the species tested (TEHP >> Tween

398

20 > Tween 80 > Span 20 = Span 80 > no adjuvant). The leaf damage values for all

399

species were averaged to compare them with the results gained from laboratory

400

experiments. By plotting the slope against the PXD flow across isolated P.

401

laurocerasus cuticles, a positive relationship was observed between the penetration

402

enhancing effect of all adjuvants and their impact on weed control efficiency of PXD



Page 18 of 40

403

in the greenhouse (Figure 6). This indicates that cuticular penetration is one of the

404

major bottlenecks for the weed control efficiency of PXD.

405

In summary, we comprehensively studied the complexity of mode of action of several

406

adjuvants from the sorbitan ester family and TEHP, by uncoupling the many

407

parameters which contribute to the spray process in the field such as droplet

408

retention, spreading, the formation of the spray deposit and also penetration of the AI

409

through the plant cuticle.

410

We showed that the ethoxylated sorbitan esters Tween 20 and Tween 80 have

411

multifunctional adjuvant qualities. They aid retention, have promoting humectant

412

properties, maintaining the bioavailability of PXD in the surface residue and finally

413

increase the cuticular penetration of PXD. The combination of these factors improves

414

the efficacy of PXD in controlling the growth of grass-weed species in the

415

greenhouse compared to non-adjuvanted treatments. In contrast, the non-

416

ethoxylated sorbitan esters Span 20 and Span 80 are rather inefficient since they

417

show minimal effects on retention, droplet hydration or cuticular penetration, resulting

418

in reduced PXD effects in greenhouse experiments. In all grass-weed species, the

419

formulation containing TEHP was most efficient for the use with PXD across the

420

range of rates tested. As it was shown in the surface tension measurements and

421

spray experiments, TEHP does not contribute much to retention and spreading, but it

422

enhanced the diffusion of PXD across isolated cuticular membranes of P.

423

laurocerasus five times compared to the best performing sorbitan ester Tween 20.

424

These findings indicate that the direct effect of penetration aids on PXD plays a

425

critical role and cuticular uptake is a significant bottleneck. In fact, SOFP experiments

426

with isolated cuticles are a well-suited system to screen for potent permeation

427

enhancing adjuvants for AIs such as PXD in a small scale. Nevertheless, in practice,


Page 19 of 40


428

also retention and spreading play a significant role since spray droplets behave

429

differently and the leaf area index at the time of application differs. Therefore,

430

additional studies are required to close the gap between laboratory experiments,

431

greenhouse studies with controlled environmental conditions and the real situation in

432

the field.

433 434 435

ABBREVIATIONS USED

436

AI

Active ingredient

437

ALOMY

Alopecurus myosuroides

438

AVEFA

Avena fatua

439

CA

Contact angle

440

CM

Cuticular membrane

441

DAA

Days after application

442

DST

Dynamic surface tension

443

EC

Emulsion concentrate

444

EW

Emulsion oil-in-water

445

EO

Ethylene oxide

446

LOLMU

Lolium multiflorum

447

PXD

Pinoxaden

448

RH

Relative humidity

449

SETVI

Setaria viridis

450

SOFP

Simulation of foliar penetration

451

SST

Static surface tension

452

TEHP

Tris(2-ethylhexyl)phosphate 19 ACS Paragon Plus Environment


453

TRZAW

Page 20 of 40

Triticum aestivum

454 455

Author Information

456

*Corresponding author

457

e-mail: [email protected], phone + 49 931 31 86200

458

§

459

Katja Arand and Elisabeth Asmus contributed equally to this work

Author Contribution

460 461

#

462

Bayer AG, Research & Development / Crop Science, Building H872, 65926 Frankfurt

463

am Main, Germany

Present address

464 465

Funding

466

This work was partially supported by a grant from Syngenta Crop Protection AG.

467

Notes

468

The authors declare no competing financial interest.

469 470

ACKNOWLEDGEMENTS

471

The authors deeply express their thanks to René Jaun, Markus Krischke, Andreas

472

Krommrei and James Murdock for their valuable technical assistance.

473 474

SUPPORTING INFORMATION DESCRIPTION

475

Supplementary data associated with this article can be found online. This file

476

includes a comparison of freshly settled droplets (3 µl volume) containing the

477

respective adjuvant and the strawberry-red azo dye Sanolin Ponceau and the droplet


Page 21 of 40


478

residue after water evaporation on adaxial Triticum aestivum cv. Arina leaves (Figure

479

S1). This material is available free of charge via the Internet at http://pubs.acs.org

480



481

Page 22 of 40

FIGURE CAPTIONS

482 483

Figure 1. Dynamic surface tension as a function of surface age (ms) and static

484

surface tension (mN m-1). The surface age ranges from 10 ms to 30000 ms. Static

485

surface tension values (mN m- 1) indicate the equilibrium-state of surface tensions.

486 487

Figure 2. Box-plots of flow rates of cuticular permeation (F) of PXD in the presence

488

of varying surfactants at 50% RH. Continuous horizontal lines within the boxes

489

represent the median and dotted lines the mean. Whiskers indicate the 5th to 95th

490

percentiles and dots the minimum and maximum values observed. Box-plots

491

indicated with * are significantly different from the non-adjuvanted control (Kruskal-

492

Wallis Test with Dunn’s Test, p < 0.001).

493 494

Figure 3. Photographs of plant troughs 14 days after application. Plants sprayed with

495

non-adjuvanted Axial EC 100 containing 15 g PXD ha-1 water or the respective

496

adjuvant. Plants from left to right: Triticum aestivum, Avena fatua, Lolium multiflorum,

497

Setaria viridis, Phalaris paradoxa and Alopecurus myosuroides. On Triticum

498

aestivum (left) no damages could be observed. The most significant damage was

499

visible for Setaria viridis (fourth from left).

500 501 502

Figure 4.

503

Dose-response curves of mean grass-weed control by PXD with different adjuvant

504

treatments on (A) Setaria viridis (SETVI), (B) Avena fatua (AVEFA), (C) Lolium


Page 23 of 40


505

multiflorum (LOLMU) and (D) Alopecurus myosuroides (ALOMY). In some cases,

506

error bars (SD) are smaller than the symbols (mean).

507 508

Figure 5. Relationship between the retention (ng Tinopal OB per mm2 leaf area) on

509

Triticum aestivum cv. Arina leaves and the dynamic surface tension (DST) at 94 ms

510

surface age of selected adjuvants. Dots indicate mean values and error bars

511

represent the standard deviation, n=10.

512 513

Figure 6.

514

Correlation between the slope of the initial linear part of the dose response

515

relationship (Figure 4) and the mean cuticular flow rates (F) of PXD with different

516

adjuvants at 50% RH (Figure 2). Mean values of the slope were calculated across all

517

grass-weed species. Error bars represent the standard deviation. Dots for Span 20

518

and Span 80 overlap each other. The non-adjuvanted control is excluded from the

519

regression line (dashed line, R² = 0.99).

520



Page 24 of 40

521

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522

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668 669



Page 32 of 40

Table 1. Contact angles (°) at three respective times and spread area (mm2) after the evaporation of water on the upper side of Triticum aestivum cv. Arina leaves of selected adjuvants. Retention (ng Tinopal OB per mm2 leaf area) and leaf coverage (%) of selected adjuvant solutions on both sides of Triticum aestivum cv. Arina leaves. Mean (SD) n=10.

Tradename

Water Span 20 Span 80 Tween 20 Tween 80 TEHP EW400 Trend 90

Spread area (mm2)

Contact angle (°)

initial 151.9 (4.9) 136.9 (6.3) 138.2 (5.8) 129.7 (4.7) 137.1 (5.1) 132.9 (3.3) 56.4 (5.7)

30 s 151.8 (5.9) 128.6 (10.6) 136.7 (6.9) 129.2 (4.3) 136.0 (5.5) 132.4 (3.4) 30.4 (6.8)

60 s 151.2 (5.6) 124.5 (10.2) 135.8 (7.0) 129.0 (4.2) 136.0 (5.7) 131.7 (3.4) 24.5 (6.3)

0.6 (0.4) 63.2 (23.9) 6.8 (1.3) 1.9 (0.5) 1.8 (0.2) 2.9 (0.7) 17.2 (2.0)

Retention (ng Tinopal OB per mm2 leaf area) 0.20 (0.15) 0.28 (0.08) 0.18 (0.03) 0.82 (0.25) 0.57 (0.14) 0.40 (0.12) 0.95 (0.22)

ACS Paragon Plus Environment

Leaf coverage (%) 0.96 (1.04) 4.49 (1.05) 2.23 (0.74) 5.31 (2.19 5.34 (1.52) 2.97 (0.74) 13.70 (4.27)

Page 33 of 40


Figure 1 static surface tension (mN m-1) 72.3

surface tension (mN m-1)

70

29.3

60

48.0

50

46.0

40

38.0 32.0

Water Tween 20 Tween 80 Span 20 Span 80 Trend 90 TEHP EW400

30

20

101

27.8

102

103

104

surface age (ms)


105


Figure 2


Page 34 of 40

Page 35 of 40


Figure 3



Figure 4


Page 36 of 40

Page 37 of 40


Figure 5

1.0

2

retention (ng Tinopal OB per mm leaf area)

1.2

0.8

0.6

Water Tween 20 Tween 80 Span 20 Span 80 Trend 90 TEHP EW400

0.4

0.2

0.0 30

40

50

60

70

80

DST at 94 ms surface age



Figure 6

30

slopedose response

25

20

15

10 No Adjuvant Tween 20 Tween 80 Span 20 Span 80 TEHP

5

0 0

5

10

15 6

20

25

30

-1

FPXD * 10 (µg s )


Page 38 of 40

Page 39 of 40


GRAPHICS FOR TABLE OF CONTENTS



85x48mm (150 x 150 DPI)


Page 40 of 40

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