Use of Mg–Al Nanoclay as an Efficient Vehicle for the Delivery of the

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Research Article Cite This: ACS Sustainable Chem. Eng. 2019, 7, 10962−10970

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Use of Mg−Al Nanoclay as an Efficient Vehicle for the Delivery of the Herbicide 2,4-Dichlorophenoxyacetic Acid Pavani P. Nadiminti,#,† Hemant Sharma,#,† Sitarama R. Kada,‡ Frederick M. Pfeffer,† Luke A. O’Dell,‡ and David M. Cahill*,† †

School of Life and Environmental Sciences, Deakin University, Geelong Waurn Ponds Campus, Victoria 3216, Australia Institute for Frontier Materials, Deakin University, Geelong Waurn Ponds Campus, Victoria 3216, Australia

‡

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ABSTRACT: Anionic exchange materials, such as layered double hydroxides (or nanoclays) are established delivery vehicles for bioactives in the pharmaceutical industry. In contrast, the use of these same nanoclays for the delivery of agrochemicals is not well explored yet has great potential for transporting and transferring bioactives to plants that compromise agricultural crop productivity. Herein, we have prepared a MgAl-layered double hydroxide (MgAl-LDH) nanoclay loaded with 2,4-dichlorophenoxyacetic acid (2,4-D) and characterized this material using Fourier-transform infrared, nuclear magnetic resonance, X-ray powder diffraction and Transmission electron microscopy. Spray application of agriculturally relevant doses of bare nanoclay (from 0.2% w/v to 1.0% w/v) on to Arabidopsis thaliana plants did not induce cellular damage or stress when tested for the site-specific stress marker, callose (a β-1,3 glucan). In vitro studies showed that the 2,4-D was slowly released from the preloaded nanoclay over 18 h. When compared to positive controls, spray-applied at equivalent doses, the 2,4-D loaded nanoclay applications (from 0.6 to 1.0% w/v) clearly showed a stronger and irreversible herbicidal effect on the test plants. We therefore propose that nanoclay materials have distinct advantages over conventional surfactant-based agrochemical spray applications and can contribute toward advanced agricultural practices. KEYWORDS: Nanoclay, 2,4-D, Arabidopsis thaliana, Slow release, Weeds, Delivery vehicle, Herbicide

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INTRODUCTION

overcome the problems associated with conventional surfactant-/adjuvant-based agrochemical spray formulations. The use of nanotechnology for chemical delivery in agriculture is still in its infancy but has a range of advantages over traditional approaches including reduced off-target effects, reduction in water use and lower costs to farmers.5,6 To date, a number of different types of nanoparticles have been designed or repurposed for agricultural applications. For example, positively charged mesoporous silica nanoparticles have been successfully used for the delivery of 2,4-D to cucumber ( Cucumis sativus) seedlings. Similarly, perylene-3-ylmethanol nanoparticles have been used for controlled release of 2,4-D to brown pea seedlings (Cicer arietinum).7 Nanostructured liquid crystalline particles (NLCP)8 and self-assembling lipid-based nanoparticles9 were used to deliver 2,4-D and picloram respectively to wild radish (Raphanus raphanistrum) in field trials and the model plant Arabidopsis thaliana in laboratory studies. Though NLCP efficiently delivered 2,4-D for weed eradication, dose dependency was not demonstrated, limiting their use as a commercial agrochemical carrier vehicle.

Increased crop production necessitates high yields and agricultural practices that prevent loss of productivity. Protection of crops from yield loss is a significant challenge faced by farmers across the globe and typically involves the prevention and control of invasive weeds, damaging insects and other pests. For example, in Australia, it was recently estimated that due to weed infestation there was a yield loss to grain growers of 2.76 million tonnes and that weed management cost AUD $3,318 million.1 For the control of weeds, herbicides are often spray-applied with chemical additives such as surfactants and adjuvants.2 For example, commercial 2,4-D formulations (that contain unspecified amounts of surfactants and adjuvants) are often spray-applied to control dicot weeds in cereal crops.3 Along with the costs associated with herbicidal spray applications, there is increasing concern about the toxicity of formulations that contain surfactants and adjuvants, not only to the crop plants but also to the environment and to human health.4 A recent report has shown that volatile organic compounds including agrochemical spray volatiles are globally one of the most important air pollutants that may cause adverse human health effects.4 Therefore, there is an urgent need to provide strategies that © 2019 American Chemical Society

Received: April 10, 2019 Revised: May 13, 2019 Published: May 22, 2019 10962

DOI: 10.1021/acssuschemeng.9b02001 ACS Sustainable Chem. Eng. 2019, 7, 10962−10970

Research Article

ACS Sustainable Chemistry & Engineering

The precipitates were dried at 100 °C in a hot air oven for 16 h to obtain the pale yellow colored 2,4-D loaded nanoclay. Characterization. X-ray diffraction (XRD) measurements were performed on a Panalytical XPert Powder diffractometer (Malvern Products, Taren Point, NSW, Australia) equipped with Cu Kα X-rays and operated with an applied voltage and current of 40 kV and 30 mA, respectively. The crystallite sizes of the powders were calculated using the Scherrer’s equation. The fine structure of the nanoclay particles was examined using a JEOL 2100 transmission electron microscopy (TEM) instrument (JEOL Ltd., Tokyo, Japan). The nanoclay suspension was first sonicated in 60% ethanol and then a copper grid (ProSciTech, Qld, Australia) was loaded with 10 μL of the suspension and allowed to dry at room temperature before imaging at 200 kV. Energy-dispersive Xray spectroscopy (EDS) measurements were performed using a fieldemission gun scanning electron microscopy instrument (Supra 55 VP, Carl Zeiss Pty Ltd., NSW, Australia) operating at an applied voltage of 5 keV. Spectrophotometric studies were carried out using a Cary 300 UV−visible spectrophotometer (Agilent Technologies Australia, Mulgrave, Vic, Australia). The emission spectra were recorded on a Cary eclipse fluorescence spectrophotometer using a 10 mm quartz cell. The excitation and emission slit width was 10 nm and excitation wavelength was 283 nm. Fourier-transform infrared (FTIR) spectra were recorded on a Bruker Alpha FTIR spectrometer (Bruker Pty. Ltd., Preston, Vic, Australia). Solid state 27Al NMR was recorded on a Bruker Avance III 500 MHz wide bore spectrometer and 2.5 mm HX MAS probe (Bruker Pty. Ltd.) with a sample spinning rate of 25 kHz, an excitation pulse width of 1 μs, a recycle delay of 5 and 52 scans acquired per spectrum. To determine accurately the slow release of the 2,4-D from the nanoclay, UV−visible spectroscopy (Agilent Technologies Australia) was performed. A small quantity of 2,4-D loaded nanoclay (3.06 mg) was carefully packed in a dialysis membrane and then placed in a cuvette containing 60% ethanol in water. A wire mesh was placed in the upper half of the cuvette to prevent the dialysis membrane from sinking to the bottom while the UV−visible spectra were recorded. The experiment was replicated three times. Plant Growth and Maintenance. Arabidopsis thaliana ecotype Col-0 seeds were stratified for 2 days at 4.0 °C and then grown in Petri plates containing MS basal media (Sigma-Aldrich) for 14 days as previously described.24 Five seedlings were then transferred to clean plastic pots (10 cm high ×10 cm diameter) filled with sterile soil (Terracotta and Tub Superior Potting Mix, Debco, Tyabb, VIC, Australia). Petri plates and the plants (in the pots) were maintained in growth cabinets (Thermoline Scientific, Wetherill Park, NSW, Australia) under cool fluorescent light (120 μmol/m2/s) with a 16/ 8 light/dark photoperiod at 21 °C. Plant Treatment with Nanoclay Formulations. In order to assess any impact of application of the nanoclay particles on A. thaliana plants, they were spray applied with nanoclay using a handheld spray atomizer (New Directions Australia, Sydney, NSW, Australia) at increasing concentrations of 0.2% (w/v) increments to 1.0% (w/v). Two higher concentrations were also applied [20% (w/ v) and 50% (w/v)], by placing with the aid of a pipette, a 20 μL drop of the nanoclay solution directly on the leaf surface. As a positive control, 0.1% (v/v) of a commercial surfactant “empigen”, an alkyl dimethyl betaine (C12−14) (a gift of Nufarm Australia Ltd.,) was used and water was used as the untreated control. Five, three-week-old A. thaliana plants were treated for each concentration and the experiment was replicated two times. For testing the efficiency of the nanoclay to deliver the herbicide, 2,4-D loaded nanoclay (2,4-D + NC) was mixed in water and spray applied to three week old plants of A. thaliana. Prior to spray application, the amount of 2,4-D released from the nanoclay at zero hours was individually calculated for each of the dosages (0.2%−1.0% (w/v) using a standard curve obtained for the pure 2,4-D in 60% (EtOH:H2O; v/v). For each dose of 2,4-D + NC, the respective amount of 2,4-D released was calculated.

Recently, and like other nanoparticles that were originally designed for use in the pharmaceutical industry, nanoclays have begun to be explored for use in agriculture.10 Nanoclays or layered double hydroxides are lamellar sheets with a net positive charge that is balanced by intercalated anions (active ingredients in this case) in the hydrated interlamellar space.11−13 Nanoclay is often represented by the x+ m− x− 3+ chemical formula [M2+ 1−x Mx (OH)2] [(A )x/m]·nH2O] , 2+ 3+ where M and M are respectively divalent and trivalent cations, while Am− is an exchangeable anion.14,15 Hydroxide anions typically form an octahedral complex with the metal and these octahedra, through edge sharing, extend to form an infinite sheet. The intercalating anions are electrostatically bound between the layers. Further, the interlayer anions are easily exchangeable with other anionic species16 and this presents a means to load a range of agronomically important chemical moieties. The application of nanoclays as anionic-exchange materials, catalysts and as delivery vehicles for bioactive materials is well recognized11−13 and the use of nanoclay as a carrier vehicle has been of particular interest.17 For example, nanoclay materials were used to deliver DNA and proteins to animal models to protect them from various types of cancers,18,19 and their biosafety has been established for human blood cells and vascular cells.20 Despite this potential nanoclays have only been used to a limited extent in relation to plants.21,22 For example, lactate-nanoclay composites were used to deliver fluorescent dyes and ssDNA (60mer) to Nicotiana tobacum cv Bright Yellow 2 (BY-2) cells.15 To date, the potential of nanoclay based delivery of an agrochemical active has not been investigated especially in regards to impacts on stress physiology when applied to plant surfaces. In the present work, an MgAl layered double hydroxide (nanoclay) was synthesized through coprecipitation. Interlayer anions were then exchanged with one of the most widely used phenoxy herbicides, 2,4-D, with the aim to develop a surfactant free, environmentally safe yet potent agrochemical formulation. When the nanoclay alone was applied to leaves of the model plant species A. thaliana, it did not induce the cellular stress marker callose (a β-1,3 glucan). The loaded 2,4-D was slowly released into solvent for up to 18 h. A strong irreversible herbicidal response was found when the loaded nanoclay was applied to plants at agriculturally relevant doses showing that nanoclays are an efficient and cost-effective agrochemical delivery vehicle.

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MATERIALS AND METHODS

Nanoclay Synthesis. The nanoclay was synthesized as previously reported23 with minor modification. In brief, under vigorous stirring, a mixture of Mg(NO3)2·6H2O (3 M) (Sigma-Aldrich, Castle Hill, NSW, Australia), Al(NO3)3·9H2O (1 M) (Sigma-Aldrich) and methanol (150 mL) was added dropwise to alkaline methanol (200 mL) solution that contained NaOH (1.6 M) (Sigma-Aldrich) and Na2CO3 (0.1 M) (Sigma-Aldrich). The mixture was then refluxed at 80 °C for 24 h. After the solution was cooled to room temperature, the precipitate was collected using vacuum filtration and washed thrice with distilled water. The resultant white colored nanoclay was calcinated at 100 °C in a hot air oven for 16 h. Loading 2,4-D on MgAl-LDH clay (2,4-D loaded NC). Nanoclay and 2,4-dichlorophenoxyacetic acid ethylhexyl ester (2,4D) (gifted by Nufarm Ltd., Laverton, Vic, Australia) were mixed in a 1:3 ratio (w/w) in ethanol (150 mL), and the solution was stirred overnight. The resultant precipitate was collected using vacuum filtration and washed thrice with ethanol to remove unbound 2,4-D. 10963

DOI: 10.1021/acssuschemeng.9b02001 ACS Sustainable Chem. Eng. 2019, 7, 10962−10970

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Figure 1. Functional group characterization of nanoclay. (a) FTIR spectra of (i) nanoclay, (ii) 2,4-D and (iii) 2,4-D loaded nanoclay in the range of 400−4000 cm−1. (b) Absorbance spectra of 2,4-D and 2,4-D loaded nanoclay in ethanol. The inset represents the absorbance peak of 2,4-D loaded nanoclay between 250 and 330 nm. (c) Emission spectra of 2,4-D and 2,4-D loaded nanoclay in ethanol (λext 283 nm; slit width ext. and em. 10 nm). Callose Visualization. Callose was visualized in A. thaliana leaves to provide a measure of any cellular damage induced by nanoclay spray application. One week after spray application of various formulations, excised leaves from the treated A. thaliana plants were transferred to a tube containing 80% (v/v) ethanol. The ethanol was intermittently changed to remove chlorophyll and the remaining plant tissue was then transferred to a callose staining solution that contained 0.5% (w/v) aniline blue (Sigma-Aldrich) in 0.15 M K2HPO4 (Sigma-Aldrich). After overnight incubation, callose was visualized in the leaves by placing each leaf in water on a microscope slide and examining using epifluorescence microscopy (Axio Imager M2 microscope, Zeiss, North Ryde, NSW, Australia) with an excitation wavelength of 365 nm and emission wavelength of 420 nm.24 In order to estimate the amount of callose distributed in the leaves, a previously described robust digital quantification method was adopted.8 For this, ten images collected over the two replicates were used and data was subjected to statistical analysis using Duncan’s posthoc tests (IBM, SPSS Version 19, NSW, Australia). Phytotoxicity Assessment. For the quantification of the herbicidal effect, a robust and routinely used single-blind assessment method was adopted.9,25 Images collected after day three and seven for all the spray applications were subjected to a process as described earlier.19 Briefly, all the images obtained for various treatments of the two replicates were assessed by three independent reviewers on a scale between 0 and 5 where, 0 = healthy plants with no visual signs of tissue damage; 1 = minor chlorosis, tissue necrosis; 2 = minor chlorosis and necrosis along with lamina and petiole curling along the axis of the stem; 3 = moderate chlorosis and necrosis, leaf and petiole curling, first signs of apical bud deformation; 4 = severe chlorosis,

necrosis, leaf lamina and petiole curling associated with deformation of apical buds and 5 = very severe phytotoxicity associated with complete necrosis and death. After confirming uniform distribution, the ranked data was subjected to statistical analysis using Duncan’s posthoc tests (IBM, SPSS Version 19, NSW, Australia).

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RESULTS Physical Characterization of the Nanoclay. FTIR spectroscopy was used for both the identification of key functional groups and to investigate interlayer anions interactions (Figure 1a). The FTIR spectrum of the nanoclay alone has four main peaks at 3407, 1352, 641 and 447 cm−1 (Figure 1a[i]). In addition, weak bands were also observed at 1643 and 447 cm−1. The band at 3407 cm−1 was attributed to the stretching vibration of the O−H group and the weak band at 1643 cm−1 to bending vibrations (Figure 1a[i]). The strong band at 1352 cm−1 represents symmetrically hydrogen bonded carbonate anions while the bands at 780, 641 and 447 cm−1 were assigned to M−O vibrations and M−O−H bending (Figure 1a[i]).26 The FTIR spectrum of 2,4-D (Figure 1a[ii]) was also recorded and compared with that of the 2,4-D loaded nanoclay (Figure 1a[iii]) and bare nanoclay. For the 2,4-D loaded nanoclay, the above-mentioned characteristic peaks of nanoclay were all observed along with additional peaks at 2962, 2931 and 2865 cm−1 that represent −C−H stretching 10964

DOI: 10.1021/acssuschemeng.9b02001 ACS Sustainable Chem. Eng. 2019, 7, 10962−10970

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ACS Sustainable Chemistry & Engineering

Figure 2. Physical characterization of nanoclay. (a)27Al SSNMR of nanoclay and 2,4-D loaded nanoclay (n.b. small peaks at around +200 and −200 ppm are spinning sidebands). (b) XRD patterns from bare nanoclay and 2,4-D loaded nanoclay powders. (c) TEM images of bare nanoclay (d) and 2,4-D loaded nanoclay. Scale bar is 100 nm.

vibrations and at 1744 cm−1 which shows a carbonyl stretching vibration. The UV−vis absorption spectra of 2,4-D showed three peaks at 228, 283, and 291 nm (Figure 1b) and loading of 2,4-D on nanoclay resulted in a hypsochromic shift (Δ11 nm) in the peak at 228 nm, however peaks at 283 and 291 nm remained unchanged (Figure 1b insert). Similarly the emission intensity at 329 nm of 2,4-D loaded nanoclay was enhanced compared with that for 2,4-D alone (Figure 1c).

To investigate the effect of 2,4-D on the local aluminum environment of the MgAl-LDH crystal, 27Al solid-state MAS NMR was recorded and compared with nanoclay alone. Both the bare nanoclay and 2,4-D loaded nanoclay had a single sharp peak at a chemical shift of ∼7 ppm, which is typical for an octahedrally coordinated AlO6 environment (Figure 2a).27 The formation of the nanoclay was confirmed by matching the observed diffraction peaks with “Magnesium Aluminum Nitrate Hydroxide Hydrate” phase (ICDD No. 00-062-0583) 10965

DOI: 10.1021/acssuschemeng.9b02001 ACS Sustainable Chem. Eng. 2019, 7, 10962−10970

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ACS Sustainable Chemistry & Engineering

Figure 3. Callose visualization in A. thaliana leaves. (a) Similar to water treated controls increasing doses of nanoclay treatment from (b) 0.2% (w/ v) to (c) 1.0% (w/v) did not induce the production of the site specific stress marker. (d) Punctate distribution of callose is evident after treating the leaves with 20% (w/v) nanoclay. (e) Large aggregates of callose were observed on leaves treated with 50% (w/v) nanoclay. (f) For the application of positive control, 0.1% (w/v) empigen, callose production was very intense where large aggregates of callose coalesced to cover the leaf. (a to f) Brightly stained trichomes on the leaves can be seen. Scale bar equals to 850 μm.

of callose aggregates that formed a continuous layer within affected leaf tissues (Figure 3f). To quantify the distribution of callose, the collected images were subjected to image analysis. Compared to that of untreated controls, callose distribution did not differ significantly for plants treated with nanoclay between doses 0.2% (w/v) to 1.0% (w/v) (Figure 4). For the nanoclay doses

in the PDF-4+ database. The observed peaks for 2,4-D loaded nanoclay are slightly broader than those of bare nanoclay while the reported phase has a rhombohedral crystal system (space group of R3̅m). By subtracting the instrumental contribution of ∼0.35° 2θ, the crystallite size decreased from 14 to 8 nm following the addition of 2,4-D into nanoclay (Figure 2b). In the TEM images, the nanoclay appeared as near hexagonal sheets of size less than 40 nm (Figure 2c). After loading 2,4-D, nanoclay retained this shape and there were no observed differences in the morphology and size of 2,4-D loaded nanoclay (Figure 2d). The hydrodynamic diameter measured using dynamic light scattering (DLS) was found to be 79.4 and 81.0 nm for bare and loaded nanoclay, respectively (Figure S1). The elemental concentrations (wt %) of Mg, Al and Cl for bare nanoclay were respectively recorded as 27.3 ± 1.2, 15.1 ± 0.9 and chlorine was not detected, while those of the 2,4-D loaded nanoclay are respectively 24.2 ± 2.6, 27.9 ± 3.5 and 8.1 ± 1.1 (Figure S2). The loading efficiency of nanoclay for 2,4-D was found to be 76.4%.28 Callose Visualization and Quantification in Isolated Leaves of A. thaliana. To visualize any impact on the leaves of the plants after application of the nanoclay, they were stained for callose with aniline blue. For the control A. thaliana leaves that were treated with only water, there was no observed callose production (Figure 3a). Similarly, application of the nanoclay from 0.2% (w/v) to 1.0% (w/v) did not induce callose production (Figure 3b,c). With a further increase in the concentration of applied nanoclay to 20% (w/v) and 50% (w/ v), callose production was increased. For the nanoclay treatments at 20% (w/v) callose formation appeared as flecking along the cell walls of both epidermal and mesophyll cells, which coalesced to form larger aggregates (Figure 3d). The distribution and production of callose after application of 50% (w/v) nanoclay was similar to that of the 20% (w/v) nanoclay application but the aggregates were larger and their density higher (Figure 3e). The positive control of commercial surfactant [0.1% (v/v) empigen] also produced abundant callose deposits as had been previously observed.8 Such an effect was characterized by the presence of a very high density

Figure 4. Quantification of callose distribution in nanoclay spray treated leaves. Consistent with the microscopy images, callose production is not induced in untreated control (UTC) and in plants spray applied with nanoclay in a dose range of 0.2% (w/v) and 1.0% (w/v). Note: Tiny bars between UTC and 1.0% (w/v) correspond to the brightly stained trichomes that are present on the plant leaf surface but not callose. Though a slight increase in the callose distribution was observed for 20% (w/v) and 50% (w/v) nanoclay treatments, it is significantly less than that of the positive control (Pctrl) treatments.

of 20% (w/v) and 50% (w/v), callose distribution was high and dose dependent, while the positive controls exhibited a very high and extensive distribution of callose (Figure 4). All these observations were consistent with the results observed for callose visualization. Release of 2,4-D from Nanoclay Particles. To evaluate herbicide release, the UV−visible spectra of a 2,4-D loaded nanoclay solution were recorded at 283 nm for 18 h. From the standard calibration curve obtained for 2,4-D in 60% (EtOH:H2O; v/v) ethanol, the amount of 2,4-D released 10966

DOI: 10.1021/acssuschemeng.9b02001 ACS Sustainable Chem. Eng. 2019, 7, 10962−10970

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ACS Sustainable Chemistry & Engineering

overall induced herbicidal response was dose-dependent with plants treated with 1.0% (w/v) showing the most pronounced effect (Figure S3f). Corresponding to 0.2% (w/v), 0.4% (w/v), 0.6% (w/v), 0.8% (w/v) and 1% (w/v) 2,4-D loaded nanoclay, the amount of 2,4-D released from nanoclay within the first hour was estimated to be 0.36, 0.72, 1.08, 1.44, and 1.8 μM, respectively. Positive controls were therefore set up at these concentrations to make a direct comparison (Figure S3g−i). The herbicidal effect for the positive controls that were spray applied with 2,4D in water were similar to those of the nanoclay treated plants as indicated by the presence of leaf curling and chlorosis (Figure S3h−j). Compared to the herbicidal effect induced by nanoclay formulations, spray applications of 2,4-D at higher concentrations of 1.44 μM (Figure S3k) and 1.86 μM (Figure S3l), did not induce plant wilting. Seven days after the spray application of the 2,4-D nanoclay formulations, the overall herbicidal effect was more pronounced when compared to the positive controls (Figure 6). The early leaf curling observed for 0.2% (w/v) and 0.4% (w/v) 2,4-D loaded nanoclay applications progressed to a severe wilting (Figure 6b,c). Plant death was evident for the spray applications above 0.4% (w/v) (Figure 6d−f). Further, to assay the effect of 2,4-D nanoclay formulations, five-week-old A. thaliana plants were sprayed with the same, at doses ranging between 0.2% (w/v) and 1.0% (w/v) and then observed for 12 days (Figure S4). A dose response consistent with our earlier observations was noted. Quantification of the Herbicidal Effect of 2,4-D. Corresponding to 0.2% (w/v), 0.4% (w/v), 0.6% (w/v), 0.8% (w/v) and 1% (w/v) 2,4-D loaded nanoclay, the amount of 2,4-D released from nanoclay was estimated and positive controls were set up at concentrations, 0.36, 0.72, 1.08, 1.44 and 1.8 μM, respectively. Single-blind assay, a routinely used method to quantify the herbicidal effect between 2,4-D loaded nanoclay treatments and respective positive controls was used to rank the images collected after various spray applications. The average phytotoxicity rating 3 days after the spray applications, (excluding the untreated controls) ranged between 2.3 ± 0.3 and 3.0 ± 0.6. Although there were differences in the individual ratings obtained for 2,4-D loaded

from 1.0% (w/v), 0.80% (w/v), 0.60% (w/v), 0.40% (w/v) and 0.20% (w/v) 2,4-D loaded nanoclay measured within the first hour was 1.80, 1.44, 1.08, 0.72 and 0.36 μM, respectively. To obtain the total in vitro cumulative release profile, 2,4-D released from nanoclay was monitored for 18 h. The amount of 2,4-D released was estimated as 0.03 μM at the end of the first hour increasing to 1.38 μM by the end of the sixth hour. At 18 hours, 2,4-D release was 1.64 μM (Figure 5).

Figure 5. Change in the absorbance of 2,4-D loaded nanoclay (at 283 nm) with time (h). Cumulative release of 2,4-D from nanoclay. The amount of 2,4-D released in the first 6 h was rapid, which then steadily increased until the end of 18 h.

Herbicidal Efficacy of Nanoclay Loaded 2,4-D. To determine the effect of nanoclay for the slow delivery of 2,4-D, increasing doses of 2,4-D loaded nanoclay were spray applied to A. thaliana and images of the plants were captured 3 days after spray application (Figure S3). Application of 0.2% (w/v) 2,4-D loaded nanoclay clearly induced an herbicidal effect characterized by the presence of lamina and petiole curling (Figure S3b). Leaf curling was consistent with further increases in the dosage from 0.4% (w/v) through to 1.0% (w/v) and the herbicidal effect advanced to plant wilting (Figure S3c−f). The

Figure 6. Herbicidal effect of 2,4-D loaded nanoclay compared to positive controls. Compared to (a) water treated A. thaliana plants, strong herbicidal effects were observed on plants 7 days after the application of the agrochemicals. (b and c) Plants appeared to wilt along with chlorosis and leaf curling symptoms. (d, e and f) A dose responsive trend of the strong herbicidal effect along with chlorosis, wilting and death was clear. (g) Water treated control. (h−l) The herbicidal symptoms such as leaf curling and chlorosis are observed consistently on all the treated plants. NC = nanoclay. Scale bar equals to 3.0 cm. 10967

DOI: 10.1021/acssuschemeng.9b02001 ACS Sustainable Chem. Eng. 2019, 7, 10962−10970

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ACS Sustainable Chemistry & Engineering

From the FTIR spectrum, the formation of the nanoclay was confirmed by the stretching and bending vibrations of O−H groups, which in turn correspond to the brucite-like layer of the nanoclay and the adsorbed interlayer water molecules.30 The bending vibrations also confirm the presence of both the interlayer and adsorbed H2O molecules of the brucite-like layer.26 The symmetrical hydrogen bonded carbonate anions, M−O vibrations and M−O−H bending along with the characteristic peak of Mg2AL-LDH hydroxides at 447 cm−1 established that we had successfully synthesized nanoclay.31 The loading of 2,4-D on the surface of nanoclay was examined by comparing the FTIR spectra of 2,4-D, bare nanoclay and 2,4-D coated nanoclay. The 2,4-D loading on to the nanoclay was shown by the presence of both the −C−H symmetric and asymmetric vibrations that are exclusive to 2,4-D and were observed in the 2,4-D loaded nanoclay spectrum. Also, the presence of a strong absorption, which is specific to the carbonyl group of 2,4-D, in the loaded nanoclay spectrum, further confirmed the agrochemical loading. Furthermore, to understand the level of interaction between 2,4-D and nanoclay, optical properties of 2,4-D loaded nanoclay were studied. As observed in the absorption spectra, π−π* (228 nm) and n−π* (283 and 291 nm) electronic transitions are specific for 2,4-D.32 The blue shift (Δ11 nm) for π−π* after loading 2,4-D on to nanoclay was indicative of the electronic interaction between hydroxyl groups and 2,4-D molecules.32 The electronic interaction between 2,4-D and nanoclay was further supported by emission spectra collected for 2,4-D loaded nanoclay. The increment in the emission intensity for 2,4-D loaded nanoclay likely arose from charge transfer from 2,4-D through to nanoclay. The charge transfer promoted the radiative transition that further enhanced the emission spectra.33 To understand the effect of loading on the structural orientation, 27Al SSNMR of nanoclay was measured before and after loading. The peak commonly observed for both the nanoclay and 2,4-D loaded nanoclay corresponded with an octahedral Al in brucite pattern that is surrounded by six Mg2+ ions. This further indicated that electronic interactions facilitated loading of 2,4-D onto the nanoclay.34 On the other hand, for the X-ray diffraction studies, a reduction in the crystallite size was observed for 2,4-D loaded nanoclay and yet TEM investigations revealed no apparent difference in the particle morphology. Together, these findings confirm the presence of 2,4-D in between layers of nanoclay that caused lattice distortion and a simultaneous decrease in the crystallite size. For the use of nanoclay as an agrochemical delivery vehicle, it would be pragmatic to consider its physiological interaction with plants but nanoclay induced stress in plants has not been described in the literature. Therefore, we visualized the production of callose that modulates or is found in conjunction with biotic or abiotic stress in plants.8,35 Callose, a sensitive site-specific β-1,3-glucan, is formed in plant cells after induction of stress. It is thus, routinely visualized and quantified in plant cells as a robust marker for induced physiological stress.36 In our study, for nanoclay application at 1.0% (w/v) or lower, callose production was insignificant and clearly indicated that physiological stress was not induced in those plants. Very high doses did induce callose production, but those doses were unrealistic for common agricultural practises.

nanoclay treatments and respective positive controls they were not significantly different (Figure S5). However, there were marked differences in the observations that were made 7 days after the different agrochemical applications (Figure 7). While at lower doses 0.2% (w/v)

Figure 7. Quantification of the herbicidal effect 7 days after agrochemical spray application. The herbicidal effect for nanoclay loaded 2,4-D (orange bars) and 2,4-D (in water) (blue bars) formulations was statistically similar at doses 0.36 and 0.72 μM. For doses 1.08, 1.44 and 1.86 μM, the consolidated herbicidal effect observed for nanoclay based delivery is statistically different from that of 2,4-D treatments. Note, 0.36 μM 2,4-D applied in water = 2,4-D released from 0.2% (w/v) 2,4-D loaded nanoclay; 0.72 μM = 0.4 (w/ v); 1.08 μM = 0.6% (w/v); 1.44 μM = 0.8% (w/v) and 1.86 μM = 1.0% (w/v). Bars ± SE with the same letters are statistically similar after Duncan’s posthoc test (p < 0.05) performed on uniformly distributed data.

(=0.36 μM 2,4-D in water, positive control) and 0.4% (w/v) (=0.72 μM), the effect of both the agrochemical formulations, which is 2,4-D loaded nanoclay and 2,4-D in water, appeared to be statistically similar on the treated A. thaliana plants. After the application of nanoclay formulation at 0.6% (w/v) (=1.08 μM), 0.8% (w/v) (=1.44 μM) and 1.0% (w/v) (=1.86 μM), the phytotoxicity ratings were respectively recorded as 4.3 ± 0.5, 4.0 ± 1.1 and 4.7 ± 0.5. These ratings are statistically different from that of 3.8 ± 0.4, 3.3 ± 0.5 and 3.8 ± 0.8, respectively obtained for 2,4-D in water treatments at 1.08, 1.44 and 1.86 μM (Figure 7).

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DISCUSSION Nanoclays are well-known as anionic exchange materials,12 as pharmaceutical delivery vehicles17 and their biosafety to animal cell lines has been well established. 29 However, the physiological stress response induced in-planta by their exposure to nanoclay and the capacity of the nanoclay to deliver agrochemical actives has not been investigated. We have demonstrated here that spray application of agriculturally relevant doses of nanoclay to plants does not induce production of callose, a site-specific stress marker. Simultaneously, we determined that 2,4-D was slow released from loaded nanoclay for up to 18 h in the solvent (60% v/v ethanol). A 2,4-D loaded nanoclay (spray applied in water) between doses of 0.6% (w/v) to 1.0% (w/v) induced a greater herbicidal effect on the test plants when compared to that of the positive controls (2,4-D spray applied in water) at equal doses. We clearly demonstrate that nanoclay can be an alternative for the current surfactant-based agrochemical delivery and can significantly contribute to the goals associated with sustainable agricultural practice. 10968

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ACS Sustainable Chemistry & Engineering 2,4-D is one of the first commercialized37 and extensively used auxin mimic herbicide belonging to the class of phenoxycarboxylic acids.38 Often, 2,4-D is selectively used in broad acre cereal crops such as Triticum aestivum (wheat), Hordeum vulgare (barley) and Avena sativa (oats) to control broad leaf weeds such as Sinapis arvensis (wild mustard) and Raphanus raphanistrum (wild radish).1 Auxin mimic herbicides block the proteins (F-box proteins) that regulate auxin responsive gene transcription resulting in over production of plant hormones such as ethylene and abscisic acid.38 Hormone over production disrupts the in planta hormonal balance causing, epinasty, chlorosis, tissue swelling and heightened production of reactive oxygen species (ROS) that ultimately lead to plant death.39,40 The observed herbicidal symptoms in our experiments with the model plant corresponded very well to the known herbicidal activity of 2,4-D. Through the in vitro release assay, we could demonstrate that 2,4-D was rapidly released into the solvent (60% (v/v) ethanol) for 6 h, which then gradually increased over the time course of the experiment (18 h). For 2,4-D loaded nanoclay that is spray-applied to plants, we could expect much slower rates of release on the plant surface. The release of 2,4-D on a leaf surface would solely depend on the decomposition of the nanoclay that is facilitated by carbonic acid formation from atmospheric CO2 and moisture.10 In laboratory studies, sustained release of phenoxy herbicides into water, for up to 7 days, was shown for herbicide nanoclay complexes.41 The overall herbicidal effect observed for applied doses of 2,4-D loaded nanoclay correlated well with a slow release of the herbicide. Such an effect was, however, not observed for 2,4-D (in water) applications made at equal concentrations. There is a great need for investigation of the impacts of nanoparticles, including nanoclays, on the physiological performance of plants. To address this shortfall in many related studies, we investigated callose, a robust, sensitive stress marker in plants, in response to the application of nanoclay to leaves. We have shown that nanoclay applications induce very little if any stress in plants at agriculturally relevant doses. In a way similar to liquid crystalline particles that are able to stick to plant surfaces,25 nanoclays are also able to strongly adhere to leaf surfaces due to their electronic properties.10 Thus, when nanoclay materials are used as a delivery vehicle their inherent physicochemical properties, eliminate the use of surfactants and other coformulants, that are commonly used to deliver agrochemcials. In addition, aluminum and magnesium nanoclays degrade to liberate important plant nutrients such as magnesium, 10 which play central roles in plant cell biochemistry and physiology.42 The ability of nanoclays to retain anionic active ingredients strongly by electronic interactions also means that the amount of 2,4-D that leaches into soil would be reduced which in turn would reduce soil and groundwater contamination.41 We, thus propose that nanoclays are ideal candidates for sustainable delivery of agrochemicals and associated green chemistries.

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loaded nanoclay on A. thaliana plants; Figure S4 herbicidal effect of 2,4-D loaded nanoclay on five-weekold A. thaliana plants; Figure S5 - evaluation of day three herbicidal effect (PDF)

AUTHOR INFORMATION

Corresponding Author

*D. M. Cahill. Email: [email protected]. ORCID

Pavani P. Nadiminti: 0000-0002-6175-7731 Luke A. O’Dell: 0000-0002-7760-5417 Author Contributions #

P.P.N. and H.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.S. thanks Deakin University for an Alfred Deakin Postdoctoral Fellowship. We acknowledge Dr. Rosey Squire, Advanced Characterisation Facility; Deakin University, Geelong, for assistance with TEM.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.9b02001. Figure S1 - dynamic light scattering (DLS) analysis; Figure S2 - EDS spectra; Figure S3 - effect of 2,4-D 10969

DOI: 10.1021/acssuschemeng.9b02001 ACS Sustainable Chem. Eng. 2019, 7, 10962−10970

Research Article

ACS Sustainable Chemistry & Engineering

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