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Synthesis of (Hexaconazole-Zinc/Aluminium-Layered Double Hydroxide Nanocomposite) Fungicide Nanodelivery System for Controlling Ganoderma Disease in Oil Palm Isshadiba Mustafa, Mohd Zobir Hussein, Bullo Saifullah, Abu Seman Idris, Nur Hailini Zainol Hilmi, and Sharida Fakurazi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04222 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Synthesis of (Hexaconazole-Zinc/Aluminium-Layered Double Hydroxide Nanocomposite)

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Fungicide Nanodelivery System for Controlling Ganoderma Disease in Oil Palm

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Isshadiba F. Mustafa1, Mohd Zobir Hussein1*, Bullo Saifullah1, Abu Seman Idris2, Nur

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Hailini Z. Hilmi2 and Sharida Fakurazi3,4

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7 8

Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia 2

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Malaysian Palm Oil Board (MPOB), No.6, Persiaran Institusi, Bandar Baru Bangi, 43000,

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Kajang, Selangor, Malaysia 3

11 12 13

Materials Synthesis and Characterization Laboratory, Institute of Advanced Technology,

Laboratory of Vaccine and Immunotherapeutics, Institute of Bioscience (IBS), Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

4

Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra

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Malaysia, 43400 UPM Serdang, Selangor, Malaysia

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Corresponding author: [email protected]

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Abstract: Fungicide, namely hexaconazole was successfully intercalated into the

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intergalleries of zinc/aluminium-layered double hydroxide (ZALDH) using ion exchange

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method. Due to the intercalation of hexaconazole, the basal spacing of the ZALDH was

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increased from 8.7 Å in ZALDH to 29.45 Ǻ in hexaconazole-intercalated ZALDH

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(HZALDH). The intercalation of hexaconazole into the interlayer of the nanocomposite was

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confirmed using the Fourier-transform infrared (FTIR) study. This supramolecular chemistry

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intercalation process enhanced the thermal stability of the hexaconazole moiety. The

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fungicide loading was estimated to be 51.8 %. The nanodelivery system also shows better

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inhibition towards the Ganoderma boninense growth than the counterpart, free hexaconazole.

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The results from this work have a great potential to be further explored for combating basal

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stem rot (BSR) disease in oil palm plantation.

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Keywords: Nanocomposite, layered double hydroxide, hexaconazole, nanodelivery,

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agronanochemical.

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1. Introduction

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Ganoderma boninense is a wood-rotting fungus that has caused basal stem rot (BSR)

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disease in oil palm. This disease is one of the critical issue causing low yields in the oil palm

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industry in Malaysia.

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In controlling the BSR disease, the fungicides such as hexaconazole and dazomet were

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used. However, it was reported that the use of the fungicides had increased soil acidity since

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the residue of hexaconazole fungicide in the soil sample was found to be at the double

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recommended dosage1. There is currently no effective way to ensure that the fungicides was

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only released at the fungal site instead of going downward in soil profile through leaching. In

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this work, a fungicide controlled release formulation was designed and synthesized in

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controlling the release of fungicides and subsequently reduce the acidity problem.

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Lately, many researchers were attracted on layered double hydroxides or also called

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hydrotalcite-like compounds. LDHs are a group of inorganic nanolayers with structurally

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positively charged layers and interlayer balancing anions. This inorganic nanolayer also

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known as anionic clay, with formula (MII

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referring to divalent while MIII is trivalent cations and An- represents the anions which are able

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to balance the electro-neutrality of the positive charged layers2. The superior properties of

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LDH such as its biocompatibility, its ability to act as removal agents of pesticides, slow

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release system and low toxicity makes it suitable to be used for various agriculture

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applications 3.

1-x

MIII

x

[OH]2 ).(Ax/nn-).yH2O in which MII is

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It was reported that hexaconazole was intercalated into Mg/Al-LDH exhibited a

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potential pesticide controlled release4, but currently, no study on antifungal potential towards

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Ganoderma boninense by using Zn/Al LDH as a carrier. In this work, hexaconazole was

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accommodated in intergalleries of Zn/Al-LDH (ZALDH) so that the release of fungicide can

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be occurred in a sustained manner. The use of Zn/Al-LDH is hoped to have beneficial effect

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as Zn is an essential element for plant.

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Here we discuss our work on the intercalation of hexaconazole into the interlamellae of

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ZALDH to form a nanocomposite of hexaconazole-ZALDH 2D layered structure and

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subsequently study its physico-chemical and phytotoxicity properties. Hexaconazole was

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selected in this work because it is widely introduced as a preventive treatment and prolonging

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the productive life of infected palms5.

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2. Materials and methods

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2.1 Materials

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Hexaconazole, with 95 % pure (Changzhou, China) was used as received. All chemical

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reagents involved were obtained from Sigma-Aldrich. All experiments were conducted using

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deionized water.

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2.2 Method

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2.2.1

Synthesis of Zn/Al-NO3-LDH

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The salts of Zn(NO3)2 and Al(NO3)3 were dissolved in 250 mL deionised water with a

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molar ratio of 2:1. After 15 minutes, the NaOH solution with concentration of 2 M, was

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dropped slowly to the mixture until the solution was achieved to pH 7-7.5 with vigorous

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stirring under nitrogen environment. Then, the sample was kept into an oil bath at 70 °C for

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18 h, washed three times with deionised water and centrifuged 6. After drying in an oven for

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two days, the sample obtained was labeled as ZALDH.

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2.2.2 Synthesis of hexaconazole micelle

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An amount of 1.223 g of anionic surfactant, sodium dodecylbenzenesulfonate was

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dispersed into 250 mL deionised water, 100 mL acetone solution containing 0.2 mol

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fungicide, hexaconazole was mixed with the surfactant and stirred at 40-45 °C. The fungicide

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micelle was obtained once the acetone was fully evaporated 7.

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2.2.3 Synthesis of Zn/Al-hexaconazole by the ion-exchanged method

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About 0.5 g ZALDH was added into a 250 mL solution containing approximately 150

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mL, 0.2 M hexaconazole micelle. Then, the mixture was stirred at 75 ºC for 72 h. After that,

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the sample was obtained after centrifugation process using deionised water and acetone. The

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fungicide-LDHs, labeled as HZALDH nanocomposite was obtained after drying at 70 ºC in an

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oven for about 72 h.

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2.3

Characterizations

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Powder X-ray diffraction (PXRD) pattern was recorded using a Shidmadzu

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diffractometer, with 2-60° in range, by a CuKα radiation source (λ=1.5405 Å) driven at 40 kV

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and 30 mA. The FTIR spectra were obtained using a Perkin-Elmer 1725X spectrophotometer

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by ATR technique with wavelength of 400-4000 cm-1. Thermogravimetric analyses (TGA)

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was carried out using a Mettler Toledo instrument with 50 mL/min nitrogen flow and 10

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⁰C/min heating rate, between 25-1000 ⁰C. The surface morphology of the nanocomposite was

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studied using a scanning electron microscope (SEM), JEOL JSM – 6400 model. A Perkin

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Elmer ultraviolet-visible spectrophotometer (Lambda 35) was used in determination of

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controlled release property. The high-performance liquid chromatography (HPLC), equipped

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with Sykam S3250 UV/Vis detector was used to determine the percentage of the

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hexaconazole loading.

129 130

2.4

Measurements of release amount of hexaconazole from HZALDH

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The release of fungicide from HZALDH nanocomposite was conducted in a pH 5.5

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(soil pH) of phosphate-buffered solution. HZALDH nanocomposite (10 mg) was put into 20

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mL phosphate buffer solution. At preset interval times, the sample was taken (2-3 mL) and

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replaced with new solution. After the aliquot was filtered, the fungicide content was

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determined at the maximum wavelength of 202 nm using an ultraviolet-visible

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spectrophotometer.

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2.5

Loading amount of hexaconazole in HZALDH

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HPLC analysis of the hexaconazole fungicide in the HZALDH nanocomposite was

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carried out using the method as previously described elsewhere8. Two mobile phases were

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used, namely acetonitrile and 0.1 % orthophosphoric acid. The isocratic mobile phase of

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acetonitrile and orthophosphoric acid was fixed at a ratio of 75:25, with a flow rate of 1

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mL/min. It was shown that the retention time was 2.9 minutes. A calibration curve was

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obtained by running a standard at different concentrations of hexaconazole (0, 50, 100, 150,

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and 200 ppm), resulted in a good R2 value of 0.98. Approximately, 10 mg of the HZALDH

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was dissolved in 50 mL (5 mL of 1 molar HCl and the remaining volume was composed of

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the mobile phase) and standard hexaconazole solutions were also prepared in the same way.

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The percentage loading of hexaconazole in HZALDH nanocomposite was calculated to be

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51.8 %.

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2.6

Assessment of antifungal activity

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2.6.1 Culture of Ganoderma boninense

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Pathogenic G. boninense culture (PER71) was obtained from the Malaysian Palm Oil

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Board (MPOB), Bangi, Malaysia. The culture was maintained in Petri dishes (diameter 10

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mm) on potato dextose agar (PDA) media (Oxoid, Thermo Scientific) (pH 5.5) and incubated

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at 28 ± 2 °C prior to further usage.

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2.6.2 Determination of antifungal activity of the nanocomposites against G. boninense

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The antifungal activity of the nanocomposites was tested for their antibiosis properties

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through poison food agar assay. Mycelial discs (5 mm) of 7 days old fungal culture were sub-

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cultured in the middle of PDA agar plates containing different concentrations of

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nanocomposites. The PDA has been previously prepared by incorporating the desired

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concentration of fungicide, i.e. 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 ppm into

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sterilized PDA. The inoculated plates were sealed and incubated at 28 ± 2 °C for 7 days.

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The growth of G. boninense in the agar plate was measured through the radius growth

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and the measurements were taken throughout the 7 days. Using the Equation 19, the

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percentage inhibition of radical growth (PIRG) was calculated;

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PIRG = (R1 – R2) / R1 x 100

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(1)

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where R1 is the radius growth of G. boninense in a control plate and R2 is the radius growth of

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G. boninense in the fungicides treated plate.

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2.6.3 Statistical analysis

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All data are presented as mean ± standard deviation for 5 independent tests. The

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comparison of values obtained was analyzed using a Minitab 16 statistical analysis software

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(Minitab Inc., State College, PA, USA) by one way and two way analysis of variance

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(ANOVA) followed by the Tukey’s test. The significant value was considered when p-value

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is less than 0.05.

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3.

Results and Discussion

185 186

3.1 Powder x ray diffraction analysis

187 188

The XRD patterns of HZALDH nanocomposite, prepared using ion exchange method

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was shown in Figure 1(A). Based on the figure, pure ZALDH has a basal spacing of 8.7 Ǻ,

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consistent with a monolayer of nitrate as the counter anion. This is because the brucite-like

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layer has a thickness of 4.8 Ǻ and the remaining 3.9 Ǻ corresponds to a monolayer of the the

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nitrate anion 10. The resulting nanocomposite has expanded from 8.7 to 28.9 Ǻ, which indicate

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that the newly exchanged anion, hexaconazole has been intercalated into the intergalleries of

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ZALDH to replace nitrate. It was obviously seen that the hexaconazole has higher affinity to

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be intercalated into the LDH intergalleries compared to the nitrate, due to its higher

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concentration or bigger size11.

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The results showed that the preparation using 0.2 M HZALDH displayed a sharp,

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symmetric, high crystallinity, showing the pure product has been obtained with no left over

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adsorbed Zn(NO3)2 and Al2(NO3)3. After several optimisations using various concentrations

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of hexaconazole micelle, this sample was then chosen for further studies. A slow scan PXRD

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of the sample, HZALDH exhibits 8 harmonics; 29.45, 14.83, 9.65, 7.30, 5.84, 4.96, 4.25, and

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3.67 Ǻ as shown in Figure 1D, which producing an average basal spacing of 29.42 Ǻ. This

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value was determined by dividing sum of reflections (nxd) with the total number of

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reflections. This value was then used to predict the plausible arrangement of hexaconazole in

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the intergallery of HZALDH nanocomposite.

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3.2 Spatial orientation of the hexaconazole moiety in the ZALDH interlayers.

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Figure 2 (A) and (B) show the three dimensional molecular size of hexaconazole and

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sodium dodecylbenzesulfonate (SDBS) using a Chemoffice software. The x, y and z axes of

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hexaconazole and SDBS were calculated and were found to be 10 Å, 8 Å, 5 Å and 24 Å, 6 Å,

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4 Å, respectively. Based on the XRD pattern, HZALDH nanocomposite synthesized at 0.2 M

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hexaconazole has a mean of basal spacing (d) with value of 29.42 Å. Therefore by subtracting

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the layer thickness of the ZALDH layer which is 4.8 Å, a value of 24.62 Å was obtained.

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Therefore, 24.62 Å is a space that can be allocated for the spatial orientation of hexaconazole

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molecule in the interlayer of ZALDH. The plausible arrangement of hexaconazole is found

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to be oriented in a biomolecular vertical form along with sodium dodecylbenzene sulfonate

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and water molecules, as shown in Figure 2 (C)12.

219 220

3.3 Fourier-transform infrared analysis

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The presence of hexaconazole moiety in the ZALDH nanolayers has been confirmed

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by the FTIR spectroscopy (see Fig. 3). The hydroxyl group of the LDH layers and the nitrate

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anion stretching vibration can be seen at 3440 and 1378 cm-1 as shown in infrared spectrum of

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ZALDH (Fig. 3B). The 1638 cm-1 band was due to H-OH bending vibration. Fig. 3A shows

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that the hexaconazole displays a band at 3217 cm-1, which represents hydroxyl group

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vibrational mode. The C=C and C-H stretching from the aromatic ring were detected at 1431

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and 847 cm-1. FTIR spectra of HZALDH nanocomposite (Fig. 3C) shows the characteristic

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bands of hexaconazole alone with slightly shift in the wavenumber position, which indicates

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the incorporation of hexaconazole into the ZALDH intergalleries.

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After the intercalation of hexaconazole, the 1601 cm-1 band has been formed, which

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indicates that C=N stretching from hexaconazole molecule has present. The band assigned to

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the nitrate anions (1378 cm-1) was absent in this resulting nanocomposite, which strongly

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support that the NO3- anions have been replaced by the fungicide moiety, hexaconazole. The

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existence of hexaconazole in the new nanodelivery system and sulfonate ions can be observed

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at 2956 and 687 cm-1 13.

237 238

3.4 Thermal Studies

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Fig. 4 illustrates the TGA/DTG thermograms of free hexaconazole, ZALDH and

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HZALDH nanocomposite. . For hexaconazole, the thermogram (Fig. 4A) shows a sharp peak

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at 283 °C with 100 % weight loss, due to complete combustion of hexaconazole moiety. In

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Fig.4B, ZALDH shows four stages of weight loss, observed at 108, 244, 310 and 510 °C, with

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percentage loss of 7.0, 16.7, 4.0 and 5.5 %, respectively. The first one was associated with

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loss of water molecule. The second one is due to strongly held water molecules and the third

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and fourth weight losses are almost completed at 510 °C, which referring to dehydroxylation

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of brucite-like layers and removal of the interlayer anions14.

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Due to the intercalation of hexaconazole into the interlayer of ZALDH (Fig. 4C),

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TGA/DTG curves show five thermal events at 90, 229, 322, 425 and 888 °C with weight

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losses of 6.9, 8.3, 10.9, 30.3 and 6.9 %, respectively. The first and the second stage of weight

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losses are the same as that for ZALDH, which is due to the removal of adsorbed water and

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dehydroxylation of the hydroxyl layer, respectively. The third stage at 322 °C is a result of the

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organic moiety decomposition in the interlayer of the nanohybrid, leaving only a relatively

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less volatile metal oxide15. The weight loss was increased to 30.3 % at 425 °C in HZALDH

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nanocomposite because of the hexaconazole anions combustion. The last stage of weight loss

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that occurred at around 888 °C was due to the formation of the spinel (ZnAl2O4) phase16. This

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was presumably due to the high electrostatic force in the molecule as the thermal

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decomposition affected by the substituents on the ligand17.

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It was obviously seen that the thermal stability of HZALDH nanocomposite was

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greatly increased after the intercalation which is at 425 °C compared to 283 °C (for the free

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hexaconazole). The result had shown that ZALDH has a potential to be used as a carrier to

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store fungicide with good thermal stability.

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3.5 Surface morphology

265 266

Figs. 5 (A) and (B) show the field emission scanning electron micrograph, showing

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the surface morphology of HZALDH at 50,000x and 100,000x magnifications, respectively.

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The HZALDH nanocomposite shows agglomerated non uniform granule structure, similar to

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the other nanocomposites previously prepared by other works18.

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3.6 Release behavior of hexaconazole from HZALDH nanocomposite

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The release behaviors of both pure hexaconazole and HZALDH nanocomposite

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were studied in PBS solution at pH 5.5 as shown in Fig. 6. The release behavior of pure

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hexaconazole was very fast for the first 420 min, and become slower thereafter, before it

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achieved 100 % complete release in 2000 min. The rapid release of the hexaconazole from the

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nanocomposite was observed initially, then followed with a slower one thereafter and finally

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the sustained release was achieved at 62 % after 3000 min. It is obviously seen that the

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hexaconazole released from the nanocomposite was slower than the release of free

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hexaconazole. This indicates that the nanocomposite served a role as a fungicide controlled

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release system. The electrostatic interaction between the positively charged ZALDH

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nanolayers and the negatively charged hexaconazole anions have influences the release

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property of hexaconazole from its HZALDH interlayers.

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3.7 Release kinetics of hexaconazole from the nanocomposite

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The release kinetics of the hexaconazole from HZALDH nanocomposite was

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analyzed using the pseudo-first order Eq. (2)19, pseudo-second order Eq. (3)20, Hixon-Crowell

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Eq. (4)21, Higuchi Eq. (5)22, and Korsmeyer Peppas model Eq. (6)23. The equations are given

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below;

291 292

ln (qe-qt) = ln qe-kt

(2)

293

t/qt = 1/kqe2 + t/qe

(3)

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qo 1/3 – qt 1/3 = kt

(4)

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qt = k(t)0.5

(5)

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qt = ktn

(6)

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where qo, qe and qt are the initial amount of anions, equilibrium release amount and release

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amount at time t, respectively with k is the corresponding release rate constant.

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Fig. 7 displays the five kinetic models used to fit the release data of hexaconazole from

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nanocomposite. The parameters such as the rate constant, k and the correlation coefficient

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value, R2 obtained from the five models are shown in Table 2. As shown in results above, the

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best fitted of the release of hexaconazole from the pure phase, ZALDH inorganic host is the

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pseudo-second order kinetic model.

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3.8 Antifungal activity of the nanocomposite against the G. boninense

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The antifungal activity of free hexaconazole, ZALDH and HZALDH sample of different

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concentrations (0.001 to 10 ppm) towards G. Boninense were tested and are represented in

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Fig. 8 (A to D) with the error bar as standard deviation. The inhibition zone of G. boninense

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by the samples are also illustrated in Fig. 8 (E). (Based on Fig. 8(A) and 8(C), it can be seen

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that the HZALDH is fully inhibited the G. boninense growth at lower concentration, 0.1 ppm,

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compared to hexaconazole alone, which is at 0.5 ppm. On the other hand, for the ZALDH

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(Fig. 8B), the radial growth of G. boninense steadily increased to the seventh days, which

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shows that it gives no effect on inhibition towards the G. boninense growth. This study also

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revealed that the as-synthesized nanocomposite exhibited significant anti-fungal activity as

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shown in Fig. 8(D).

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As a result of using probit analysis of Sigma Plot 10.0, the half maximal effective

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concentration, EC50 was obtained. The value of EC50 for hexaconazole, ZALDH and

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HZALDH was found to be 0.05, 2.03 and 0.03 ppm, respectively. These findings indicate that

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the resulting nanodelivery system of hexaconazole developed in this work is more effective in

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combating G. boninense compared to its counterpart, the free hexaconazole as indicated by

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the lower EC50 value, 0.03 compared to 0.05 ppm, respectively.

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Table 3 and Table 4 represent the significant effects of pure hexaconazole, ZALDH,

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HZALDH concentrations under one – way ANOVA and the interaction between the treatment

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and concentration towards the growth of Ganoderma boninense under two-way ANOVA,

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respectively. The mean and standard deviation of different concentrations of pure

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hexaconazole and HZALDH are significant, while for the ZALDH, it is insignificant to

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growth as shown in Table 3. In comparing both factors of treatment and concentration, the

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treatment give highly significant effect towards the growth, about seven times compared to

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concentration, as shown in Table 4.

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This finding has showed that zinc/aluminium layered double hydroxide can be used

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as a nanocarrier for a fungicide, hexaconazole in developing new environmental-friendly

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agronanochemicals.

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Acknowledgement

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This project was supported by the Universiti Putra Malaysia and the Ministry of Higher

338

Education of Malaysia (UPM-MOHE) grants under the NANOMITE vot no. 9443100 and

339

5526300. The G. boninense studies were accomplished at the Malaysian Palm Oil Board

340

(MPOB) laboratory and facilities.

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References

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Barahuie F, Hussein MZ, Gani SA, Fakurazi S, Zainal Z. Synthesis of protocatechuic

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Hussein MZ, Hashim N, Yahaya AH, Zainal Z. Synthesis of Dichlorprop-Zn/Al-

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Cheng X, Huang X, Wang X, Sun D. Influence of calcination on the adsorptive removal of phosphate by Zn – Al layered double hydroxides from excess sludge liquor.

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formulation of an herbicide, 2,4-dichlorophenoxyacetate incapsulated in zinc –

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Fig. 1. PXRD patterns of free hexaconazole (A), ZALDH (B) and HZALDH nanocomposite (C) and the slow scan with a dwell time of 0.5°/min (D) and from the 8 reflections, the average value of the interlamellae (nxd) was found to be 29.42 Å.

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C

A

B

Carbon

Nitrogen

Sulphur

Chlorine

Hydrogen

Oxygen

Fig. 2. Three–dimensional structure of hexaconazole (A), sodium dodecylbenzenesulfonate (B) and plausible arrangement of hexaconazole and sodium dodecylbenzenesulfonate in the intergallery of HZALDH nanocomposite (C)

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Fig. 3. Fourier transformed infrared (FTIR) spectra of free hexaconazole (A), ZALDH (B) and HZALDH (C).

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A

a

B B

C

Fig. 4. TGA/DTG thermograms of hexaconazole (A), ZALDH (B) and HZALDH nanocomposite (C).

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A

B

Fig. 5. Field emission scanning electron micrographs of HZALDH nanocomposites (A and B) at 50,000x and 100,000x magnifications.

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Table 1. The thermal properties of pure hexaconazole, ZALDH and HZALDH nanocomposite.

Sample name

T1-T2

Ts (⁰C )

∆m (mg)

Weight loss (%)

Pure hexaconazole

160-328

283

8.34

100

34-160

108

0.40

7.0

160-285

244

1.57

16.7

285-355

310

0.33

4.0

355-560

510

0.45

5.5

30-146

90

0.58

6.9

146-265

229

0.77

8.3

265-361

322

1.07

10.9

361-587

425

3.08

30.3

778-994

888

0.41

6.9

ZALDH

HZALDH

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Fig. 6. Release profiles of hexaconazole from pure hexaconazole and HZALDH nanocomposite at pH 5.5.

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Fig. 7. Fitting the release data of hexaconazole from nanocomposite using the pseudo-first order, pseudo-second order kinetics, Higuchi, Hixon-Crowell and Korsmeyer Peppas models.

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Table 2. Rate constant and correlation coefficient (R2) value of the release data of hexaconazole from the nanocomposite using pseudo-first order, pseudo-second order, Higuchi model, Hixon-Crowell and Korsmeyer Peppas kinetic models.

Sample

HZALDH

C

Saturation release (%) 62

R2 Pseudofirst order 0.9166

Korsmeyer -Peppas model 0.8101

Pseudo-second order Higuchi model 0.8743

HixonCrowell model 0.7227

D

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R2

0.9975

Rate constant, k (mg/min) 1.54x10-2

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E

I

II

III

IV

Fig. 8. The growth curves of G. Boninense treated with hexaconazole (A), ZALDH (B), HZALDH (C) for seven days and the percentage of inhibition radical growth (PIRG) against concentration (ppm) of free hexaconazole, HZALDH and the control after 7 days (D) where *p >0.05 and **p