Aluminum-Layered Double Hydroxide

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Synthesis of Dazomet-Zinc/Aluminium Layered Double Hydroxide Nanocomposite and its Phytotoxicity Effect on Oil Palm Seeds Growth Isshadiba Mustafa, Mohd Zobir Hussein, Idris Abu Seman, Nur Hailini Zainol Hilmi, and Sharida Fakurazi ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b02584 • Publication Date (Web): 18 Oct 2018 Downloaded from http://pubs.acs.org on October 25, 2018

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ACS Sustainable Chemistry & Engineering

Synthesis of Dazomet-Zinc/Aluminium Layered Double Hydroxide Nanocomposite and its Phytotoxicity Effect on Oil Palm Seeds Growth Isshadiba F. Mustafa1, Mohd Zobir Hussein1*, Sharida Fakurazi2,3, Abu Seman Idris4 and Nur Hailini Z. Hilmi4

1Materials

Synthesis and Characterization Laboratory, Institute of Advanced Technology,

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

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

3Department

of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

4Malaysian

Palm Oil Board (MPOB), No.6, Persiaran Institusi, Bandar Baru Bangi, 43000, Kajang, Selangor, Malaysia Corresponding author: [email protected]

Abstract: A fungicide nanodelivery system had been synthesised by the intercalation of dazomet into the interlayer galleries of the zinc/aluminium–layered double hydroxide (Zn/Al-LDH) using the ion-exchanged method. The basal spacing expansion from 8.9 Å in the layered double hydroxide (LDH) to 29.7 Å in the nanocomposite was observed. Fourier-transform infrared (FTIR) study has shown that the absorption bands of the resulting nanocomposite were composed of both dazomet and Zn/Al-LDH characteristics, which confirmed the intercalation of dazomet into the Zn/Al-LDH interlayers with enhanced thermal stability of the guest, dazomet. Further studies on oil palm seedlings were also conducted on the dazomet-intercalated Zn/Al-LDH

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(DZALDH) and compared with the hexazonazole-intercalated Zn/Al-LDH (HZALDH). Both of them have shown positive effects on oil palm seeds growth. This work has shown that the resulting agronanochemicals have a dual-modal fungicide nanodelivery system, as a fungicide and a micro-nutrient supplier to support early plant growth and has the potential to avoid direct contact of the fungicides with the users due to the intercalation process. Keywords: Layered double hydroxide, Dazomet, Hexaconazole, Ganoderma boninense, Nanodelivery, Nanocomposite.

Introduction

Basal stem rot disease was associated with a pathogen, Ganoderma boninense which has shortened the productive life of oil palm and caused serious economic losses to the oil palm industry. Thus, two commercially available fungicides; hexaconazole and dazomet were used by oil palm industry as an initiative to lengthen the life of oil palms. Nevertheless, the disadvantage of using bare hexaconazole has been discussed 1 as it caused a threat to marine communities. For dazomet, methylisothiacynate (MITC) release not only has potential to eradicate Ganoderma inoculum in oil palm but also harmful towards the living organisms especially human 2 due to its highly volatilization. Previous study on different carriers such as nanohexaconazole based on chitosan

3,

polyethyleneglycol 4 and their biological study against bacteria, green algae 5, maize plant 6 and dazomet-chitosan nanoparticles and its fungicidal activity 7 have been documented. These studies have shown that the conversion of both fungicides to their nano-formulations have attributed to better efficiency against fungi and nitrogen fixation.


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A need was felt for studying a new formulation based on layered double hydroxide, as this designation able to increase solubility, enhance bioavailability and sustained release which results in reduced amount of applied active ingredients and finally decrease the dose-dependent toxicity for non-target organism and the environmental burden. Layered double hydroxide (LDH) or the so called hydrotalcite-like compound has become one of the effective nanomaterials which can be used as a carrier in agro-chemicals because of their ease of preparation, low cost and environmentally compatible. The capability of inorganic nanolayer as a nutrient storage, adsorbent and reduce leaching in the soil columns also contributed to the maintenance of soil fertility, as well as the plant, palm oil 8. Therefore, here we discuss our attempt to design and synthesise a fungicide nanodelivery system based on a dazomet-intercalated Zn/Al LDH (DZALDH) which has potential as dual-modal applications; a fungicide and a micro-nutrient supplier to support plant growth. This is because the micro-nutrients; namely Zn and Al which are the main composition of the LDH brucite-like inorganic structure could contribute to oil palm growth by supplying zinc and/or Al in shoots and roots 9. In this report, we discuss the synthesis and physico-chemical properties of DZALDH. In addition, its phytotoxicity studies on oil palm seedling in comparison with hexaconazole-Zn/Al LDH (HZALDH) and several commercial formulations will be presented. Our work on the synthesis and physico-chemical properties of hexaconazole-Zn/Al-LDH (HZALDH) was previous reported elsewhere 10.

Materials and methods



Materials

Zinc and aluminium nitrate hexahydrate were purchased from Sigma Aldrich. Dazomet with 98 % purity was obtained from Changzhou, China. Deionised water was used throughout all the experiments.

Method

Synthesis of Zn/Al-NO3 - LDH

The zinc nitrate and aluminium nitrate salts with a ratio of 4:1 were dissolved into a beaker containing 250 mL deionised water. After both salts were completely dissolved, 2M sodium hydroxide (NaOH) was added slowly to the solution under vigorous stirring supplied with nitrogen gas. After pH 7-7.5 was achieved, the suspension was agitated for 18 hours. The suspension was then centrifuged and washed three times with deionised water. The sample was then labeled as ZALDH after it was dried for two days in an oven.

Synthesis of fungicides micelle

Sodium dodecylbenzenesulfonate (SDBS), as a surfactant was mixed with the fungicide, dazomet to produce dazomet micelle. Dazomet micelle was obtained by mixing 0.4 M dazomet into 100


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mL acetone solution and was mixed with 1.226 g SDBS and dispersed into 250 mL deionised water. The solution then was left stirring at 40 - 45 °C to allow the acetone to evaporate completely.

Synthesis of fungicides – LDH nanocomposites via the ion exchanged method

The prepared 150 mL of 0.4 M dazomet micelle was mixed with 0.5 g ZALDH and left stirred at 75 °C for three days. Then, the sample was washed two times with deionised water and rinsed once with acetone before drying in an oven at 70 °C. The fungicide-LDH obtained was labeled as DZALDH nanocomposite.

Characterisation

X-ray diffraction (XRD) pattern was obtained using a diffractometer (Shidmadzu) with CuKα radiation at 40 kV and 30 mA. Infrared spectra were obtained by an attenuated total reflection (ATR) technique using a Perkin-Elmer 1725X spectrophotometer. Thermogravimetric analysis (TGA/DTG) was recorded using a Mettler Toledo instrument in the range of 25 – 1000 °C, at a heating rate of 10 oC/min with 50 mL/min nitrogen flow rate. In order to examine the surface morphology of the nanocomposite, the sample was scanned under a field emission scanning electron microscope (FESEM), JEOL JSM – 6400 model.



Loading percentage of fungicides in DZALDH nanocomposite

Approximately 10 mg of DZALDH was dissolved into 5 mL of 1.0 M hydrochloric acid, diluted up to 20 mL with acetone. The acid was used in order to dissolve the layered double hydroxide so that the fungicide can be released completely from the nanolayer. An ultraviolet – visible (uv-vis) spectrophotometer was used to determine the percentage loading of dazomet at a maximum wavelength of dazomet (max) at 281 nm.

In vivo phytotoxicity evaluation of the nanocomposites

Nursery study and experimental design

The nursery study was conducted at the nursery of Malaysian Palm Oil Board (MPOB), Section 15, Bandar Baru Bangi, Selangor, Malaysia. One week old germinated oil palm seeds were obtained from MPOB Research Station Kluang, Johor. The experiment involved eight treatments with thirty germinated seeds for each treatment. The treatments used in this study are presented in Table 1. Each germinated seed was put into a polybag (18 x 12 inch) containing top soil, organic and sand with a ratio of 3:2:1. The germinated seeds were arranged in completely randomized block design (CRBD) and the seeds were watered daily to maintain the growth development as previously discussed elsewhere 11.


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Table 1. List of treatments conducted on germinated oil palm seeds

Treatment label

Treatment agent for the germinated seeds

T1

Tap water

T2

Hexaconazole-based commercially available fungicide (HC)

T3

Hexaconazole

T4

Dazomet

T5

Dazomet-based commercially available fungicide (DC)

T6

Zinc/aluminium-LDH (ZALDH)

T7

Hexaconazole-based ZALDH nanodelivery system (HZALDH)

T8

Dazomet-based ZALDH nanodelivery system (DZALDH)

Preparation of treatments solutions

The treatment solutions comprising HC, hexaconazole, dazomet, DC, ZALDH, HZALDH as previously described elsewhere

10

and DZALDH nanocomposite, were prepared in an acetone -

deionised water solution with a ratio of 1:10 at 5000 mg/L 12. Each germinated seed was sprayed first with the treatment solution separately, before it was placed and grown in a polybag. The treatments were applied monthly and NPK green fertilizer were applied according to the standard procedures by the Malaysian Palm Oil Board (MPOB) 13 up to three months period.



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Sampling and data measurement of toxic effects

The growth of germinated seeds were evaluated based on several parameters; height of seedling, root length, leaf width, wet and dry weight 14. The germinated seeds were harvested at 4, 8 and 16 weeks. At week 16, the photosynthesis and transpiration rate were measured using the CIRAS-3 portable photosynthesis system (PP Systems, USA).The photosynthetic chamber provided a leaf area of 4.5 cm2, leaf temperature of 25 °C, relative humidity of 70 % and 390 μmol mol-1 CO2 concentration. Soil plant analysis development, SPAD-502 (Minolta) was used to measure the chlorophyll content of leaves. The degree of toxicity effect also was determined using the severity of foliar system formula as given in Equation 1 15 in indicating the leaf dessication.

Leaf dessication (%) = [(a x 1) + (b x 0.5)] x100/c

Equation 1

where a is the number of desiccated (browned/wilted) leaves, 1 is the index for desiccated leaves, b is number of yellowing leaves, 0.5 is index for yellowing leaves and c is the total number of leaves

Statistical analysis


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All treatments were conducted with three replicates and the results were analyzed using a Minitab 16 software (USA) and presented as mean ± SD (standard deviation). The differences between the treatments and parameters used were compared using the analysis of variance (ANOVA), followed by Tukey’s test in determining the significance difference between treatments, where p < 0.05.

Results and discussion

Powder X-Ray diffraction

Figure 1. PXRD patterns of pure dazomet (A), ZALDH (B), DZALDH nanocomposite (C) and the slow scan of DZALDH nanocomposite (D).

Based on the XRD patterns shown in Figure 1 (A), the intercalation of dazomet into the ZALDH interlayers had taken place to form a host-guest intercalation complex, DZALDH nanocomposite



which was accomplished by indirect, ion-exchanged method. The intercalation of 32 % dazomet into ZALDH has resulted in the increased of the basal spacing of ZALDH from 8.7 to 29.65 Å. In order to get the pure phase DZALDH nanocomposite, the concentration of dazomet micelle was varied from 0.05 to 0.6 M. Finally, the preparation using 0.05 M dazomet micelle has resulted in pure phase nanocomposite with good, 2-dimensional (2D) layered crystalline nanostructure. This sample was then subsequently used for further characterizations and studies.

As shown in Figure 1 (D) the slow scan of PXRD shows that DZALDH has well-ordered 2D nanolayered structure with 8 reflections at 28.69, 14.44, 9.65, 7.16, 5.68, 4.76, 4.06 and 3.55 Ǻ. These reflections were due to a well-ordered 2D inorganic nano-layered structure of DZALDH corresponding to the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th reflection, respectively. Based on these 8 basal spacing values, the average was calculated to be 28.62 Å. Subsequently, the value was used to determine the plausible arrangements of dazomet moiety together with sodium dodecylbenzene sulfonate (SDBS) and water in the ZALDH interlayers.


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Spatial orientation of the guests molecules in the ZALDH interlayers.

A

C

B

Carbon

Figure

2.

Nitrogen

Three-dimensional

(3D)

Sulphur

molecular

Sodium

structure

Hydrogen

of

dazomet

Oxygen

(A),

sodium

dodecylbenzenesulfonate (B) and plausible spatial orientation of dazomet molecule together with sodium dodecylbenzene sulfonate (SDBS) and water in the 2D, ZALDH interlayers.

As mentioned earlier, using XRD pattern the average basal spacing for DZALDH nanocomposite was found to be 28.62 Å. After subtracting the thickness of ZALDH, which is 4.8 Å, a value of 23.82 Å was obtained. This can be allocated as the space occupied by dazomet together with



sodium dodecylbenzene sulfonate (SDBS) and water in the ZALDH interlayers. The molecular size with 3D structure of dazomet and SDBS was calculated using a Pymol Software as shown in Figure 2 (A) and (B). The size of dazomet and SDBS calculated based on the x, y and z axes were found to be 7x4x3 Å and 23x5x4 Å, respectively. As a result, the proposed plausible spatial orientation of dazomet and SDBS together with water molecules in ZALDH interlayers is as shown in Figure 2 (C).

Surface morphology

Figure 3. Field emission scanning electron micrographs of DZALDH nanocomposite at magnifications of 100,000x.

The surface morphology of DZALDH nanocomposite was observed under a field emission scanning electron microscope at 100 000x magnifications as shown in Figure 3. The DZALDH nanocomposite showed agglomerated, granular structure, which has similar morphology to the sample obtained by previous study 16.


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Infrared spectroscopy

Figure 4. FTIR spectra of pure dazomet (A), ZALDH (B) and DZALDH (C).

Figure 4 shows the FTIR spectra of pure dazomet, ZALDH and DZALDH. Based on the FTIR spectrum of pure dazomet (Figure 4A), the vibrational modes of C-H stretching and C=S (thiocarbonyl) functional groups are seen at 2856 and 1176 cm-1, respectively 17. For the ZALDH (Figure 4B), a broad band at 3440 cm-1 was observed which indicating the presence of O-H in nanolayers. The nitrate anion and OH bending vibration for water molecules also were observed as indicated by bands observed at 1378 and 1638 cm-1.

As a result of the intercalation of dazomet into the ZALDH interlayers, some characteristic bands due to the intercalation episode were observed (Figure 4C) and this was supported by the presence of dazomet intercalated in between of the 2D ZALDH nanolayers. Two bands at 2956 and 1634 cm-1 were associated with the functional groups in dazomet chemical structure, since the vibrations were referring to C-H stretching and C-N stretching vibrations. The interaction



between the LDH and sulfonate ions also can be seen as indicated by the presence of a band at 689 cm-1.

Thermal stability

A

B


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C

Figure 5. TGA/DTG thermograms of pure dazomet (A), ZALDH (B) and DZALDH nanocomposite (C).

Figure 5 and Table 2 show the thermal properties of pure dazomet, ZALDH and DZALDH nanocomposites. The thermogram in Figure 5 (A) shows that the dazomet was 100 % thermally decomposed at 193 oC. The four thermal events at 108, 244, 310 and 510 oC occurred as shown in Figure 5 (B) were referring to the weight loss in ZALDH with 7.0, 16.7, 4.0 and 5.5 %, respectively. The first and second weight losses were related to the removal and the strong bond of water molecules while the third and fourth weight losses were due to the hydroxyl group that was released from the nanolayers and the elimination of interlayer anions, respectively.

Figure 5 (C) represents four major thermal degradations in DZALDH, which are 9.4, 6.9, 24.0 10.9 % weight losses at 109, 230, 454, 879 °C, respectively. The first step weight loss was due to the elimination of surface physisorbed water molecules, followed by the dehydroxylation of the hydroxyl layer. The decomposition of dazomet was occurred at 454 °C with 24.0 % mass reduction in DZALDH nanocomposites, thus proving that the thermal stability of DZALDH had



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increased compared to dazomet alone (Figure 5A). It was also observed that at 879 oC, ZnAl2O4 phase was formed 18.

Table 2. The thermal decomposition of pure dazomet, ZALDH and DZALDH nanocomposite.

Sample name

Trange (oC )

Tmax (oC )

Δm (mg)

Weight loss (%)

Dazomet

85-316

193

6.72

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

33-178

109

0.52

9.4

178-316

230

0.40

6.9

318-644

454

2.01

24.0

654-966

879

0.92

11.0

ZALDH

DZALDH


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Phytotoxicity (in vivo) studies of different treatments towards oil palm seed germination

The growth parameters such as height, root length, leaf width, wet weight and dry weight of seedlings were recorded every month. In order to obtain better insights into the actual seed germination and comparison, the results were collected at early stage (Week 4), middle stage (Week 8) and the final stage (Week 16) after the planting process. At the final stage, the physiological parameters namely the SPAD chlorophyll value, transpiration and photosynthesis rate and pathological data such as leaf dessication and dead seedlings were recorded and analysed. Effect of the DZALDH nanocomposite on oil palm seed germination

The result of the growth of the oil palm seeds subjected to different treatments are presented in Figure 6 (A to E) with the error bar showing the standard deviation. Figure 6 (A) shows the height of seedlings which shows no much significant difference between the treatments at early stage (week 4). All treatments show slightly increased in the height when the seedlings were reached Week 8, except for HC treatment, (T2). When the treatment time was increased to Week 16, the ZALDH (T6) and DZALDH (T8) did not show any significant difference from the control (T1). Treatments of hexaconazole (T3), dazomet (T4), DC (T5) and HZALDH (T7) show no significant difference among their seedlings height, where all of these five treatments gave almost the same, which was around 50 % compared to the control (T1). However, the HC treatment (T2) was significantly affected, as the maximum height observed was only 4.6 cm, which is 6 folds lower than the control (T1). This observation shows that HC treatment (T2) resulted in no much



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difference in term of height throughout the planting time. This finding indicates that HC treatment has suppressed the growth development of the seedlings very much.

The histogram in Figure 6 (B) shows the relation between the root length of the seedlings against treatments. Based on the data presented for all three weeks, the root length for most of the treatments increased with the treatment period, indicating that all the seedlings growth did not show much significant difference except for HC (T2) and ZALDH treatments (T6). As planting time was increased to Week 8, seedlings treated with ZALDH (T6) achieved the highest root length, followed by the seedling treated with hexaconazole (T3) and the control (T1). This trend shows that the root length of seedlings under treatment with dazomet (T4) and DC (T5) are always 50 % lower than the three treatments, T6, T3 and T1. At Week 16, it was clearly observed that the ZALDH (T6) and HC treatment (T2) has the highest and lowest root length, which was 30.5 and 0.8 cm, respectively. The root length of HC (T2) was significantly inhibited, showing that this treatment delayed the growth development and contributed to negative effect on the seedling growth.

Not much significant difference was observed at the early stage of treatment due to the seedlings were still young and the leaves were just started to grow (Figure 6C)

14

.Starting from Week 8,

with the exception of treatments with hexaconazole (T3) and dazomet (T4), a steady growth of leaves and seedlings was observed for the samples treated under five treatments; control (T1), DC (T5), ZALDH (T6), HZALDH (T7) and DZALDH (T8). As the treatment period was increased to the final stage, a more significant difference was observed between all the treatments. The highest


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leaf width was achieved for seedlings treated with DZALDH (T8) with a value of 52.8 cm2, followed by the control (T1) with a value of 51.6 cm2. This indicates that the growth of the seedlings in DZALDH (T8) was significantly better than the control (T1). The seedlings treated with HZALDH (T7) also produced healthy leaves with the leaf width of 48.8 cm2. However, treatments with bare hexaconazole (T3) and dazomet (T4) had resulted in smaller leaves area compared to other treatments, with the leaf area of only 15.9 and 21. 5 cm2, respectively. There was no leaf growth observed for seedlings with HC treatment (T2) from Week 4 to Week 16, which revealed that HC has totally retarded the seedlings growth since at the early stage.

Based on Figure 6 (D), it was seen that there are some significant differences among the eight treatments with minor difference in value during the early stage of recorded wet weight. The wet weight achieved by all the treatments were in the range of 3.24 to 3.63 g, except for dazomet (T4) which is only 2.72 g. At Week 8, only the control (T1) showed significant result by having different significant letter compared to rest of the treatments. After 16 weeks, the wet weight of all the treatments increased to almost double compared to Week 4. This result is slightly differ compared to the other parameters, as HC treatment (T2) which always has the least value for all the other parameters. However, in this case, the wet weight of HC treatment (T2) was the second highest with 9.64 g compared to the control, which is 10.52 g. The increasing of wet weight under HC treatment (T2) until week 16 can be correlated with the cytokinin, carbohydrates and moisture contents in seeds. The active ingredient in HC, which is triazole compound has ability to enhance the carbohydrates synthesis 19, cytokinin production and to partially closing the stomata by the synthesis of more abscisic acid (ABA) which lead to high moisture content in the seeds resulting in the contribution to higher value of wet weight

20.


The seeds treated with HC were


found to have physical dormancy due to their hard and dense endocarps, which blocking the oxygen absorption for embryo development. With high moisture content in seeds, these will block the intercellular spaces and imbibe the embryo, resulting the growth of oil palm seeds to be unconsistent with the low germination rate 21. The seedlings under treatments of HZALDH (T7) and DZALDH (T8) shows higher wet weight, with value of 8.86 and 7.57g, respectively compared to the other treatments (T3 to T6). This indicated that wet weight value of treatments (T3 to T6) were proportionally correlated with dry weight, in agreement with the previous finding 22.

Figure 6 (E) shows the dry weight for all the eight treatments which shows significant difference between the groups. At early stage, the highest dry weight was observed under dazomet treatment (T4), then followed by DZALDH (T8), control (T1) and ZALDH (T2) with 0.22, 0.20 and 0.18 g for both T1 and T2, respectively. At this stage, HC treatment (T2) has the lowest dry weight with a value of 0.09 g. After 8 weeks planting, dry weight for most treatments except the control (T1) and HC (T2) did not show much difference with a value in the range of 0.2 – 0.4 g. This trend indicates that dry weight for all treatments were increased up to the final stage of planting. At Week 16, the dry weight of the control (T1) reached the highest level with a value of 2.3 g, increased 4 folds to the dazomet (T4), with the lowest value of 0.48 g. Based on the results, it was found that the dry weight of ZALDH (T6), HZALDH (T7) and DZALDH (T8) were significantly higher than the one using the commercially available fungicides (T2 to T5). This clearly indicates that the healthy seedlings have resulted to higher dry weight of the seeds germination 23.


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Physiological parameters

The photosynthesis is a physiological process where plants convert the light energy, water and carbon dioxide to form glucose (food). The environmental factors such as concentration of carbon dioxide in air, light, humidity and internal factors like chlorophyll content (SPAD value) and stomatal distribution (stomatal conductance) play very important roles in determining the rate of photosynthesis 24.

Table 3 shows, most of the treatments showing that the increase of the stomatal conductance contribute to the high photosynthetic and transpiration rate 25. The HC treatment (T2) shows zero value since the seedlings under this treatment has very small leaf, thus the chlorophyll content cannot be detected. However, in the case of commercial DC (T5), the highest stomatal conductance was achieved at 202.88 molm-2s-1, but both the photosynthetic rate and SPAD value recorded were the second lowest with a value of 5.12 μmol CO2 m-2s-1 and 26.80, respectively. This result is parallel with the previous study which showed that SPAD value was linearly correlated with photosynthesis rate

26.

The ZALDH treatment (T6) gave the highest value of

SPAD and photosynthetic rate among all the treatments which indicates that ZALDH promotes the oil palm seedlings growth. The results also suggest that both HZALDH (T7) and DZALDH (T8) treatments promote the production of glucose which could increase their photosynthetic rate.



The leaf dessication

The leaf desiccation is the common foliar symptoms of oil palm seedlings, where it happens at the leaf tip and then become yellowing and finally drying

27.

During the 12 weeks treatment,

some yellowing leaves were observed only in dazomet (T4) and DC (T5) treatments. After 15 weeks, the symptoms also appeared in all the treatments, and the dessication symptoms of dazomet (T4) and DC (T5) were quite severe, where the symptoms were visible for all the seedlings in the treatments.

Based on Table 4, the dazomet treatment (T4) shows the highest dessication symptom with 83.33 %, followed by treatment with DC (T5), 75.3 %. Both treatments were significantly different with the other treatments. The HZALDH treatment (T7) was similar to the control (T1) as both treatments did not show any symptoms, indicating that the treatment applied did not give any negative effect towards the growth. For the ZALDH (T6) and DZALDH (T8) treatments, they show low severity with 1.28 and 0.60 %, respectively. Interestingly, most of dessication symptoms were observed on the seedlings treated with the commercial fungicides, while those treated with ZALDH-fungicide nanocomposites; HZALDH (T7) and DZALDH (T8) resulted in low dessication symptoms

28.

These findings suggest that fungicides in combination with LDH

via intercalation process for the formation of their host-guest complexes has delayed the infection and lowered the dessication percentage of oil palm seedlings.


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Percentage of dead seedlings

The percentage of dead seedlings in four treatments; HC (T2), hexaconazole (T3), dazomet (T4) and DC (T5) is shown in Table 4, while all the seedlings subjected to other treatments were still alive. The dead seedlings were seen started from Week 13 until Week 16. Similar to the leaf dessication percentage, the dazomet treatment (T4) was significantly affected the seedlings growth and achieved the highest dead seedlings with 71.43 % and followed by the DC treatment (T5) with 57.14 %. We also observed that some seedlings in HC (T2) and hexaconazole (T3) treatments were totally dead. However, there was no statistical difference between both treatments with the seedlings that are still alive since the number of seedlings affected was only 4.76 and 3.57 %, respectively. This statistical test confirmed that dazomet (T4) and hexaconazole T5) treatments had contributed to significant toxicity towards the seedling growth, while the rest of the treatments were insignificant, as there was no seedlings death 29. The typical of dead and alive oil palm seedlings were divided in two categories, as shown in Figures 7 and 8.

The results obtained in this study has shown that the elements, namely zinc and aluminium which are two main composition of the inorganic 2D brucite-like layered structure of the layered double hydroxide of the nanocomposites, DZALDH and HZALDH has promoting the growth of oil palm seedlings better than the commercially available counterparts, dazomet and hexaconazole fungicide alone. The phytotoxicity results also convinced that the combination of fungicide and layered double hydroxide has very good potential to be used as supplements for the



oil palm seedlings at the early stage of treatment compared to the commercially available counterparts, which will be only applied when the oil palms were infected with the disease.

A

B


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C

D



E

Figure 6. The effect of various treatments on seedling height (A), root length (B), leaf width (C), wet weight (D) and dry weight (E) of oil palm seedlings at 4, 8 and 16 weeks ; Note: means that do not share a letter are significantly different, where p

Aluminum-Layered Double Hydroxide

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