Pesticidal activity of nanostructured metal oxides for generation of

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Agricultural and Environmental Chemistry

Pesticidal activity of nanostructured metal oxides for generation of alternative pesticide formulations Haytham Ayoub, Mohamed Khairy, Salaheldeen Elsaid, Farouk Abdallah Rashwan, and Hanan F. Abdel-Hafezb J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01600 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018

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Journal of Agricultural and Food Chemistry

Pesticidal activity of nanostructured metal oxides for generation of alternative pesticide formulations

Haytham A. Ayoub,a,b Mohamed Khairy,a* Salaheldeen Elsaid,c Farouk A. Rashwan,a Hanan F. Abdel-Hafezb

a

Chemistry Department, Faculty of Science, Sohag University, Sohag, 82524, Egypt.

b

Plant Protection Research Institute, A. R. C., Dokki, Giza, 12311, Egypt.

c

Zoology Department, Faculty of Science, Sohag University, Sohag, 82524, Egypt.

Email: [email protected] Tel: +(02)01092099116

1 ACS Paragon Plus Environment


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ABSTRACT Herein, nanostructured metal oxides of essential soil nutrient elements (i. e. CuO and CaO) with definite shape and size were simply synthesized and their pesticidal activities against cotton leafworm (Spodoptera littoralis) were explored for the first time. These metal oxide nanostructures represented novel economic and eco-friendly nano-pesticides for sustainable plant protection and might boost the nutrient content of soil. The results showed that CuO NPs and CaO NPs exhibited potential entomotoxic effects against S. littoralis. Interestingly, CuO NPs exhibited fast entomotoxic effect with LC50 = 232.75 mg/L after 3 days, while CaO NPs showed a slow entomotoxic effect with LC50 = 129.03 mg/L after 11 days of posttreatments. The difference in the pesticidal activity of the metal oxides is related to their physical characteristics and interfacial surfaces upon insect mid-gut and cuticle layer of insect body wall. Thus, nano-engineered metal oxides might be utilized to generate an alternative and cost effective pesticide formulation in the next future.

Keywords: Inorganic pesticides, Metal oxides nanostructures, Nanocides, Plant Protection, Spodoptera littoralis


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1. INTRODUCTION In modern agriculture, organic pesticides are playing a significant role for plant protection and production due to their powerful biological activity to control the pests.1 Large agronomic and economic benefits have been achieved through the enhancement of plant production to meet the continuous increases in global population and therefore an increased need for food production demand.2,3 Over than 2.8 million tons of organic pesticides are used annually over the world and about 0.5 million tons (as 12.5 billion dollar industry) in United States alone.4 However, it has been estimated that only 0.1% of applied organic pesticides reach the target pests, leaving their bulk (99.9%) to cause deleterious impact for the environment including water supplier and soil.5 As a consequence; 25 million agricultural workers worldwide experience unintentional pesticide poisonings each year.4 Therefore, several agriculture and environmental strategies have been developed recently to reduce the agrochemicals as well as control the harmful pests as follows; (i) Genetically engineered crops designed to produce their own insecticides or exhibit resistance to broad spectrum herbicide products or pests. (ii) Integrated pest management (IPM) systems which discourage the development of pest populations. Although all these strategies found good impacts in plant production, still more environment and public health effects need more scientific and technological considerations. The application of nanotechnology in the area of plant and agriculture sciences has attracted great attention in last decades.6,7,8 The functional nanomaterials might offer many possible interventions to mitigate the environmental and human risks and consequently increases profitability, yields, and sustainability within the agricultural and food industries. Nanomaterials have been recently utilized in insecticides,9 fungicides,10 and fertilizers.6,8 However, internalization and subsequent toxicity of nanoparticles in vitro microenvironments depend on their chemical composition, particle size, and surface characteristics. Therefore,



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great efforts have been devoted to control the nanomaterial characteristics to meet with diverse applications.11 In our previous work, silica nanoparticles (SiO2-NPs) have been successfully applied as alternative pesticides against cotton leaf worm.12 It was found that, the particle size is a key factor that control the pesticidal activity of SiO2-NPs.12 Although, silica-based material is considered as a major continent of agriculture soil, it caused a significant influence on Bt-transgenic and non-transgenic shoot biomass. They promote the transport of Mg in Bt-transgenic xylem sap and Fe in both the xylem sap of non-transgenic and Bt-transgenic. The bioaccumulation of SiO2-NPs in plants showed likely harmful effects which impacted directly on food crops and human health. For example, The Si contents increased up to 2000 mg/L in both shoots and roots of Bt-transgenic cotton.13 Copper is an essential micronutrient for plant growth, widely distributed in plant tissues and involved in many physiological processes.14 it is available in two oxidation states Cu+ and Cu2+, thus it act as reducing or oxidizing agent in biochemical reactions. This behavior makes Cu also potentially toxic because copper ions may catalyze the production of free radicals.15,16 It was found that, the toxicity of copper oxide nanoparticles (CuO-NPs ) depends on plant species, growth conditions, exposure time, concentration and size of CuO-NPs.17 The CuO-NPs caused salient toxic effects on seedling, length, morphology, photosynthetic efficiency, and carbon dioxide fixation in rice.18 CuO-NPs also postulated to inhibit the growth and development of transgenic and conventional cotton plants, 19 However, CuO-NPs significantly enhanced the expression of the Bt-toxin protein in the leaves and roots of Bttransgenic cotton. Therefore, the uses of CuO-NPs with adjustable concentration might offer a promising technology for improving pest resistance of transgenic insecticide crops.19,20 Calcium is also an essential plant macronutrient that plays structural role in the cell wall, membranes and regulates plant growth and development.21 Calcium availability is strongly limited the structural integrity of stems that hold flowers and fruit, as well as the quality of


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the produced fruit. Furthermore, calcium enhances the disease resistance in plants against bacterial and viral diseases.22 Calcium oxide nanoparticles (CaO-NPs) have been used for the correction of calcium deficiency in groundnut.23 Foliar application of CaO-NPs significantly increased germination and growth of groundnut; compared to their bulk counterparts calcium oxide nanoparticles transmit into the phloem.23 In the present manuscript, we aim to take the advantage of nanostructured engineering surfaces contributed in soil nutrients and explore the impacts of metal oxide nanostructures (CuO and CaO) for development of an alternative and efficient pesticides against cotton leafworm which commonly distributed in Mediterranean area.11,12 2. MATERIALS AND METHOD 2.1.

Chemicals

All chemicals were of the highest analytical grades used as received without further purification. Copper acetate monohydrate (Cu(CH3COO)2.H2O; 98%), Citric acid (99%), ammonium hydroxide (NH4OH, 28%), sodium hydroxide (NaOH), calcium chloride (CaCl2; 97%) were purchased from Sigma-Aldrich Co. LtD, Germany and imported by International Egyptian Centre. The aqueous solutions were freshly prepared using bi-distilled water with resistivity > 18.2 MΩ/cm at 25°C.

2.2.

Synthesis of CuO with flower-like morphology

Direct precipitation method was used for synthesis of copper oxide nanostructures with flower-like morphology. In typical synthesis, 0.1 mol/L (50 mL) of Cu(CH3COO)2.H2O was prepared in 250 mL beaker, the Cu(CH3COO)2 solution was heated and maintained under magnetic stirring at 60oC. Then, ammonia solution (28%) was added dropwise until the appearance of pale blue colour of [Cu(NH3)6]2+, excess amount of ammonia solution was introduced to dissolve the formed complex. A solution of 0.1 mol/L of NaOH was added 5 ACS Paragon Plus Environment


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dropwise until formation of black precipitate of CuO. Finally, the mixture was left to cool, filtered and dried at 60oC (Supporting information; Fig. S1).

2.3.

Synthesis of CaO with hexagonal sheet-like morphology

Hexagonal Calcium oxide sheets were synthesised via sol-gel process in the presence of citric acid as capping agent. In typical synthesis, 1.0 g of citric acid was dissolved in 50 mL of distilled water and the solution maintained under magnetic stirring at 100oC for 15 min. A 50 mL of 0.2 mol/L CaCl2 was introduced dropwise to citric acid solution followed by ammonia solution (28%) until a white precipitate was formed. The precipitate was filtrated, washed several times by ethanol/water mixture and dried at 60oC overnight. Hexagonal CaO sheets were obtained after thermal treatment at 350oC for 3 hours (Fig. S2).

2.4.

Characterization of metal oxide nanostructures

Wide-angle powder X-ray diffraction (XRD) patterns were measured by using X-ray diffractometer (Model FW 1700 series, Philips, Netherlands) with monochromatic CuKα radiation (λ=1.54 Å), employing a scanning rate of 0.060 min-1. The diffraction data were analysed using PDF-2 Release 2009. Fourier transform-infrared (FTIR) spectroscopy of the CuO and CaO samples were recorded using Bruker Alpha FTIR instrument.

The textural surface properties of metal oxides were determined by N2 adsorption/desorption isotherms at 73 K with a NOVA 3200 apparatus, USA. The specific surface area (SBET) was calculated using the Brunauer–Emmett–Teller (BET) method with multipoint adsorption data from the linear segment of the N2 adsorption isotherm. The pore size distribution was determined from the analysis of desorption branch of isotherm using Barrett-Joyner-Halenda (BJH) method.


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The morphology of metal oxides samples were investigated using scanning electron microscopy (SEM, JEOL model 5400 LV). Metal oxide powders were grinded and fixed onto a specimen stub using double-sided carbon tape. To obtain high-resolution micrographs, a 10 nm Pt film was coated on the metal oxide using anion sputtering (Hitachi E-1030) at room temperature. The SEM was operated at 15 kV to obtain high-resolution SEM images.

Further the morphology of metal oxide also investigated using transmission electron microscopy (TEM) (JEOL-JEM model 2100). TEM was conducted at an acceleration voltage of 200 kV. The TEM images were recorded using a CCD camera. Metal oxides samples were dispersed in ethanol solution using an ultrasonic bath, and then dropped on a copper grid. Prior to inserting the samples into the TEM column, the grid was vacuum dried for 20 min.

2.5. Insects

Laboratory strain of cotton leaf worm (Spodoptera littorals) was cultured on leaves of the castor oil plant (Ricinus communis L.). The strain was reared under constant laboratory conditions in incubator at 25 ± 2°C and 65 ± 5% relative humidity with 8 hour light: 16 hour darkness photoperiod in Plant Protection Research Institute, A. R. C., Dokki –Giza, Egypt. The second instar larval stage of the insect was used in the insecticidal bioassay.

2.6. Bioassays

The pesticidal activity of metal oxide nanostructures and methomyl pesticide were evaluated via leaf-dip bioassay method under recommended experimental conditions. Briefly, different concentrations of CuO and CaO nanostructures (150, 300, 450 and 600 mgl/L) were dispersed in distilled water containing 0.1% Triton X100 with measurable pH of 6.0 and 8.5, respectively.24 Then, leaf discs of castor oil plant with the same sizes were cleaned and dipped into the dispersed metal oxides solutions. For comparison, methomyl was utilized and 7 ACS Paragon Plus Environment


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test under the same experimental conditions. The treated and untreated (control) leaves were introduced into drying container containing equal numbers of 2nd instar larvae of S. littoralis. The containers were maintained and covered with muslin cloth to allow aeration as shown in Scheme 1. To control the bioassay experiment, we have repeated the experiments three times. The mortality percentages were calculated by using Equation (1), and natural mortality was corrected by using Abbott’s formula (Equation 2) as follows;

Mortality percentage =

×100

(1)

Corrected percentages of mortality = ⦋1 −

⦌ ×100

(2)

where T is treated larvae and C is control larvae.

2.7.

Histological study

The castor oil leaves were immersed into LC50 of CuO and CaO nanostructure samples. The treated leaves were introduced to 2nd instar larvaes of S. littoralis via feeding bioassay method for three days. Then, the survived larvaes of S. littoralis were collected and allowed to grow on untreated/normal leaves till reach to 6th instar larvae. The insects were washed in ethanol (70%) and then dehydrated in according series (70% ˗ 100%) of ethanol. Infiltration embedding of samples was carried out in paraffin wax. Sections (5.0 µm) were stained with Haematoxylin-Eosin (H-E). The morphological alterations of the insect mid-gut and cuticle abrasion were analysed by microscopic examination and compared to the tissues taken from the control.


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2.8.

Biochemical analysis

The castor oil leaves were immersed in LC50 of CuO and CaO nanostructures. The treated leaves have been introduced to 2nd instar larvaes of S. littoralis via feeding bioassay method for three days. Then, the survived larvaes of S. littoralis were collected and allowed to grow on untreated/normal leaves till reach to 6th instar larvae. The insects were homogenized for biochemical analyses in a chilled glass Teflon tissue homogenizer (ST-2 Mechanic-Preczyina, Poland). The supernatant was kept in a deep freezer at -20 ºC for further biochemical analyses. Carbohydrates, proteins, lipids, phenol oxidase and chitinase activity were estimated. The absorbance measurement of coloured substances or metabolic compounds was performed by using double beam UV/Vis spectrophotometer (Spectronic 1201, Milton Roy Co., USA).

2.9.

Statistical analysis

The median lethal concentration (LC50) and the median lethal time (LT50) values were calculated by using probit analysis program.25 Prior analysis; all natural mortalities were corrected by using Abbott’s formula as shown in Equation (2). The data are presented as the mean ± standard error (SE). Statistical analyses were performed using one-way analysis of variance (ANOVA) and multiple comparisons tukey test using SPSS 10.1 software. A confidence interval of 95% (p < 0.05) was considered significant in all cases.



3.

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RESULTS AND DISCUSSION

3.1. Characterization of metal oxide nanostructures

Crystal structure, functionality and surface textural characteristics of CuO and CaO samples were carried out using wide angle X-ray diffraction, fourier transform infrared spectra (FTIR) and N2 adsorption/desorption isotherm (Fig. 1(a-d)). The appearance of sharp peaks in the diffraction patterns of CuO and CaO indicates the formation of highly crystalline materials. CuO exhibited typical diffraction peaks of monoclinic Tenorite CuO phase (JCPDS card no. 02-1040).26 While CaO revealed the characteristic diffraction peaks of cubic-pure CaO (JCPDS card no. 82-1691). Figure 1b showed the FTIR analysis of CuO and CaO samples. They represented strong absorption bands below 1000 cm-1, which revealed the formation of the CuO and CaO crystals. The characteristic absorption peaks at 477.0 and 594.82 cm-1 are due to the vibrational modes of Cu–O bond. 26While the band at 551 and 659 assigned to vibrational modes of Ca-O bond. The sharp absorption bands near 858, 1403 and 1546 cm-1 assigned to vibrational modes of C-O bond in citrate groups. This evidence indicated the formation of citrate groups attached to the calcium atoms.27, 28

According to IUPAC classifications, CuO and CaO samples revealed type IV isotherm with pronounced H3 hysteresis loop which could characterize slit-like mesopore entrances (Fig. 1c). The specific surface areas of CuO and CaO were SBET = 40.54 and 23.27 m2/g, respectively. The CuO and CaO samples exhibited mesoporous network architectures with pore diameters of 3.38 and 3.8 nm, respectively (Fig. 1d). These mesoporous structure might be suitable for adsorption of biomolecules (proteins, fats and carbohydrates) of the insect exoskeleton. These features are very helpful to examine the entomotoxic effects related to the surface area.29

Fig. 1 10 ACS Paragon Plus Environment

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Figure 2 shows scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of CuO and CaO samples. The as-synthesized CuO sample is composed of aggregated nanoneedles with an average diameter of 200 - 300 nm arranged in hierarchical beautiful microflower-like structures with average size of 1.0 µm (Fig. 2a). The CaO sample exhibited polygonal particles with sharp edges with an average size 2.0 µm and thickness about 100 nm (Fig. 2b). Further, the TEM image clearly indicates that, the CuO microflower is composed of assembled nanoneedles with worm-like mesopores (Fig. 2) according to the distinct colour contrast of TEM image. The needle has a micrometer length and average diameter is about 300 nm. Interestingly, this hierarchical porous needle is originally formed through interconnected nanoparticles as shown in Fig. 2d. These CuO nanoparticles have average size of 20 nm. Figure 2(e, and f) showed TEM images of CaO sample, and it can be found that the CaO polygonal particles have mostly hexagonal morphology in the internal angle between adjacent well-faced edges of 120˚ and wormlike porous network. The hexagonal sheets are very thin and obviously rough with size about 1.0 µm.

Fig. 2

3.2.

Biological potency

3.2.1. Pesticidal activity of metal oxide nanostructures

CuO and CaO NPs exhibit low cost, biocompatible and available starting materials for commercialization. Interestingly, they have different mechanisms of antimicrobial action. CuO NPs cross the cell membrane and then damaging the vital enzymes, however, CaO NPs can generate a superoxide on the particles surface which damages the cell membrane and causing leakage of intracellular contents.30 The pesticidal activity of these metal oxides was investigated by utilizing leaf-dip bioassay protocol (feeding method) as shown in Scheme 1. 11 ACS Paragon Plus Environment


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The CuO and CaO NPs solutions of 150, 300, 450 and 600 mg/L were prepared in presence of 0.1% (V/V) Triton X-100 at 25 ± 2°C. The mortality was monitored after 3 days and 11 days post-treatment with CuO NPs and CaO NPs, respectively. In order to calculate the median lethal concentration LC50, the corrected mortality percentages were statistically estimated by using probit analysis program.25 The mortality percentages of CuO NPs and CaO NPs treated larvae showed positive correlation with metal oxides concentrations (Fig. 3, Table S1 and TableS2). The LC50 values for the metal oxides NPs against S. littoralis were calculated as recommend for pesticide formulations.24 Interestingly, CuO NPs exhibit fast entomotoxic effect with LC50 = 232.75 mg/L after 3 days. On the other hand, CaO show slow entomotoxic effect with LC50 = 129.03 mg/L after 11 days post-treatment. Despite CaO NPs takes long time, it exhibited lower LC50 value. Based on these results, CaO NPs is the best choice for slow control efficiency. Methomyl LC50 value was estimated and it was 434.49 mg/L after one day of exposer Methomyl is a broad-spectrum carbamate insecticide. It has been used in a wide range of agriculture products although it was considered as highly toxic organic pesticide for birds and mammals.31 The LT50 of CaO and CuO at 600 mg/L were 7.15 and 2.69 days, respectively. Thus, urgent control needs CuO NPs with relatively low eco-toxic effect compared to methomyl while CaO is more bio-save (Figs. S3 and S4). To investigate the effect of morphology and particle size on the pesticidal activity, rectangular CuO microstructure was synthesised (Fig. S5). Rectangular CuO microstructure (0.50 ×1.0 µm) was synthesised in alkaline media in presence of citric acid as a template. The FTIR and XRD confirmed the formation of highly crystalline CuO with monoclinic structure (JCPDS card no. 02-1040). The rectangular CuO showed also fast entomotoxic effect with LC50 = 205.63 mg/L after 3 days and LT50 was 2.13 days. These results confirmed that the morphology and particle size are significant key factors that control the pesticidal activity of metal oxides.11, 12


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Scheme 1

Fig. 3

3.2.2. Histological changes 3.2.2.1. Mid-gut The mid-gut is the main origin for digestion and absorption of ingested food.32 Insects mid-gut wall comprises of two muscular layers and an epithelial layer lining the lumen. The intestinal epithelium contains four types of cells; digestive, regenerative, endocrine, and goblet cells which are a characteristic feature of Lepidoptera.32 The most frequent cell type is the digestive epithelium cell which is responsible for enzyme production, digestion and absorption. Mid-gut epithelium is the prime target for many insecticides.33 Previously, we have found that, the pesticidal activity of silica NPs might be attributed to impairment of the digestive tract, integument overgrowth, dehydration and blockage of the respiratory system (spiracles and trachea).12,34 Further, the results showed that the pesticidal activity is mainly dominated with particle size with LC50 = 327.7 mg/L after eleven days post-treatment.34 Sorption and dissolution of silica NPs in aqueous medium generate reactive oxygen radicals that are responsible for this damage.34 In the mid-gut, silica NPs cleaves hemolytic (Si•, SiO•) or heterolytic (Si+, SiO-) due to the cracking of silicon-oxygen bond.33 Both CuO and CaO NPs might generate superoxide on particle surface that consequently causes the impairment of intestinal epithelium cells.30 As observed in Fig. 4(a - c), CuO and CaO NPs caused remarkable histopathological changes in the mid-gut epithelium.

The normal histological structure of the mid-gut that consists of tall columnar cells which contain large nuclei in the middle of the apical region interspersed apically with the goblet cells in-between and basally with regenerative cells. Intensive histopathological



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changes in term of losing the compact appearance of the muscular layer, exfoliation and vacuolization were observed for both CuO and CaO NPs treatments. Significantly, CuO NPs showed a complete disruption of the peritrophic membrane compared to CaO NPs. 32, 33, 34

3.2.2.2. Cuticle layer The cuticle consists of epicuticle, exocuticle, and endocuticle layers.36 The underlying epidermal cells are responsible for its production. These cells are metabolically active as their shape and number are changed during development.37 Basal lamina or basement membrane separates the body wall (cuticle and epidermis) from the hemolymph.36 Fig. 4 (d - f) showed the cuticle structure of both control and treated S. littoralis larvae. A normal histological structure of the body wall with intact layers was observed (Fig. 4d). The treated larvae suffered from cuticle abrasion; partially destroyed and slightly separated from the epidermal cells.38 CuO NPs caused greater damage to the insect exoskeleton through the damage of its cuticle water barrier mostly by abrasion. Further, reactive oxygen species of both CuO and CaO NPs create pore network on the cell membrane which in turn cause leakage of intracellular contents (i.e. lipids) and deterioration of the protective wax layer (Table S3).39 Thus, the larvae start to lose water from their bodies and die due to desiccation process as shown in the photographic images of control and treated insects (Fig. S6).40, 41

Fig. 4

3.2.3. Biochemical aspects Biochemical alterations of 6th instars larvae of S.littoralis treated with CuO and CaO NPs were estimated (Fig. 5).42-46 Carbohydrates, proteins and lipids are major components necessary for an organism development, growth and performance for various vital activities. As shown in the histological observations, the treated larvae showed a remarkable aberration 14 ACS Paragon Plus Environment

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in the epithelium lining the mid-gut. This damage is directly responsible for the poor digestion and absorption of ingested food. In this respect, there was a high significant decrease in carbohydrates and lipids of larvae in both CuO and CaO NPs treatments. The total proteins of CaO NPs treated larvaes showed no significant decrease compared to the control set. Interestingly, the total proteins of CuO NPs treated larvae decreased by one-third than that treated with CaO NPs (Fig. 5, Table S3 and Table S4).42- 44 Our results are in a good agreement with the previous studies that report the direct effect of CuO NPs on the metabolism of carbohydrates, lipids, and proteins of Galleria mellonella (Lepidoptera).35 Further, there is a direct relationship between the level of carbohydrates, lipids and proteins and the activity of chitinase enzyme, which is responsible for chitin synthesis.44 This finding is perfectly matched with our results. Both chlorfluazuron and flufenoxuron are insecticides that act as chitin synthesis inhibitors.47 The treated larvae of S.littoralis with these insecticides showed a drastic increase in the enzymatic activities of phenoloxidase and chitinase.48 On the other hand, treatment with either CuO or CaO NPs, up-regulated phenoloxidase activity significantly but not for chitinase in CaO NPs treatment. This finding matched well with the histological observation that shows an overgrowth of the cuticular layer resulting in its detachment from the underlying epidermis.38,

39

According to the

multiple comparisons (Tukey test), we can observe that the activity of chitinase enzyme is greatly affected by the changes in carbohydrates, proteins and lipids and that for all treatments. While phenol oxidase affected only by chitinase change. Phenol oxidase for CuO NPs treated sample affected by the change in proteins, lipids and chitinase change. Phenol oxidase and chitinase activity for CaO NPs treated sample didn’t affect and look like the control set. Based on these observations, the biochemical profile of CaO NPs did not differ greatly from the control set compared to CuO NPs that caused drastic biochemical alterations (Table S5). Thus, we are suggesting CuO and especially CaO NPs as eco-friendly



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insecticides other than the traditional organic pesticides such as methomyl, Chlorfluazuron and flufenoxuron.31, 49

Fig. 5

In summary, we have explored alternative pesticides for S. littoralis control based on metal oxides nanostructures derived from essential soil nutrient elements. Large scale production of CuO and CaO nanostructures has been synthesized via simple wet-chemical methods. The metal oxides nanoparticles showed an interesting pesticidal activity against S. littoralis with different pesticidal behavior. CuO NPs exhibited fast entomotoxic effect, while CaO NPs revealed slow entomotoxic effect. The difference in the pesticidal activity is related to their physical surface characteristics upon the insect mid-gut and the cuticle layer in the insect body wall. These interesting features indicated that the metal oxides can play a significant role for next generation of pesticide formulations.

Supporting Information Available: Synthesis steps of metal oxides, toxicity curves, photographic images of treated larvae, rectangular CuO data, descriptive analysis, ANOVA analysis and tukey test.


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4. REFERENCES (1)

Al-Degs, Y. S.; Al-Ghouti, M. A.; El-Sheikh, A. H. Simultaneous determination of pesticides at trace levels in water using multi walled carbon nanotubes as solid-phase extractant and multivariate calibration. J. Hazard. Mater. 2009, 169, 128–135.

(2)

Bhat, S. A.; Bhat, W. F.; Bano, B. Spectroscopic evaluation of the interaction between pesticides and chickpea cystatin: comparative binding and toxicity analyses. Sci. Processes Impacts, 2006, 18, 872–881.

(3)

Gerstl, Z.; Nasser, A.; Mingelgrin, U. Controlled release of pesticides into soils from clay−polymer formulations. J. Agric. Food Chem, 1998, 46, 3797– 3809.

(4)

Alavanja, C.R. Pesticides Use and Exposure Extensive Worldwide. Rev Environ Health, 2009, 24, 303–309.

(5)

Horrigan, L.; Lawrence, R.S.; Walker, P. How sustainable agriculture can address the environmental and human health harms of industrial agriculture. Environ Health Perspect, 2016, 110, 445 – 456.

(6)

Chen, H.; Seiber, J. N.; Hotze, M. ACS select on nanotechnology in food and agriculture: a perspective on implications and applications. J. Agri. Food Chem, 2014, 62, 1209 –1212.

(7)

Raliya, R.; Tarafdar, J.C.; Biswas, P. Enhancing the mobilization of native phosphorus in the mung bean rhizosphere using ZnO nanoparticles synthesized by soil fungi. J. Agric. Food Chem, 2016, 64, 3111–3118.

(8)

Wang, P.; Lombi, E.; Zhao, F. J.; Kopittke, P. M. Nanotechnology: a new opportunity in plant sciences. Trends Plant Sci, 2016, 21, 699–712.

(9) Wibowo, D.; Zhao, C. X.; Peters, P. C.; Middelberg, A. P. J. Sustained release of fipronil insecticide in vitro and in vivo from biocompatible silica nanocapsules. J. Agri. Food Chem, 2014, 62, 12504 –12511.



(10)

Page 18 of 30

Capaldi Arruda, S. C.; Diniz Silva, A. L.; Moretto Galazzi, R.; Antunes Azevedo, R.;

Zezzi Arruda, M. A. Nanoparticles applied to plant science: a review. Talanta, 2015, 131, 693–705. (11)

Khairy, M.; Ayoub, H. A.; Rashwan, F. A.; Abdel-Hafez, H. F. Chemical

modification of commercial kaolin for mitigation of organic pollutants in environment via adsorption and generation of inorganic pesticides. Appl. Clay Sci., 2018, 153,124–133. (12)

Ayoub, H. A.; Khairy, M.; Rashwan, F. A.; Abdel-Hafez, H. F. Synthesis and

characterization of silica nanostructures for cotton leaf worm control. J Nanostruct Chem, 2017, 7, 91–100. (13)

Le, V. N.; Rui, Y.; Gui, X.; Li, X.; Liu, S.; Han, Y. Uptake, transport, distribution and

Bio-effects of SiO2 nanoparticles in Bt-transgenic cotton. J Nanobiotechnology, 2014, 12, 50. (14)

Sommer, A. L. Copper as an essential for plant growth. Plant Physiology, 1931, 6,

339–345. (15)

Hänsch, R.; Mendel, R. R. Physiological functions of mineral micronutrients (Cu, Zn,

Mn, Fe, Ni, Mo, B, Cl). Curr Opin Plant Biol, 2009, 12, 259–266. (16)

Ivask, A.; Bondarenko, O.; Jepihhina, N.; Kahru, A. Profiling of the reactive oxygen

species related ecotoxicity of CuO, ZnO, TiO2, silver and fullerene nanoparticles using a set of recombinant luminescent Escherichia coli strains: differentiating the impact of particles and solubilised metals. Anal. Bioanal. Chem, 2010, 398,701–716. (17)

Karlsson, H. L.; Gustafsson, J.; Cronholm, P.; Möller, L. Size-dependent toxicity of

metal oxide particles a comparison between nano- and micrometer size. Toxicol Lett, 2009, 188,112–118.


Page 19 of 30


(18)

Costa, M.V. J.D.; Sharma, P. K. Effect of copper oxide nanoparticles on growth,

morphology, photosynthesis, and antioxidant response in Oryza sativa. Photosynthetica, 2016, 54, 110–119. (19)

Vana, n. L.; Mac, Ch.; Shanga, J.; Rui,Y.; Liua,S.; Xingc, B. Effects of CuO

nanoparticles on insecticidal activity and phytotoxicity in conventional and transgenic cotton. Chemosphere, 2016, 144,661–670. (20)

Katsumiti, A.; Thorleyb, A. J.; Arostegui,I.; Reipd, P.; Valsami-Jones, E.; , Tetley,T

D.; Cajaravillea, MP. Cytotoxicity and cellular mechanisms of toxicity of CuO NPs in vitro and comparative sensitivity with human cells. Toxicol In Vitro, 2018, 48, 146–158. (21)

Helper, P.K. Calcium: a central regulator of plant growth and development. Plant cell,

2005, 17, 2142–2155. (22)

Usten, N.H.; Yokas, A. L.; Saygili, H. Influence of potassium and calcium level on

severity of tomato pith necrosis and yield of greenhouse tomatoes. ISHS Acta Hortic, 2006, 808, 345–350. (23)

Deepa, M.; Sudhakar, P.; Nagamadhuri,K.V.; Reddy, K.B.; Krishna,T.G.;

Prasad,T.N.V.K.V. First evidence on phloem transport of nanoscale calcium oxide in groundnut using solution culture technique. Appl Nanosci, 2015, 5, 545–551. (24) Memarizadeh, N.; Ghadamyari, M.; Adeli, M.; Talebi, K. Linear-dendritic copolymers/indoxacarb supramolecular systems: biodegradable and efficient nanopesticides. Environ. Sci, 2014, 16, 2380–2389. (25)

Finney, D. J. 1971. Probit Analysis, 3rd Ed. Cambridge

University Press, London,

333. (26)

Khairy, M.; Mahmoud, B. G. Copper oxide microstructures with hemisphere

pineapple morphology for selective amperometric determination of vitamin C (L-ascorbic acid) in human fluids. Electroanalysis, 2016, 28, 1– 8.



(27)

Page 20 of 30

Liu, T.; Zhu, Y.; Zhang, X.; Zhang, T.; Zhang, T.; Li, X. Synthesis and

characterization of calcium hydroxide nanoparticles by hydrogen plasma-metal reaction method Materials. Mater. Lett, 2010, 64, 2575–2577. (28)

Roy, A.; Bhattachary, J. Microwave-assisted synthesis and characterization of CaO

nanoparticles, Int. J. Nanosci, 2011,3 , 413– 418. (29) El-Safty, S.A.; Shenashen, M.A.; Ismael, M.; Khairy, M. Encapsulation of proteins into tunable and giant mesocage alumina. Chem. Commun, 2012, 48, 6708 – 6710. (30) Dizaj, S. M.; Lotfipour, F.; Barzegar-Jalali, M.; Zarrintan, M. H.; Adibkia, K. Antimicrobial Activity of the Metals and Metal Oxides Nanoparticles. Mater. Sci. Eng. C, 2014, 14, 516 – 521. (31) U.S. Environmental Protection Agency. 1987. Health Advisory Summary: Methomyl. Office of Drinking Water, Washington DC, 3–40. (32)

Lehane, M.J.; Billingsley, P.F. 1996. Structure and ultrastructure of the insect midgut .Biology of the insect mid gut. Chapman & Hall, London, 3-25.

(33) Forkpah, C.; Dixon, L.R.; Fahrbach, S.E.; Rueppell, O. Xenobiotic Effects on Intestinal Stem Cell Proliferation in Adult Honey Bee (Apis mellifera L.) Workers, PLoS ONE, 2014, 9, e91180. (34) Fubini, B.; Hubbard, A. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radical Biol Med, 2003, 34, 1507–1516. (35) Tuncsoy, B.S.; Ozalp, P. Toxic effects of copper oxide nanoparticles in midgut and fat body of Galleria mellonella (Lepidoptera: Pyralidae). Toxicol. Lett, 2016, 258, 17–29. (36) Kramer, K J., Hopkins, T L., Schaefer, J. 1988. Insect Cuticle Structure and Metabolism, Biotechnology for Crop Protection. ACS Symposium Series; American Chemical Society: Washington DC, 160-185.


Page 21 of 30


(37)

Binnington, K., Retnakaran, A. 1991. Cuticular permeability and its control. Physiology of the Insect Epidermis: CSIRO Publications, Melbourne, Australia, 252283.

(38)

Amer, M. M.; El-Sayed, T. I.; Bakheit, H. K.; Moustafa, S. A.; El-Sayed, Y. A. Pathogenicity and genetic variability of five entomopathogenic fungi against Spodoptera littoralis. Res. J. Agric. Biol. Sci, 2008, 4, 354–367.

(39) Bi, J. L.; Felton. G. W.

Foliar oxidative stress and insect herbivory: primary

compounds, secondary metabolites, and reactive oxygen species as components of induced resistance. J. Chem. Ecol, 1995, 21, 1511–1530. (40) Debnath, N.; Das, S.; Seth, D.; Chandra, R.; Bhattacharya, R. C.; Goswami, A. Entomotoxic effect of silica nanoparticles against Sitophilus oryzae (L.) . J. Pest Sci, 2011, 84, 99 – 105. (41)

Debnath, N.; Mitra, S.; Das, S.; Goswami, A. Synthesis of surface functionalized silica nanoparticles and their use asentomotoxic nanocides. Powder Technol, 2012, 221, 252 – 256.

(42) Chippendale, G. M. 1978. The function of carbohydrate in insect life processes. Biochemistry of Insects, Academic Press, New York, 2 –54. (43) Knight, J.A.; Anderson, S.; Rawle, J. M. Chemical basis of the sulfo-phospho-vanillin reaction for estimating total serum lipids. Clin. Chem, 1972, 18, 199 – 202. (44)

Yu, S. J.; Terriere, L.C. Ecdysin metabolism by soluble enzymes from three species of Diptera and its inhibition by the insect growth regulatar TH-6040. Pestic. Biochem. Physiol, 1977, 17, 48–55.

(45) Nigam, Y. I.; Maudlin, S. W.; Ratcliffe, N. A. Detection of phenoloxidase activity in the hemolymph of tsetse flies, refractory and susceptible to infection with Trypanosoma brucei rhodesiense. J. Invertebr. Pathol, 1997, 69, 279–281.



(46)

Page 22 of 30

Rawi, S.M.; El-Gindy, H.; Haggag, A. M.; Elhassan, A. A.; Abdel-Kader, A. New possible molluscicides from calendula micranthaofficinalis and Ammi majus plants. Physiological effect on Biomphalaria alexandrina and Bulinus truncatus. J. Egypt. Geriatr.Soc. Zool, 1995, 16 , 69–75.

(47) Taha, H. S.; Al-Hadek. M. K. Biochemical studies of chlorfluazuron and diflubenzuron effect on chitinase and phenol oxidase and biological studies on the black cutworm agrotis ipsilon (HUFN.) (Lepidoptera:Noctuidae). J.Agric.Chem.and Biotechn, 2017, 8, 143–148. (48) Bayoumi, A.E.; Balana-fouce,R.; SobeihaA.K.; Hussein, E. M. K.The biochemical influences of some chitin synthesis inhibitors against the cotton leafworm Spodoptera littoralis (Boisd.). Bol. San. Veg. Plagas, 1997, 23, 583–593. (49)

Shauruba, H. El-Sayed.; Zohdya, Z. N.; Abdel-Aal, E.A.; Emarab, A.S. Effect of chlorfluazuron and flufenoxuron on development and reproductive performance of the black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae). Invertebr. Reprod. Dev, 2018, 62, 27-34.


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FIGURES AND SCHEME CAPTIONS

Scheme 1. Schematic representation of application of metal oxide nanostructures on cotton leaf worm by using feeding bioassay.

Fig. 1. (a) XRD, (b) FTIR (c) N2 adsorption/desorption isotherms and (d) corresponding pore size distribution curves of CuO and CaO nanostructures.

Fig. 2. SEM images of CuO (a) and CaO (b) nanostructures, and TEM micrographs of CuO (c and d) and CaO (e and f) synthesised by wet-chemical methods. Fig. 3. Mean mortality of 2nd instar larvae of S.littoralis exposed to different concentrations of CuO and CaO NPs via feeding bioassay method. Each value is the mean of three independent replicates and vertical bars represent the standard error, Comparison between the two metal oxides was statistically analysed using one-way analysis of variance (ANOVA) ),[ ♣ insignificant at P < 0.05, ⃰ insignificant at P < 0.05]. Fig. 4. Mid-gut cross sections of 6th instar larvae for control and treated S. littoralis with LC50 of CuO and CaO NPs via feeding bioassay (20 × H-E) (a, b and c) (L; lumen, PM; peritrophic membrane, EL; epithelial layer, GC; goblet cells), and the corresponding cuticle structures (100 × H-E) (d, e and f). Fig. 5. Carbohydrates, proteins, lipids, phenol oxidase and chitinase activity of 6th instars of S. littorals treated with LC50 of CuO and CaO NPs via feeding bioassay method; Values are mean ± standard error of three independent replicates, statistical analysis were performed by using one-way analysis of variance (ANOVA),[ ♣ insignificant at P < 0.05, ⃰ insignificant at P < 0.05].



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LIST OF FIGURES

Scheme 1


Transmittance (%)


10

20

30

40

60

70

594.82

0.12

SBET m2/g

V cm3/g

D nm

23.267

0.0281

3.379

40.545

0.106

3.801

858

427.27

500

1000 1500 2000 2500 3000 Wavenumber (cm-1)

CuO CaO

0.10

dVp/dlog(dp)

20

(b)

80

2θ

30

Va/cm3 (STP).g-1

50

CaO

1403 1546

(a)

CuO 477.08

CuO CaO

551 659

Intensity (a.u)

Page 25 of 30

0.08 0.06

10

0 0.0

(d)

0.04

(c)

CuO CaO 0.2

0.4

0.6

0.8

1.0

0.02 0

5

p/pο

10

15

20

25

30

dp(nm) Figure 1



Page 26 of 30

Figure 2


Page 27 of 30


100

CuO NPs after 3days CaO NPs after 11days

90

♣

Mortality (%)

80

♣

♣

70 60 50 40

∗

30 20 10 0 150

300

450

600

[MO]mg/L Figure 3



Page 28 of 30

Figure 4


Page 29 of 30


50

Carbohydrates

12

∗

10

∗

8 6 4 2

♣

40

∗

30 20 10 0

control

CuO

CaO 55 50 45 40 35 30 25 20 15 10 5 0

control Phenoloxidase

∗

∗

CuO (ug NAGA /min/g.b.wt)±SE

0

8

Proteins

(mg/g.b.wt ) M± SE

(mg/g.b.wt ) M± SE

14

(O.D units /min/g.b.w)±SE

(mg/g.b.wt ) M± SE

16

CaO 1800

Lipids

7

♣

6 5

∗

4 3 2 1 0

control

CuO

CaO

Chitinase activity

∗

1600 1400

♣

1200 1000 800 600 400 200 0

control

CuO

CaO

control

CuO

CaO

Figure 5



Page 30 of 30

TOC Graphic


Pesticidal activity of nanostructured metal oxides for generation of

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