**Particulate Nanoinsecticides: A New Concept in Insect Pest Management Pest Management**

**Particulate Nanoinsecticides: A New Concept in Insect** 

DOI: 10.5772/intechopen.72448

Teodoro Stadler, Micaela Buteler, Susana R. Valdez and Javier G. Gitto Javier G. Gitto Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Teodoro Stadler, Micaela Buteler, Susana R. Valdez and

http://dx.doi.org/10.5772/intechopen.72448

#### **Abstract**

Nanostructured alumina (NSA) has insecticidal properties and has been demonstrated to be effective against stored product insect pests in laboratory bioassays. NSA is a nanoengineered material synthesized by oxidation of metals, and resulting particles show fixed electric charges. On the other hand, insects exhibit their own electric charges generated by triboelectrification. We propose that the mechanism of action of NSA involves two steps occurring in sequential order. First, a strong electrical binding between negatively charged NSA particles and positively charged insect. Next, dehydration of the insect occurs due to the strong sorbtive action of the NSA particles that remove the insect cuticular, leading to death by dehydration. As postulated for insecticidal inert powder in generals, particles attach to the insect cuticle surface disrupting water balance, and effectiveness decreases as ambient humidity increases, given that electrostatic bond forces are reduced by electrostatic discharge. The high insecticidal efficacy of NSA is a result of its intrinsic electric charge, small particle size and high sorptive potential due to its large specific surface area. NSA could provide an alternative to conventional synthetic organic insecticides due to its strong insecticidal properties with the advantage that its mechanism of action involves physical and electrostatic phenomena.

**Keywords:** nanoinsecticides, mode of action, triboelectrification, *Sitophilus oryzae*, insecticide powders

### **1. Introduction**

The advent of synthetic organic pesticides by mid-1950s made the control of insect pests highly effective and despite their drawbacks, most of these active principles are still used in modern agriculture. The use of synthetic insecticides has allowed an increase in yields and lowered the

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

cost of farming. Synthetic organic pesticides also remain an important tool to control vectors of infectious diseases of both humans and domestic animals, leading to a great reduction in their incidence in many areas of the world. However, synthetic organic insecticides may impact negatively on human health and ecosystems, affecting populations of non-target organisms and biodiversity [1]. Moreover, the accumulation of active ingredients or their metabolites in the environment as well as in organisms, may lead to bioaccumulation, where these pollutants enter the food chain, posing a serious threat to both wildlife and humans [2, 3].

change in the physicochemical properties of nano-pesticides [16, 17]. Compared to larger particles of the same chemical substance, nanoparticles are more reactive, more biologically active and have a more catalytic action [18, 19]. Nanoparticles could help use pesticides and fertilizers more effectively [16], for example, by reducing agrochemical components to nanosize or to pack the active ingredients in nanocapsules, which release them selectively, would allow for lower amounts with the same effect, only under certain conditions of heat, sunlight or pH [20, 21].

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The current levels of application of nanoparticles and the expected developments to come, suggest that nanotechnology will have a direct impact on the evolution of pest management practices in agriculture [16, 22–25]. Recently, the discovery of nanoinsecticides brings new alternatives to expand the spectrum of applications of inorganic powders [6, 24, 26, 27]. Nanoengineered aluminium oxide as nanostructured alumina (NSA), has been shown to have insecticidal properties, low non target toxicity, non-reactivity, low cost and reduced prob-

In a previous work, Stadler et al. [6, 24, 28] assumed that "*rice weevil*" (*S. oryzae*) adults acquire electrostatic charge by triboelectrification when walking on a dielectric surface such as wheat kernels, and that these charges on the insect body lead NSA particles from the treated substrate to the insect body surface. In order to verify this phenomenon empirically, studies were undertaken to examine and model the tribo-charging of *S. oryzae* adults on a dielectric surface and to identify the type and magnitude of the electrostatic charge on NSA, diatomaceous

A reduction in particle size of a substance results in increased surface/volume ratio per unit weight, which generally correlates with increased toxicity [29]. This characteristic has been exploited by some researchers to control various microorganisms and insects by applying nanoparticles [26, 28]. For example, nano-engineered alumina (NSA) is the result of combustion synthesis, using a redox mixture, with glycine as fuel and aluminum nitrate as oxidizer, where the final product is a homogeneous powder of high purity with uniform characteristics and specific physicochemical properties [30]. During the combustion process, alumina nanoparticles (40–60 nm) aggregate in primary clusters of approximately equal diameter building electrically loaded amorphous micrometric agglomerates/aggregates with a specific surface area of 14 m<sup>2</sup>

ranging in size from 0.1 to a few micrometers [31]. Due to its special characteristics as kinetics and bioactivity, it allows for varied and novel uses as a pesticide for human, livestock and agricultural use, stored product protection, treatments for wood preservation, carriers of pheromone or virus for insect pest control, etc. [32]. NSA has been shown to have strong insecticidal properties to several insect species through a mechanism of action different from conventional pesticides. Nanostructured alumina (NSA) has been shown to be an effective contact insecticide

In nanomaterials synthesized by oxidation of metals, such as the NSA, the resulting particles are electrically charged, showing a dipole-dipole interaction that promotes aggregate formation with resistance to dissociation forces [34]. Depending on the synthesis procedure, the greater part of the aggregates charged either positively or negatively and only some of these

for several species of stored grain insect pests [6] as well as for leaf cutting ants [33].

/g

earth (DE) samples and the net electrostatic charge density of wheat kernels.

**1.2. Nanostructured alumina: a novel nano-engineered insecticidal powder**

abilities of generating resistance in insects [26].

Caught in a vicious circle? Agriculture has waged a costly struggle fighting insects by constantly rotating obsolescent pesticides in a desperate strategy of chemical warfare. However, a comprehensive and successful strategy for minimizing acute and chronic risk from pesticide use should be based on research initiatives aimed at radical changes in pest management strategies and the replacement of the synthetic organic pesticides with effective but less hazardous substances [4]. Part of the research on new biorational pesticides focuses on natural products such as plant extracts, oils and inorganic products. These are frequently a source of new chemical classes of insecticides, as well as environmentally and toxicologically less hazardous active ingredients than many of the conventional products used for insect pest control. Furthermore, new active ingredients often have mechanisms of action or molecular target sites which still remain unexploited by conventional marketed pesticides [5]. Hence, substances with new properties are promising tools for crop protection and food production, opening new frontiers in pest management [6]. However, only 14% of the pesticides on the market are biorational products and only 1% consists of natural products like plant extracts, essential oils and insecticide powders [7].

#### **1.1. Nanotechnology as a source of modern pesticides**

Nanotechnology is a collective term for a wide range of technologies that deal with structures and processes at the nanometer scale. The transition to the nanometer scale (10−9 m), leads to an increase in dominance of quantum-physical effects, optical, magnetic, electronic, mechanical and chemical properties [8]. Because of its potential for the fundamental transformation of entire technology fields, nanotechnology will not only influence technological development in the near future, but also have economic, ecological and social implications [9]. The size reduction to the nanometer range often leads to characteristic properties of materials which are useful for new applications and which do not occur in the case of macroscopic pieces of the same material. These include, for example, higher breaking strength at low temperatures as well as superplasticity at high temperatures, formation of additional electronic states, high chemical selectivity of the surface structures and a markedly increased surface energy [10].

Nanotechnology has advanced rapidly over the last 10 years and numerous nanomaterials, with a variety of potential applications, have been developed. For instance, improvements in medical science through nanotechnology offer the possibility to develop novel diagnostics and therapeutics [11], as well as new nano-engineered products with pesticide properties which have shown to be promising as tools for low impact or alternative organic agriculture and food production [12–15]. Engineered versions of conventional pesticides, growth regulators and seed treatment agents are among the first nano-chemicals that could be used in agriculture [13]. The use of nanoparticles could make pesticides more effective by reducing particle size to the nanoscale given the associated increase in surface area which introduces a fundamental change in the physicochemical properties of nano-pesticides [16, 17]. Compared to larger particles of the same chemical substance, nanoparticles are more reactive, more biologically active and have a more catalytic action [18, 19]. Nanoparticles could help use pesticides and fertilizers more effectively [16], for example, by reducing agrochemical components to nanosize or to pack the active ingredients in nanocapsules, which release them selectively, would allow for lower amounts with the same effect, only under certain conditions of heat, sunlight or pH [20, 21].

cost of farming. Synthetic organic pesticides also remain an important tool to control vectors of infectious diseases of both humans and domestic animals, leading to a great reduction in their incidence in many areas of the world. However, synthetic organic insecticides may impact negatively on human health and ecosystems, affecting populations of non-target organisms and biodiversity [1]. Moreover, the accumulation of active ingredients or their metabolites in the environment as well as in organisms, may lead to bioaccumulation, where these pollutants

Caught in a vicious circle? Agriculture has waged a costly struggle fighting insects by constantly rotating obsolescent pesticides in a desperate strategy of chemical warfare. However, a comprehensive and successful strategy for minimizing acute and chronic risk from pesticide use should be based on research initiatives aimed at radical changes in pest management strategies and the replacement of the synthetic organic pesticides with effective but less hazardous substances [4]. Part of the research on new biorational pesticides focuses on natural products such as plant extracts, oils and inorganic products. These are frequently a source of new chemical classes of insecticides, as well as environmentally and toxicologically less hazardous active ingredients than many of the conventional products used for insect pest control. Furthermore, new active ingredients often have mechanisms of action or molecular target sites which still remain unexploited by conventional marketed pesticides [5]. Hence, substances with new properties are promising tools for crop protection and food production, opening new frontiers in pest management [6]. However, only 14% of the pesticides on the market are biorational products and only 1% consists of natural products like plant extracts, essential oils and insecticide powders [7].

Nanotechnology is a collective term for a wide range of technologies that deal with structures and processes at the nanometer scale. The transition to the nanometer scale (10−9 m), leads to an increase in dominance of quantum-physical effects, optical, magnetic, electronic, mechanical and chemical properties [8]. Because of its potential for the fundamental transformation of entire technology fields, nanotechnology will not only influence technological development in the near future, but also have economic, ecological and social implications [9]. The size reduction to the nanometer range often leads to characteristic properties of materials which are useful for new applications and which do not occur in the case of macroscopic pieces of the same material. These include, for example, higher breaking strength at low temperatures as well as superplasticity at high temperatures, formation of additional electronic states, high chemical selectivity of the surface structures and a markedly increased surface energy [10]. Nanotechnology has advanced rapidly over the last 10 years and numerous nanomaterials, with a variety of potential applications, have been developed. For instance, improvements in medical science through nanotechnology offer the possibility to develop novel diagnostics and therapeutics [11], as well as new nano-engineered products with pesticide properties which have shown to be promising as tools for low impact or alternative organic agriculture and food production [12–15]. Engineered versions of conventional pesticides, growth regulators and seed treatment agents are among the first nano-chemicals that could be used in agriculture [13]. The use of nanoparticles could make pesticides more effective by reducing particle size to the nanoscale given the associated increase in surface area which introduces a fundamental

enter the food chain, posing a serious threat to both wildlife and humans [2, 3].

**1.1. Nanotechnology as a source of modern pesticides**

84 Insecticides - Agriculture and Toxicology

The current levels of application of nanoparticles and the expected developments to come, suggest that nanotechnology will have a direct impact on the evolution of pest management practices in agriculture [16, 22–25]. Recently, the discovery of nanoinsecticides brings new alternatives to expand the spectrum of applications of inorganic powders [6, 24, 26, 27]. Nanoengineered aluminium oxide as nanostructured alumina (NSA), has been shown to have insecticidal properties, low non target toxicity, non-reactivity, low cost and reduced probabilities of generating resistance in insects [26].

In a previous work, Stadler et al. [6, 24, 28] assumed that "*rice weevil*" (*S. oryzae*) adults acquire electrostatic charge by triboelectrification when walking on a dielectric surface such as wheat kernels, and that these charges on the insect body lead NSA particles from the treated substrate to the insect body surface. In order to verify this phenomenon empirically, studies were undertaken to examine and model the tribo-charging of *S. oryzae* adults on a dielectric surface and to identify the type and magnitude of the electrostatic charge on NSA, diatomaceous earth (DE) samples and the net electrostatic charge density of wheat kernels.

#### **1.2. Nanostructured alumina: a novel nano-engineered insecticidal powder**

A reduction in particle size of a substance results in increased surface/volume ratio per unit weight, which generally correlates with increased toxicity [29]. This characteristic has been exploited by some researchers to control various microorganisms and insects by applying nanoparticles [26, 28]. For example, nano-engineered alumina (NSA) is the result of combustion synthesis, using a redox mixture, with glycine as fuel and aluminum nitrate as oxidizer, where the final product is a homogeneous powder of high purity with uniform characteristics and specific physicochemical properties [30]. During the combustion process, alumina nanoparticles (40–60 nm) aggregate in primary clusters of approximately equal diameter building electrically loaded amorphous micrometric agglomerates/aggregates with a specific surface area of 14 m<sup>2</sup> /g ranging in size from 0.1 to a few micrometers [31]. Due to its special characteristics as kinetics and bioactivity, it allows for varied and novel uses as a pesticide for human, livestock and agricultural use, stored product protection, treatments for wood preservation, carriers of pheromone or virus for insect pest control, etc. [32]. NSA has been shown to have strong insecticidal properties to several insect species through a mechanism of action different from conventional pesticides. Nanostructured alumina (NSA) has been shown to be an effective contact insecticide for several species of stored grain insect pests [6] as well as for leaf cutting ants [33].

In nanomaterials synthesized by oxidation of metals, such as the NSA, the resulting particles are electrically charged, showing a dipole-dipole interaction that promotes aggregate formation with resistance to dissociation forces [34]. Depending on the synthesis procedure, the greater part of the aggregates charged either positively or negatively and only some of these are dipoles [31]. Thus, the combustion manufacturing process is the main factor responsible for the affinity of particles with the triboelectrically charged body surface of different insect species (**Figure 1**) and as a consequence, also responsible for insecticidal activity. However, the morphology of nanoalumina agglomerates can be influenced by different variables during the synthesis such as substrate concentration, additives and calcination temperature which play a decisive role in the final morphology and characteristics of nanoalumina [35].

to diatomaceous earth and *R. dominica* is among the least susceptible ones. Chemical makeup of epicuticular waxes varies across insect species [37], and this should translate into differences in susceptibility to nanoalumina and other inert powders due to differences in wetting. Treatment with NSA as well as Protect-It® also reduced progeny production although NSA powder was more effective in eliminating F1 adults than Protect-It®, for both species of insects tested. NSA reduced F1 progeny drastically at concentrations as low as 62.5 ppm for *S. oryzae* for high, medium and low humidity levels, and ranging from 250 to 500 ppm for *R. dominica* depending on the humidity level [32]. These results obtained with NSA are encouraging given

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that Protect-It® is one of the most effective DE-based products in the market [36, 38].

experiments on delivery options to further enhance NSA products.

*1.3.2. The intake toxicity of NSA in stored product insect pests*

Comparison of these results with recommended rates for commercial insecticidal powders suggests that inorganic nanostructured alumina may prove a good alternative or complement to DE-based products, and encourage further testing with other insect pests and systems, plus

Although dehydration appears to be the main cause of mortality due NSA exposure, but it cannot be assumed as the only one, for it is found that at sub-lethal concentrations, insecticide powders in general exert further noxious effects on the insect [33, 36, 39]. Our studies revealed that intake toxicity is a significant mortality factor that occurs simultaneously with contact toxicity during insect exposure to NSA. Dietary intake of exposure to concentrations lower than 75 ppm caused sub-lethal effects in *S. oryzae* and adult mortality occurred after only 7 days exposure to NSA in food. These results indicate that toxicity due to ingestion is also a relevant mortality factor. There was a delayed response to NSA intake through ingestion which occurred up to 39 days of continuous exposure to NSA-treated flour discs. Mortality of adult *S. oryzae* was dose-dependent reaching up to 100% at concentrations of 250, 350 and 500 ppm, and up to 40% for concentrations below 125 ppm in wheat discs. The LC50 value calculated from intake bioassays on NSA-treated flour discs was 180.97 ppm [(CI = 167.07; 195.91); Slope = 0.01; Intercept = −1.68] and the LT50 calculated for the maximum dose concentration tested of 500 ppm was 23.82 days [(CI = 22.05; 25.17); Slope = 0.13; Intercept = −3.04]. The body weight of live individuals fed with NSA-treated wheat kernels (TPP plate No. 3), also presented a substantial reduction, of 51.6 (± 2.51)% on average [28]. These findings are similar to what Alexander et al. [40] observed after the treatment of *S. granarius* with various insecticide powders and by Trewin et al. [41] after treatment of *Ephestia kuehniella*, *Oryzaephilus surinamensis*, *Tenebrio molitor* and *Tribolium castaneum* with Aerosil® dispersions, a silica product. Results demonstrate that ingestion toxicity is a relevant long-term mortality factor that

should be taken into account when assessing the efficacy of NSA and inert powders.

An important characteristic of nanomaterials is their extremely large surface. For example, the same mass of material in the form of nanoparticles has a specific surface area which is many times larger than a coarse powder. This large surface can chemically react with materials that are otherwise non-reactive and non-toxic. However, it is not the size alone that contributes to the potential toxicity of nanomaterials. Rather, it has been shown that the toxicity of

**1.4. The** *in vivo* **toxicity and the** *in vitro* **cytotoxicity of NSA particles**

The effect of NSA on insects has been investigated through contact as well as dietary intake toxicity bioassays. Also, the *in vivo* toxicity and the *in vitro* cytotoxicity of NSA particles were screened as reviewed below.

#### **1.3. Insecticidal effect of NSA on stored product insect pests**

#### *1.3.1. The contact toxicity of NSA against stored product insect pests*

The contact toxicity of NSA was first investigated using dry powder applications at three different relative ambient humidity levels [6]. Tests were conducted simultaneously with enhanced diatomaceous earth, Protect-It®, to compare the efficacy of NSA to that of commercial insecticide powders. Two major stored grain pests *Rhyzopertha dominica* and *Sitophilus oryzae* were tested and significant delayed mortality was observed. Both species experienced significant mortality after 3 days of continuous exposure to treated wheat. Nine days after treatment, the median lethal doses (LD50) observed ranged from 127 to 235 mg kg−1. Results showed that NSA was more effective in killing *S. oryzae* than Protect-It® and was equally toxic to *R. dominica*. *R. dominica* was, less susceptible to inert powders than *S. oryzae* [32]. According to Subramanyam and Roesli [36], *S. oryzae* is among the most susceptible species

**Figure 1.** SEM images of individuals of three stored insect pest species [(a, b) *Sitophilus oryzae* (Coleoptera: Curculionidae); (c, d) *Ceratitis capitata* (Dipetara: Tephritidae); (e, f) *Oryzaephilus surinamensis* (Coleoptera: Silvanidae)] showing the affinity of NSA particles to triboelectrically charged insect body surfaces. (a, c, e) after exposure to untreated wheat kernels; (b, d, f) after exposure to 125 ppm NSA-treated wheat kernels. Fformat JEOL/EO, version 1.0; instrument JSM-6610.

to diatomaceous earth and *R. dominica* is among the least susceptible ones. Chemical makeup of epicuticular waxes varies across insect species [37], and this should translate into differences in susceptibility to nanoalumina and other inert powders due to differences in wetting. Treatment with NSA as well as Protect-It® also reduced progeny production although NSA powder was more effective in eliminating F1 adults than Protect-It®, for both species of insects tested. NSA reduced F1 progeny drastically at concentrations as low as 62.5 ppm for *S. oryzae* for high, medium and low humidity levels, and ranging from 250 to 500 ppm for *R. dominica* depending on the humidity level [32]. These results obtained with NSA are encouraging given that Protect-It® is one of the most effective DE-based products in the market [36, 38].

Comparison of these results with recommended rates for commercial insecticidal powders suggests that inorganic nanostructured alumina may prove a good alternative or complement to DE-based products, and encourage further testing with other insect pests and systems, plus experiments on delivery options to further enhance NSA products.

#### *1.3.2. The intake toxicity of NSA in stored product insect pests*

are dipoles [31]. Thus, the combustion manufacturing process is the main factor responsible for the affinity of particles with the triboelectrically charged body surface of different insect species (**Figure 1**) and as a consequence, also responsible for insecticidal activity. However, the morphology of nanoalumina agglomerates can be influenced by different variables during the synthesis such as substrate concentration, additives and calcination temperature which

The effect of NSA on insects has been investigated through contact as well as dietary intake toxicity bioassays. Also, the *in vivo* toxicity and the *in vitro* cytotoxicity of NSA particles were

The contact toxicity of NSA was first investigated using dry powder applications at three different relative ambient humidity levels [6]. Tests were conducted simultaneously with enhanced diatomaceous earth, Protect-It®, to compare the efficacy of NSA to that of commercial insecticide powders. Two major stored grain pests *Rhyzopertha dominica* and *Sitophilus oryzae* were tested and significant delayed mortality was observed. Both species experienced significant mortality after 3 days of continuous exposure to treated wheat. Nine days after treatment, the median lethal doses (LD50) observed ranged from 127 to 235 mg kg−1. Results showed that NSA was more effective in killing *S. oryzae* than Protect-It® and was equally toxic to *R. dominica*. *R. dominica* was, less susceptible to inert powders than *S. oryzae* [32]. According to Subramanyam and Roesli [36], *S. oryzae* is among the most susceptible species

**Figure 1.** SEM images of individuals of three stored insect pest species [(a, b) *Sitophilus oryzae* (Coleoptera: Curculionidae); (c, d) *Ceratitis capitata* (Dipetara: Tephritidae); (e, f) *Oryzaephilus surinamensis* (Coleoptera: Silvanidae)] showing the affinity of NSA particles to triboelectrically charged insect body surfaces. (a, c, e) after exposure to untreated wheat kernels; (b, d,

f) after exposure to 125 ppm NSA-treated wheat kernels. Fformat JEOL/EO, version 1.0; instrument JSM-6610.

play a decisive role in the final morphology and characteristics of nanoalumina [35].

**1.3. Insecticidal effect of NSA on stored product insect pests**

*1.3.1. The contact toxicity of NSA against stored product insect pests*

screened as reviewed below.

86 Insecticides - Agriculture and Toxicology

Although dehydration appears to be the main cause of mortality due NSA exposure, but it cannot be assumed as the only one, for it is found that at sub-lethal concentrations, insecticide powders in general exert further noxious effects on the insect [33, 36, 39]. Our studies revealed that intake toxicity is a significant mortality factor that occurs simultaneously with contact toxicity during insect exposure to NSA. Dietary intake of exposure to concentrations lower than 75 ppm caused sub-lethal effects in *S. oryzae* and adult mortality occurred after only 7 days exposure to NSA in food. These results indicate that toxicity due to ingestion is also a relevant mortality factor. There was a delayed response to NSA intake through ingestion which occurred up to 39 days of continuous exposure to NSA-treated flour discs. Mortality of adult *S. oryzae* was dose-dependent reaching up to 100% at concentrations of 250, 350 and 500 ppm, and up to 40% for concentrations below 125 ppm in wheat discs. The LC50 value calculated from intake bioassays on NSA-treated flour discs was 180.97 ppm [(CI = 167.07; 195.91); Slope = 0.01; Intercept = −1.68] and the LT50 calculated for the maximum dose concentration tested of 500 ppm was 23.82 days [(CI = 22.05; 25.17); Slope = 0.13; Intercept = −3.04]. The body weight of live individuals fed with NSA-treated wheat kernels (TPP plate No. 3), also presented a substantial reduction, of 51.6 (± 2.51)% on average [28]. These findings are similar to what Alexander et al. [40] observed after the treatment of *S. granarius* with various insecticide powders and by Trewin et al. [41] after treatment of *Ephestia kuehniella*, *Oryzaephilus surinamensis*, *Tenebrio molitor* and *Tribolium castaneum* with Aerosil® dispersions, a silica product. Results demonstrate that ingestion toxicity is a relevant long-term mortality factor that should be taken into account when assessing the efficacy of NSA and inert powders.

#### **1.4. The** *in vivo* **toxicity and the** *in vitro* **cytotoxicity of NSA particles**

An important characteristic of nanomaterials is their extremely large surface. For example, the same mass of material in the form of nanoparticles has a specific surface area which is many times larger than a coarse powder. This large surface can chemically react with materials that are otherwise non-reactive and non-toxic. However, it is not the size alone that contributes to the potential toxicity of nanomaterials. Rather, it has been shown that the toxicity of nanomaterials is influenced by many parameters like size, shape, surface area, electric charge and texture. Moreover, it is possible to design a basic nanomaterial in its morphology and surface properties on the nanotechnology. This results in new properties of a new material but it is unclear to what extent significant changes in toxicological properties can result from changes in the morphology [42]. Soluble nanomaterials lose their nanostructure features after they have come into contact with in biological fluids and a non-specific toxicity could possibly arise. However, insoluble nanomaterials that retain their nanostructures which are wrapped in a stable material and thus cannot come into contact with biological fluids or those that can be absorbed into organism are of lesser importance. Therefore, the free nanomaterials, which can be absorbed in different ways, are of particular toxicological significance. These nanomaterials are likely to lead to a higher exposure of humans and the environment [43].

mononuclear cells (PBL) were isolated from a healthy donor venous blood. PBL were exposed for 24 hours to increasing concentrations of NSA (50, 100 and 200 μg/mL) and then collected. Concentrations used were the same as those tested by Pochettino [46]. DNA and chromosomal damage was assessed throughout the alkaline comet assay and micronuclei (MIN) test, respectively, and cell viability was tested with the resazurin assay. The comet assay allowed to quantify DNA damage and revealed no significant increase in DNA damage induced by NSA. No statistical significant differences were found in terms of cellular viability and NSA

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Regarding animal experiments (*in vivo*), the acute oral toxicity and the acute inhalation toxicity of engineered aluminum oxide nanostructured particles (avg. 100 nm) were assessed in Wistar albino rats [48]. Acute oral toxicity was assessed by a limit test at a test dose of 2000 mg/ kg b.wt that was administrated in a single dose. No mortality was observed in treated animals and no significant differences in body weight where observed (p < 0.05) either. No morphological changes where observed through pathological examinations. After inhalation exposure (0.02 mg/L air), respectively, during 4 hours, no changes in body weight gain were noted. A decrease in body weight gain was observed after inhalation exposure with 0.07 mg/L. No morbidity or mortality was observed in inhalation NSA exposed rats. These studies provide information applicable to the early stage in the hazard identification process for this type of nanomaterials that could be useful in risk management in the context of production, handling and use of nanomaterials. These results show that acute oral and inhalation exposure to NSA

The rapid proliferation of engineered nanomaterials and the limited toxicological data currently available on it presents a dilemma to regulators regarding risk assessment processes for these materials [49]. For recently developed nanomaterials, there are in many cases insufficient investigations into health effects. Therefore, no sufficiently reliable statement can be made about these nanomaterials. There is a need to determine the extent of absorption, systemic availability, accumulation and excretion of nanomaterials after inhalation and oral exposure. However, the necessary in vivo studies should be integrated into any toxicological studies to avoid unnecessary animal experiments. The influence of modifications in the NSA synthesis on the kinetic parameters [26] as well as on the toxicological properties of the nanomaterials should also be examined. Finally, oxidative stress and the formation of reactive oxygen species (ROS) are fundamental key mechanisms of cellular defense after particle capture. In order to develop biorational pesticides through design of NSA synthesis, further research is necessary on the complex relationships between its physical and chemical parameters and its toxicity.

**2. Identifying and understanding the mechanism of pesticide action** 

Tribo-charging is the advent of electric charges based on the mechanisms of charge transfer which occurs when two different non-conductive bodies (materials) are brought into contact

had no significant effect on MIN induction.

did not result in morbidity or mortality in male rats.

**of nanostructured alumina**

**2.1. Triboelectric charging in insects**

Each nanostructured material has to be individually tested for its potential toxicity, since the knowledge about the complex relationships between physical and chemical parameters and a possible toxicity is missing. The toxicity of aluminum oxide nanoparticles has been discussed in many publications providing mixed results [44, 45]. On the other hand, toxicity of nanostructured aluminum oxide particles (NSA) [26] remains still an object of experimental work. Pochettino et al. [46] evaluated *in vitro* effect of the NSA on macrophages from the THP-1 cell line, exposed during two different time periods (6 and 24 hours) to different NSA concentrations (5, 25, 100 and 250 μg/mL). Cell cultures exposed to the lower concentrations of NSA during 6 hours show increased levels of the proinflammatory cytokine synthesized by macrophages IL-1β and a significant reduction of catalase (CAT) antioxidant enzyme activity. The two highest concentrations of NSA induced a decrease in cell viability (MTT assay) and an increase in lactate dehydrogenase activity (LDH: cytotoxicity indicator) and IL-1β release, in exposed cell cultures, and a decrease in CAT activity and thiol groups (−SH: thiols groups, antioxidants properties). These changes observed in CAT, LDH, −SH are indicators of oxidative stress. After NSA treatment, mitochondria lost their filamentous shape and displayed several morphological alterations. The effect of NSA on cell cultures after 24 hours of exposure was similar to that observed at 6 hours.

The exposure of THP-1 macrophage cell cultures to high NSA concentrations induces the release of IL-1β but also causes cell death, where NSA-mediated oxidative stress could play an important role. The generation of a controlled oxidative stress leads to the activation of intracellular mechanisms to compensate the production of reactive oxygen species (ROS), however a continuous overproduction of these species causes the onset of pathological states. Further studies should address the mechanisms involved in the oxidative stress caused by NSA in order to characterize and limit these. Also, further studies on the balance between pro- and anti-inflammatory molecules in *vitro* cell cultures exposed to NSA will be necessary looking for the mechanisms involved in acute effects of NSA exposure. On the other hand, low NSA concentrations raise the IL-1β levels without inducing changes in cell viability; so, this could be of relevance to enhance triggering immune responses. These results motivate further research on the mechanisms underlying the observed effects of NSA on THP-1 macrophage cells, as well as to analyze other mediators and immunological parameters in order to evaluate the potential of the NSA at low dose as a modulator of the immune response.

Deepening at the cellular level, Nadin [47] studied the genotoxicity effects of NSA at the cellular level. To determine whether NSA induces DNA damage, human peripheral blood mononuclear cells (PBL) were isolated from a healthy donor venous blood. PBL were exposed for 24 hours to increasing concentrations of NSA (50, 100 and 200 μg/mL) and then collected. Concentrations used were the same as those tested by Pochettino [46]. DNA and chromosomal damage was assessed throughout the alkaline comet assay and micronuclei (MIN) test, respectively, and cell viability was tested with the resazurin assay. The comet assay allowed to quantify DNA damage and revealed no significant increase in DNA damage induced by NSA. No statistical significant differences were found in terms of cellular viability and NSA had no significant effect on MIN induction.

Regarding animal experiments (*in vivo*), the acute oral toxicity and the acute inhalation toxicity of engineered aluminum oxide nanostructured particles (avg. 100 nm) were assessed in Wistar albino rats [48]. Acute oral toxicity was assessed by a limit test at a test dose of 2000 mg/ kg b.wt that was administrated in a single dose. No mortality was observed in treated animals and no significant differences in body weight where observed (p < 0.05) either. No morphological changes where observed through pathological examinations. After inhalation exposure (0.02 mg/L air), respectively, during 4 hours, no changes in body weight gain were noted. A decrease in body weight gain was observed after inhalation exposure with 0.07 mg/L. No morbidity or mortality was observed in inhalation NSA exposed rats. These studies provide information applicable to the early stage in the hazard identification process for this type of nanomaterials that could be useful in risk management in the context of production, handling and use of nanomaterials. These results show that acute oral and inhalation exposure to NSA did not result in morbidity or mortality in male rats.

The rapid proliferation of engineered nanomaterials and the limited toxicological data currently available on it presents a dilemma to regulators regarding risk assessment processes for these materials [49]. For recently developed nanomaterials, there are in many cases insufficient investigations into health effects. Therefore, no sufficiently reliable statement can be made about these nanomaterials. There is a need to determine the extent of absorption, systemic availability, accumulation and excretion of nanomaterials after inhalation and oral exposure. However, the necessary in vivo studies should be integrated into any toxicological studies to avoid unnecessary animal experiments. The influence of modifications in the NSA synthesis on the kinetic parameters [26] as well as on the toxicological properties of the nanomaterials should also be examined. Finally, oxidative stress and the formation of reactive oxygen species (ROS) are fundamental key mechanisms of cellular defense after particle capture.

In order to develop biorational pesticides through design of NSA synthesis, further research is necessary on the complex relationships between its physical and chemical parameters and its toxicity.
