**2. Application of insecticides nanoformulations**

#### **2.1. Nanoemulsions**

merous losses that are caused by insects on agriculture, several chemicals have been used to kill them or inhibit their reproduction and feeding habits. Those classes of compounds are collectivity known as insecticides. These molecules are able to interfere in the insect metabo‐ lism. They alter is in such a way that the plague cannot feeds on the crop or the harvest or even reproduce anymore. The use of insecticides is described since ancient times, with docu‐ ments providing evidences as far as in the 16th century BC. The *Ebers Papyrus*, wrote by the Egyptians, reports several chemical and organic substances used against overcome fleas, gnats and biting flies among others [4]. Nowadays, the insecticides are widely employed around the world. Several known substances are extremely effective in controlling or even wiping out almost all important agricultural plagues. This multi-billion-dollar has an esti‐ mated production of 2 million metric tons of hundreds of chemical and biological different

Insecticides are used in different ways, based on the physical-chemical characteristics of the each chemical substance, the area that needs to be covered and the target. Typical applica‐ tion of insecticides in crops is made by spraying a solution, emulsion or colloidal suspension containing the active chemical compound, which is made by a vehicle which may be a hand pump, a tractor or even a plane. This mixture is prepared using a liquid as a carrier, usually water, to ensure a homogenous distribution. Other methods for applying insecticides are through foggers or granule baits embedded with the active compound, among others that are less used. However, due to several degradation processes, such as leaching or destruc‐ tion by light, temperature, microorganism or even water (hydrolysis), only a small amount of these chemical products reaches the target site. In this case, the applied concentrations of these compounds have been much higher than the required. On the other hand, the concen‐ tration that reaches its target might be lower than the minimum effective one. In general, de‐ pending of the weather and method of application, the amount of applied agrochemicals, as much as 90%, may not reach the target and so do not produce the desired biological re‐ sponse. For this reason, repeated application of pesticides become hence necessary to effi‐ cient control of target plagues, which increase the cost and might cause undesirable and serious consequences to the ecosystems, affecting human health [6]. Due to the lack of selec‐ tivity, their unrestrained use can also lead to the elimination of the natural enemies, what implies in the fast growth of plague population. Moreover, it often makes the insects resist‐

Another important point that needs attention is the formulation for the application of the in‐ secticide on the crops. There are several different classes of compounds, which sometimes do not match with a simple dilution in water and must be prepared by other means such as powders, emulsions or suspensions. Some kinds of formulations must be handled with more precaution, since it can severely contaminate workers on the field with small airborne

The advances in science and technology in the last decades were made in several areas of insecticide usage. It includes either the development of more effective and non-persistent pesticides and new ways of application, which includes controlled release formulations (CRFs). The endeavors are direct towards the successful application of those compounds on

products, with a budget of a US\$35 billion dollars worldwide [5].

522 Insecticides - Development of Safer and More Effective Technologies

ant to the pesticides.

solid particles that can be inhaled [7].

Casanova *et al*. [9] evaluated the production of a nicotine carboxylate nanoemulsion using a series of fatty acids (C10 – C18) and surfactant. The oil-in-water nanoemulsion showed a monomodal distribution of size, with mean particle sizes of 100nm. The bioactivity of the insecticide formulations was evaluated against adults of *Drosophila melanogaster* by assessing the lethal time 50 (LT50). They observed that the encapsulation efficiency decreased with in‐ creasing size of the fatty acids tested. The bioactivity followed the same trend, with better bioactivity when the chain length decreased. This would be readily attributed to the higher amount of active compound inside the nanoemulsion. For the smallest fatty acid emulsion used, the capric acid (C10) one, the greatest encapsulation efficiency was observed, but it had the lowest bioactivity. The results were explained in terms of lesser bioavailability of the insecticide in its active form due to increased stability of the organic salt formed between the insecticide and the fatty acid. This experiment highlights the necessity of developing differ‐ ent kinds of possible assembles between the active compounds and matrix, and extensively studying the interactions in nanoscale formulations, where sometimes nontrivial effects might be unexpectedly observed.

Wang *et al.*[10] developed an assemble of oil-in-water nanoemulsion (O/W) with 30 nm droplets by careful control of experiment conditions, using the neutral surfactant poly(oxy‐ ethylene) lauryl ether and methyl decanoate to encapsulate highly insoluble β-cypermeth‐ rin. The dissolution of the insecticide was enhanced. The stability tests were performed by spraying nanoemulsion in a glass slide and observing under polarizing light microscopy. They showed no apparent precipitate in nanoemulsions samples. These results were differ‐ ent from the ones obtained using a commercial β-cypermethrin formulation, with apparent signs of solid residues after 24 hours. This enhanced stability may be used to decrease the concentration of insecticides in commercial spray applications, without losing efficiency.

#### **2.2. Classical micro and nanoparticles**

Allan *et al.* [11] published the first report on a controlled release system of an insecticide through a polymeric encapsulation. Even so, at first the encapsulated systems were not so effective. Problems associated with controlled release and particle stability hindered their practical field application for some decades. In one of the first successful works in the field of pesticides encapsulation, Greene *et al.* [12] used poly (n-alkyl acrylates) (Intelimer®) to produce temperature-sensitive microcapsules of the organophosphate insecticide diazinon.. The active chemical was controlled release by increasing the ambient temperature above 30ºC, which is the melting temperature of the polymer,. Experiments were performed with Banded cucumber beetle *Diabrotica balteata* and Western corn rootworm *Diabrotica virgifera* as target insects at 20ºC and 32ºC, under and above the polymer melting point respectively. Mortality was compared to commercial granular formulation. At lower temperatures, the commercial formulation showed the best mortality. At higher temperatures the activity of the encapsulated formulation was better, showing about 90% of mortality for over 8 weeks. The commercial formulation had indeed lost some of its activity, presumably due to heat degradation.

clusion of poly(ε-caprolactone) (PCL) into the matrix in 50-80% weight were analyzed, with formation of microspheres of PLA/PCL blend with 20-120µm of the diameter, showing up to

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525

In contrast to conventional desire to produce compounds with extended residual activity, quick-release microcapsules are demanded in certain areas of agriculture. However, some‐ times it is also necessary a quick liberation of the active compound from the matrix after the application. The strong backbone might pose as a problem to effectively deliver. Studies per‐ formed by Tsuda *et al.* [18,19] have shown that is possible to assemble "self-bursting" micro‐ capsules that retain its form in water suspension, but easily burst after solvent evaporation. They used the interfacial polymerization method to assemble spherical polyurethane micro‐ capsules containing the insecticide pyriprofixen, obtaining particles with mean diameter of 23µm. The entrapment ratio was 99% for all formulations tested, greatly improving the solu‐ bility of the pesticide in water. According to the results, there is a correlation between the wall thickness of the microcapsules and the self-bursting phenomenon. Tuning this property

The effectiveness of encapsulated formulations, it is not restricted to extend the residual activi‐ ty of insecticides, but should also include the overcoming of problems associated with accu‐ mulation of recalcitrant organic pollutants that remains in ecosystems in amounts above the Maximum Residual Level (MRL). Therefore, it can be harmful to the environment and to peo‐ ple who might consume the treated crops. For instance, Guan *et al.* [20] encapsulated imidaclo‐ prid, a chloro-nicotinyl systemic and broad spectrum insecticide in a mixed sodium alginate/ chitosan microparticle through self-assembly layer-by-layer (LbL) methodology. The capsules showed a mean diameter of 7µm. Particles were impregnated with a photocatalyst made of SDS/TiO2/Ag, and the photocatalytic property and the insecticidal activity of the microcap‐ sule was evaluated. Prolonged residual activity of the encapsulated formulation was ob‐ served. The toxicity was higher in the *Martianus dermestoides* adult stage compared to the one of pure insecticide. In a field test with soybean [21], the nano-imidacloprid formulation prevent‐ ed the accumulation of the pesticide on the soybean leaves and soil. The results showed pro‐ nounced degradation over 25 days of trials when compared to commercial concentrate formulations, even though the initial concentration of both formulations was equivalent. In this way, regardless the initial effectiveness of the insecticide, safer levels of agrochemicals can

Besides the chemical compounds, the micro- and nanotechnology have also been developed and applied to microorganisms that need special protection or to improve their solubility in aqueous phase. For instance, Ramírez-Lepe *et al.* [22] developed an aluminium-carboxyme‐ thylcellulose microcapsule with photoprotective agents for holding a *Bacillus thuringiensis* serovar *israelensis* (B.t.i.) spore-toxin complex named δ-endotoxin. The protein produced by this gram-positive bacterium during sporulation is extremely toxic to larval stage of some mosquitoes and flies which are vectors for important tropical diseases such as malaria and dengue. The encapsulated formulation was tested for its UV irradiation protective efficiency

be obtained in less time, improving the safety of insecticide application.

**2.3. Entomopathogenic microorganisms encapsulated**

88,5% of insecticide release in aqueous media over a 48h period.

a controlled released can be achieved.

Latheef *et al.* [13] tested several different polymers such as poly (methyl methacrylate) (PMMA), ethyl cellulose, poly(α-methylstyrene) and cellulose acetate butyrate to produce microcapsules of the insecticide sulprofos. Ethyl cellulose formulations were the only ones that had shown good results against eggs and larvae of the tobacco budworm *Heliothis vires‐ cens* in cotton plants. The results were comparable to the ones obtained with the use of an emulsifable-concentrate (EC) commercial formulation of sulprofos.

In other to develop commercial formulation containing microencapsulated cyfluthrin, Ar‐ thur[14] evaluated its use against the rice weevil *Sitophilus oryzae* in stored wheat, for a peri‐ od of 8 months. Survival of beetles was statistically correlated with the concentration of the pyrethroid insecticide in the formulation. The average survival rate was only 12% when 4ppm was used, with constant activity throughout the entire experiment. This evidenced the controlled release of the substance over a long period of time.

In the work carried out by Quaglia *et al.* [15], a hydrophobic waxy prepared through a mix‐ ture of di- and triglycerides of PEG esters was used to construct microspheres containing the insecticide carbaryl. Microparticles was obtained with particle size ranging from 16 to 20µm. Controllable release dynamics depended on the amount of gelucire used, Studies of release profiles from the encapsulated formulation showed a lower vertical mobility of the insecti‐ cide when compared to a commercial nonencapsulated formation. This suggested that the controlled release profile of the microcapsules may be useful to avoid or minimize ground‐ water contamination.

Cao *et al.* [16] produced diffusion-controlled microcapsules with diameter ranging from 2 to 20µm with encapsulated acetamiprid, an alkaline and high temperature-sensitive insecti‐ cide, using tapioca starch as matrix with urea and sodium borate as additives. The particle showed increased degradation resistance by heat for 60 days, and UV radiation over 48h, with no more than 3% of degradation. This represents less than one tenth when compared to the UV degradation of commercial emulsifable concentrate. Even in those conditions, it was also able to promote controlled liberation of the active compound for up to 10 weeks de‐ pending on the formulation used.

In another work with acetamiprid, Takei *et al.* [17]produced microparticles with diameter of 30-150µm using poly-lactide (PLA) as the polymeric matrix. Initial results showed that mi‐ crospheres containing only PLA did not have a good release kinetic of the active chemical compound from its interior. It is presumably due to their tight structure and high hydropho‐ bicity, which hinders water diffusion and therefore limits the insecticide liberation. The in‐ clusion of poly(ε-caprolactone) (PCL) into the matrix in 50-80% weight were analyzed, with formation of microspheres of PLA/PCL blend with 20-120µm of the diameter, showing up to 88,5% of insecticide release in aqueous media over a 48h period.

30ºC, which is the melting temperature of the polymer,. Experiments were performed with Banded cucumber beetle *Diabrotica balteata* and Western corn rootworm *Diabrotica virgifera* as target insects at 20ºC and 32ºC, under and above the polymer melting point respectively. Mortality was compared to commercial granular formulation. At lower temperatures, the commercial formulation showed the best mortality. At higher temperatures the activity of the encapsulated formulation was better, showing about 90% of mortality for over 8 weeks. The commercial formulation had indeed lost some of its activity, presumably due to heat

Latheef *et al.* [13] tested several different polymers such as poly (methyl methacrylate) (PMMA), ethyl cellulose, poly(α-methylstyrene) and cellulose acetate butyrate to produce microcapsules of the insecticide sulprofos. Ethyl cellulose formulations were the only ones that had shown good results against eggs and larvae of the tobacco budworm *Heliothis vires‐ cens* in cotton plants. The results were comparable to the ones obtained with the use of an

In other to develop commercial formulation containing microencapsulated cyfluthrin, Ar‐ thur[14] evaluated its use against the rice weevil *Sitophilus oryzae* in stored wheat, for a peri‐ od of 8 months. Survival of beetles was statistically correlated with the concentration of the pyrethroid insecticide in the formulation. The average survival rate was only 12% when 4ppm was used, with constant activity throughout the entire experiment. This evidenced the

In the work carried out by Quaglia *et al.* [15], a hydrophobic waxy prepared through a mix‐ ture of di- and triglycerides of PEG esters was used to construct microspheres containing the insecticide carbaryl. Microparticles was obtained with particle size ranging from 16 to 20µm. Controllable release dynamics depended on the amount of gelucire used, Studies of release profiles from the encapsulated formulation showed a lower vertical mobility of the insecti‐ cide when compared to a commercial nonencapsulated formation. This suggested that the controlled release profile of the microcapsules may be useful to avoid or minimize ground‐

Cao *et al.* [16] produced diffusion-controlled microcapsules with diameter ranging from 2 to 20µm with encapsulated acetamiprid, an alkaline and high temperature-sensitive insecti‐ cide, using tapioca starch as matrix with urea and sodium borate as additives. The particle showed increased degradation resistance by heat for 60 days, and UV radiation over 48h, with no more than 3% of degradation. This represents less than one tenth when compared to the UV degradation of commercial emulsifable concentrate. Even in those conditions, it was also able to promote controlled liberation of the active compound for up to 10 weeks de‐

In another work with acetamiprid, Takei *et al.* [17]produced microparticles with diameter of 30-150µm using poly-lactide (PLA) as the polymeric matrix. Initial results showed that mi‐ crospheres containing only PLA did not have a good release kinetic of the active chemical compound from its interior. It is presumably due to their tight structure and high hydropho‐ bicity, which hinders water diffusion and therefore limits the insecticide liberation. The in‐

emulsifable-concentrate (EC) commercial formulation of sulprofos.

524 Insecticides - Development of Safer and More Effective Technologies

controlled release of the substance over a long period of time.

degradation.

water contamination.

pending on the formulation used.

In contrast to conventional desire to produce compounds with extended residual activity, quick-release microcapsules are demanded in certain areas of agriculture. However, some‐ times it is also necessary a quick liberation of the active compound from the matrix after the application. The strong backbone might pose as a problem to effectively deliver. Studies per‐ formed by Tsuda *et al.* [18,19] have shown that is possible to assemble "self-bursting" micro‐ capsules that retain its form in water suspension, but easily burst after solvent evaporation. They used the interfacial polymerization method to assemble spherical polyurethane micro‐ capsules containing the insecticide pyriprofixen, obtaining particles with mean diameter of 23µm. The entrapment ratio was 99% for all formulations tested, greatly improving the solu‐ bility of the pesticide in water. According to the results, there is a correlation between the wall thickness of the microcapsules and the self-bursting phenomenon. Tuning this property a controlled released can be achieved.

The effectiveness of encapsulated formulations, it is not restricted to extend the residual activi‐ ty of insecticides, but should also include the overcoming of problems associated with accu‐ mulation of recalcitrant organic pollutants that remains in ecosystems in amounts above the Maximum Residual Level (MRL). Therefore, it can be harmful to the environment and to peo‐ ple who might consume the treated crops. For instance, Guan *et al.* [20] encapsulated imidaclo‐ prid, a chloro-nicotinyl systemic and broad spectrum insecticide in a mixed sodium alginate/ chitosan microparticle through self-assembly layer-by-layer (LbL) methodology. The capsules showed a mean diameter of 7µm. Particles were impregnated with a photocatalyst made of SDS/TiO2/Ag, and the photocatalytic property and the insecticidal activity of the microcap‐ sule was evaluated. Prolonged residual activity of the encapsulated formulation was ob‐ served. The toxicity was higher in the *Martianus dermestoides* adult stage compared to the one of pure insecticide. In a field test with soybean [21], the nano-imidacloprid formulation prevent‐ ed the accumulation of the pesticide on the soybean leaves and soil. The results showed pro‐ nounced degradation over 25 days of trials when compared to commercial concentrate formulations, even though the initial concentration of both formulations was equivalent. In this way, regardless the initial effectiveness of the insecticide, safer levels of agrochemicals can be obtained in less time, improving the safety of insecticide application.

#### **2.3. Entomopathogenic microorganisms encapsulated**

Besides the chemical compounds, the micro- and nanotechnology have also been developed and applied to microorganisms that need special protection or to improve their solubility in aqueous phase. For instance, Ramírez-Lepe *et al.* [22] developed an aluminium-carboxyme‐ thylcellulose microcapsule with photoprotective agents for holding a *Bacillus thuringiensis* serovar *israelensis* (B.t.i.) spore-toxin complex named δ-endotoxin. The protein produced by this gram-positive bacterium during sporulation is extremely toxic to larval stage of some mosquitoes and flies which are vectors for important tropical diseases such as malaria and dengue. The encapsulated formulation was tested for its UV irradiation protective efficiency in laboratory conditions. While the protein in its natural form had lost all of its activity after 24 hours of exposure, encapsulated formulations showed up to 88% of larvae mortality.

released was proportional to the initial oil concentration on particles, with less than 20% of liberation for low oil concentrations, and about 60% liberation for high oil concentration.

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527

Yang *et al.* [32] assembled polyethylene glycol (PEG) nanoparticles loaded with garlic essen‐ tial oil using a melt-dispersion method, reaching over 80% of encapsulation efficiency, with round shaped nanoparticles of lower 240nm of average diameter. The encapsulated formu‐ lations had their insecticidal activity evaluated against adult red flour beetle *Tribolium casta‐ neum*. While the control experiment done with free garlic oil showed only 11% of efficiency over a five month period, the encapsulated formulation efficiency remained over 80% after five months. This was attributed to the slow and controlled release of the essential oil, and

The basic structure of the polymer chitosan was used by Lao *et al*. [33] to build the amphi‐ philic-modified *N*-(octadecanol-1-glycidyl ether)-*O*-sulfate chitosan (NOSCS). Octadecanol glycidyl ether and sulfate were the hydrophobic and the hydrophilic groups sources respec‐ tively. They successfully entrap the herbal insecticide rotenone in the polymer. This chemi‐ cal compound has been allowed for application in organic crop production due to its natural origin, short persistence in the environment, safety to non-target organisms and low resist‐ ance development. The encapsulation was necessary to defeat the problems of chemical sta‐ bility of the substance to environmental effects and also to improve the solubility of this pesticide in water, which is usually quite low (2.0x10-6g.L-1). Using the reverse micelle meth‐ od, the authors have assembled nanometric micelles with 167.7-214.0 nm of diameter, with values of critical micellar concentration (CMC) of those chitosan derivatives ranging from 3.55×10−3 to 5.50×10−3 g.L-1. Although the entrapment efficiency was not very high, they also improved the aqueous solubility of the chemical compound in 13,000 fold, up to 0.026g.L-1, favoring a controlled release of the substance in aqueous media. The complete controlled re‐ lease took more than 230 hours, almost 10 times more when compared to the chemical com‐

Chitosan derivatives were prepared [34]. They synthesized 6-*O*-carboxymethylated chitosan with anchorage of ricinoleic acid at the *N*-linkage, which further improve its solubility at neu‐ tral water (pH = 7.0), to encapsulate the herbal insecticide azadirachtin. Nanoparticles of 200-500nm were obtained by water dispersion with more than 50% of loading efficiency and tested for their stability in outdoor as controlled release systems. Results were compared against simple azadirachtin water dispersion and modified dispersion containing ricinoleic acid and azadirachtin. In 5 days of sun exposure, all content of control samples were lost, while the encapsulated formulation had a nearly constant residual concentration detected through‐ out the 12 days of the experiment, indicating that the nanoparticles produced were effective at controlling the degradation rate and the release mechanism of the botanical insecticide.

Extracts of Neem were prepared contend high concentration of azadirachtin being nanoen‐ capsulated by Forim *et al.* [35]. Through the use of poly-(ε-caprolactone) polymer, they pre‐ pared nanocapsules and nanospheres with average diameter of 150.0 and 250.0nm, respectively. The morphological analysis revealed spherical nanoparticles (Figure 1). The azadirachtin was used as reference. The nanoformulations showed high entrapment efficien‐

thus could be used as an effective pest control to stored products.

pound without nanoencapsulation.

In their turn, Tamez-Guerra *et al.* [23] also tested the encapsulation of the spore-toxin of *Ba‐ cillus thuringiensis* Berliner, evaluating over 80 formulations of spray-dried microcapsules made of lignin and corn flour with and without photoprotective agents. The best formula‐ tions showed improved insecticidal activity in laboratory tests against neonates of European corn borer *Ostrinia nubilalis* when compared to nonencapsulated or commercial formula‐ tions of the same endotoxin. In a field test, the microcapsules showed increased residual in‐ secticide activity in cabbage after 7 days against neonates of the cabbage looper *Trichoplusia ni* when compared to commercial formulations.

Very promising results have been obtained by the Agricultural Research Service of the US‐ DA regarding the encapsulation of biopesticides made of species-specific nucleopolyhedro‐ viruses (NPV) isolated from several insects, including celery looper *Anagrapha falcifera* (Tamez-Guerra *et al*., 2000 [24-26]), alfalfa looper *Autographa californica* [27], codling moth *Cydia pomonella* [28] and fall armyworm *Spodoptera frugiperda* [29]. In these works, formula‐ tions were developed using different mixtures of corn flour and lignin, through spray-dry‐ ing technique to encapsulate the viruses. All results obtained in laboratory and field tests performed have shown improvements in insecticidal activity, resistance to environmental conditions, like rain and UV light exposure, and a prolonged residual activity against pests in field studies. Samples were kept in storage for up to 12 months and maintained their in‐ secticidal activity.

#### **2.4. Novel micro and nanoparticles for bioinseticides**

Conventional protocols for encapsulation usually run under relatively high temperatures, which might be inadequate for preserving plant-derived essential oils integrity. Processes which use high pressure instead of temperature can be an alternative for encapsulating these sensible extracts. Varona *et al.* [30,31] developed new methods to produce stable parti‐ cles of lavandin (*Lavandula hybrida*) essential oil, using polyethylene glycol 9000 (PEG9000) or n-octenyl succinic (OSA) modified starches as the shell material. The methods for prepar‐ ing the microcapsules were based on PEG precipitation from a mixture of molten polymer and essential oil in supercritical CO2, and PGSS-drying an oil-in-water emulsion of the es‐ sential oil with OSA starch. The difference between these processes is the presence of water on the latter, which needs to be removed by carefully tuning the equipment conditions to promote water evaporation. Microcapsules produced by these methods show a mean parti‐ cle size of 10-500µm for PGSS, and 1-100µm for PGSS-drying. One important observation by scanning electron microscopy (SEM) images is that the experimental conditions can influ‐ ence the shape of the microparticles. While PEG particles were only spherical (the best shape for controlled release mechanism), in PGSS-drying needle-like structures are formed,, de‐ pending on the pre-expansion temperatures of the mixtures, The last one, probably does not hold the active ingredient, presenting some limitations to this specific method without fur‐ ther improvements. Release kinetics were evaluated over a 20-day period. The amount of oil released was proportional to the initial oil concentration on particles, with less than 20% of liberation for low oil concentrations, and about 60% liberation for high oil concentration.

in laboratory conditions. While the protein in its natural form had lost all of its activity after 24 hours of exposure, encapsulated formulations showed up to 88% of larvae mortality.

In their turn, Tamez-Guerra *et al.* [23] also tested the encapsulation of the spore-toxin of *Ba‐ cillus thuringiensis* Berliner, evaluating over 80 formulations of spray-dried microcapsules made of lignin and corn flour with and without photoprotective agents. The best formula‐ tions showed improved insecticidal activity in laboratory tests against neonates of European corn borer *Ostrinia nubilalis* when compared to nonencapsulated or commercial formula‐ tions of the same endotoxin. In a field test, the microcapsules showed increased residual in‐ secticide activity in cabbage after 7 days against neonates of the cabbage looper *Trichoplusia*

Very promising results have been obtained by the Agricultural Research Service of the US‐ DA regarding the encapsulation of biopesticides made of species-specific nucleopolyhedro‐ viruses (NPV) isolated from several insects, including celery looper *Anagrapha falcifera* (Tamez-Guerra *et al*., 2000 [24-26]), alfalfa looper *Autographa californica* [27], codling moth *Cydia pomonella* [28] and fall armyworm *Spodoptera frugiperda* [29]. In these works, formula‐ tions were developed using different mixtures of corn flour and lignin, through spray-dry‐ ing technique to encapsulate the viruses. All results obtained in laboratory and field tests performed have shown improvements in insecticidal activity, resistance to environmental conditions, like rain and UV light exposure, and a prolonged residual activity against pests in field studies. Samples were kept in storage for up to 12 months and maintained their in‐

Conventional protocols for encapsulation usually run under relatively high temperatures, which might be inadequate for preserving plant-derived essential oils integrity. Processes which use high pressure instead of temperature can be an alternative for encapsulating these sensible extracts. Varona *et al.* [30,31] developed new methods to produce stable parti‐ cles of lavandin (*Lavandula hybrida*) essential oil, using polyethylene glycol 9000 (PEG9000) or n-octenyl succinic (OSA) modified starches as the shell material. The methods for prepar‐ ing the microcapsules were based on PEG precipitation from a mixture of molten polymer and essential oil in supercritical CO2, and PGSS-drying an oil-in-water emulsion of the es‐ sential oil with OSA starch. The difference between these processes is the presence of water on the latter, which needs to be removed by carefully tuning the equipment conditions to promote water evaporation. Microcapsules produced by these methods show a mean parti‐ cle size of 10-500µm for PGSS, and 1-100µm for PGSS-drying. One important observation by scanning electron microscopy (SEM) images is that the experimental conditions can influ‐ ence the shape of the microparticles. While PEG particles were only spherical (the best shape for controlled release mechanism), in PGSS-drying needle-like structures are formed,, de‐ pending on the pre-expansion temperatures of the mixtures, The last one, probably does not hold the active ingredient, presenting some limitations to this specific method without fur‐ ther improvements. Release kinetics were evaluated over a 20-day period. The amount of oil

*ni* when compared to commercial formulations.

526 Insecticides - Development of Safer and More Effective Technologies

**2.4. Novel micro and nanoparticles for bioinseticides**

secticidal activity.

Yang *et al.* [32] assembled polyethylene glycol (PEG) nanoparticles loaded with garlic essen‐ tial oil using a melt-dispersion method, reaching over 80% of encapsulation efficiency, with round shaped nanoparticles of lower 240nm of average diameter. The encapsulated formu‐ lations had their insecticidal activity evaluated against adult red flour beetle *Tribolium casta‐ neum*. While the control experiment done with free garlic oil showed only 11% of efficiency over a five month period, the encapsulated formulation efficiency remained over 80% after five months. This was attributed to the slow and controlled release of the essential oil, and thus could be used as an effective pest control to stored products.

The basic structure of the polymer chitosan was used by Lao *et al*. [33] to build the amphi‐ philic-modified *N*-(octadecanol-1-glycidyl ether)-*O*-sulfate chitosan (NOSCS). Octadecanol glycidyl ether and sulfate were the hydrophobic and the hydrophilic groups sources respec‐ tively. They successfully entrap the herbal insecticide rotenone in the polymer. This chemi‐ cal compound has been allowed for application in organic crop production due to its natural origin, short persistence in the environment, safety to non-target organisms and low resist‐ ance development. The encapsulation was necessary to defeat the problems of chemical sta‐ bility of the substance to environmental effects and also to improve the solubility of this pesticide in water, which is usually quite low (2.0x10-6g.L-1). Using the reverse micelle meth‐ od, the authors have assembled nanometric micelles with 167.7-214.0 nm of diameter, with values of critical micellar concentration (CMC) of those chitosan derivatives ranging from 3.55×10−3 to 5.50×10−3 g.L-1. Although the entrapment efficiency was not very high, they also improved the aqueous solubility of the chemical compound in 13,000 fold, up to 0.026g.L-1, favoring a controlled release of the substance in aqueous media. The complete controlled re‐ lease took more than 230 hours, almost 10 times more when compared to the chemical com‐ pound without nanoencapsulation.

Chitosan derivatives were prepared [34]. They synthesized 6-*O*-carboxymethylated chitosan with anchorage of ricinoleic acid at the *N*-linkage, which further improve its solubility at neu‐ tral water (pH = 7.0), to encapsulate the herbal insecticide azadirachtin. Nanoparticles of 200-500nm were obtained by water dispersion with more than 50% of loading efficiency and tested for their stability in outdoor as controlled release systems. Results were compared against simple azadirachtin water dispersion and modified dispersion containing ricinoleic acid and azadirachtin. In 5 days of sun exposure, all content of control samples were lost, while the encapsulated formulation had a nearly constant residual concentration detected through‐ out the 12 days of the experiment, indicating that the nanoparticles produced were effective at controlling the degradation rate and the release mechanism of the botanical insecticide.

Extracts of Neem were prepared contend high concentration of azadirachtin being nanoen‐ capsulated by Forim *et al.* [35]. Through the use of poly-(ε-caprolactone) polymer, they pre‐ pared nanocapsules and nanospheres with average diameter of 150.0 and 250.0nm, respectively. The morphological analysis revealed spherical nanoparticles (Figure 1). The azadirachtin was used as reference. The nanoformulations showed high entrapment efficien‐ cy (> 95%) for this compound and a UV stability at least of 30 times more when compared with commercial products.

ruling the (bio) availability of the active compound after the application [48]. They find the greatest applicabilities in two major agricultural fields: nutrition and protection. In the first one, CRFs are employed in the delivery of fertilizers [49-51]. In the second one, CRFs are mostly used to target plague insects in a sustainable way [52,53], but they can also be ap‐ plied to block the growth of weeds [54]. Tomioka *et al.*, 2010. Controlled release formula‐ tions become especially interesting in cases of antagonist activity of biocides, what can naturally leads to a lower in effectiveness of one or both compounds. In this case the formu‐ lation should be "programmed" to release each one at different times [55,56]. Furthermore, still talking about protection, the application of CRFs in wood surfaces, like furniture or floor covering, helps to prevent the deterioration. Van Voris *et al.* [57] patented a formula‐ tion in which an insecticide is continually released in a minimum level for a long period of time and is absorbed by the wood. It thus creates a "chemical barrier", blocking the insect

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Most of those controlled release biocides applications were and still are successfully made

Micro- and nanomaterials-based formulations are known for some decades. The first micro‐ capsule-based formulation became commercially available in the 1970s [58]. Nanocapsules have been widely used in medicinal area as drug carrier in treatment of diverse diseases

Microencapsulation has been used as a versatile tool for hydrophobic pesticides, enhancing their dispersion in aqueous media and allowing a controlled release of the active compound. The use of nanotechnology is a recent approach, and has been a growing subject on several different areas of the science, with an overwhelming perspective. In general, materials that are assembled in nanometric scales (<1000nm) have distinct and almost always better char‐ acteristics when compared to the same material built in a conventional manner [62]. One

rials in nanometric scale may be controlled to promote an efficient assemble of a structure which could present several advantages, such as the possibility to better interaction and mode of action at a target site of the plant or in a desired pest due to its tunable controlled release system and larger superficial area, acting as an artificial immune system for plants [34,63]. As smart delivery systems, they confer more selectivity, without hindering in the bi‐ oactive compounds towards the target pathogen [65]. Other advantages of the use of nano‐ particle insecticides are the possibility of preparing formulations which contain insoluble compounds that can be more readily dispersed in solution. It reduces the problems associat‐ ed with drifting and leaching, due to its solid nature, and leads to a more effective interac‐ tion with the target insect. These features enable the use of smaller amount of active compound per area, as long as the formulation may provide an optimal concentration deliv‐ ery for the target insecticide for longer times. Since there is no need for re-applications, they also decrease the costs), reduce the irritation of the human mucous-membrane, the phyto‐ toxicity, and the environmental damage to other untargeted organisms and even the crops themselves [65,66]. In a few words, nanotechnology can be applied in several ways in order

m). In general, the chemical properties of mate‐

attacks.

due to the advances in nanotechnology area.

[59], from tropical ones [60] up to cancer [61].

nanometer is a billionth of a meter (1nm = 109

to enhance efficacy of insecticides in crops.

**Figure 1.** Scanning electron microscopy images of nanoparticles containing extracts of Neem.

#### **2.5. Commercial products**

The interesting results obtained in academic researches over the last few decades have been closely followed by several companies. Nevertheless, R&D in nano-based agrochemicals is led mainly by world's largest agroscience companies, further enhancing their market share and consolidating the market structure based on oligopoly that have been seen in late 20th century and early 21th century, when the 10 biggest companies hold around 80% of market [36].

Some companies over the last decade, such as Syngenta, Bayer, Monsanto, Sumitomo, BASF, and Dow Agrosciences have already deposited several different patents comprising a wide range of protocols for production and application of encapsulated formulations, which can be used to produce nanoinsecticides [37-46]. Despite the hard work and heavy investment, no commercial nano-insecticide formulation has been extensively commercialized up to 2012.

Along with those big industries, several other companies, as well as individual researches have been actively depositing patents in the area, thus promoting even more the research and investments in this new field of applied technology. However, as strongly reinforced throughout the world by dozens of organizations such as the ETC Group, the impact of nanotechnology is still unclear, and care should be taken to assure that its use will not bring more problems than solutions [47].
