Photocatalytic Degradation of Carbamate Pesticide (methomyl) Using Synthesized TiO2 Nanoparticles Against the Cotton Leafworm S. littoralis

The journal of Toxicology and pest control is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers related to the interaction between insects and their environment. The goal of the journal is to advance the scientific understanding of mechanisms of toxicity. Emphasis will be placed on toxic effects observed at relevant exposures, which have direct impact on safety evaluation and risk assessment. The journal therefore welcomes papers on biology ranging from molecular and cell biology, biochemistry and physiology to ecology and environment, also systematics, microbiology, toxicology, hydrobiology, radiobiology and biotechnology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
The Egyptian cotton leafworm, S. littoralis (Boisd.)(Noctuidae: Lepidoptera) is an economically important pest with a wide range of host plants.This species has acquired resistance to many insecticides (Hassanein, 1999).The increased use of several groups of chemical pesticides to control this insect has led to environmental pollution causing danger to all organisms including man Intense and massive agricultural methods ultimately lead to accumulation of pesticides in high concentrations for long durations of time in soil.Also, pesticides can enter the water through spills, leaks, and back siphoning from nearby mixing, loading, storage, and equipment cleanup sites.
The conventional insecticide, methomyl was used for the lepidopterous pests control (Kassem et al., 1986).Methomyl, which has been classified by the World Health Organization, Environmental Protection Agency, USA, and European Commission as a very toxic and hazardous pesticide (Strathmann et al., 2001) is highly soluble in water (57.9 g/l, 20°C).It also has a low sorption affinity to soil and can therefore easily cause groundwater contamination in the agricultural areas (Malato et al., 2002).In addition, various amounts of methomyl have been detected in surface and ground waters across Europe and America, not only during actual insecticide application but also after a long period of use (Strathmann et al., 2001).Methomyl is widely used all over the world because of its powerful control of many different pests (Fernández et al., 2002).Studies on the use of various sorbets for the adsorption of methomyl have been conducted, including activated carbon (Chang et al., 2012), marine sediments (Yang et al., 2005), and hypercrosslinked polymers (Chang et al., 2008).Traditional and more expensive methods such as cation exchange (Hesketh et al., 1996) and dialysis (Devitt et al., 1998) have been also introduced to remove other pesticides.The use of enzymes to detoxify wastewater failed to attract much attention due to the high cost of enzyme-based systems (Kauffmann et al., 2000).Filtration through membranes needs another method such as oxidation reaction catalyzed by enzymes to transform the pesticide into an insoluble product; therefore this method is highly expensive (Boussahel et al., 2000).Photocatalytic degradation processes introduced themselves as effective strategies to remove the organic pollutants from the wastewaters.The main advantage of these processes is a complete degradation of contaminants to harmless compounds such as CO 2 , water and inorganic salts.Photocatalysis proved to be an excellent new advanced oxidation technology to eliminate methomyl present in the water.Moreover, the photocatalysis process is economically acceptable.The conventional photocatalysts such as TiO 2 and ZnO could be used with low initial concentration not only with methomyl (Tamimi et al., 2008 andPoulios et al., 2006) but also with other organic pollutants (Yousef et al., 2012 andYousef et al., 2012b).Nano crystalline TiO 2 which is prepared in an eco-friendly method and using visible light for the decomposition of organic pollutants is potential in the view of green chemistry.
The principle aim of the present study was to estimate the residual effect of methomyl mixed with nanomaterials against the S. littoralis larvae as evident for the detoxification of the pesticide.

Insect rearing
The cotton leaf worm, Spodoptera littoralis was reared in the laboratory at room temp.ranged between 25±2 -28 ±2 0 C and 60±5 -65±5% R.H. Larvae were fed on castor bean leaves, Ricinus communis (L.) in a wide glass jars until pupation period and adults emergence.The newly emerged adults were mated inside glass jars supplied with a piece of cotton wetted 10% sugar solution as feeding source for the emerged moths and branches of Tafla (Nerium oleander L.) or castor bean leaves as an oviposition site according by (El-Defrawi et al., 1964).Egg masses were kept in plastic jars until hatching.The obtained second and fourth instar larvae were used for bioassay tests.The bioassay evaluations were performed under the same laboratory condition, at temp.25±2− 28±2 o C and 60±5 -65±5% R.H.

Synthesis of nanomaterial
Analytical grade titanium tetrachloride was adopted as the source material and sodium hydroxide as mineralizer.An aqueous solution of titanium was obtained by mixing one molar stoichiometric ratio of titanium tetraisopropoxide (TTIP) in 50 ml of distilled water.The solution 2-3 mol of NaOH with stirring at several minutes, resulting in a white colloidal sol.The final volume was adjusted to 90 ml using distilled water.Therefore, 90ml sol was transferred to a 100 ml Teflon lined auto clave vessel.The sealed vessel was heated to 240 o C for 12 hrs and the resultant precipitate was dried at 450 o C for 2 hrs to obtain TiO 2 nanoparticles (Mahshid et al., 2007) .

Characterization of the Samples
XRD pattern of the investigated TiO 2 sample is illustrated in Fig. ( 1), the sample of TiO 2 revealed anatase form with excellent crystallinity as obvious from the peak intensities.It crystallized in the well-known tetragonal symmetry with 4 molecules per unit cell.The data were compared and indexed with the ICDD card no 21-1272.The crystal size was calculated using Scherer's formula (Klug et al., 1974) and was found to be 95nm.The FESEM image of the spherical TiO 2 nanoparticles is shown in Fig .(2), the TiO 2 grains appeared to be with homogenous distribution with a small degree of coalescence.The phase of TiO 2 was confirmed from FT-Raman analysis and the spectrum is shown in Fig. (3), where active peaks near 142, 305, 510 and 624 cm -1 are prominent for the anatase phase TiO 2 NPs because of Eg, B1g, A1g and Eg vibrational changes respectively.
Neither signal characteristics of brookite nor rutile phases of TiO 2 having Raman shifts in the range of 249 to 826 cm -1 respectively, appear in the spectra.and 50% for second and fourth instars respectively compared with 80 and 75% for methomyl nanoparticles mixture.
Table 1: Residual effect of methomyl mixed with TiO 2 nanoparticles against the 2 nd and 4 th instars of laboratory strain of Spodoptera littoralis larvae at 0, 3, 5, 7, 9 and 12 days after the treatment in relative to control.
The obtained results agree with those obtained by (Santhanalakshmi et al., 2012) studied the catalytic degradation of the four organopesticides, Chloropyrifos, Quinolphos, Imidocloprid and Endosulphon in water by Nano crystalline TiO2.They found that the increase in the amount of the catalyst generally increases the number of active sites on the catalyst surface and the mass or concentration of the catalyst when exceeds the optimum value, the degradation rate decreases.They indicated that the four pesticides are effectively and photo catalytically degraded at lower pH itself.Also, (Zaman et al., 2012) recorded that an efficient catalytic effect of petals and flowers like CuO nanostructures (NSs) on the degradation of two organic dyes, methylene blue (MB) and rhodamine B (RB).They indicated highest degradation of 95% in CuO and 72 % in flowers for MB at 24 h., while for RB, the degradation was 85 % and 80 % in petals and flowers, respectively for 5h.They observed that CuO petals appeared to be more active than flowers for degradation of both dyes associated to high specific surface area.Also, Barakat et al. (2014) reported that CdSO4-doped TiO2 nanoparticles are introduced as a powerful and reusable photocatalyst for the photocatalytic degradation of methomyl pesticide in concentrated aqueous solutions.They mentioned that the introduced nanoparticles could successfully eliminate the harmful pesticide under the sunlight radiation within a very short time (less than 1 h), with a removal capacity reaching 1,000 mg pesticide per gram of the introduced photocatalyst.They added that the increase in the initial concentration of the methomyl did not affect the photocatalytic performance; typically 300, 500, 1,000, and 2,000 mg/l solutions were completely treated within 30, 30, 40, and 60 min., respectively, using 100 mg catalyst, the photocatalytic efficiency was not affected upon multiple use of the photocatalys.Also, Khan et al. (2015) mentioned that Nanobased membrane filtration technology design and developed were found effective and efficient for the degradation of pesticides under UV light irradiation, as compared to photocatalytic degradation carried out using ZnO.

Latent effect: Larval and pupal durations:
Data presented in Tables (2 and 3) demonstrate the residual effect of TiO 2 nanoparticles methomyl mixture and methomyl alone reflect the biological activities of S. littoralis.The feeding of 2 nd and 4 th instar larvae on leaves previous sprayed with methomyl nanoparticles mixture after 12 days of application, significant (p<0.05)prolonged the larval duration to average 19 and 15days compared with 16.1 and 11.3 of control for both 2 nd and 4 th instar respectively.But the results differ for methomyl treatment alone as the larval duration of 2 nd instar decrease to 15.9days compared with 16.1 for control while it was found 16.8days for 4 th instar compared with 11.3 for control.
On the other hand data presented in Tables (2 and 3) demonstrate that the feeding of 2 nd and 4 th instar larvae on leaves previous sprayed with methomyl nanoparticles mixture after 12 days of application, decrese the pupal duration to average 13.3 for 2 nd instar compared with 16.1 for control.And for the 4 th instar found 13.9days compared with 12.7days of control.But the results differ for methomyl treatment alone as the pupal duration of 2 nd instar non significance as 9.1days compared with 8.7 for control while it was found that pupal duration decrease for 4 th treatment to 8.3days compared with 12.7 for control.
The obtained results agree with those obtained by (Morillo et al., 2004)who found that the duration of the larval and pupal stages and the developmental period from egg to adult of Spodoptera frugiperda was significantly longer in the lambdacyhalotrin-selected strain and the methomyl-selected strain compared to the control strain, from the first to the last generation.Moreover, (Ahmed 2004) mentioned that the larval period was elongated and the pupal period shorted for the new hatched larvae of pink and spiny bollworms (Laboratory strain) treated with the higher concentrations of Spinosad when compared with untreated larvae.

Pupation and Pupal weight:
Data presented in Tables (2 and 3) showed that the second instar larvae of S. littoralis which fed on the sprayed leaves after 12days from treatment with methomyl nanoparticles mixture treatments induced a highly significant (p<0.01)decrease of the pupation percentage in respect to control.Which caused pupation % decrease to average 39 and 30 for both 2 nd and 4 th instar respectively as compared to 100 %pupation of control.On the other hand, the larval feeding of 2 nd instar on the leaf residues aged 12 days with methomyl TiO 2 nanoparticles mixture highly significantly (p<0.01)only reduced the pupal weight of the resulting pupae to average182mg as compared to217mg of that of control.While, the feeding of the 4 th instar with methomyl nanoparticles mixture highly significant (p<0.01)decreased the pupal weight to average 168.9mg as compared to 272 mg of the pupal weight produced from untreated 4 th instar larvae.The obtained results are in harmony with that obtained by ( Swelam et al., 2006) who reported that at different combinations of insecticides, methomyl, carbaryl, esfenvalerate and profenofos used by mixing at the level of LC 25 with the ratios of 1: 2, 1: 1 and 2: 1 against S. littoralis appeared significant changes in the pupa weight compared with the control.Also, Ahmed (2004) found that the average of pupation percentages for pink and spiny bollworms gradually decreased with increasing concentrations of the tested compounds (Agerin, Diple 2x Naturalis L, Spinosad) in laboratory and field strains, respectively.

Moths emergence:
Results show that both of the 2 nd instar larvae fed on the sprayed leaves after 12days of application with the feeding of the instars with methomyl nanoparticles mixture and methomyl alone gave none significant decrease of the adult emergence averaged 80 and 85.7%, respectively, as relative to100%of that of control Tables (2 and 3).While the larval feeding of the 4 th instar on methomyl nanoparticles mixture had the effect in the adult emergence inhibition to 80%, as compared to 100%of that of control.
These results are agreement to those obtained by Ahmed (2004) who found that adult emergence for pink and spiny bollworms grdually decreased with increasing concentrations of the tested compounds (Agerin, Diple 2x Naturalis L , Spinosad) in laboratory strain.

Morphogenetic abnormalities:
Data presented in Tables (2 and 3) demonstrated that the larval feeding of 2 nd instars of S. littoralis on the leaf residues, methomyl TiO 2 nanoparticles mixture and methomyl alone didn't affect the pupae stage.But the larval treatment of the 4 th instar with methomyl alone induced the highest percentage to reach 33.3%, respectively, as compared to 0% of control.Also, the 2 nd instar larvae treated with methomyl TiO 2 nanoparticles mixture gave percent reached 20% adult malformation percentages as, compared with 0% of that of control, and the malformations of S .littoralispupae resulting from the larval treatment of the 2 nd and 4 th instars with mixtures and methomyl alone mostly as appeared as pupal moth intermediates as moth failed to emerge from the pupal skin at head and thorax (Fig. 6), or pupae with shrinkage body (Fig. 5), or moths had weakly developed wings with short body (Fig. 4), or pupal adult intermediate as moth failed to emerge (Fig. 3).As compared to normal pupae (Fig. 1) and Normal adult (Fig. 2).
These results are in agreement with to those obtained by (Javier et al., 2008) demonstrated that Align when administered orally Lobesia botrana gave phenotypic effects included inability to molt properly and deformities.Also, (Swelam et al., 2006) reported that at different combinations of insecticides, methomyl, carbaryl, esfenvalerate and profenofos by mixing at the level of LC 25 with the ratios of 1: 2, 1: 1 and 2: 1 used against S. littoralis produced some malformations in the pupae and moths stages.Also, Ahmed (2004) indicated that Spinosad gave malformed pupal and adults in both laboratory and field strains of both Pink and Spiny bollworms.

Adult fecundity and fertility
Data presented in Table (4) demonstrated that the larval feeding of S. littoralis on the leaf residues aged 12days sprayed with methomyl nanoparticles mixture and even the methomyl alone violent reduce the adult fecundity to reach zero eggs/female, as compared to206 eggs/female of that of control.
Therefore, the larval feeding of S. littoralis on the leaf residues aged12days sprayed with the tested methomyl TiO 2 nanoparticles mixture and even the methomyl alone inhibited the total number of eggs laid by adult females fed as 4 th instar larvae with these treatments and also the eggs hatching to zero, as compared to that of control(100%).These results are agreement with those obtained by (Javier et al., 2008) recorded that Align When administered orally, reduced the fecundity and fertility of adults of Lobesia botrana treated with 1, 5, and 10 mg litre -1 and at the highest doses, fecundity and fertility were zero.Also, (Pineda et al., 2007) demonstrated that spinosad and methoxyfenozide reduced in a dose-dependent manner the fecundity and fertility of S. littoralis adults when orally and residually treated.They reported that the combination of lethal and sublethal effects of methoxyfenozide and Spinosad might exhibit significant effects on the population dynamics of S. littolaris.Likewise, (Swelam et al., 2006).Found that some of the mixtures of insecticides, methomyl, carbaryl, esfenvalerate and profenofos at the level of LC25 with the ratios of 1: 2, 1: 1 and 2: 1 against S. littoralis showed sterility effect.Also, (Morillo et al., 2004) mentioned that the fertility of eggs of S. frugiperda diminished to 50.61 and 47.31% in the last generation, in the lambdacyhalotrinselected strain and the methomylselected strain, respectively.They indicated that the differences in the duration of some of the insect phases represent a reproductive deterioration in compensation of the survival to the process of selection pressure with the insecticides lambdacyhalotrin and methomyl.

Adult longevity
Data presented in Table (4) showed that feeding of the fourth instar larvae on the leaf residues aged 12days sprayed with methomyl nanoparticles mixture gave none significant decrease of the adult longevity, as respect of that control.These results contracted with those obtained by (Javier et al., 2008) who recorded that Align when administered orally, the longevity of Lobesia botrana adults was not affected.While (Morillo et al., 2004) found that the longevity of males and females of Spodoptera frugiperda only showed differences in some generations in the strains exposed to insecticides.

Adult sex ratio:
Data in Table (4) indicated that the 4 th instar larvae of S. littoralis fed on the leaf residues aged 12days sprayed with, methomyl nanoparticles mixture and methomyl alone gave adult males increase to 66.7%, and decreased the adult females to 33.3% as compared to50:50% adult females: males ratios, respectively, of that of control.

CONCLUSION
The results of the present work demonstrated that by adding the TiO 2 nanoparticles to methomyl pesticide decrease its toxicity effect and decrease the toxic residual period as TiO 2 cause photodegredation to the methomyl pesticide.By using the 2 nd and 4 th instars larvae of S. littoralis as toxic residual indicator.thus in agriculture process we can use these nanoparticles by mixing it with pesticides by this way we can reduce pollution effects resulted from using these pesticides.

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Table 2 :
the latent effect of TiO 2 nanoparticles methomyl mixture, on the larval and pupal durations, pupal weight, pupation and adult emergence percentage of the 2 nd instar larvae of the lab.strain of S. littoralis at 12 d. of the treatment in relative to control.

Table 3 :
the latent effect of TiO 2 nanoparticles methomyl mixture on the larval and pupal durations, pupal weight, pupation and adult emergence percentage of the4 th instar larvae of the lab.strain of S. littoralis at 12 d. of the treatment in relative to control.

Table 4 :
Latent effect of methomyl TiO 2 nanoparticles mixture, as compared to methomyl alone on fecundity, eggs hatching, and adult longevity and sex ratios against the 4 th instar larvae of the lab.strain of S. littoralis at 12 d. of the treatment in relative to control.