الفهرس 
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Abstract
The need to protect plants against environmental challenges, abiotic and biotic, leads to the application of nanomaterials and pesticides in the environment. Recently, nanopesticides have been developed to replace classic pesticides. Their wide application in the agricultural practice leads to deposition of nanomaterials (and potential residuals) in the natural environment. The use of nanopesticides is a great challenge for lack of information about their potential ecotoxicity, animal, human and other non-target organisms toxicity.
This study was carried out to, prepare, formulate and characterize the novel nano-emulsion compound of lambda-cyhalothrin, to estimate and compare the larvicidal activity of the novel nano-emulsion lambda-cyhalothrin with, the conventional compound of lambda- cyhalothrin, and to investigate the oxidative stress and genotoxicity of nano lambda- cyhalothrin in albino rats.
Preparation and characterization of the novel compound of lambda-cyhalothrin, Nano- emulsion Formulation of Lambda – Cyhalothrin was obtained. The lambda-cyhalothrin- loaded nano-delivery system was developed, using Tween 80 as a hydrophobic surfactant through a solvent evaporation method. The novel compound of lambda-cyhalothrin has unique chemical, physical, and biological properties. The recorded properties of lambda- cyhalothrin nano-emulsion showed spherical particles having an average size of 70.3 nm using transmission electron microscopy. Dynamic light scattering Zetasizer reached 77.51 nm and Zeta potential has a negative surface charge value of -17.8 mV. Lambda-cyhalothrin and lambda-cyhalothrin nano-emulsion showed a strong absorption band at different wavelengths. Fourier transform infrared spectroscopy analysis revealed the addition of new bonds in the lambda-cyhalothrin nano-emulsion compound. The larvicidal activity of the novel nano-emulsion of lambda-cyhalothrin was estimated and compared with larvicidal activity of the conventional compound of lambda-cyhalothrin and indicated a significant difference between the mortality means in the larvae of Culex pipiens. The produced lambda-cyhalothrin nano-emulsion has a new hydrophilic attendance and better efficacy for pest control resulting in promising nano-pesticide formulations to improve agricultural practice.
Building on the superiority and higher efficacy of the produced lambda-cyhalothrin nano- emulsion on target organisms, emerging promise for addressing the problems in agriculture
and food production. A great potential of the nanopesticides to partially or totally substitute the conventional agrochemicals, establishes a new era of nano pesticide.
The investigation of the oxidative stress and genotoxicity of nano lambda-cyhalothrin in albino rats by, a total number of 72 healthy adult Sprague–Dawley male rats aged 8–10 weeks with an average body weight of 150–200g were divided randomly into three groups,
Conventional-treated rats: 18 rats were given 1/20 of LD50 (79 mg/kg b.wt.) LC which equal to 4mg/100gm b.wt. dissolved in 1 ml corn oil as a vehicle, orally twice weekly (El- Masry, et al., 2014).
Lambda-cyhalothrin nano- treated rats: 18 rats were given nano λ cyhalothrin which equal 1/80 of LD50 (El-Masry et al., 2014) prepared by dilution of 1ml of nano emulsion in 100 ml distilled water which equal (0.01mg/100gm b. wt.) orally, twice weekly.
Control group 1,2: 36 rats kept as control and divided into two sub-group have18 rat each. First and second sup-groups administered 1 ml corn oil and 1 ml distilled water, respectively, orally, twice weekly.
The samples were collected according to specific schedule, every 15 days. Three randomly selected rats from each group were euthanized under diethyl ether anesthesia. Blood samples with and without anticoagulant were collected from the descending aorta, and serum samples were harvested and kept at - 20 °C until analysis. Serum samples were prepared for biochemical assay. The liver specimens were collected, and divided into two parts; the first part was used for comet assay, and the second part was preserved in 10% neutral buffered formalin for histopathological examination. Other organs like brain, testis and kidneys samples for histopathological investigation.
Blood collected with EDTA was used for haematological analysis. Haematological variables measured were, total red blood cells count (RBCs), haemoglobin concentration (HGB), red blood cells distribution width (RDW), haematocrit (HCT), main corpuscular volume (MCV) mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets count (PLT), total white blood cells count (WBCs), lymphocytes count and per cent, neutrophils count and per cent, monocytes count and per cent.
Serum samples were obtained from clotted blood collected without anticoagulant after centrifugation. The serum samples were used for determination Superoxide Dismutase (SOD) activity and serum malondialdehyde (MDA) level.
The expression of the inflammatory cytokines TNF-α average level concentration in the serum was determined by enzyme-linked immunosorbent assay (ELISA). DNA damage in the brain and liver was detected using comet assay. Sections from the brain, liver, kidneys and testes were used, immediately after sacrifice for histopathological examination.
Body weight, organ absolute and relative weight, biochemical parameters, comet assay as well as histopathological changes were the criteria used to evaluate the oxidative stress and genotoxicity of nano lambda-cyhalothrin in albino rats.
In the present study, the results of body weight revealed that in both groups conventional and group of nano ʎ cyhalothrin treated rats, showed non-significant change during the whole period of experiment in comparison with control group. Also, the comparison between the results obtained of two groups, group of conventional and group of nano ʎ cyhalothrin treated rats, showed non-significant change during the whole period of experiment. This could be attributed to the effects of insecticide exposure on body weight are contradictory, probably depending on various factors, such as dose and route of administration, species, sex, and treatment duration. The non-significant differences observed in the body weight of the experimental animals shows that lambda cyhalothrin is not a systemic toxin. Therefore, it can silently destroy the vital organs of the body without conspicuous changes in the bodyweight. The results of brain absolute and relative weight revealed that in both groups conventional and nano ʎ cyhalothrin treated rats, the absolute weight of brain of male rats showed non-significant change during the whole period of experiment in comparison with control group. But the group of conventional ʎ cyhalothrin treated rats showed a significant decrease in the value of relative brain weight at 4th̰ week of the experiment in comparison with control group. The significant decrease observed in the brain relative weight in this study could be attributed to vacuolization in the cerebral hemisphere as insecticide produced neuronal degeneration and necrosis. The results of liver absolute and relative weight revealed that a significant increase in the group of conventional ʎ cyhalothrin treated rats in comparison with control group. In the group of nano ʎ cyhalothrin treated rats, the value of liver absolute weight showed a significant increase in comparison with control group and in comparison with group of conventional ʎ cyhalothrin treated rats. Increased weight of liver recorded in this study might be because of elevated circulation due to raised requirements for the detoxifying compounds that are toxic.
The results of kidney absolute weight revealed a significant increase in the group of conventional ʎ cyhalothrin treated rats in comparison with control group. Also, showed highly significant increase in comparison with group of nano ʎ cyhalothrin treated rats. The results of kidney relative weight of the group of nano ʎ cyhalothrin treated rats revealed a significant increase in comparison with control group. The kidneys contributed to the maintenance of the body’s homeostasis thanks to the excretion of these unnecessary metabolic products. Concentrating urine in the tubular fluid also increases the concentration of xenobiotics in it. Renal transport and the accumulation and biotransformation of pyrethroid metabolites contribute to the susceptibility of the kidneys to damage by this group of xenobiotics. The results of testis absolute and relative weight in both groups of conventional and nano ʎ cyhalothrin treated rats, showed a non-significant change along the time of the experiment in comparison with control group and in comparison with each other. Our results could be contributed to cyhalothrin may not have obvious effect on the gonadosomatic indices but silently destroy target cells of the body over time. histopathological analysis of the testicles revealed reproductive toxicity.
This study revealed that the value of RBC’s counts, Hb content, Hct and MPV values in both groups conventional ʎ cyhalothrin treated rats and group of nano ʎ cyhalothrin treated rats, showed non-significant change during the whole period of experiment in comparison with control group and with each other. The inconsistent and the observed differences could be attributed to various factors influencing hematological parameters such as feeding pattern, food utilization, fluids and salts balance, blood sampling and experimental variables.
This study revealed that, the value of RDW in group of conventional ʎ cyhalothrin treated rats showed a significant decrease in comparison with control group. In a group of nano ʎ cyhalothrin treated rats, showed a significant decrease in comparison with control group. This study revealed that the value of platelets count in the group of conventional ʎ cyhalothrin treated rats showed a significant increase in comparison with control group. In a group of nano ʎ cyhalothrin treated rats, showed a significant increase in comparison with control group. This study revealed that the value of total WBCs count in the group of conventional ʎ cyhalothrin treated rats showed a significant increase in comparison with control group, and in comparison with nano ʎ cyhalothrin treated rats. In a group of nano ʎ cyhalothrin treated rats, showed a significant increase at whole period of experiment in comparison with control group. This study revealed that the value of lymphocytes count in
the group of conventional ʎ cyhalothrin treated rats showed a significant increase in comparison with control group, and in comparison with nano ʎ cyhalothrin treated rats. In a group of nano ʎ cyhalothrin treated rats, showed a significant increase in comparison with control group. This study revealed that the value of the peripheral neutrophils percentage in the group of conventional ʎ cyhalothrin treated rats showed a significant increase in comparison with control group. In a group of nano ʎ cyhalothrin treated rats, showed a significant increase in comparison with control group. This study revealed that, the value of the peripheral monocytes percentage in group of conventional ʎ cyhalothrin treated rats showed a significant increase in comparison with control group. In a group of nano ʎ cyhalothrin treated rats, showed a significant increase in comparison with control group.
An increase in the number of leukocytes in the blood of animals irrespective of the pyrethroid applied for intoxication, may result from the mobilization of the immunological system and /or a shift in the leukocytic pool from the spleen to peripheral blood. The increase in WBC may be indicative of activation of defense and immune system of the body. This might results in increase in the release of WBCs from bone marrow storage pool into the blood. The rise in WBCs count suggests the increase in the defense mechanism against probable attack of toxic molecules.
This study revealed that, the value of total superoxide dismutase activity (SOD), in the group of conventional ʎ cyhalothrin treated rats showed a significant decrease during the whole period of experiment in comparison with control group. Also, showed a significant decrease at 8th̰ week of the experiment in comparison with group of nano ʎ cyhalothrin treated rats. In the group of nano ʎ cyhalothrin treated rats, showed a significant decrease in comparison with control. This study revealed that the value of MDA in the group of conventional ʎ cyhalothrin treated rats showed a significant increase in comparison with control group. In the group of nano ʎ cyhalothrin treated rats, showed a significant increase in comparison with control group. Our study results revealed the elevated level of lipid peroxidation indicator MDA and the decline of SOD activity which could be attributed to, the decline of antioxidant capacity (TAC), thus triggering the peroxidation of unsaturated fatty acids in the cell membrane of RBCs and WBCs, thus inducing cytotoxicity. This is contributed to LCH is a type II pyrethroid, which has a α-cyano moiety, therefore the release of cyanohydrins, which are unstable under physiological conditions and further decompose to cyanides and aldehydes, which in turn could act as a source of free radicals. The lowered TAC may indicate a deficiency in the defense against ROS, resulting in H2O2 accumulation,
which in turn can inhibit superoxide dismutase (SOD) activity, resulting in superoxide radical accumulation. Thus, the excess radicals could readily oxidize the membrane lipids, which culminated in the expressive MDA increase, observed in the LCH-exposed group. Lipid peroxidation arises from the reaction of free radicals with lipids and this is considered to be an important feature of the cellular injury brought by free radical attack. The results in our study could be contributed to pesticides induce oxidative stress which leads to the generation of free radicals, changes in antioxidants levels and lipid peroxidation. In accordance with the data obtained from this study, pyrethroids have been found to induce oxidative damage in various tissues owing to the formation of ROS and impairments in free oxygen radicals scavenging enzyme systems. Antioxidant enzymes cause a primary defense that prevents oxidative damage of biological macromolecules. According to the obtained results the activities of serum SOD treated rats were significantly decreased. These results suggested that LCT has the capability to induce free radicals and oxidative damage as evidenced by alterations in various antioxidant enzymes.
This study revealed that the mRNA expression levels of the inflammatory cytokines TNF-α average level in the group of conventional ʎ cyhalothrin treated rats showed a highly significant increase and up-regulation in comparison with control group. In a group of nano ʎ cyhalothrin treated rats, the value of TNF α expression showed a significant up-regulation in comparison with control group. Also showed a highly significant increase in comparison with group of conventional ʎ cyhalothrin treated rats. The recorded impairment suggested the immunosuppressive effect of these pesticides. The expression of cytokines (TNF-α) as a vital part of the immune system is modulated by infection or inflammation, so assessing their expression levels may help explain the mechanisms of specific contaminants’toxicity. These findings suggested that ROS produced by LCT and its metabolites activated genes involved in inflammation through the stimulation of a transcription factor, NF-κB, which is situated in the crossover of inflammatory and oxidative stress. LCT and its metabolites may mediate the inflammation process. The positive correlation between LCT metabolites (CFMP and 3-PBA) and inflammatory gene expressions (TNF-α, IL-6, and IL-1β) were observed. Based on these observations, it is suggested that the increased activity of cytokines following LCT exposure is an evidence of the oxidative process and the presence of inflammation. These results were correlated with histopathology results, which revealed portal infiltration with inflammatory cells, perivenular sinisoidal dilatation, parenchyma inflammation, and congestion of central veins were seen in LC-treated groups.
The evaluating data of genotoxicity and DNA damage in both blood and liver samples revealed a non-significant change in the value of comet assay parameters including tail DNA%, tail length (µm), tail moment % and olive tail moment% in the conventional ʎ cyhalothrin treated rats along the whole time of the experiment in comparison with the control group. On the other hand, the group of nano ʎ cyhalothrin treated rats showed a significant increase in the value of comet assay parameters which indicate DNA damage in both blood and liver samples including significant increase in tail DNA%, tail length (µm), tail moment % and olive tail moment% at 12th̰ week of the experiment in comparison with control group and with the group of conventional ʎ cyhalothrin treated rats. The results of conventional ʎ cyhalothrin could be attributed to, LCT genotoxicity is dose-dependent. Genotoxicity of nanomaterials (NMs) could be induced either through direct binding of NMs to chromosomes or DNA or by the generation of ROS inducing DNA lesions. Based on molecular dynamics simulations it has been shown that NMs can bind to nucleotides at the free ends and minor grooves of double stranded A-form DNA, thereby permanently disrupting hydrogen bonds and causing deformation. More recently other mechanism, NPs with high binding affinity to DNA have a greater potential to interfere with DNA replication. For a great number of NMs the induction of ROS has been shown to be a mode of action through which primary genotoxicity is induced. A possible mechanism of genotoxicity, due to nanomaterials have a greater affinity toward the lipid molecules present in the biological environment. Their interaction with lipid bilayers reduce the lipid transition temperature and the membrane fluidity of all types of lipid vesicles. Secondly, these nanomaterials were found capable of covalently binding with the nucleic acid by electrochemical interaction. This results in a reversible electron transfer at the interfacial level, generating holes on the nucleic acid bases thereby affecting the length of the strand, terminal phosphates and different types of DNA strands.
The result of histopathological examination revealed that, liver of conventional and nano ʎ cyhalothrin treated rats showed marked hydropic degenerated hepatocytes, inflammatory cell infiltration with mono nuclular cells in portal triad, inter lobular fibrosis, and Kupffer cells activation. Kidney of conventional and nano ʎ cyhalothrin treated rats showed hypercellularity in the glomeruli, necrosis of some renal tubular epithelium and intertubular mononuclear cell infiltration, intratubular hemorrhage, perivascular fibrosis, renal tubular cast, sever degeneration and necrosis of renal tubular epithelium. Brain of conventional and nano ʎ cyhalothrin treated rats showed perivascular hemorrhage in the cerebral cortex,
pyknotic and degenerated Purkinje cells in cerebellum, congestion of blood vessels, degenerative neurocytes, degenerated and pyknotic pyramidal cells. Testis of conventional and nano ʎ cyhalothrin treated rats showed hyperplasia of Leydig cells, disorganization of spermatogonia, spermatocytes and nuclear pyknosis of germ cells, degeneration of Leydig cells and marked interstitial edema.
It could be concluded that, this study represents an advanced work to investigate the toxic effect of a nano pesticide formulation on non-target vertebrate animal, laboratory animal (rat). This study provides the environmental impact of some novel nano-pesticides and points to their possible toxic effects on non-target organisms. The results of the present study represent a guide of the eco-toxicological studies for the application of some novel nano-pesticides in the environment.
The finding of this study suggested that there was no significant difference between the oxidative effect of conventional and nano form of the insecticide lambda cyhalothrin in all parameters, including organ weight index, hematological, biochemical and histopathological but the result of comet assay which indicate DNA damage revealed that the nano form cause genotoxic effect and DNA damage after 12 week of exposure.
Finally, there are scarce studies that have been done on the toxicity of nano pesticides. Therefore, the present work was undertaken to understand the relative toxicological differences between these novel nano pesticides and the conventional one. So, what combined toxicity of nanopesticides can produce and what effects of these combined toxicity have on organisms are still poorly studied. Thus we have to raise concerns about this issue, because its potential impact on the environment and humans is far-reaching.