Capes; CNPq; International Foundation for Science (IFS) / O alto consumo de pesticidas no Brasil tem levado à preocupação pública sobre sua disposição, contaminação e acúmulo no ambiente. A flubendiamida, N2 - [1,1- dimethyl-2- (methylsulfonyl) ethyl] - 3- iodo-N1-[2-methyl- 4- [1,2,2,2-tetrafluoro-1-(trifluoromethyl) ethyl]phenyl]-1,2- benzenedicarboxamide, é um inseticida persistente ao meio ambiente (classe III) e também tóxico para os microcrustáceos. Diante disto, este trabalho teve como principais objetivos: selecionar fungos isolados de solo pelo teste de inibição de crescimento com inseticidas Actara e Belt; analisar os produtos da biotransformação do inseticida flubendiamida por fungos isolados do solo, pelo Botryosphaeria rhodina MAMB-05 e pela enzima lacase; e avaliar se os inseticidas Belt®, Actara® e o padrão flubendiamida da Bayer® atuam como indutores de lacase. O teste de inibição de crescimento foi realizado em meio sólido BDA com os fungos isolados de solo e com o B. rhodina. Este fungo e os que demonstraram melhores resultados de crescimento foram conduzidos ao experimento de biotransformação da flubendiamida, junto com o extrato de lacase do fungo B.rhodina. Os metabólitos foram avaliados por LC-MS/MS. A atividade lacase foi quantificada utilizando o ABTS como substrato. O resultado do teste de inibição destacou o crescimento dos fungos JUSOLCL039 e JUANT070, além do fungo B. rhodina MAMB-05. No experimento de biotransformação, foram detectados 47 metabólitos. Entre eles, a rota metabólica do íon m/z 587 obtida pelo fungo MAMB-05 foi proposta, através da perda do iodo e uma reação de hidroxilação. O metabólito m/z 663 foi encontrado no extrato do fungo JUANT070 e no extrato da enzima lacase. Sua rota metabólica foi proposta, indicando uma reação de desalogenação do flúor, confirmada pela espectrometria de massas em Tandem. O metabólito de íon m/z 697 foi formado pela reação de hidroxilação e identificado no extrato da enzima lacase. Os resultados do experimento de biotransformação apresentaram metabólitos formados pela enzima lacase e por outras enzimas presentes nos fungos JUANT070, JUSOLCL039 e B.rhodina. Estes fungos mostraram-se produtores constitutivos de lacase, na presença dos inseticidas Actara, Belt e no padrão analítico de flubendiamida. Entretanto, não demonstraram atuarem como indutores de lacase para os três fungos. / The high consumption of pesticides in Brazil has led to public concern about their disposal, contamination and accumulation in the environment. Flubendiamide, N2 - [1,1- dimethyl-2- (methylsulfonyl) ethyl] - 3- iodo-N1-[2 methyl-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]-1,2-benzenedicarboxamide is toxicologically classified as persistent to the environment (class III) and also toxic to microcrustaceans. This study mainly aimed to: selecting fungi isolated from soil by growth inhibition test using Belt and Actara insecticides; analyze the flubendiamide biotransformation product by soil fungi isolated, by Botryosphaeria rhodina MAMB-05 and by the laccase enzyme fungi; and evaluete if Belt®, Actara® and flubendiamide Bayer standard ® insecticides act as laccase inducers. The growth inhibition test was on solid medium PDA with the fungi isolated from soil and with B. rhodina. This fungus and those which showed the best growth results were conducted to the flubendiamide biotransformation experiment, together with the laccase extract of the fungus B. rhodina.The metabolites were assessed by LC-MS / MS. Determination of laccase activity was measured using ABTS as substrate. The result of the inhibition test emphasizes the growth of JUSOLCL039 and JUANT070 fungi. In addition to B. rhodina MAMB-05, biotransformation experiment detected 47 metabolites. The metabolic route from ion m / z 587 by the fungus MAMB-05 was proposed, through loss of iodine and hydroxylation reaction. The metabolite m / z 663 was found in the extract of JUANT070 fungi and in the laccase extract. The metabolic route suggested a dehalogenation reaction of fluoride, following by its confirmation through Tandem mass spectrometry. The metabolite m / z 697 formed by the hydroxylation reaction was identified in the laccase enzyme extract. The results showed metabolites which were formed by the laccase enzyme, or by JUANT070, JUSOLCL039 and B.rhodina other enzymes. These fungi were shown to be constitutive producers of laccase in the presence of the insecticide Actara, Belt and the analytical standard flubendiamide. However, these insecticides demonstrated not act as laccase inducers for the three fungi.

Organophosphates and carbamates are potent inhibitors of the neurotransmitter acetylcholinesterase. This inhibition results in the blocking of nerve signal transference into the post synaptic neuron leading to loss of muscle action and death. Because of the universal mechanisms of signal transduction in animals, these inhibitors have been widely used as agricultural pesticides as well as chemical warfare agents (nerve agents). Health issues associated with pesticide usage result from the fact that both the pesticides and their breakdown products often end up in water and food sources as well as in the soil. As a result, there has been an increase in the number of studies aimed at the detection of these pesticides in the environment. One popular research area is enzyme based biosensor construction. Some important criteria for consideration during the construction of biosensors are the importance of a suitable solid support as well as the enzyme immobilisation method. Recently, there has been increased interest in using nano-scale material e.g. using nanoparticles as enzyme support material. This is largely due to their advantages such as large surface area to volume ratio as well as reduced mass transfer resistance. Electrospinning is a straight forward and cost effective method for producing nanofibres from any soluble polymer(s). The applications of electrospun nanofibres have been reported in clinical studies, biofuel production as well as bioremediation. In this study two polymers were selected: nylon for its mechanical stability and chitosan for its biocompatibility and hydrophilicity, for the fabrication of electrospun nanofibres which would function as immobilisation support material for acetylcholinesterase. The first objective of this study was to electrospin nanofibres from a nylon-6 and chitosan blend solution. A binary solvent system consisting of formic acid and acetic acid (50:50) successfully dissolved and blended the polymers which were subsequently electrospun. Scanning electron microscopy characterisation of the nanofibres showed that (i) a nylon-6: chitosan ratio of 16%: 3% resulted in the formation of bead free nanofibres and (ii) the fibres were collected in non-woven mats characterised by different size nanofibres with average diameters of 250 nm for the main fibres and 40 nm for the smaller nanofibres. Fourier transform infra-red (FT-IR) analysis of the nanofibres indicated that a new product had been formed during the blending of the two polymers. The second aim of the study was to carry out a facile immobilisation of electric eel acetylcholinesterase via glutaraldehyde (GA) cross-linking. Glutaraldehyde solution 5% (v/v) resulted in the immobilisation of 0.334 mg/cm² of acetylcholinesterase onto the nanofibres. The immobilisation procedure was optimised with reference to acetylcholinestease and crosslinker concentrations, incubation time and the cross-linking method. A comparative investigation into the optimum pH and temperature conditions, pH and thermal stabilities, substrate and inhibition kinetics was then carried out on free and immobilised acetylcholinesterase. The final objective of this study was to determine the storage stabilities of the immobilised and free enzymes as well as the reusability characteristics of the immobilised acetylcholinesterase. Several conclusions were drawn from this study. Acetylcholinesterase was successfully immobilised onto the surface of nylon-6:chitosan nanofibres with retention of its activity. There was a shift in the pH optimum of the immobilised acetylcholineseterase by 0.5 units towards a neutral pH. Although both free and immobilised acetylcholinesterase exhibited the same optimum temperature, immobilised acetylcholinesterase showed enhanced thermal stability. In terms of pH stability, immobilised acetylcholinesterase showed greater stability at acidic pH whilst free acetylcholinesterase was more stable under alkaline pH conditions. Relative to free acetylcholinesterase, the immobilised enzyme showed considerable storage stability retaining ~50% of its activity when stored for 49 days at 4°C. Immobilised acetylcholinesterase also retained > 20% of its initial activity after 9 consecutive reuse cycles. When exposed to fixed concentrations of carbofuran or demeton-S-methyl sulfone, immobilised acetylcholinesterase showed similar inhibition characteristics to that of the free enzyme. The decrease in enzyme activity observed after immobilisation to the nanofibres may have been due to several reasons which include some enzyme molecules being immobilised in structural conformations which reduced substrate access to the catalytic site, participation of the catalytic residues in immobilisation and enzyme denaturation due to the reaction conditions used for acetylcholinesterase immobilisation. Similar observations have been widely reported in literature and this is one of the major drawbacks of enzyme immobilisation. In conclusion, nylon-6:chitosan electrospun nanofibres were shown to be suitable supports for facile acetylcholinesterase immobilisation and the immobilised enzyme has potential for use in pesticide detection. Future recommendations for this study include a comparative study of the GA cross-linking method for AChE immobilisation which will lead to more intensely bound enzyme molecules to prevent non-specific binding. An investigation into the effect of inhibitors on stored immobilised AChE, as well as reactivation and reuse studies, may also be useful for determining the cost-effectiveness of reusing immobilised AChE for pesticide detection in environmental water samples. Several models have been designed for the determination of the kinetic parameters for immobilised enzymes. These take into account the mass transfer resistance as well as the overall charge of the immobilisation matrix. The use of these models to analyse experimental data will give a clear understanding of the effects of immobilisation on enzyme activity

Orientador: Luiz Lonardoni Foloni / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Agricola / Made available in DSpace on 2018-08-04T06:05:15Z (GMT). No. of bitstreams: 1 Plese_LuisPedrodeMelo_D.pdf: 1119151 bytes, checksum: 71b0dc99bac0c1f913f9f77bc3014bc4 (MD5) Previous issue date: 2005 / Resumo: A utilização de produtos fitossanitários para o controle de plantas daninhas, pragas ou doenças têm sido muito difundido em função da necessidade crescente da oferta de alimentos, limitação de áreas agricultáveis e disponibilidade de mão-de-obra. Os problemas gerados com a aplicação de produtos fitossanitários, sem o devido conhecimento, podem causar riscos ao meio ambiente e à saúde humana. A tendencia é órgão fiscalizador é exigir que antes do lançamento de um novo produto no mercado e antes de sua aplicação em áreas agricultáveis seja feito estudo rigoroso de seu comportamento no meio ambiente. Quando existe um bom banco de dados, modelos matemáticos são desenvolvidos objetivando prever comportamento ambiental do produto fitossanitário. O conceito de fugacidade, embora não seja novo, praticamente não tem sido estudado no Brasil na áreadas ciencias agrárias. A fugacidade pode ser um novo caminho para quantificar o transporte, a biocumulação e transferência entre os compartimentos ambientais. Este trabalho teve por objetivos: (a) desenvolver uma metodologia preliminar de previsão de destino ambiental dos inseticidas carbosulfan e carbofuran no cultivo de arroz irrigado, através de modelo matemático baseado no conceito de fugacidade, para delimitar os compartimentos mais vulneráveis; (b) prever o comportamento e o destino ambiental das moléculas do carbosulfan e carbofuran no cultivo de arroz irrigado, aplicando o conceito de fugacidade; (c) verificar, em condições de campo, o comportamento e o destino ambiental do carbosulfan e do carbofuran, comparando-os com a previsão feita. A fase experimental foi desenvolvida na Fazenda Varjão, município de Bariri-SP, coordenadas (21º59'47¿S e 48º36'41¿LGr) em solo Gleissolos. A cultura foi implantada no dia 22/11/02, utilizando-se a cultivar IRGA-420. Para evitar possíveis contaminações, a área experimental foi locada em um tabuleiro de 2 ha a montante da propriedade. A propriedade foi dividida em tabuleiro com dimensões variáveis de 1,5 a 2,5 ha, totalizando 200 tabuleiros, separados por canais de irrigação e drenagem. Tradicionalmente, para o controle da larva da bicheira-da-raiz é recomendado o uso do inseticida carbosulfan, na dose de 400 g i.a. ha-1, no sistema de benzedura. Foram determinados 8 pontos ao acaso no tabuleiro estudado, para coleta de amostras. Os compartimentos amostrados foram água laminar, solução do solo e do próprio solo. As seqüências de tempo escolhidas para as coletas de amostras de água laminar e solução do solo foram de 0, 24, 48, 96, 192, 378 e 678 horas após a aplicação. As amostras de solo foram realizadas em 0, 24, 48, 96, 192, 378, 678, 1536 e 1656 horas após a aplicação. As cinéticas de degradação do carbofuran e do carbosulfan foram avaliadas em condições de campo através da análise dos resíduos das amostras coletadas. Utilizou-se da identificação e quantificação dos resíduos foi realizada através de um detector seletivo de massas, acoplado ao cromatógrafo. Este foi operado no modo de monitoramento de íons (SIM), sendo utilizados os fragmentos 160 e 164 para a quantificação de carbofuran e carbosulfan, respectivamente. Os dados experimentais e as equações diferenciais que descrevem a cinética do carbosulfan e do carbofuran permitiram estimar a meia-vida dos inseticidas na água e na solução do solo e no próprio solo. Os valores estimados das meias-vidas para o carbosulfan na água laminar, no solo e na solução do solo foram 1, 25 e 21 dias, respectivamente. Para o carbofuran as meias-vidas na água laminar e na solução do solo foram 3 e 10 dias, respectivamente. Os resultados indicam os compartimentos preferenciais do carbofuran, facilitando a tarefa de amostragens em programas do monitoramento da qualidade ambiental, e permite antecipar o destino ambiental do inseticida carbofuran. Experimentos de campo foram conduzidos para verificar a proximidade entre os valores simulados e os valores observados de concentrações do carbofuran na água e no solo / Abstract: Pesticide use to control pests, diseases and weeds is steadily increasing due to a general food demand increase, cropping area limitation, and manpower availability. Pesticide application without a sound knowledge may hazard environment and human health. Regulatory agencies enforces that, before a new product releasing to the market for agricultural use, intensive studies on its environment impacts ought to be developed. When a large and useful database is available, mathematical models are developed to estimate or predict product environmental behavior. In Brazil, although the available knowledge, practically there are no studies on fugacity concept applied to agricultural subjects. Fugacity in the proposed form might be a new tool in quantifying the movement, bioaccumulation and transfer of pesticides betweeen environment compartments. The objectives of this research work were: (a) to develop a preliminary method for predicting the environmental destination of carbosulfan and carbofuran insecticides applied to field irrigated rice, using a model based on the fugacity concept to delimit most vulnerable compartments; (b) to predict the behavior and environmental destination of carbosulfan and carbofuran molecules applied on field irrigated rice, using the fugacity concept; (c) to validate the prediction model by comparison with experimental data obtained under field conditions. The experimental data was obtained from a field irrigated rice experiment carried out at 'Varjão¿ Farm, district of Bariri, State of São Paulo, Brazil (21º59'47¿S and 48º36'41¿LGr) in a Gleissol type soil. Rice cv IRGA-420 was seeded in 11.22.2004, using the uppermost rice field area to avoid eventual contaminations. The property is divided in 1.5-2.5 ha-field areas, separated by rrigation or drainage channels. Traditionally, rice root weevil is controlled through carbosulfan application at the rate of 400 g a.i. ha-1. Eight sampling points were randomly located in the rice fields. The sampled compartments were: laminar water, soil solution and soil. The time sequence for water and soil solution samplings were 0, 24, 48, 96, 192, 378 and 678 hours after pesticide application, and for soil, 0, 24, 48, 96, 192, 378, 678, 1536 and 1656 hours after pesticide application. The carbofuran and carbosulfan degradation kinetics were evaluated through residue sample analysis. Residue identification and quantification were made through a mass selective detector (MSD) device coupled to a chromatograph. This equipment was operated in the system of ion monitoring mode (SIM), utilizing 160 and 164 fragments for the carbofuran and carbosulfan quantification, respectively. The experimental data and differential equations that describe the carbofuran and carbosulfan kinetics in the field rice water, allowed the insecticide half-life estimation in the three compartments: water, soil solution and soil. The estimated values for carbosulfan half-lives were: 1, 25 and 21 for water, soil and soil solution, respectively, and 3 and 10 days for carbofuran half-lives for the water and soil solution, respectively. The proposed model showed to be viable for the evaluation of insecticide behavior inthe irrigated field rice. High correlation was observed between data obtained by simulation and that from field experimentation, through residue analyses in the water and soil compartments. In an evaluation of environmental risks, the fugacity model level IV was adequate to estimate or predict the insecticide product destination / Doutorado / Agua e Solo / Doutor em Engenharia Agrícola

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The first objective of this research was to determine if pesticides were leaching into the shallow groundwater beneath agricultural sites, and if so, to determine a correlation between soil and groundwater pesticide concentrations. The second was to examine the correlation between pesticide concentrations measured by gas chromatography with electron capture detector (GC/ECD) and an immunoassay method developed by OHMICRON Corporation. Samples from four agricultural and one reference (undeveloped) site were analyzed for pesticides over an 11 month period from April, 1992 to February, 1993. One hundred and nineteen separate groundwater samples were analyzed for: alachlor, atrazine, carbofuran, cyanazine, and metolachlor. Pesticide analysis of groundwater and seepage meter water was carried out by immunoassay and by solid phase extraction (SPE) with octadecyl bonded extraction disks followed by GC/ECD. Fifty-five soil and sediment samples were Soxhlet extracted followed by gas chromatography/mass spectrometry (GC/MS). Pesticides were detected in 13.4% groundwater samples by GC/ECD with only one detection being greater than 1 ppb. The immunoassay method detected pesticides in 32% of the groundwater samples with the majority of these detections also being below 1 ppb. Alachlor and/or metolachlor were detected in 44% of the soil samples at concentrations ranging from 7 ppb to 485 ppb. The study concluded that the majority of the target pesticides were being adsorbed by the soil and only limited amounts, less than 1 ppb, were being transported to the groundwater. It was also concluded that the immunoassay had lower limits of detection, but may yield some false positive results. / Master of Science

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Runoff is generally regarded as one of the most important routes of nonpoint source pesticide pollution in agricultural surface waters. Of major concern is the fact that low, sub-lethal levels of pesticide exposure are responsible for negative ecotoxicological effects, stressing the need for methods capable of identifying problem areas where populations could be at risk. Predicted average losses of three pesticides in tributaries of nine sub-catchments of the Lourens River were calculated through use of a GIS-based runoff model. There was a significant (p < 0.005) positive correlation between the predicted average loss and mean measured concentrations of the insecticides both in water and suspended sediments (R2 between 0.75 and 0.9), indicating that the model could serve as a powerful tool for the risk assessment and management of surface waters in South African orchard areas. Based on field relevant exposure scenanos, the potential effects of azinphos-methyl on macroinvertebrate communities were evaluated in a combined microcosm and field approach. Microcosms were contaminated for 1 h with AZP (control, 0.2; 1,5and 20 ug/L; three replicates each) and acute effects on survival were evaluated 6 days after exposure. The sensitivity or tolerance of 12 core taxa was determined based on their response to the exposure scenarios and compared to field tolerance/sensitivity as was established by a field investigation at a control and contaminated site of the Lourens River. The sensitivity/tolerance of ten of the 12 taxa corresponded to that which was found in the field. Thus microcosm studies employing a field relevant design can be successfully linked to field studies and indicate that transient pesticide contamination affects the aquatic communities of the Lourens River. / AFRIKAANSE OPSOMMING Afloop word oor die algemeen beskou as een van die belangrikste roetes van niepuntbron pestisiedbesoedeling in landbou oppervlakwaters. Die feit dat lae, sub-letale vlakke van pestisiedblootstelling negatiewe ektoksikologiese gevolge kan hê, is van groot belang. Dit beklemtoon die behoefte aan metodes om probleemgebiede te kan identifiseer waar bevolkings aan risiko onderhewig is. 'n GIS-gebaseerde afloopmodel is gebruik om die gemidddelde verlies van drie pestisiede in die sytakke van nege sub-opvangsgebiede van die Lourensrivier te voorspel. Daar was 'n beduidende (p < 0.005) positiewe korrelasie tussen die voorspelde gemiddelde verlies en gemete konsentrasies van insektisiede in beide die water en sediment (R2 between 0.75 and 0.9) fases, wat aandui dat die model as 'n kragtige hulpmiddel vir risikobestuur van oppervlakwaters in Suid Afrikaanse boord-gebiede kan dien. Die potensiële gevolge van azinfos-rnetiel (AZP) op makroinvertebraat gemeenskappe is deur middel van 'n gekombineerde mikrokosmos (wat op veldrelevante blootstellings gebaseer is) en veldbenadering bepaal. Mikrokosmosse is vir 1 h met AZP gekontamineer (kontrole; 1; 0.2; 1; 5 en 20 ~g1L; drie replikate elk), en die akute gevolge op oorlewing is ge-evalueer na ses dae van blootstelling. Die sensitiwiteit of toleransie van 12 sleutel taksa is deur middel van hulle respons op die blootstellingsreeks bepaal, en met hulle veldtoleransie/sensitiwiteit vergelyk wat in 'n veldstudie by 'n kontrole- en gekontamineerde gebied in die Lourensrivier bepaal is. Die sensitiwiteit/toleransie van 10 van die 12 taksa in die mikrokosmos eksperimente het ooreengestem met die wat in die veld gevind is. Mikrokosmosstudies wat op 'n veldrelevante ontwerp gebaseer is, kan dus suksesvol aan veldstudies gekoppel word, en dui aan dat oorgedraagde pestisiedkontaminasie die akwatiese gemeenskap van die Lourensrivier beinvloed.