Application of methyl parathion hydrolase (MPH) as a labeling enzyme

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 mu L hemolymph from an infected shrimp and differentiated it from a healthy shrimp.

Square wave adsorptive cathodic stripping voltarnmetry automated by sequential injection analysis - Potentialities and limitations exemplified by the determination of methyl parathion in water samples

This paper describes the development and evaluation of a sequential injection method to automate the determination of methyl parathion by square wave adsorptive cathodic stripping voltammetry exploiting the concept of monosegmented flow analysis to perform in-line sample conditioning and standard addition. Accumulation and stripping steps are made in the sample medium conditioned with 40 mmol L-1 Britton-Robinson buffer (pH 10) in 0.25 mol L-1 NaNO3. The homogenized mixture is injected at a flow rate of 10 mu Ls(-1) toward the flow cell, which is adapted to the capillary of a hanging drop mercury electrode. After a suitable deposition time, the flow is stopped and the potential is scanned from -0.3 to -1.0 V versus Ag/AgCl at frequency of 250 Hz and pulse height of 25 mV The linear dynamic range is observed for methyl parathion concentrations between 0.010 and 0.50 mgL(-1), with detection and quantification limits of 2 and 7 mu gL(-1), respectively. The sampling throughput is 25 h(-1) if the in line standard addition and sample conditioning protocols are followed, but this frequency can be increased up to 61 h(-1) if the sample is conditioned off-line and quantified using an external calibration curve. The method was applied for determination of methyl parathion in spiked water samples and the accuracy was evaluated either by comparison to high performance liquid chromatography with UV detection, or by the recovery percentages. Although no evidences of statistically significant differences were observed between the expected and obtained concentrations, because of the susceptibility of the method to interference by other pesticides (e.g., parathion, dichlorvos) and natural organic matter (e.g., fulvic and humic acids), isolation of the analyte may be required when more complex sample matrices are encountered. (C) 2007 Elsevier B.V. All rights reserved.

Electrochemical feasibility study of methyl parathion determination on graphite-modified basal plane pyrolytic graphite electrode

A low-cost electrochemical method was developed for the determination of trace-level of methyl parathion (MP) based on the properties of graphite-modified basal plane pyrolytic graphite electrode (graphite-bppg). A combination of graphite-bppg with square-wave voltammetric (SWV) analysis resulted in an original, sensitive and selective electrochemical method for determination of MP pesticide in drinking water. The electrode was constructed and the electrochemical behavior of MP was studied. Immobilization is achieved via film modification from dispersing graphite powder in deionized water and through pipeting a small volume onto the electrode surface allowing the solvent to volatilize. The strong affinity of the graphite modifier for the phosphorous group of the MP allowed the deposition of a significant amount of MP in less than 60 seconds. The cyclic voltammetric results indicate that the graphite-bppg electrode can enhance sensitivity in current intensity towards the quasi-reversible redox peaks of the products of the cathodic reduction of the nitro group at negative potential (peak I = 0.077 V and peak II = –0.062 V) and that the cathodic irreversible peak (peak III = –0.586 V) in comparison with bare bppg electrode and is also adsorption controlled process. Under optimized conditions, the concentration range and detection limit for MP pesticide are respectively 79.0 to 263.3 mmol L-1 and 3.00 mmol L-1. The proposed method was successfully applied to MP determination in drinking water and the performance of this electrochemical sensor has been evaluated in terms of analytical figures of merit.

Responsiveness of the interrenal tissue of Jundia (Rhamdia quelen) to an in vivo ACTH test following acute exposure to sublethal concentrations of agrichemicals

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Eficácia do paration metílico e do extrato aquoso de folhas secas de nim no controle de anacanthorus penilabiatus (monogenoidea) em pacu (piaractus mesopotamicus)

This work determined the efficacy of the insecticide methyl parathion and the natural pesticide azadirachtin present in the aqueous extract of dry neem leaves (AEDNL) to Anacanthorus penilabiatus (Monogenoidea) control in pacu (Piaractus mesopotamicus). The efficacy of methyl parathion was evaluated in an experiment consisting of six treatments (0.0, 3.0, 4.0, 5.0, 6.0 and 7.0 mg methyl parathion/L water) and five exposure times (2, 4, 8, 16 and 24 h). The efficacy of azadirachtin present in AEDNL was assessed in an experiment consisting of seven treatments (0,0; 25; 50; 75; 100; 125; e 150 mL/L water) and five exposure times (24, 48, 72, 96 and 120 h). The efficacy of methyl parathion increased with increasing concentration and exposure time. The highest control efficacy was obtained with a concentration of 7 mg methyl parathion/L at all exposure times. In this treatment, the highest efficacies were observed at 16 and 24 h of exposure, with a control rate of 96.2 and 97.0%, respectively. For the AEDNL, the highest control efficacy (89.2%) was obtained with a concentration of 2.9 mg/L after 120 h of exposure. The efficacy in the treatments employing 1.47 and 1.18 mg/L was 83.9 and 82.5%, respectively, after 120 h of exposure. Methyl parathion presented a higher efficacy in the control of A. penilabiatus than the AEDNL. The AEDNL was moderately effective in the control of the parasite.

Efeito de inseticidas em insetos predadores em culturas de algodão

Com o objetivo de verificar o efeito de inseticidas em insetos predadores em cultura de algodão (Gossypium hirsutum L.), instalaram-se, em 1993-1994, dois experimentos, um no campo, e outro, em laboratório. No experimento realizado no campo, os tratamentos foram: Fipronil 200 SC (75 g/ha de i.a.); Fipronil 800 WDG (64, 80 e 100 g/ha de i.a.); Endosulfan 350 CE (700 g/ha de i.a.); e testemunha. em laboratório, além das formulações à base de Fipronil foi utilizado o Paration metílico 600 CE (480 g/ha de i.a.). Fipronil foi seletivo para os artrópodes predadores (Scymnus sp., Geocoris ventralis, Cycloneda sanguinea e Doru lineare) no campo, e a Cycloneda sanguinea (L.), em laboratório, e pode ser recomendado em programas de manejo integrado de pragas na cultura do algodoeiro para o controle de Alabama argillacea (Rueb.), e Anthonomus grandis Boh. Endosulfan foi seletivo em relação a Scymnus sp., Geocoris ventralis Thomazini e Doru lineare (Eschs) no campo, com uma redução dos insetos inferior a 30%, e o Paration metílico não foi seletivo para C. sanguinea em laboratório.

Tetanorhynchus leonardosi (Mello-Leitão) (Orthoptera: Proscopiidae), nova praga em eucaliptos

We report for the first time the attack of the walking stick Tetanorhynchus leonardosi (Mello-Leitão) (Orthoptera: Proscopiidae) on young eucalyptus, Eucalyptus urophylla, in Brazil. The upper third of the trees was attacked (apical meristems and new leaves). During the hottest hours of the day the insects were found sheltered between leaves on the median third of the plants, and feeding when temperatures were milder. The attack was heterogeneous in the stand, being more concentrated when it was neighbouring an area of native 'Cerrado' vegetation. Mean defoliation was 5.8%, with an average of 5.2% of plants with apical meristems cut by the walking sticks. An application of Methyl Parathion was not effective against the pest. Apparently plants older than 6 months were less attacked by the insects, that preferred 3-month old trees. The reduction of native vegetation may explain the dispersion of T. leonardosi from its native host plants to the exotic eucalyptus.

Cortisol response to acute stress in jundiá Rhamdia quelen acutely exposed to sub-lethal concentrations of agrichemicals

Exposure to agrichemicals can have deleterious effects on fish, such as disruption of the hypothalamus-pituitary-inter-renal axis (HPI) that could impair the ability of fish to respond to stressors. In this study, fingerlings of the teleost jundiá (Rhamdia quelen) were used to investigate the effects of the commonly used agrichemicals on the fish response to stress. Five common agrichemicals were tested: the fungicide - tebuconazole, the insecticide - methyl-parathion, and the herbicides - atrazine, atrazine + simazine, and glyphosate. Control fishes were not exposed to agrichemicals and standard stressors. In treatments 2-4, the fishes were exposed to sub-lethal concentrations (16.6%, 33.3%, and 50% of the LC50) of each agrichemical for 96 h, and at the end of this period, were subjected to an acute stress-handling stimulus by chasing them with a pen net. In treatments 5-7 (16.6%, 33.3%, and 50% of the LC50), the fishes were exposed to the same concentrations of the agrichemicals without stress stimulus. Treatment 8 consisted of jundiás not exposed to agrichemicals, but was subjected to an acute stress-handling stimulus. Jundiás exposed to methyl-parathion, atrazine + simazine, and glyphosate presented a decreased capacity in exhibiting an adequate response to cope with stress and in maintaining the homeostasis, with cortisol level lower than that in the control fish (P < 0.01). In conclusion, the results of this study clearly demonstrate that the acute exposure to sub-lethal concentrations of methyl-parathion, atrazine + simazine, and glyphosate exert a deleterious effect on the cortisol response to an additional acute stressor in the jundiá fingerlings. © 2008 Elsevier Inc. All rights reserved.

Resposta cortisolêmica e sensibilidade ao hormônio adrenocorticotrófico (ACTH) de jundiá (Rhamdia quelen) em exposição sub-letal a agrotóxicos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Validação e aplicação de método para análise de pesticidas em água para consumo humano de dourados (ms) por CLAE/UV e CG/DTE

Indiscriminate and inappropriate use of pesticides in agriculture has been pointed out for increasing health problems and environmental damage. Considering that water resources are the principal destiny of those compounds after application, the present study presents optimization and validation of two simple and effi cient analytical methods for pesticides quantifi cation in both surface and groundwater. Were selected the pesticides more commonly used at Dourados (MS - Brazil), region with intense agricultural activity. Pesticides were preconcentrated by solid-phase extraction using C18 (500 mg) cartridges and then divided in two groups for elution and quantifi cation: 2.4-D and 2.4-DCP were eluted with methanol and quantifi ed by high performance liquid chromatography with ultra-violet detector (HPLC-UV) while atrazine, DIA, DEA, trifl uralin and methyl parathion were eluted with ethylacetate (1:1, v/v) and quantifi ed by gas chromatography with thermionic specifi c detector (GC-TSD). The methods showed satisfactory accuracy (76-107%) and precision (<12%) for the substances analyzed at the fortifi ed levels selected for the study, except for DIA (<51%). Study of pesticide stability also presented good results: C18 cartridges could be stored for at least for 21 days at -20ºC with no signs of the compounds degradability. Both methods limits of quantifi cation of the pesticides (0.22 - 0.48 μg L-1) are in accordance to the levels currently established by the Brazilian national legislation for pesticides in water. Although only the pesticide 2.4-D has been detected in two distinct collection points in the study period of time, this work warns for the requirement of systematical analysis of pesticides presence in water destined to human consume, principally in areas of intense agriculture activity. Such monitoring can provide subsidies for public environmental policies.

Seletividade de inseticidas a alguns dos inimigos naturais na cultura do algodão

The aim of this work was study the selectivity of insecticides in favor of natural enemies in cotton (Gossypium hirsutum latifolium Hurtch Lr), DeltaOpal cultivar, in the city of Malhada (BA), and to know the associated beneficial fauna. The study was conducted at the agricultural year of 2010/2011. The design was conducted in randomized blocks with six treatments and four replications. The treatments were: (1) Fipronil 200 SC (0.38 L.ha-1); (2) Alphacypermethrin 100 SC (0.30 L.ha-1), (3) Lufenuron 50 EC (0.30 L.ha-1), (4) Imidacloprid 200 SC (0.30 L.ha-1), (5) Methyl parathion 600 EC (1.00 L.ha-1), and (6) control (water). The product was applied 80 days after emergence, and the evaluations were performed one day before application and 1, 7 and 14 days after application (DAA). The samples were taken using the sampling method beating cloth and Moericke traps. Natural enemies were brought to the laboratory for sorting, counting and identification by family. The toxicity of the products ranged according to the group of natural enemies. Imidacloprid is selective to the spiders and insecticides are moderately toxic (Methyl Parathion and Alphacypermethrin: 1 and 14th DAA; Lufenuron: 14th DAA) or toxic (Fipronil and Alphacypermethrin: 7th DAA). Fipronil (1 DAA), Alphacypermethrin (7th DAA) and Methyl  Parathion (14th DAA) are moderately toxic to adult ladybirds. The analyzed insecticides are toxic to the larvae of ladybirds, with more impact until seven days after the application, with the exception of Methyl Parathion classified, as innocuous until this period. The occurrence of 13 families of spiders and 18 families of parasitic Hymenoptera is registered in cotton agroecosystems in the region of Malhada, in the state of Bahia.

Remoção de metil paration e atrazina em reatores de bancada com fungos

Neste estudo foi avaliada a remoção de metil paration - inseticida e atrazina - herbicida presentes em água, em reatores de bancada, com fungos. A pesquisa foi dividida em quatro etapas: operação em batelada com metil paration e micélio fúngico, com e sem glicose; teste de toxicidade em placas com Aspergillus niger AN400; operação em batelada com os pesticidas atrazina e metil paration e esporos de Aspergillus niger AN400, com e sem glicose; e operação em reatores de leito fixo e fluxo ascendente. Na primeira etapa, a remoção de metil paration foi de 97% nos reatores sem glicose e 94% nos reatores com glicose com 32 dias de reação. Na operação em batelada, com esporos, um modelo cinético de primeira ordem representou bem a velocidade de decaimento de metil paration nesta fase, principalmente, nos reatores que continham glicose. Para os experimentos sem adição de glicose, a constante cinética foi de 0,063 ± 0,005/h, enquanto que para os experimentos com glicose a constante foi de 0,162 ± 0,014/h. Dessa forma, a adição de glicose resultou efetivamente em aumento na velocidade de conversão do inseticida. Na fase experimental, com atrazina e esporos de Aspergillus niger AN400, a presença do substrato primário (glicose) não teve influência na remoção de atrazina, sendo que os percentuais de remoção foram muito próximos aos percentuais encontrados nos reatores sem glicose. O estudo cinético, nessa fase com atrazina e esporos, revelou que para os experimentos sem a adição de glicose, o valor da velocidade de conversão de atrazina (RATZo) foi de 0,023/d, enquanto que para os experimentos com glicose (RATZo) foi 0,022/d. Portanto, a adição de glicose parece não ter influenciado significativamente a velocidade de remoção do herbicida por Aspergillus niger AN400. O teste de toxicidade demonstrou que metil paration e atrazina não inibiram o crescimento do fungo nas várias concentrações testadas, inclusive nas mais elevadas, que foram 60 mg/L e 25 mg/L para metil paration e atrazina, respectivamente. No reator de leito fixo a remoção de metil paration foi de 40% com 12 h de tempo de detenção hidráulica, e 0,5 g glicose/L. Porém, quando a concentração de glicose foi duplicada a remoção de metil paration diminuiu para 35%. Neste reator o pH se manteve na faixa ácida 3,4 a 5,2, considerada ideal para os fungos. Os resultados encontrados mostram a viabilidade dos fungos para remoção desses pesticidas, considerados persistentes no ambiente.