Laccase–Prussian blue film–graphene doped carbon paste modified electrode for carbamate pesticides quantification

A novel enzymatic biosensor for carbamate pesticides detection was developed through the direct immobilization of Trametes versicolor laccase on graphene doped carbon paste electrode functionalized with Prussianblue films (LACC/PB/GPE). Graphene was prepared by graphite sonication-assisted exfoliation and characterized by transmission electron microscopy and X-ray photoelectron spectro- scopy. The Prussian blue film electrodeposited onto graphene doped carbon paste electrode allowed considerable reduction of the charge transfer resistance and of the capacitance of the device.The combined effects of pH, enzyme concentration and incubation time on biosensor response were optimized using a 23 full-factorial statistical design and response surface methodology. Based on the inhibition of laccase activity and using 4-aminophenol as redox mediator at pH 5.0,LACC/PB/GPE exhibited suitable characteristics in terms of sensitivity, intra-and inter-day repeatability (1.8–3.8% RSD), reproducibility (4.1 and 6.3%RSD),selectivity(13.2% bias at the higher interference: substrate ratios tested),accuracy and stability(ca. twenty days)for quantification of five carbamates widely applied on tomato and potato crops.The attained detection limits ranged between 5.2×10−9 mol L−1(0.002 mg kg−1 w/w for ziram)and 1.0×10−7 mol L−1 (0.022 mg kg−1 w/w for carbofuran).Recovery values for the two tested spiking levels ranged from 90.2±0.1%(carbofuran)to 101.1±0.3% (ziram) for tomato and from 91.0±0.1%(formetanate)to 100.8±0.1%(ziram)for potato samples.The proposed methodology is appropriate to enable testing pesticide levels in food samples to fit with regulations and food inspections.

Detection of carbamate pesticides in vegetable samples using cholinesterase-based biosensors

Biosensors for determination of carbamates in vegetables based on five different cholinesterases as biorecognition elements and a screen-printed electrode system as an amperometric transducer were developed. Measurements were simply performed by dropping solutions (either sample or substrate) directly on the biosensor. The response of biosensors to selected carbamates (aldicarb, carbaryl, carbofuran, methomyl and propoxur) was characterized. The performance was evaluated on extracts of potatoes and carrots, the results from the AChE biosensor and a standard HPLC procedure were compared. Finally, the biosensor was used for the direct analysis of vegetable juices without any pretreatment steps. In this case, 10 mu g/L levels of added carbofuran and propoxur were reliably identified. The whole procedure takes less than 20 min including 10 min incubation with samples. The concentrations of carbamates determined with biosensor agreed well for carbofuran. Lower response was observed for propoxur.

Electrochemical detection of carbamate pesticides in fruit and vegetables with a biosensor based on acetylcholinesterase immobilised on a composite of polyaniline-carbon nanotubes

A sensitive electrochemical acetylcholinesterase (AChE) biosensor was successfully developed on polyaniline (PANI) and multi-walled carbon nanotubes (MWCNTs) core-shell modified glassy carbon electrode (GC), and used to detect carbamate pesticides in fruit and vegetables (apple, broccoli and cabbage). The pesticide biosensors were applied in the detection of carbaryl and methomyl pesticides in food samples using chronoamperometry (CA). The GC/MWCNT/PANI/AChE biosensor exhibited detection limits of 1.4 and 0.95 mu mol L-1, respectively, for carbaryl and methomyl. These detection limits were below the allowable concentrations set by Brazilian regulation standards for the samples in which these pesticides were analysed. Reproducibility and repeatability values of 2.6% and 3.2%, respectively, were obtained in the conventional procedure. The proposed biosensor was successfully applied in the determination of carbamate pesticides in cabbage, broccoli and apple samples without any spiking procedure. The obtained results were in full agreement with those from the HPLC procedure. (C) 2012 Elsevier Ltd. All rights reserved.

Organophosphate and carbamate poisonings in the northwest of Parana state, Brazil from 1994 to 2005: clinical and epidemiological aspects

In the present study, clinical and epidemiological aspects of 529 intoxication cases of organophosphate or carbamate pesticides in the northwest of the state of Parana, Brazil, over a twelve-year period (1994-2005), are presented. One hundred-five of 257 patients (40.8%) who attempted suicide were admitted to Intensive Care Units (ICUs), with an average hospital stay of two days (range 1-40 days). Men corresponded to 56.4% of the cases of suicide attempts and sixteen individuals died. One hundred-forty patients intoxicated due to occupational exposure were all young adults and nine of them were admitted to ICU, with average hospital stays of eight days (range 1-16 days). Of these cases, two patients died. One hundred twenty-four patients intoxicated due to accidental exposure were mainly children and had a hospital average stay of four days. Twenty patients were admitted to the ICU, and one of them died. Overall complications included respiratory failure, convulsions, and aspiration pneumonia. Deliberate ingestion of organophosphates and carbamates Was much more toxic than occupational and accidental exposure. Men aged 15-39 years were the most likely to attempt suicide with these agents and had more prolonged ICU with significant complications and mortality

The development of automated methods for the determination of trace concentrations of carbomate pesticides in water using solid sorbent pre-concentration methods and high performance liquid chromatography /

Several automated reversed-phase HPLC methods have been developed to determine trace concentrations of carbamate pesticides (which are of concern in Ontario environmental samples) in water by utilizing two solid sorbent extraction techniques. One of the methods is known as on-line pre-concentration'. This technique involves passing 100 milliliters of sample water through a 3 cm pre-column, packed with 5 micron ODS sorbent, at flow rates varying from 5-10 mUmin. By the use of a valve apparatus, the HPLC system is then switched to a gradient mobile phase program consisting of acetonitrile and water. The analytes, Propoxur, Carbofuran, Carbaryl, Propham, Captan, Chloropropham, Barban, and Butylate, which are pre-concentrated on the pre-column, are eluted and separated on a 25 cm C-8 analytical column and determined by UV absorption at 220 nm. The total analytical time is 60 minutes, and the pre-column can be used repeatedly for the analysis of as many as thirty samples. The method is highly sensitive as 100 percent of the analytes present in the sample can be injected into the HPLC. No breakthrough of any of the analytes was observed and the minimum detectable concentrations range from 10 to 480 ng/L. The developed method is totally automated for the analysis of one sample. When the above mobile phase is modified with a buffer solution, Aminocarb, Benomyl, and its degradation product, MBC, can also be detected along with the above pesticides with baseline resolution for all of the analytes. The method can also be easily modified to determine Benomyl and MBC both as solute and as particulate matter. By using a commercially available solid phase extraction cartridge, in lieu of a pre-column, for the extraction and concentration of analytes, a completely automated method has been developed with the aid of the Waters Millilab Workstation. Sample water is loaded at 10 mL/min through a cartridge and the concentrated analytes are eluted from the sorbent with acetonitrile. The resulting eluate is blown-down under nitrogen, made up to volume with water, and injected into the HPLC. The total analytical time is 90 minutes. Fifty percent of the analytes present in the sample can be injected into the HPLC, and recoveries for the above eight pesticides ranged from 84 to 93 percent. The minimum detectable concentrations range from 20 to 960 ng/L. The developed method is totally automated for the analysis of up to thirty consecutive samples. The method has proven to be applicable to both purer water samples as well as untreated lake water samples.

A greener and highly sensitive flow-based procedure for carbaryl determination exploiting long pathlength spectrophotometry and photochemical waste degradation

An environmentally friendly analytical procedure with high sensitivity for determination of carbaryl pesticide in natural waters was developed. The flow system was designed with solenoid micro-pumps in order to improve mixing conditions and minimize reagent consumption as well as waste generation. A long pathlength (100 cm) flow cell based on a liquid core waveguide (LCW) was employed to increase the sensitivity in detection of the indophenol formed from the reaction between carbaryl and p-aminophenol (PAP). A clean-up step based on cloud-point extraction was explored to remove the interfering organic matter, avoiding the use of toxic organic solvents. A linear response was observed within the range 5-200 mu g L(-1) and the detection limit, coefficient of variation and sampling rate were estimated as 1.7 mu g L(-1) (99.7% confidence level), 0.7% (n=20) and 55 determinations per hour, respectively. The reagents consumption was 1.9 mu g of PAP and 5.7 mu g of potassium metaperiodate, with volume of 2.6 mL of effluent per determination. The proposed procedure was selective for the determination of carbaryl, without interference from other carbamate pesticides. Recoveries within 84% and 104% were estimated for carbaryl spiked to water samples and the results obtained were also in agreement with those found by a batch spectrophotometric procedure at the 95% confidence level. The waste of the analytical procedure was treated with potassium persulphate and ultraviolet irradiation, yielding a colorless residue and a decrease of 94% of total organic carbon. In addition, the residue after treatment was not toxic for Vibrio fischeri bacteria. (c) 2010 Elsevier B.V. All rights reserved.

Cloud point extraction and concentration of carbaryl from natural waters

An improved procedure is proposed for determination of the pesticide carbaryl in natural waters based on double cloud point extraction. The clean up step was carried out only with Triton X-114 in alkaline medium in order to avoid the use of toxic organic solvents as well as to minimise waste generation. Cloud point preconcentration of the product of the reaction of the analyte with p-aminophenol and cetyltrimethylammonium bromide was explored to increase sensitivity and improve the detection limit. Linear response was achieved within 10 and 500 mu g L-1 and the apparent molar absorptivity was estimated as 4.6 x 105 L mol-1 cm-1. The detection limit was estimated as 7 mu g L-1 at the 99.7% confidence level and the coefficient of variation was 3.4% (n = 8). Recoveries within 91 and 99% were estimated for carbaryl spiked water samples. The results obtained for natural water samples were in agreement with those achieved by the batch of spectrophotometric procedure at the 95% confidence level. The proposed procedure is then a simple, fast, inexpensive and greener alternative for carbaryl determination.

Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: characterisation and role in organophosphate resistance

Acetylcholinesterase is the target of organophosphate and carbamate pesticides. Organophosphate resistance is widespread in the cattle tick, Boophilus microplus, in Australia. We have isolated a cDNA of acetylcholinesterase from B. microplus and show that it would encode a protein 62 kDa in size. The predicted amino acid sequence contains all the residues characteristic of an acetylcholinesterase. Alternative splicing of the transcript was detected at both the 5' and 3' ends. Alternative splicing at the 5' end would result in two proteins differing by six amino acids. This is the first report of alternative splicing of the N-terminal coding region in a cholinesterase. No point mutations were detected in the acetylcholinesterase gene from organophosphate resistant strains of B. microplus. Alternative explanations for resistance to organophosphates in B. microplus are discussed. (C) 1998 Elsevier Science Ltd. All rights reserved.

Effect of Community of Residence on Neurobehavioral Development in Infants and Young Children in a Flower-Growing Region of Ecuador

In this study we compared neurobehavioral development in Ecuadoran children living in two communities with high potential for exposure to organophosphate (OP) and carbamate pesticides to that of children living in a community with low potential for exposure. Residence in communities with high potential for exposure to OP and carbamate pesticides was associated with poorer neurobehavioral development of the child even after controlling for major determinants of delayed development. Malnourished populations may be particularly vulnerable to neurobehavioral effects of pesticide exposure.

Determination of carbaryl in tomato ""in natura"" using an amperometric biosensor based on the inhibition of acetylcholinesterase activity

This work reports the utilization of two methodologies for carbaryl determination in tomatoes. The measurements were carried out using an amperometric biosensor technique based on the inhibition of acetylcholinesterase activity due to carbaryl adsorption and a HPLC procedure. The electrochemical experiments were performed in 0.1 mol L-1 phosphate buffer solutions at pH 7.4 with an incubation time of 8 min. The analytical curve obtained in pure solutions showed excellent linearity in the 5.0 x 10(-5) to 75 x 10(-5) mol L-1 range, with the limit of detection at 0.4 x 10(-3) gL(-1). The application of such a methodology in tomato samples involved solely liquidising the samples, which were spiked with 6.0 x 10(-6) and 5.0 x 10(-5) mol L-1 carbaryl. Recovery in such samples presented values of 99.0 and 92.4%, respectively. In order to obtain a comparison, HPLC experiments were also conducted under similar conditions. However, the tomato samples have to be manipulated by an extraction procedure (MSPD), which yielded much lower recovery values (78.3 and 84.8%, respectively). On the other hand, the detection limit obtained was much lower than that for the biosensor, i.e., 3.2 x 10(-6) g L-1. Finally, the biosensor methodology was employed to analyze carbaryl directly inside the tomato, without any previous manipulation. In this case, the biosensor was immersed in the tomato pulp, which had previously been spiked with the pesticide for 8 min, removed and inserted in the electrochemical cell. A recovery of 83.4% was obtained, showing very low interference of the matrix constituents. (C) 2007 Elsevier B.V. All rights reserved.

Evaluation of a highly sensitive amperometric biosensor with low cholinesterase charge immobilized on a chemically modified carbon paste electrode for trace determination of carbamates in fruit, vegetable and water samples

A highly sensitive amperometric biosensor for determination of carbamate pesticides directly in water, fruit and vegetable samples has been evaluated, electrochemically characterized and optimized. The biosensor strip was fabricated in screen printed technique on a ceramic support using silver-based paste for reference electrode, and platinum-based paste for working and auxiliary electrodes. The working electrode was modified by a layer of carbon paste mixed with cobalt(II) phthalocyanine and acetylcellulose. Cholinesterase (ChE) enzymes with low enzymatic charge were immobilized on this layer. The operational simplicity of the biosensor consists in that a small drop (similar to 50 mu l) of substrate or sample is deposited on a horizontally positioned biosensor strip representing the microelectrochemical cell. The working potential of the biosensor was 370 mV versus Ag/AgI on a ship reference electrode preventing the interference of electroactive species which are oxidable at more positive potentials. The biosensor was applied to investigate the degradation of two reference ChE inhibitors in freeze dried water under different storage conditions and for direct determination of some N-methylcarbamates (NMCs) in fruit and vegetable samples at ppb concentration levels without any sample pretreatment. A comparison of the obtained results for the total carbamate concentration was done against those obtained using HPLC measurements. (C) 1999 Elsevier B.V. B.V. All rights reserved.

Conventional (MG-BR46 Conquista) and Transgenic (BRS Valiosa RR) Soybeans Have No Mutagenic Effects and May Protect Against Induced-DNA Damage In Vivo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In vitro and in vivo inhibition of acetylcholinesterase and carboxylesterase by metals in zebrafish (Danio rerio)

Metals are natural components in ecosystems; however, if these elements are in excess, they can have adverse effects on living organisms. This study analyzes the interference of copper, lead, iron and cadmium in acetylcholinesterase (AChE) and carboxylesterase (CbE) activities in zebrafish. AChE was significantly inhibited in vitro by copper, iron, lead and cadmium at higher concentrations (10 and 20 mmol/L), whereas CbE was inhibited only at a concentration of 20 mmol/L. In vivo, only lead and cadmium were able to cause AChE inhibition at higher concentrations, while iron didn't cause any changes, and copper promoted an increase in AChE activity at a concentration of 0.06 mg/L. CbE activity did not change at any of the times (two and seven days) and concentrations tested, except in the case of copper exposure, which resulted in a decrease in CbE activity. Indeed, iodoacetamide treatment didn't changed AChE neither CbE activities, results which indicate that the metal inhibiting effect is probably not due to its biding to thiol groups close the active site of the enzyme. This outcome reveals that metals are important esterase inhibitors in zebrafish, and should be considered in environmental monitoring studies that use esterase inhibition as exposure biomarkers of organophosphate and carbamate pesticides. © 2012 Elsevier Ltd. All rights reserved.

Desenvolvimento de sensor biomimético para detecção de resíduos de pesticidas organofosforados e carbamatos em alimentos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A multiresidue method for the analysis of carbamate and urea pesticides from soils by microwave-assisted extraction and liquid chromatography with photodiode array detection

A multiresidue approach using microwave-assisted extraction and liquid chromatography with photodiode array detection was investigated for the determination of butylate, carbaryl, carbofuran, chlorpropham, ethiofencarb, linuron,metobromuron, and monolinuron in soils. The critical parameters of the developed methodology were studied. Method validation was performed by analyzing freshly and aged spiked soil samples. The recoveries and relative standard deviations reached using the optimized conditions were between 77.0 ± 0.46% and 120 ± 2.9% except for ethiofencarb (46.4 ± 4.4% to 105 ± 1.6%) and butylate (22.1 ± 7.6% to 49.2 ± 11%). Soil samples from five locations of Portugal were analysed.