Air, Dermal, and Urinary Metabolite Levels of Backpack and Tractor Sprayers Using the Herbicide Acetochlor in Thailand.

Acetochlor is a chloroacetanilide selective pre-emergent herbicide used for controlling grass and broadleaf weeds in crops. This study compared the acetochlor exposures of backpack and tractor sprayers and assessed whether dermal or air exposures were more important contributors to the overall body burden as measured by urinary metabolites. Sixty sugarcane farmers in Nakhonsawan province, Thailand participated in the study, and breathing zone air and dermal patch samples were collected during spraying. Urine samples were collected before spraying, at the end of the spraying task, and on the day after spraying. For backpack and tractor sprayers, there was no significant difference in their breathing zone air concentrations, total body dermal samples, or urinary 2-methy-6-methyaniline (EMA) concentrations on the day after spraying. In addition, although most backpack and tractor sprayers wore long pants and long sleeve shirts, they were still exposed to acetochlor, as evidenced by a significant increase in the urinary EMA from before spraying (GM = 11.5 µg/g creatinine) to after spraying (GM = 88.5 µg/g creatinine) to the next day (GM = 111.0 µg/g creatinine). Breathing zone air samples were significantly correlated with those of total body dermal patch samples and with urinary EMA concentrations after spraying. This suggests that both air and dermal exposure contribute to urinary EMA levels.

Urinary Cypermethrin Metabolites among Conventional and Organic Farmers in Thailand.

Cypermethrin, a pyrethroid insecticide, is frequently spread on agricultural farmlands and is also used in households in Thailand. Conventional pesticide-using farmers (n = 209) were recruited from the Phitsanulok and Nakornsawan provinces. Certified organic farmers (n = 224) were also recruited in the Yasothorn province. The farmers were interviewed via a questionnaire and the urine from their first morning void was collected. The urine samples were analyzed for 3-phenoxybenzoic acid (3-PBA), cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DCCA), and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (trans-DCCA). The results showed no significant difference in the urinary cypermethrin metabolites between the conventional farmers and the organic farmers, for whom the usage of cypermethrin was not accounted for. However, when conventional farmers who used cypermethrin on the farm and in the home were compared with conventional farmers who did not use any cypermethrin or with organic farmers, a significant difference was noted for all metabolites except for trans-DCCA. These findings show that the most significant exposures to cypermethrin are among conventional farmers who use the insecticide on their farms or in their homes. However, measurable levels of all metabolites were found among both conventional and organic farmers who only used cypermethrin in the home or not at all, suggesting that the at-home use of pyrethroids and other possible exposures from pyrethroid residues on market-bought food may contribute to urinary levels of pyrethroids that exceed those of the general population in the US and Canada.

Urinary Organophosphate Metabolites and Metabolic Biomarkers of Conventional and Organic Farmers in Thailand.

Organophosphate (OP) pesticides are used by most farmers to remove insects and to increase productivity; however, questions remain on the long-term health impacts of their use. This study assessed the relationship between OP biomarker levels and metabolic biomarker parameters. Conventional farmers (n = 213) and organic farmers (n = 225) were recruited, interviewed, and had physical health examinations. Serum glucose and lipid profiles, triglycerides, total cholesterol, high-density lipoproteins (HDL), and low-density lipoproteins (LDL) were measured. The average age, gender, education, and self-reported agricultural work time, work in second jobs, smoking status, alcohol consumption, insecticide use at home, home location near farmlands and years of pesticide use were significantly different between the conventional and organic farmers. The urinary OP metabolite levels were also significantly different between the two groups. With an increase in urinary diethyl phosphate, dimethyl phosphate and dialkyl phosphate metabolites, the total cholesterol, LDL and HDL, were significantly increased for all farmers after controlling for age, gender, alcohol consumption, years of pesticide use, and home location near farmlands. The results are consistent with our previous studies which suggests that pesticide usage, especially organophosphates, may increase the risk of cardiovascular disease and stroke among Thai farmers.

Disruption of the Diurnal Cortisol Hormone Pattern by Pesticide Use in a Longitudinal Study of Farmers in Thailand.

Alteration of the hypothalamic-pituitary-adrenal (HPA) axis hormones has been associated with a range of chronic metabolic and cardiovascular health conditions. This study evaluated whether type of farming (organic versus conventional) or the number of self-reported days of spraying pesticides in the past 8 months was associated with diurnal cortisol levels. Salivary cortisol levels were measured four times a day (waking, 30 min after waking, 6 h after waking and bedtime) longitudinally, 8 months apart during three rounds of data collection. Pesticide using (conventional) and organic farmers were recruited to participate. Pesticide use in the previous 8 months was determined as the number of spray days for each type of pesticide used (herbicide, insecticide, fungicide) from self-reported questionnaires. Estimates of cortisol levels at four time points, the cortisol awakening response (CAR), and the diurnal cortisol slope (DCS) were estimated with a longitudinal mixed model that accounted for the non-linearity of cortisol levels across the day. Conventional farmers had significantly lower cortisol levels at waking than organic farmers (3.39 versus 3.86 ng ml-1), 30 min after waking (5.87 versus 6.96 ng ml-1), 6 h after waking (1.62 versus 1.88 ng ml-1), and lower diurnal cortisol slope (-2.26 versus -2.51 ng ml-1). Farmers who frequently applied herbicides (90th percentile of the number of spray days in the past 8 months) had significantly lower waking, 30 min after waking, 6 h after waking, bedtime and diurnal cortisol slopes compared with those with no spray days of herbicide in the past 8 months (organic and some of the conventional farmers). Those who frequently applied insecticides in the past 8 months had significantly lower bedtime levels and diurnal cortisol slopes, compared with those with no spray days of insecticide in the past 8 months. There were no significant differences in cortisol hormones between those who frequently applied fungicides and those who did not spray fungicides. Repeated pesticide use appears to be disrupting the HPA axis and depressing the normal diurnal cortisol rhythm among conventional Thai farmers.

Acute Changes in Thyroid Hormone Levels among Thai Pesticide Sprayers.

The objective of this study was to investigate the relationship of acute pesticide exposures and acute changes in thyroid hormones among Thai farmers. We recruited 78 farmers, who were scheduled to spray insecticides (chlorpyrifos and/or cypermethrin) or herbicides (paraquat and/or glyphosate). On the day before spraying, farmers collected their first morning void urine and went for blood collection. On the spray day, urine samples were collected at end of the spraying event and they were interviewed with questionnaires. The next morning, the first morning void urine and blood samples were collected. Blood samples were analyzed for thyroid hormones. Urine samples were analyzed for the metabolites of the pesticide sprayed. The results showed that the thyroid hormones, free triiodothyronine (FT3) and total triiodothyronine (T3) were significantly reduced as urinary chlorpyrifos metabolite increased the day after spraying. Total thyroxine (T4) significantly increased as cypermethrin metabolites increased the day after spraying. T4 significantly increased as urinary glyphosate levels increased; however, FT3 and T3 decreased significantly as urinary paraquat levels increased the day after spraying. These findings suggest that acute exposures to the pesticides chlorpyrifos, cypermethrin, paraquat and glyphosate can produce acute effects on the hypothalamic-pituitary-thyroid (HPT) axis, acutely altering thyroid hormone levels.

A Microfluidic Paper-Based Analytical Device for Type-II Pyrethroid Targets in an Environmental Water Sample.

A detection method for type-II pyrethroids in an environmental water sample using a microfluidic paper-based analytical device (µPAD) is reported here. The detection approach is based on the formation of cyanide from the hydrolysis of type-II pyrethroids and the colorimetric detection of cyanide on a layer-based µPAD. Parafilm and inexpensive laminating pouches were used to create a hydrophobic barrier for the assay on the µPAD. This detection approach was selective to type-II pyrethroids in water for which an environmental water sample was tested. The calibration curves for cypermethrin, deltamethrin, cyhalothrin, and fenvalerate ranged from 2 to 40 µg/mL without sample preconcentration. The lower concentrations of type-II pyrethroids can be assessed by including a preconcentration step prior to the detection on a µPAD. This detection system provides an alternative platform for fast, semiquantitative testing for pesticide contamination in environmental surface water by allowing for portability, low reagent/sample consumption, and low-cost testing.

A Membrane-based Disposable Well-Plate for Cyanide Detection Incorporating a Fluorescent Chitosan-CdTe Quantum Dot.

A novel approach to building a membrane-based disposable well-plate, here applied to cyanide detection, is described. Chitosan encapsulated CdTe quantum dots with a maximum emission at 520 nm (CS-QD520) were used as fluorophores. The CS-QD520 nanoparticle was specifically quenched by copper(II), and the quenched CS-QD520 (Cu-CS-QD520) was deposited onto a glass microfiber filter (GF/B). Subsequent introduction of cyanide ion resulted in fluorescence recovery. The "signal-ON" fluorescence linearly correlated to cyanide concentrations in the range of 38.7 to 200 µM with a limit of detection of 11.6 µM. The assay was incorporated into a membrane-based well-plate format to enhance sample throughput. A three-layer paper/glass microfiber well plate design was cut using a laser cutter and assembled using a polycaprolactone (PCL) as a bonding agent in a low-cost laminator. The experimental conditions were optimized and applied to detect cyanide in drinking water with rapid, high-throughput, low-cost analysis.

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library.

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.

Development of a carbon dot (C-Dot)-linked immunosorbent assay for the detection of human α-fetoprotein.

A sensitive, selective, environmentally friendly, high-throughput, well-plate-based immunosorbent assay was developed to detect human α-fetoprotein (AFP) using carbon dots (C-Dots). Highly fluorescent C-Dots were synthesized using a one-step hydrothermal reaction, with citric acid serving as the carbon source and ethylene diamine acting as the nitrogen source. The reaction conditions were optimized to obtain the desired surface functionality. Then, the C-Dots were used to label one member of the anti-AFP pair (Ab2) via amine-amine coupling using glutaraldehyde. The capture anti-AFP (Ab1) was coated onto polystyrene well plates and bovine serum albumin (BSA) was used to block unsaturated binding sites. AFP was incubated in Ab1-coated wells; unbound AFP was then washed away with Tween-20. Next, the C-Dot-labeled Ab2 was added to form a sandwich immunocomplex with the AFP bound to the Ab1-coated wells. The fluorescence intensities detected from the C-Dots on these sandwich immunocomplexes were positively correlated to the concentrations of AFP antigen. A five-parameter logistic regression calibration curve was established between fluorescence and clinically important AFP concentrations (range: 0-350 ng/mL with a correlation coefficient of R(2) = 0.995). The results from the C-Dot-based immunoassay were in agreement with results from traditional immunoassays, which used horseradish peroxidase (HRP, R(2) = 0.964) and fluorescein isothiocyanate (FITC, R(2) = 0.973). These results indicated that C-Dots have great potential to be applied as biolabels for high-throughput well-plate-based immunoassays.

Superheated water extraction and phase transfer methylation of phenoxy acid herbicides from solid matrices.

Phase transfer catalytic methylation was applied to directly derivatise chlorophenoxy acid herbicides in superheated water extracts from sand and soil samples. The extractions were carried out at 120 degrees C statically for 5 min and then dynamically for 10 min at 1.0 mL min(-1) using water at pH 11.0 for a sand matrix and a flow rate of 0.5 mL min(-1) at pH 7.0 for soil samples. The methylation was carried out on-line on the extraction solution with ultrasonication at 80 degrees C, using either 0.05 mmol tetrabutylammonium bromide (TBAB) or 0.0125 mmol cetyltrimethylammonium bromide (CTAB) as phase transfer catalysts with 0.20 mmol methyl iodide in 2.0 mL dichloromethane trapping solvent. The former catalyst provided a higher yield but the latter gave fewer interfering peaks. The recoveries of most chlorophenoxy acids using the TBAB catalyst ranged from 67 to 105% for sand and from 82 to 114% for soil sample, except phenoxyacetic acid, 2-(2, 4-dichlorophenoxy)propanoic acid and 1-naphthaleneacetic acid, while those by using CTAB were slightly lower. Detection limits of all the analytes extracted from sand using TBAB catalyst were in a range of 5.3-16 microg g(-1) analysed by using gas chromatography with flame ionization detection (GC-FID).