Efficacy of novel bacterial consortia in degrading fipronil and thiobencarb in paddy soil: a survey for community structure and metabolic pathways.

Introduction: Fipronil (FIP) and thiobencarb (THIO) represent widely utilized pesticides in paddy fields, presenting environmental challenges that necessitate effective remediation approaches. Despite the recognized need, exploring bacterial consortia efficiently degrading FIP and THIO remains limited. Methods: This study isolated three unique bacterial consortia-FD, TD, and MD-demonstrating the capability to degrade FIP, THIO, and an FIP + THIO mixture within a 10-day timeframe. Furthermore, the bioaugmentation abilities of the selected consortia were evaluated in paddy soils under various conditions. Results: Sequencing results shed light on the consortia's composition, revealing a diverse bacterial population prominently featuring Azospirillum, Ochrobactrum, Sphingobium, and Sphingomonas genera. All consortia efficiently degraded pesticides at 800 µg/mL concentrations, primarily through oxidative and hydrolytic processes. This metabolic activity yields more hydrophilic metabolites, including 4-(Trifluoromethyl)-phenol and 1,4-Benzenediol, 2-methyl-, for FIP, and carbamothioic acid, diethyl-, S-ethyl ester, and Benzenecarbothioic acid, S-methyl ester for THIO. Soil bioaugmentation tests highlight the consortia's effectiveness, showcasing accelerated degradation of FIP and THIO-individually or in a mixture-by 1.3 to 13-fold. These assessments encompass diverse soil moisture levels (20 and 100% v/v), pesticide concentrations (15 and 150 µg/g), and sterile conditions (sterile and non-sterile soils). Discussion: This study offers an understanding of bacterial communities adept at degrading FIP and THIO, introducing FD, TD, and MD consortia as promising contenders for bioremediation endeavors.

Optimizing the QuEChERS method for efficient monitoring of fipronil, thiobencarb, and cartap residues in paddy soils with varying properties.

This study aims to optimize the QuEChERS methodology for extracting three pesticides (fipronil, thiobencarb, and cartap) from two paddy soils with distinct characteristics. Various modifications were explored to enhance extraction efficiency, employing acetonitrile (MeCN) or ethyl acetate (EtOAc) for extraction and primary-secondary amine (PSA) and graphitized carbon black (GCB) for the clean-up. Assessment criteria included accuracy, precision, linearity, detection limits, uncertainty, and matrix effects. Results revealed that the clayey soil with lower organic carbon (OC) content (1.26%) and 100% moisture yielded the highest pesticide recoveries (113.72%, 115.73%, and 116.41% for FIP, THIO, and CART, respectively). In contrast, the silty clayey soil with higher OC content (2.91%) and 20% water content exhibited poor recoveries (< 60%). FIP and CART demonstrated better recoveries with MeCN, while THIO performed better with EtOAc under specific moisture conditions. Clean-up sorbents significantly reduced FIP and CART recoveries, with THIO recoveries less affected. Acidifying with HCl substantially improved CART recovery. EtOAc introduced a moderate to strong matrix effect for FIP and THIO, while MeCN in soils with 100% moisture resulted in a strong matrix effect for CART. The study highlighted the substantial impact of extraction conditions, pesticide properties, and soil conditions on the outcomes of the QuEChERS method. A comprehensive understanding of these interplays was deemed crucial for accurately quantifying pesticide residues in agricultural soils.

Urinary metabolites of diazinon and chlorpyrifos in sprayer operators and farm workers of a potato farm.

In order to investigate the effect of diazinon and chlorpyrifos on agricultural workers exposed to pesticides, urinary metabolites 2-Isopropyl-6-methyl-4-pyrimidinol (IMPy) and 3,5,6-Trichloro-2-pyridinol (TPCy) in farm workers, sprayer operators, and non-exposed people as a control group were measured. The modified QuEChERS method was applied to extract samples and was measured using a gas chromatograph/nitrogen-phosphorus detector. The obtained results showed that the median concentrations of TCPy were 36.92-547.7 and 7.7-49.58 ng/mL for sprayer operators and farm workers, respectively. Moreover, the median concentrations of IMPy were 81.66-593.1 ng/mL for sprayer operators and 40.6-66.1 ng/mL for farm workers. The control group had no measurable metabolites. The IMPy level of 60% of sprayer operators was significantly higher (P ˂ 0.05) than the TCPy level. The analysis of variance highlighted the significant relationship (P ˂ 0.05) between the levels of each metabolite and the use of safety gloves, respiratory masks, safety goggles, working time per week, and type of insecticide exposure. Our findings revealed the need to measure the urinary metabolites of these insecticides in other exposed workers. Also, workers should be taught the impact of using personal protective equipment.

Physiological Differences Between Seasonal Dimorphs of Agonoscena pistaciae (Hemiptera: Aphalaridae) Elicit Distinct Host Plant Responses, Informing Novel Pest Management Insights.

We examined differences in the physiology and life history between dimorphs of the common pistachio psyllid, Agonoscena pistaciae (Burckhardt and Lauterer) (Hemiptera: Aphalaridae), and how they differ in elicitating host plant production of key metabolites and volatile compounds involved in the recruitment of herbivores and natural enemies. Summer morphs had higher activities of glutathione S-transferase, carboxylesterase, acetylcholinesterase, and cytochrome P450 monooxygenase, superoxide dismutase, catalase, peroxidase, phenoloxidase, and a higher total protein content compared to winter morphs, whereas the latter had higher amounts of lipid, carbohydrate, and glycogen. Winter morphs were heavier, with a higher chitin content and longer preoviposition period, but greater fecundity and longevity than summer morphs. A lower LC50 to thiamethoxam for winter morphs resulted in higher mortality following exposure to the recommended rate of this insecticide in a greenhouse trial. Feeding by winter morphs elicited more strongly the release of volatile compounds known to be attractive to other herbivores, whereas feeding by summer morphs elicited more strongly the release of volatiles implicated in the attraction of natural enemies. Feeding by psyllids increased the concentrations of nitrogenous compounds, carbohydrates, vitamins, and amino acids in plants, the winter morph eliciting larger changes and more improved host plant quality. We conclude that winter morphs are more vulnerable targets for chemical control in early spring, whereas management of summer morphs could rely more on conservation biological control.

Salicylic Acid Induced Resistance in Drought-Stressed Pistachio Seedlings Influences Physiological Performance of Agonoscena pistaciae (Hemiptera: Aphalaridae).

Induced host plant resistance is a potential approach to insect and disease management. Salicylic acid (SA) acts as a signal molecule to induce resistance in plants against sap-sucking insects. The effects of salicylic acid-induced resistance against common pistachio psylla, Agonoscena pistaciae Burckhardt and Lauterer, were investigated in well-watered and drought-stressed pistachio, Pistacia vera L. cv. Akbari, seedlings. Agonoscena pistaciae exhibited a significant preference for plants treated with SA as compared with untreated controls or those subjected to drought stress. Plants subjected to both drought stress and SA treatment were equivalently colonized as compared with control plants but were more attractive than those subjected to drought stress alone. Psyllid mortality increased on plants subjected to simultaneous drought stress and SA treatment as compared with controls. Salicylic acid treatment mediated production of defensive enzymes in plants, including superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol peroxidase (GPX) and polyphenol oxidase (PPO), as well as that of other metabolites such as phenol, malondialdehyde (MDA), H2O2, free amino acids, and pigments via phenylpropanoid pathways under conditions of drought. Despite increases in activity of detoxification (glutathione S transferase, carboxylesterase) and antioxidative (SOD, CAT, APX, phenoloxidase, GPX) enzymes in psyllids, reduced survival of A. pistaciae on drought stressed and SA-treated plants was likely caused by excessive H2O2 and high phenolic content in treated plants. Based on our results, we postulate that salicylic acid-induced defense against A. pistaciae under drought conditions could be manipulated to enhance antibiosis against this key pest in pistachio orchards.

Evaluation of physiological and biochemical responses of pistachio plants (Pistacia vera L.) exposed to pesticides.

Pesticides may manipulate plant physiology as non-target organisms. In this study, we examined biochemical responses of pistachio plants (Pistacia vera L.) to imidacloprid and phosalone as common pesticides used to control pistachio psyllids. Enzymatic characterization in treated plants with pesticides showed greater specific activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, phenylalanine ammonia-lyase, glutathione reductase, and glutathione S-transferase compared with untreated plants during 14 days after treatment. Further experiments displayed elevated levels of total phenols and total proteins coupled with significant increases in proline and total soluble carbohydrate contents in treated plants in comparison to untreated plants. Moreover, pesticide treatment leads to a significant decrease in polyphenol oxidase activity. Nevertheless, no significant changes in contents of hydrogen peroxide, malondialdehyde, total chlorophyll, and electrolyte leakage index were obtained in treated plants. Pesticides' impacts on host plant physiology resulted in similar responses between two pesticides with differences in peak days. Overall, the findings of this study provide an insight into the side effects of phosalone and imidacloprid, chemicals with no specific target site in plants, on the physiology and biochemistry of pistachio plants at recommended rates.

Physiological responses of plants and mites to salicylic acid improve the efficacy of spirodiclofen for controlling Tetranychus urticae (Acari: Tetranychidae) on greenhouse tomatoes.

Salicylic acid (SA) is a signaling molecule that can induce plant resistance to certain herbivores. Although the role of jasmonic acid in mediating mite-tomato plant interactions has been well studied, the role of salicylic acid has not. This study examined how the application of exogenous SA, via its effects on tomato plant physiology, alters the activity of mite digestive enzymes, mite energy reserves, and mite susceptibility to spirodiclofen. Enzymatic activity-including superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase-along with contents of total phenolic, hydrogen peroxide, and total chlorophyll significantly increased in plants 24 h after treatment with 2 mM of SA. In contrast, catalase activity significantly decreased in treated plants, and malondialdehyde content was unaffected. Mites fed on tomato plants treated with SA had significantly lower glutathione S-transferase, esterase, α-amylase, and aminopeptidase activities than those fed on control plants. Energy reserve analyses demonstrated a significant decrease in contents of lipid, protein, and glycogen in mites fed on SA-treated plants, whereas carbohydrate content significantly increased. The LC50 of spirodiclofen was decreased 1.8-fold for Tetranychus urticae fed on SA-treated tomato plants compared to controls. Treatment of adult mites with 2 mM SA on leaf discs did not cause any direct mortality after 24 h. Finally, a greenhouse bioassay confirmed that spider mite mortality following exposure to spirodiclofen was significantly higher on SA plants than on control plants. Mortality of mites exposed to half of the recommended rate of spirodiclofen was similar to those exposed to the recommended rate when they were held on treated plants. These results have valuable implications for T. urticae management programs in tomato production.

Micronutrient Fertilization of Greenhouse Cucumbers Mitigates Pirimicarb Resistance in Aphis gossypii (Hemiptera: Aphididae).

The nutritional status of host plants can have direct impacts on herbivore physiology and insect-plant interactions. We investigated the effect of micronutrients, including manganese, iron, zinc, and copper, on cucumber plant physiology, and on the biology and physiology of a strain of Aphis gossypii Glover selected over 12 generations to be resistant to pirimicarb. The micronutrient treatment increased the activity of superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase in cucumber plants, and also increased levels of total phenolics, hydrogen peroxide, salicylic acid, and total chlorophyl, whereas malondialdehyde levels were unaffected. Pirimicarb-resistant cotton aphids that fed on micronutritient-amended cucumber plants expressed significantly decreased levels of acetylcholinesterase and detoxifying enzymes, specifically glutathione S-transferase, and carboxylesterase. Analysis of energy reserves in resistant A. gossypii fed on micronutritient-amended plants revealed decreases in the lipid and protein contents of aphids, whereas glycogen and carbohydrate contents showed no response. Resistant cotton aphids fed on micronutritient-amended plants showed significantly reduced fecundity, longevity, and reproductive periods, and a 1.7-fold reduction in pirimicarb LC50 compared with those fed on control plants. We conclude that micronutrient amendment negatively impacts the biological performance of insecticide-resistant cotton aphids, and diminishes their resistance to pirimicarb. Both direct effects on plant health, such as enhanced inducible defenses, and indirect effects on aphid fitness, such as reduced biological performance and detoxification abilities, were implicated. Therefore, optimization of micronutrient amendments could be a useful complement to other tactics for managing insecticide-resistant A. gossypii on cucumbers, and warrants exploration in other contexts.

Diazinon degradation by bacterial endophytes in rice plant (Oryzia sativa L.): A possible reason for reducing the efficiency of diazinon in the control of the rice stem-borer.

It is well known that microorganisms can reduce the effectiveness of organophosphate pesticides after their application. But, little information is available concerning the effect of rice endophytic bacteria on the degradation of diazinon, an organophosphate pesticide used in control of the rice stem-borer, absorbed by the rice plant. Thus, aim of this study was to characterize the endophytic bacterial isolates, isolated from diazinon-treated and non-treated rice plants in paddy fields, in terms of diazinon degradation and to investigate whether potent isolates that degrade diazinon in vitro might have the same effect in the rice plant. The results showed that all endophytic isolates, isolated from both groups of rice plants (diazinon-treated and non-treated rice plants), could grow in mineral salt medium (MSM) supplemented with diazinon (20 mg L-1) as a sole carbon source, and 3.79-58.52% of the initial dose of the insecticide was degraded by the isolates within 14 d of incubation. Phylogenetic analysis based on 16 S rRNA sequencing indicated that the potent isolates (DB26-R and B6-L) clearly belonged to the Bacillus genus. The diazinon concentrations in rice plants co-inoculated with B. altitudinis DB26-R and B. subtilis subsp. Inaquosorum B6-L and single-inoculated with these strains were reduced significantly compared with endophyte-free rice plants. These results provide unequivocal evidence that the rice endophytic bacteria, in addition to in vitro degradation of diazinon, are also involved in the rapid inactivation of diazinon in rice plants treated with diazinon (in vivo degradation of diazinon).

Diazinon dissipation in pesticide-contaminated paddy soil: kinetic modeling and isolation of a degrading mixed bacterial culture.

Dissipation kinetics of diazinon was investigated in soils culled from a paddy field with a long history of the pesticide application. Goodness of fit statistical indices derived from several fitted mono- and bi-exponential kinetic models revealed a bi-phasic pattern of the diazinon dissipation curve at 15 and 150 mg kg-1 spiking levels, which could be described best by the first-order double exponential decay (FODED) model. Parameters obtained from this model were able to describe the enhanced dissipation of diazinon as the result of repeated soil applications, where a larger fraction of the pesticide readily available in the solution phase was dissipated with a fast rate. Cluster and principal component analysis (PCA) of denaturing gradient gel electrophoresis (DGGE) obtained from soil bacterial populations revealed that they were only affected at the 150 mg kg-1 diazinon concentration. This was also supported by the phylogenetic tree obtained from sequences of the main gel bands. Accordingly, bacterial populations belonging to Proteobacteria were enriched in the soil following three treatments with diazinon at 150 mg kg-1. The Shannon's index revealed a nonsignificant increase (P ≤ 0.05) in overall diversity of soil bacteria following diazinon application. Diazinon-degrading bacteria were isolated from the paddy soils in a mineral salt medium. Results showed that the isolated mixed culture was able to remove 90% of the pesticide at two concentrations of 50 and 100 mg L-1 by 16.81 and 19.60 days, respectively. Sequencing the DGGE bands confirmed the role of Betaproteobacteria as the main components of the isolated mixed culture in the degradation of diazinon.

Circadian Rhythmicity of Diazinon Susceptibility, Detoxifying Enzymes, and Energy Reserves in Aphis gossypii (Hemiptera: Aphididae).

The daily susceptibility rhythm of the cotton aphid, Aphis gossypii Glover, to diazinon and the corresponding changes in the activity of three xenobiotic detoxifying enzymes-cytochrome P450 monooxygenases (P450), glutathione S-transferases (GSTs), and esterases-were investigated. Bioassays were conducted to estimate the median lethal doses (LD50) of diazinon at six different zeitgeber times (ZT0, 4, 8, 12, 16, and 20) under constant light (LL) and lighting conditions of 16 h of light and 8 h of darkness (LD). The results showed that the highest susceptibility occurred at the onset of night (ZT16) and 4 h before this time point (ZT12) under the LD condition. The endogenous rhythmicity of susceptibility was ensured, as the highest susceptibility occurred at the same time under the LL condition. The circadian changes in susceptibility to diazinon were almost coincident with changes in esterase and GSTs activity, but not in P450 activity. We also found rhythmic changes in energy components of whole-body aphids, with similar patterns of circadian changes of proteins, lipids, and soluble carbohydrates, but not glycogen, under LL and LD conditions. These photoperiod conditions (LD and LL) showed different fluctuation in trends of energy resources and of course, different quantities. Our study represents the first report of circadian control of insecticide susceptibility in aphids and provides insights into more efficient control of these pests by unveiling the times of day during which aphids are more susceptible to insecticides with attention to endogenous physiological phenomena.

The dissipation kinetics of malathion in aqueous extracts of different fruits and vegetables.

The dissipation of malathion in 5% aqueous extracts of some fruits and vegetables including bell pepper, tomato, cucumber, cantaloupe, carrot, and also buffer (control) was investigated at 37 °C for 4 h. The dissipation trend of malathion in the fruit/vegetable samples and buffer followed first-order double-exponential decay (FODED) and simple first-order kinetic (SFOK) models, respectively. The initial dissipation rate of malathion in tomato (DT10=0.05 h), bell pepper (DT10=0.06 h), and carrot (DT10=0.07 h) was faster compared to the other samples. The slowest rate of pesticide decline belonged to cantaloupe (DT50=1.92 h) with a significant difference from the other samples (p≤0.01), whereas tomato (DT50=0.43 h) and carrot (DT50=0.53 h) showed the fastest dissipation rate. DT90 values derived from the models revealed no significant difference between the samples except for cantaloupe which had the slowest rate of dissipation (DT90=8.27 h) with a significant difference compared to others (p≤0.01). A direct correlation was observed between protein content of the samples and the rate of malathion decline which indicates the role of plant enzymes in degrading malathion residues.

Linear-dendritic copolymers/indoxacarb supramolecular systems: biodegradable and efficient nano-pesticides.

Photodegradable and biocompatible nano-indoxacarb was prepared successfully by encapsulation of indoxacarb with poly(citric acid)-poly(ethylene glycol)-poly(citric acid) (PCA-PEG-PCA) ABA type linear-dendritic copolymers both with (nano-IND/TiO2) and without (nano-IND) TiO2 nanoparticles via supramolecular interactions. Preparation of nano-indoxacarb by both formulae was confirmed using spectroscopy and microscopy analyses. TEM images showed small particles with average sizes of 10 nm for nano-IND and 12 nm for nano-IND/TiO2. Interestingly, the rate of degradation of indoxacarb in both nano-IND/TiO2 and nano-IND exposed to UV and natural light was higher than that for free indoxacarb in the presence of TiO2 nanoparticles. Furthermore, results of bioassay experiments on the model pest showed that the essential dosage of pesticide for pest control significantly decreased when nano-IND and nano-IND/TiO2 were used. Because of the higher loading capacity and slower release rate for indoxacarb from nano-IND than from nano-IND/TiO2, and because TiO2 nanoparticles show toxicity in bioassay experiments, nano-IND is introduced as a promising and eco-friendly pesticide system.

Preparation, characterization and efficiency of nanoencapsulated imidacloprid under laboratory conditions.

In this work, nano-imidacloprid was prepared by direct encapsulation with ABA triblock linear dendritic copolymers composed of poly(citric acid) (PCA) as A block and poly(ethylene glycol) (PEG) as B block. Nanocapsules of imidacloprid were characterized using spectroscopy, microscopy and thermal analysis. The encapsulation process was performed by self-assembly of PCA-PEG-PCA in the presence of imidacloprid in different solvents. Comparison of the TEM images of nano-imidacloprid prepared in ethanol and water showed that, during the first day, self-assemblies appeared as small particles with an average size of 10-20nm. Depending upon the type of solvent, the time and concentration, morphology and size of the nano-imidacloprid varied from fiber-like to globular to tubular from 10nm to several mm in size. Higher loading capacity and slower release rate of imidacloprid from nano-imidacloprid at optimum pH of Glyphodes pyloalis׳s gut (pH=10) compared to neutral pH confirmed the selective and controllable action of nano-imidacloprid. Results of bioassays on the model insect showed that by using the nanoform of imidacloprid, essential dosage of pesticide and environmental risk decreased significantly and indicated good performance for this formulation.

Diazinon residues and degradation kinetics for grapes under field conditions.

The dissipation of diazinon (O,O-diethyl O-2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate) in grapes was investigated to determine its pre-harvest interval (PHI). Diazinon was applied to grapes at the recommended dosage (0.9 g a.i. L(-1)) and twice the recommended dosage (1.8 g a.i. L(-1)) three times, at the fruit formation stage, the sour stage, and the ripening stage, in a field trial with three replications. Samples were taken at 0, 1, 2, 4, 7, 11, 15, 20, 26 and 32 days after spraying. The residue was extracted using a water/methanol/acetonitrile (1:1:1, v/v/v) solvent and solid phase extraction was employed for cleanup. Quantitative analysis was performed using a gas chromatograph equipped with a nitrogen-phosphorus detector. The dissipation trend for the recommended dosage and twice the recommend dosage followed the simple first-order kinetic model (SFOK) (DT(50) = 3.29 days, DT(90) = 11 days, PHI = 13.5 days) and first-order double-exponential decay (FODED) model (DT(50) = 1.08 days, DT(90) = 5.82 days, PHI = 15.29 days), respectively. The average initial deposit of diazinon at the recommended dosage was 9.04 mg kg(-1)and for twice the recommended dosage was 27.38 mg kg(-1)and it dissipated rapidly within days of spraying.

Demographic traits of Tetranychus urticae (Acari: Tetranychidae) on leaf discs and whole leaves.

Life tables of twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), were studied on leaf discs and whole leaves of the common bean plant, Phaseolus vulgaris L. Data were analyzed based on the age-stage, two-sex life table theory. Durations of deutonymph stage, total preadult stage, and preoviposition period of mites reared on whole leaves (1.22, 7.6, and 0.29 d, respectively) were significantly shorter than those reared on leaf discs (1.73, 8.2, and 0.89 d, respectively). The lifetime fecundities were 22.81 and 12.05 offspring on whole leaves and leaf discs, respectively. Although hooked trichomes on the lower surface of the bean leaf contribute to adult mortality, spider mites successfully survive and reproduce on bean plants. The intrinsic rate of increase on whole leaves (0.235 d(-1)) was higher than that on leaf discs (0.159 d(-1). Higher intrinsic rate and fecundity found in mites reared on whole leaves may be due to the higher nutritional quality of whole leaves over leaf discs. We suggest that whole leaves should be used in future studies to better simulate realistic life history characteristics. The advantages of using age-stage, two-sex life tables over female age-specific life tables are discussed.

Dissipation of phosalone and diazinon in fresh and dried alfalfa.

The objectives of this study were to determine the persistence of phosalone (S-6-chloro-2,3-dihydro-2-oxobenzoxazol-3-ylmethyl O, O-diethyl phosphorodithioate) and diazinon (O,O-diethyl O-2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate) residues in fresh and baled alfalfa under field conditions. Plots of alfalfa were sprayed with each insecticide. Fresh alfalfa was sampled up to 20 days after treatment, and dried alfalfa was sampled up to 25 weeks after baling. Samples were analyzed for residues using high performance liquid chromatography (HPLC) equipped with a UV detector. The half-lives of diazinon and phosalone in fresh alfalfa were 1.8 and 3.3 days, respectively. In baled alfalfa the half-life of diazinon and phosalone were 2.8 and 16.7 weeks, respectively. No diazinon residues were detected in baled alfalfa, sampled after week 9, although the concentration of phosalone found at week 25 was 5.51 mg/kg.

Effects of two different formulations of metalaxyl on their residues in cucumber.

Investigations were carried out on the presence of metalaxyl residues in field grown cucumbers. In one experiment, cucumber plants underwent once and twice sprayings by Ridomil-MZ 72 WP at 2.5 g/litre. In another, single and double applications of metalaxyl granule (5G) were carried out at 5 g/m2. Fungicide application performed every 14 days. Samples were taken at different time intervals and metalaxyl level was determined by HPLC. The results showed variable dissipation rates of metalaxyl residues in cucumber. In samples from single and double sprayed plots, the residues were 0.19 and 0.08 mg/kg 14 days after treatment respectively. In single granulated plots, the residues increased during the first 7 days and then decreased to 0.85 mg/kg, 14 days after granule application. However, in double granulated plots, the residues decreased to 0.02 mg/kg after the same period of time. In all treatments except single granulated plots, the residues declined below the MRL (0.5 mg/kg), 7 days after application. These experiments showed that two times spraying and granule applications did not cause the fungicide accumulation in cucumber.