Ethylenebisdithiocarbamate Fungicides Research Articles

Management of Xanthomonas Leaf Blight of Onion with Bacteriophages and a Plant Activator.

Xanthomonas leaf blight of onion (Allium cepa), caused by Xanthomonas axonopodis pv. allii, continues to be a challenging and yield-threatening disease in Colorado and other regions of onion production worldwide. Studies were conducted to develop management strategies for this disease that are equally effective and more sustainable than the current practices of making multiple applications of copper bactericides. Mixtures of bacteriophages and the plant defense activator, acibenzolar-S-methyl, were evaluated under field and greenhouse conditions for their abilities to reduce Xanthomonas leaf blight severity. Bacteriophage populations in the phyllosphere of onion were monitored over time. Bacteriophage populations persisted on onion leaves for at least 72 to 96 h under field and greenhouse conditions, respectively. Under field conditions at one location, biweekly or weekly applications of bacteriophages reduced disease severity by 26 to 50%, which was equal to or better than weekly applications of copper hydroxide plus mancozeb. Acibenzolar-S-methyl also successfully reduced disease severity by up to 50% when used alone preventatively or followed by biweekly bacteriophage applications. Reductions in disease severity generally were not associated with improvements in onion bulb size or yield. Integration of bacteriophage mixtures with acibenzolar-S-methyl appears to be a promising strategy for managing Xanthomonas leaf blight of onion, and could reduce grower reliance on conventional copper bactericide applied with ethylenebisdithiocarbamate fungicides.

Reactive oxygen species generation by the ethylene- bis-dithiocarbamate (EBDC) fungicide mancozeb and its contribution to neuronal toxicity in mesencephalic cells

Previous in vitro studies in our laboratory have shown that mancozeb (MZ) and maneb (MB), both widely used EBDC fungicides, are equipotent neurotoxicants that produce cell loss in mesencephalic dopaminergic and GABAergic cells after an acute 24 h exposure. Mitochondrial uncoupling and inhibition were associated with fungicide exposure. Inhibition of mitochondrial respiration is known to increase free radical production. Here the mechanism(s) of neuronal damage associated with MZ exposure was further explored by determining the role that reactive oxygen species (ROS) played in toxicity. Damage to mesencephalic dopamine and GABA cell populations were significantly attenuated when carried out in the presence of ascorbate or SOD, indicative of a free radical-mediated contribution to toxicity. ROS generation monitored by hydrogen peroxide (H 2O 2) production using Amplex Red increased in a dose-dependent manner in response to MZ. Inhibition of intracellular catalase with aminotriazole had little effect on H 2O 2 generation, whereas exogenously added catalase significantly reduced H 2O 2 production, demonstrating a large extracellular contribution to ROS generation. Conversely, cells preloaded with the ROS indicator dye DCF showed significant MZ-induced ROS production, demonstrating an increase in intracellular ROS. Both the organic backbone of MZ as well as its associated Mn ion, but not Zn ion, were responsible and required for H 2O 2 generation. The functionally diverse NADPH oxidase inhibitors, diphenylene iodonium chloride, apocynin, and 4-(2-aminoethyl)benzene-sulfonyl fluoride hydrochloride significantly attenuated H 2O 2 production by MZ. In growth medium lacking cells, MZ produced little H 2O 2, but enhanced H 2O 2 generation when added with xanthine plus xanthine oxidase whereas, in cultured cells, allopurinol partially attenuated H 2O 2 production by MZ. Minocycline, an inhibitor of microglial activation, modestly reduced H 2O 2 formation in mesencephalic cells. In contrast, neuronal-enriched cultures or cultures treated with MAC-1-SAP to kill microglia, did not show an attenuation of ROS production. These findings demonstrate that Mn-containing EBDC fungicides such as MZ and MB can produce robust ROS generation that likely occurs via redox cycling with extracellular and intracellular oxidases. The findings further show that microglia may contribute to but are not required for ROS production by MZ.

Acute neurotoxic effects of mancozeb and maneb in mesencephalic neuronal cultures are associated with mitochondrial dysfunction

Recent studies suggest that exposure to agrochemicals may contribute to the development of idiopathic Parkinson's disease. Maneb (MB), a widely used Mn-containing ethylene-bis-dithiocarbamate (EBDC) fungicide, has been implicated in selective dopaminergic neurotoxicity. In this study, we examine the potential neurotoxicity of mancozeb (MZ), a widely used EBDC fungicide that is structurally similar to MB, but contains both Zn and Mn. Primary mesencephalic cells isolated from Sprague–Dawley embryonic day 15 rat embryos were exposed in vitro to either MZ or MB to compare their cytotoxic potential. Exposure to 10–120 μM MZ or MB for 24 h resulted in a dose-dependent toxicity in both the dopamine (DA) and GABA mesencephalic populations as assessed by a functional assay for high affinity transporter activity. Consistent with this, cell viability as well as tyrosine hydroxylase-positive neurons decreased with increasing doses of MZ or MB. Toxic potencies for MZ and MB were similar and no difference in sensitivity between the DA and GABA populations was observed with the fungicides. Exposure to ethylene thiourea, the major metabolite of either MZ or MB, was not toxic, implicating the parent compound in toxicity. Both the organic and Mn metal components of the fungicides were found to contribute to toxicity. Non-toxic exposures to the fungicides decreased ATP levels in a dose-dependent manner suggesting impairment of energy metabolism. In whole mitochondrial preparations isolated from adult rat brains, MZ and MB inhibited NADH-linked state 3 respiration. Mild to moderate mitochondrial uncoupling was also observed in response to the fungicides. In conclusion, our findings indicate that acute exposure to high doses of MZ and MB produce equipotent toxic effects in both DA and GABA neurons that may be associated with perturbations in mitochondrial respiration.

Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes

In this study the effects of mancozeb, a widely used ethylenebisdithiocarbamate fungicide, on mouse oocyte meiotic maturation and fertilization were analyzed. Oocyte cumulus cell-complexes were matured in vitro with or without increasing concentrations of the fungicide (from 0.001 to 1 μg/ml) that, due to its different stability in organic solvents and in water, was resuspended either in dimethyl sulfoxide or in culture medium. Although, about 95% of oocytes reached the metaphase II stage; mancozeb-exposed oocytes showed a dose-dependent increase of alterations in spindle morphology, and this negative effect was more evident when the fungicide was resuspended in culture medium. Under the latter culture condition, oocytes matured in the presence of 0.1 and 1 μg/ml mancozeb showed a significant reduction also in the formation of male and female pronuclei. These results indicate that mancozeb can adversely affect mammalian reproductive performance, likely by perturbing microtubular organization during meiotic maturation.

Management of Xanthomonas Leaf Blight of Onion with a Plant Activator, Biological Control Agents, and Copper Bactericides.

Xanthomonas leaf blight (Xanthomonas axonopodis pv. allii) is a yield-limiting disease of onion (Allium cepa) in the western United States. Frequent applications of copper-based bactericides amended with an ethylenebisdithiocarbamate fungicide (e.g., maneb or mancozeb, class B2 carcinogens) provide some disease suppression, but strategies to reduce conventional bactericide use are needed to minimize grower costs, environmental impact, and public exposure to class B2 pesticides. Applications of acibenzolar-S-methyl reduced in planta and epiphytic populations of X. axonopodis pv. allii as effectively as applications of copper hydroxide-mancozeb in growth chamber studies. Under field conditions, four weekly applications of acibenzolar-S-methyl reduced severity of Xanthomonas leaf blight as or more effectively than 9 to 12 weekly applications of copper hydroxide or copper hydroxide-mancozeb. Acibenzolar-S-methyl applications did not increase bulb yield or grade compared with copper bactericide treatments. However, bulb yield was reduced 22 to 27% when 10 weekly applications of acibenzolar-S-methyl were made in the absence of disease. Application of a commercial formulation of both Pantoea agglomerans strain C9-1 and Pseudomonas fluorescens strain A506 reduced severity of Xanthomonas leaf blight in field experiments. Weekly copper hydroxide applications starting 1 to 2 weeks before bulb initiation were as effective as weekly applications started 3 to 4 weeks before bulb initiation, irrespective of the maneb rate used. Integration of acibenzolar-S-methyl and biological control agents with copper hydroxide in a carefully timed spray program may eliminate the use of the class B2 carcinogens maneb and mancozeb on onion without compromising efficacy for management of Xanthomonas leaf blight.

The decrease in pesticides in fruit and vegetables during commercial processing

SummarySamples of tomatoes, peppers, asparagus, spinach and peaches were exposed to three insecticides (acephate, chlorpyrifos and cypermethrin), three ethylenebisdithiocarbamate fungicides (mancozeb, maneb, propineb) and the tetramethyldithiocarbamate fungicide thiram to study the effect of commercial processing on the residues. In most cases, canning operations led to a gradual decrease in residue levels in the finished products, particularly through washing, blanching, peeling and cooking processes. The results indicated that washing plus blanching led to more than 50% loss in pesticide residues, except for in peaches. Ethylenebisdithiocarbamates (EBDCs) were completely removed from tomatoes and spinach by washing followed by hot water blanching. The total amount of pesticide removed by all of the combined canning operations ranged from 90 to 100% in most products. Pepper retained 61% of chlorpyrifos but these residues disappeared during 3‐month storage of finished cans. Acephate showed a surprising tenacity in peaches, as 11% of the original residues were still present in 2‐year stored cans.

Identification and quantitation by high-performance liquid chromatography of mancozeb following derivatization by 1,2-benzenedithiol.

Ethylenebisdithiocarbamate (EBDC) fungicides are the most important class of organic fungicides and exhibit a high degree of carcinogenicity, mutagenicity, and neurotoxicity. For that reason, the safe application of these fungicides in practice requires a convenient method for their determination, applicable to biological fluids. We describe a high-performance liquid chromatography (HPLC) assay. After elimination of the metal which defines the product (maneb, mancozeb, zineb.) with EDTA, the resulting EBDC is derivatized with 1,2-benzenedithiol to yield a cyclocondensation product, 1,3-benzodithiole-2-thione, which can then be quantitated by reversed-phase HPLC at 365 nm using a microBondapak C18 column. The mobile phase was methanol/H2O (70:30, v/v). The assay was linear from 0.25 to 100 microg/mL. Within- and between-day precision and accuracy for this assay were better than 9% and 6%, respectively. The lower limits of detection and quantitation were estimated to be 0.1 and 0.25 microg/mL, respectively. This simple new method has been applied to determine mancozeb concentration in rat urine samples from urinary excretion studies.

LC/MS Studies on Characterization and Determination of N,N′-Ethylenebisdithiocarbamate Fungicides in Environmental Water Samples

Liquid chromatography/mass spectrometry (LC/MS) has been applied to analyze N,N'-ethylenebisdithiocarbamate fungicides (EBDCs: manzeb, maneb and zineb) in environmental water samples. The EBDCs that are zinc and/or manganese salts are transformed into readily water-soluble sodium salts by adding an alkaline EDTA solution. The N,N'-ethylenebisdithiocarbamate anion is extracted into chloroform-hexane (3:1) as an ion pair with tetrabutylammonium, and then S-alkylated with methyl iodide. The extraction and derivatization are carried out at room temperature. The derivatized ethylenebisdithiocarbamic acid dimethyl ester is introduced into an LC/MS equipped with a negative ion electrospray ionization interface. Identification of the compound is performed with the specific quasi-molecular ion, and the quantitative analyses are carried out using the peak areas. The average recoveries and coefficients of variation of EBDCs at sub-ppb level are 79.1% and 29.3% (n = 6), respectively. The limit of detection based on standard deviation of 0.043 microg/L for manzeb is achieved.

Potentiated and preferential effects of combined paraquat and maneb on nigrostriatal dopamine systems: environmental risk factors for Parkinson’s disease?

The absence of any compelling basis for a heritable basis of idiopathic Parkinson’s disease (PD) has focused attention on environmental exposures as causative agents. While the herbicide paraquat has repeatedly been implicated, its impact on dopamine systems following systemic exposures is equivocal. The restricted focus on paraquat also ignores the extensive geographical overlap of its use with other agrichemicals known to adversely impact dopamine systems, including ethylenebisdithiocarbamate fungicides such as maneb. The present study sought to determine whether combined exposures to paraquat and maneb would produce additive effects and support a multiple-hit environmental contribution to PD. C57BL/6 mice were exposed to either paraquat (5–10 mg/kg) or maneb (15–30 mg/kg) i.p. alone or in combination once a week for 4 weeks. Sustained decreases in motor activity immediately following injections were consistently observed only with combined exposures, with activity levels returning to control values 24 h later. Concurrently, levels of dopamine and metabolites and dopamine turnover were increased immediately post-injection only by combined exposures, and returned to control levels or below within 48 h. Reductions in tyrosine hydroxylase immunoreactivity, measured 3 days after the last injection, resulted only from combined exposure and were detected in dorsal striatum, but not in the nucleus accumbens. The fact that combined exposures resulted in potentiated effects that appear to target nigrostriatal dopamine systems suggests that these combinations may be important environmental risk factors for Parkinsonism. These findings also raise questions about the adequacy of current risk assessment guidelines for these chemicals which are based on effect levels derived from exposures to single agents.

Comparison of Micellar Electrokinetic Capillary Chromatographic Method with High-Performance Liquid Chromatographic Method for the Determination of Imidazolidine-2-thione (Ethylenethiourea) in Formulated Products

A micellar electrokinetic capillary chromatographic (MECC) method was developed for routine analysis of the carcinogenic compound imidazolidine-2-thione (ETU) in commercial ethylenebis(dithiocarbamate) fungicides (EBDC). The MECC method was compared with a previously developed high-performance liquid chromatographic (HPLC) method. Both methods demonstrated good precision, accuracy, linearity, and sensitivity. The relative standard deviations (RSD) for precision ranged from 0.04 to 5.09% for the MECC method and from 0.47−5.22% for the HPLC method. The recoveries for accuracy ranged from 98.3 to 100.6% for the MECC method and from 99.5 to 104.9% for the HPLC method. No matrix interference was observed in either method. The column efficiency of MECC (N = 12491) was 15 times higher than that of HPLC (N = 813). The instrument detection limit (IDL) and the method detection limit (MDL) of the MECC method were 0.25 and 0.30 μg/mL, which were 25 and 15 times higher than the IDL (0.01 μg/mL) and the MDL (0.02 μg/mL...

Degradation of ethylenethiourea (ETU) in oxic and anoxic sandy aquifers.

Ethylenethiourea is an important degradation product of ethylenebisdithiocarbamate fungicides, which are widely used in different kinds of crops. The ethylenebisdithiocarbamate group includes maneb, zineb and mancozeb. The ethylenebisdithiocarbamates are not highly toxic and degrade rapidly in the presence of moisture and oxygen, forming different compounds. One of these is the polar ethylenethiourea, which is relatively stable. Thus, this compound appears to be a potential contaminant for groundwater. Batch experiments were carried out under biotic as well as abiotic conditions to study the degradation dependence of concentration, temperature and organic matter. The decomposition of ethylenethiourea under abiotic conditions was found to be less than 5% of the degradation under biotic conditions. Further, ethylenethiourea showed to be stable over a period of 150 days at 20 degrees C in tap water as well as in batch with soil sterilized with NaN3. The degradation of ethylenethiourea depends on the concentration in the water implying first order reaction kinetics. The microbial degradation of ethylenethiourea is highly temperature dependent with aerobic Q10 between 2.9 and 4.2, and an anaerobic between 2.1 and 2.5. A minor increase in degradation rates was observed by application of nitrate and manure to the batches. The experiments show extremely complete degradation of ethylenethiourea in the presence of microbial nitrate reduction with pyrite which occurs in deeper parts of the aquifers.

Estimating the risks and benefits of pesticides: Considering the agroecosystem and integrated pest management in the use of EBDC fungicides on apples

Calculating the risks and benefits of agricultural pesticides determines whether a pesticide will be registered for use on commodities. Historically, the US Environmental Protection Agency (EPA) has used very conservative estimates of health risks, particularly in the absence of data regarding actual use patterns of the pesticide and actual pesticide residue at the point of food purchase. However, when grower groups and manufacturers were faced with the problem of losing the ethylene bisdithiocarbamate (EBDC) fungicides, they approached EPA offering to provide hard data in place of estimates. Plant pathologists familiar with apple agroecosystems pointed out that different fungicides have highly specific uses in integrated pest management (IPM) programs, and that banning EBDC use on apples would probably raise total pesticide use in apples by compromising the pesticide reductions obtained under existing IPM programs. In addition, an early study showed that use-patterns could significantly reduce the amount of EBDC residue on apples. This approach led to a more realistic appraisal of both risks and benefits of the fungicides, lowering risks and raising benefits. The process ended with a registration prescribing use-patterns which result in minimal residue at harvest, thus providing valuable tools for use in IPM programs.

HPLC Study of Mancozeb Degradation on Leaves

Abstract An HPLC procedure was developed for the determination of mancozeb on cucumber leaves after application of mancozeb suspension (ethylenebisdithiocarbamate fungicide). The analytical procedure consisted of a single extraction step with EDTA aqueous solution and sodium salt of ethylenebisdithiocarbamate was eluated from Separon NH2 column. Mobile phase was 5 % aceto-nitrile and 45 % methanol in aqueous solution of borate buffer at pH = 7.6. The level of mancozeb after application on leaves decreased slowly, with a half time 14 days.

High-Performance Liquid Chromatographic Determination of Ethylenethiourea in Food

Abstract An HPLC procedure was developed for the determination of ethylenethiourea (ETU) in peels of potatoes, tomatoes and apples after application of mancozeb suspension (ethyl-enebisdithiocarbamate fungicide). The analytical procedure consisted of a single extraction step with methanol and ETU was eluated from SEPARON CN column. Mobile phase was 4 % methanol in chloroform cyclohexane (1:1). The level of ethylenthiourea after application of mancozeb decreased slowly, with a half time six days in each case.

Heterogeneous Effects of Ethylenebisdithiocarbamate (EBDC) Pesticides on Oxidative Metabolism of Xenobiotics

Comparative evaluation of the acute effects of ethylenebisdithiocarbamate (EBDC) fungicides, mancozeb and zineb on microsomal mixed function oxidases (MFO) revealed marked substrate-dependent inhibition of oxidative metabolism of aminopyrine, p-nitroanisole and aniline in rats sacrificed 4 hr after oral administration of 100 mg mancozeb or zineb/kg body weight. Mancozeb inhibited p-nitroanisole O-dealkylase and aniline hydroxylase to a greater degree than zineb, whereas the inhibition of aminopyrine N-demethylase by the two fungicides was quantitatively comparable. Interestingly, aryl hydrocarbon hydroxylase (AHH) which exhibited maximum inhibition with mancozeb, remained unaffected following zineb administration. The time-course and dose-dependence of MFO inhibition examined 1 and 4 hr after a single oral dose of 100 mg or 250 mg zineb/kg was expressed as a dose-dependent decline in the rate of xenobiotic biotransformation at 4 hr. In vitro interaction of zineb with MFO resulted in slightly greater inhibition of aminopyrine, p-nitroanisole and aniline while AHH exhibited more pronounced decrease with mancozeb. The magnitude of inhibition of aminopyrine N-demethylase and AHH was independent of the time of preincubation of fungicides with the enzyme. Kinetic studies indicated the non-competitive nature of AHH inhibition. Chronic oral treatment with mancozeb and zineb at a dose of 250 mg/kg for 4 weeks did not modify xenobiotic biotransformations except for a slight induction of aminopyrine N-demethylase by mancozeb.

Testing of pesticides for side‐effects on nitrogen conversions in soil

A review is given of the state of the art of testing side‐effects of pesticides on nitrogen conversions in soil. Ammonification and nitrification are the most important processes of the nitrogen cycle. Usually tests for effects of pesticides are carried out with nitrogen‐containing amendments like lucerne meal. In soils with a low carbon content such amendments may result in disturbance of soil populations. The ammonium ions released by nitrification are converted by nitrifying bacteria via nitrite in nitrate. Nitrification can also be studied by addition of ammonium directly to the soil. In this way, ethylenethiourea, a conversion product in soil of ethylenebisdithiocarbamate fungicides (e.g. maneb, zineb) was found to be a strong inhibitor of nitrification in soil. A rapid test on ammonium oxidation was designed by inhibiting the conversion of nitrite into nitrate by potassium chlorate. Determination of the nitrite formation gives a rapid prediction of the inhibition of ammonium oxidation in soil by che...

Determination of Nabam Fungicide in Crops by Liquid Chromatography with Postcolumn Reaction Detection

The ethylenebisdithiocarbamate (EBDC) fungicide, nabam, was determined in several crop matrixes using liquid chromatography with postcolumn reaction detection. After separation by micellar liquid chromatography, nabam (EBDC sodium salt) was acid hydrolyzed to ethylenediamine and fluorigenically labeled with o-phthalaldehyde-mercaptoethanol (OPA-MERC). Standard curves were linear from the detection limit of ca 1 ng to 1000 ng. Nabam was recovered in high yield (89 plus or minus 7.7%) over a range of concentrations (0.1 to 20 ppm) from fortified samples of papaya, lettuce, cucumber, spinach, and applesauce using a simple extraction method. Efforts to convert the more popular EBDC fungicides, maneb and mancozeb, to nabam are discussed.

Ethylenethiourea in air and in urine as an indicator of exposure to ethylenebisdithiocarbamate fungicides.

Ethylenethiourea (ETU) is a ubiquitous impurity of the ethylenebisdithiocarbamate (EBDC) fungicides widely used in agriculture and forestry. In the present study, ETU was used as a measure of the exposure to EBDC on potato farms and in pine nurseries during the application of EBDC fungicides and the weeding of the sprayed vegetation. Biological and hygienic monitoring was carried out through the analysis of ETU in the breathing zone and the urine of exposed workers. Even if the concentrations of ETU in the ambient air of pine nurseries exceeded those of potato farms, the concentrations of ETU in the urine of potato farmers exceeded those of pine nursery workers. This result may have been due better protective equipment in the pine nurseries. The excretion rate was 6-10 ng/h during the first 60 h after the cessation of exposure, and it diminished thereafter to 0.2 ng/h over a 22-d observation period.

Environmental and biological monitoring of exposure to ethylenebisdithiocarbamate fungicides and ethylenethiourea.

Exposure of workers to ethylenebisdithiocarbamates (EBDCs; maneb or mancozeb) in 29 potato farms was evaluated during the control of potato late blight. Concentrations of EBDCs and ethylenethiourea (ETU), an impurity and degradation product in EBDC formulations, in ambient air were evaluated during pesticide application. Biological monitoring of exposure to EBDCs was carried out by measuring the concentrations of ETU, a metabolite of EBDCs, in urine for 22 days after the end of the exposure. The estimated inhaled doses of ETU and EBDCs during the average four hour application period were 0.07 and 1.8 micrograms/kg, respectively. Only 1-10% of ETU on the clothes reached the skin. The creatinine corrected concentrations of ETU in urine were 0.1-2.5 micrograms/mmol creatinine 24 hours after exposure ended. The estimated half life for eliminating ETU through the kidneys was close to 100 hours. These results indicate that the measurement of ETU in urine is suitable for biological monitoring of exposure to EBDCs.

Reduction of Ethylenethiourea Residues in Canned Spinach

Abstract Field-grown spinach (Spinacea oleraceae L.) sprayed with ethylenebisdi-thiocarbamate fungicides was washed to reduce the carbamate residue ethylenethiourea (ETU) in canned spinach. The spinach was washed with water, water + sodium hypochlorite, detergents, or detergents + sodium hypochlorite. After washing, the spinach was canned and evaluated for ETU residue and quality. ETU residue was 63 ppm in non-washed carbamate field-treated spinach, 6.9 ppm in water-washed samples, and only 0.1 ppm in washing treatments containing detergents. Washing treatments had little effect on the quality of the canned spinach; only titratable acidity was reduced significantly.