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The effect of home processing on postharvest fungicide residues in citrus fruit: residues of imazalil, 2-phenylphenol and thiabendazole in 'home-made' marmalade, prepared from late Valencia oranges.

The effects of domestic marmalade-making procedures on residues of imazalil, 2-phenylphenol and thiabendazole in 'sweet' oranges were investigated by comparing residue levels in raw fruit and in marmalades, prepared by heating the oranges with water and sugar for about 4 h in a preserving pan on a gas ring or for about 1 h in a microwave oven. Determination of residues in raw oranges and in marmalades was carried out by extraction with dichloromethane, followed by clean-up and reversed phase HPLC with UV detection for imazalil and fluorescence detection for 2-phenylphenol and thiabendazole. Residue analysis showed that the extent of carry-through of imazalil, 2-phenylphenol and thiabendazole residues into the marmalades depended on the method of marmalade-making and the nature of the fungicide. Persistence of fungicides was higher in the marmalade prepared in the microwave oven than in that prepared in the preserving pan. An increase (about 50%) in levels of imazalil in the fruit portions of marmalades compared with raw fruit residue levels indicated that imazalil was more readily extracted from oranges after processing. Comparison of 2-phenylphenol levels in raw fruit and fruit portions of marmalades showed that the longer exposure to heat, needed for marmalade-making in a preserving pan, reduced residue levels more severely (48%) than cooking in the microwave oven (13%). Thiabendazole levels in the fruit portion of marmalade prepared in the microwave oven stayed the same as in the raw fruit, but cooking in the preserving pan decreased residue levels by about 22%.

Biphenyl Compounds↗

Detection of aneuploidy by multicolor FISH in mouse sperm after in vivo treatment with acrylamide, colchicine, diazepam or thiabendazole.

Multicolor fluorescence in situ hybridization (FISH) was used to investigate the induction of aneuploidy during meiosis in young adult male mice treated with chemicals chosen for the EU sponsored aneuploidy project (acrylamide, colchicine, diazepam and thiabendazole). The aim of the present study was to evaluate the frequency of aneuploid sperm induced by each of these chemicals by sperm FISH. Male (102/ElxC3H/El)F1 mice were treated with acrylamide (120 and 60 mg/kg single dose i.p.), colchicine (1.5 and 3 mg/kg single dose, i.p.), diazepam (300, 150 and 75 mg/kg single dose by oral intubation) or thiabendazole (100 and 300 mg/kg daily for 11 days by oral intubation). At 22 days after the last treatment, sperm were collected from the cauda epididymis. Three chromosome FISH was applied to determine hyperhaploid and diploid sperm with DNA probes specific for the chromosomes X, Y and 8. Five animals were treated per dose group and sperm aneuploidy was evaluated in 10,000 sperm per animal. We found significant increases in the frequency of total hyperhaploidy for the males treated with 3.0 mg/kg colchicine (0.092 versus 0.056%, P < 0.05) and with 1.5 mg/kg colchicine (0.082 versus 0.050%, P < 0.05), as well for the males treated with 300 mg/kg diazepam (0.081 versus 0.050%, P < 0.05), indicating that colchicine and diazepam each induced germ cell aneuploidy. We also found significant increases in the frequency of total diploidy for the males treated with 300 mg/kg diazepam (P < 0.05) and with 300 mg/kg thiabendazole (P < 0.05). No significant effects were found for 120 and 60 mg/kg acrylamide or for the other doses of diazepam and thiabendazole. These first results indicate that the multicolor FISH method is useful to determine aneuploidy induction in sperm of mice.

Acrylamide↗

Identification of human and rabbit cytochromes P450 1A2 as major isoforms involved in thiabendazole 5-hydroxylation.

This report characterized one of the major cytochrome P450 isozyme involved in thiabendazole metabolism. This study was undertaken by using both cultured rabbit hepatocytes treated or not with drugs known to specifically induced various cytochromes P450 isoenzymes (i.e., P450 1A1/2 by beta-naphthoflavone, P450 2B4 by phenobarbital, P450 3A6 by rifampicine and P450 4A by clofibrate) and human liver (THLE-5) and bronchial (BEAS-2B) epithelial cells expressing or not the major constitutive human cytochromes P450 (i.e., CYP1A2, 2A6, 2B6, 2C9, 2D6, 2E1 or 3A4). Only hepatocytes exposed to beta-naphthoflavone and clofibrate significantly metabolized thiabendazole to 5-hydroxythiabendazole. Extensive biotransformation of this anthelmintic only occurred in human cells expressing CYP1A2. Moreover, experiments performed on rabbit preparations showed good correlations between thiabendazole 5-hydroxylase activity and both ethoxyresorufin and methoxyresorufin O-dealkylase activities. Thus, CYP1A2 is a major isoenzyme involved in thiabendazole 5-hydroxylation.

Animals↗

Comparative study of the effect of thiabendazole and fungizone on Histoplasma capsulatum in macrophages.

The antifungal properties of Fungizone (amphotericin B intravenous solution) and thiabendazole on Histoplasma capsulatum within guinea pig macrophages were compared using the staining method and a newly developed plating method to determine the viability of intracellular H. capsulatum. The two methods were compared to determine the effectiveness of Fungizone and thiabendazole on H. capsulatum within macrophages. Fungizone was fungicidal for intracellular H. capsulatum, killing 99.9% of the yeasts at a concentration of 0.5 microgram/ml. There was some indication that non-viable intracellular yeasts were stained which could result in misinterpretation of the effectiveness of Fungizone using the staining method unless the yeasts are very closely examined for staining abnormalities. There was a good correlation between the two methods to demonstrate suppression of the multiplication of intracellular H. capsulatum in macrophages treated with 50 microgram/ml of thiabendazole. Thiabendazole was lethal for some intracellular H. capsulatum.

Amphotericin B↗

Effect of thiabendazole on the migration of Toxocara canis larvae in the mouse.

The effects of thiabendazole on the survival and migration of Toxocara canis larvae in mice were studied. Although thiabendazole had a negligible larvicidal effect, it produced marked inhibition of the migration of the larvae in tissues. Larvae were arrested in the liver after oral inoculation and in the lungs after intravenous inoculation. Observations indicated that the drug may have a reversible, as well as a progressive, time-related metabolic effect on the larvae. Significant inhibition of migration required administration of the drug in medicated feed at a concentration of 0.25% or more. Periodic oral administration of a thiabendazole suspension was ineffective in inhibiting larval migration. It was concluded that the immobilizing effect of the drug on the larvae of T. canis may account for the clinical improvement observed after treatment with thiabendazole in cases of visceral larva migrans, the prevention of prenatal or mammary transmission of infection in dogs, and the suppression of tissue damage by nematode larvae in general.

Animals↗

Comparison of changes in resistance of Haemonchus contortus eggs following withdrawal of thiabendazole selection.

Two strains of Haemonchus contortus with different levels of thiabendazole resistance were developed from the same parent strains by being subjected to different selection intensities. These two strains were each subdivided into two; one selected by thiabendazole in each generation, the other unselected for four generations. Levels of thiabendazole resistance were monitored in each generation by an in vitro egg hatch technique. During this time the unselected strain with the higher level of resistance maintained an LC50 similar to its selected control. The LC50 of the unselected strain with the lower level of resistance actually increased until at the end of the fourth generation it had an LC50 similar to the thiabendazole selected strains.

Animals↗

Trichostrongylus colubriformis and Ostertagia sp resistant to levamisole, morantel tartrate and thiabendazole: isolation into pure strain and anthelmintic titration.

Strains of Trichostrongylus colubriformis (designated PF4) and Ostertagia sp (O circumcincta/O trifurcata, designated PF5), with varying degrees of resistance to levamisole, morantel tartrate and thiabendazole were isolated into pure culture. Detailed dose response studies showed that both T colubriformis and Ostertagia sp were highly resistant to levamisole and morantel with low level resistance to thiabendazole. The effective dose required to remove 80 per cent worm burdens (ED80) was calculated for each anthelmintic. For T colubriformis the ED80 for levamisole and thiabendazole were 12.6 and 40.1 mg/kg respectively. For Ostertagia sp, the ED80 for levamisole, thiabendazole and morantel were 20.4, 45.2 and 35.8 mg/kg respectively. The implications of these results are discussed with reference to alternative means of chemical control.

Animals↗

The anthelmintic efficacy of fenbendazole against thiabendazole-resistant strains of Haemonchus contortus and Trichostrongylus colubriformis in sheep.

The anthelmintic efficacy of fenbendazole (methyl 5-(phenylthio)-2-benzimidazole carbamate) was tested in sheep against standardised strains of Hcaemonchus contortus and Trichostrongylus colubriformis, known to be resistant to thiabendazole (LD90 for thiabendazole against H ontortus was 200 mg/kg bodyweight and against T colubriformis was 150 mg/kg). Fenbendazole at dose rates of 5, 10 and 20 mg/kg per os reduced total worm counts in H contortus infected sheep by 66, 90 and 100 per cent respectively, with similar reductions recorded for worm egg outputs. For the thiabendazole resistant strain of T colubriformis, fenbendazole reduced total worm counts in infected sheep by 4, 44 (40-48), 79 (75-83), 96 and 100 per cent at dose rates of 5, 10, 20, 40 and 80 mg/kg per os. Significant suppression of worm-egg production by thiabendazole resistant T colubriformis was obtained with fenbendazole at dose levels of 5 mg/kg and above. The implications of these results are discussed in the light of the increasing occurrence of strains of trichostrongylid nematodes resistant to currently available benzimidazole anthelmintics.

Animals↗

Continuous-flow separation and pre-concentration coupled on-line to solid-surface fluorescence spectroscopy for the simultaneous determination of o-phenylphenol and thiabendazole.

A novel and single flow-injection system combined with solid-surface fluorescence detection is proposed in this work for the resolution of a mixture of two widely used pesticides (o-phenylphenol and thiabendazole). The continuous-flow methodology is based on the implementation of on-line pre-concentration and separation of both analytes on the surface of C18 silica gel beads placed just inside the flow cell, implemented with gel-phase fluorimetric multi-wavelength detection (using 305/358 and 250/345 nm as excitation/emission wavelengths for thiabendazole and o-phenylphenol, respectively). The separation of the pesticides was possible owing to the different retention/desorption kinetics of their interactions with the solid support in the zone where the stream impinges on the solid material. No previous separation of the analytes before they reach the flow cell is needed thereby simplifying substantially both the procedure and the manifold. By using a sample volume of 2,600 microL, the system was calibrated in the range 0.5-16 and 5-120 ng mL(-1) with detection limits of 0.09 and 0.60 ng mL(-1) for thiabendazole and o-phenylphenol, respectively. The RSD values (n=10) were about 1% for both analytes. The proposed methodology was applied to environmental water samples and also to various commercial pesticide formulations containing both analytes. Recovery percentages were 97-103% and 98-102% for thiabendazole and o-phenylphenol, respectively.

Journal Article↗

Identification of an amino acid substitution in the benA, beta-tubulin gene of Aspergillus nidulans that confers thiabendazole resistance and benomyl supersensitivity.

We are using molecular genetic techniques to identify sites of interaction of beta-tubulin with benzimidizole anti-microtubule agents. We have developed a marker-rescue technique for cloning mutant alleles of the benA, beta-tubulin gene of Aspergillus nidulans and have used the technique to clone two mutant benA alleles, benA16 and benA19. These are the only A. nidulans alleles known to confer resistance to the benzimidazole antimicrotubule agent thiabendazole and supersensitivity to other benzimidazole antimicrotubule agents including benomyl and its active breakdown product, carbendazim. benA16 has been shown, moreover, to reduce thiabendazole binding to beta-tubulin. We have sequenced the two mutant alleles and have found that they carry different nucleotide changes that cause the same single amino acid substitution, valine for alanine at amino acid 165. Since thiabendazole and carbendazim differ at only one side chain, the R2 group, we conclude that the region around amino acid 165 is involved in the binding of the R2 group of benzimidazole antimicrotubule agents to beta-tubulin.

Alleles↗

[Analysis of thiabendazole in citrus fruits and bananas].

With regard to routine analyses in market control, the authors recommend two methods (according to the expected amounts of active principle) for the determination of thiabendazole residues on citrus fruits and bananas. The ultraviolet spectrophotometric method is preferable in determining thiabendazole contents of more than 1 p.p.m., if the cleaning operations described are respected. For the detection of thiabendazole and for the determination of amounts of less than 1 p.p.m. (the tolerance limit being 0.2 p.p.m. for pomes, berries, stone fruits, kernel fruits and also for potatoes) the thin-layer chromatographic method seems likewise to be suited (also in considering that it is semi-quantitative by nature); especially since the spectrophotometric method yields values by 0.2 p.p.m. too high (due to the measurement of residual absorption of vegetable constituents). The authors are of opinion that such an error must be considered to be too high for contents lower than 1 p.p.m.

Chromatography, Thin Layer↗

Determination of thiabendazole residues in meat by HPLC using ultraviolet and fluorometric detection.

An HPLC method is described for the residue analysis of thiabendazole in meat. The recovery varies from 62 to 75%. Thiabendazole is extracted from the tissue using 3 mol HCl, eluted from the Extrelut-20 column with dichloromethane and then injected onto a C18 column. The optimum conditions for detection are described using ultraviolet and fluorescence spectroscopy. The sensitivity is such that thiabendazole can be determined at a level of 5 micrograms/kg meat. The absolute detection limit with fluorometry is 100 pg.

Animals↗

Comparative efficacies of closantel, ivermectin, oxfendazole, thiophanate and levamisole against thiabendazole resistant Haemonchus contortus in sheep.

Forty-nine sheep artificially exposed to a thiabendazole (TBZ) resistant isolate of Haemonchus contortus were assigned to 7 groups of 7 animals each and used to conduct a controlled anthelmintic trial. One group of sheep served as untreated infected controls and 6 groups were treated as follows: closantel, 5.0 mg kg-1; ivermectin, 0.2 mg kg-1; oxfendazole, 5.0 mg kg-1; thiophanate, 50 mg kg-1, levamisole, 7.5 mgkg-1 and thiabendazole, 66 mg kg-1. Eggs per gram of faeces were determined on days 21, 24 and day 34 (10 days post-treatment) after infection and all animals were necropsied for residual worm counts. The calculated efficacies of the treatments against H. contortus as indicated by worm reduction were closantel (100%), ivermectin (99.3%), oxfendazole (35.2%), thiophanate (56.7%), levamisole (98.6%) and thiabendazole (24.3%). The data therefore indicate that the TBZ-resistant isolate of H. contortus used was highly resistant to the 2 benzimidazole (BZ) anthelmintics, oxfendazole and thiophanate. This is the first report in Kenya of a field strain of H. contortus resistant to thiophanate.

Animals↗

Effects of thiabendazole on dexamethasone-induced suppression of lymphocyte and neutrophil function in cattle.

Seven dosage levels of thiabendazole were evaluated for their ability to normalize lymphocyte or neutrophil function in dexamethasone-treated cattle. Dexamethasone administration (0.04 mg/kg intramuscularly daily for 3 days) significantly suppressed the lymphocyte blastogenic response to phytohemagglutinin, concanavalin A, and pokeweed mitogen. The lymphocyte blastogenic response to allogeneic lymphocytes was also suppressed in dexamethasone-treated cattle, but this effect was not statistically significant. Neutrophils from dexamethasone-treated cattle had a significantly enhanced ability to migrate under agarose, and significantly suppressed ability to reduce nitroblue tetrazolium, iodinate protein, and mediate antibody-dependent cell-mediated cytotoxicity. Thiabendazole when administered concurrently with dexamethasone in the dosage range from 1.0 to 25.0 mg/kg (orally) did significantly enhance lymphocyte blastogenic responsiveness to phytohemagglutinin, concanavalin A, pokeweed mitogen and allogeneic cells (in dexamethasone-treated cattle). This immunonormalizing effect was not observed at the 50 or 100 mg/kg dosage levels. Thiabendazole did not produce a consistent significant enhancement of any parameter of neutrophil function in dexamethasone-treated cattle.

Animals↗

The contribution of a partial tricarboxylic acid cycle to volatile end-products in thiabendazole-resistant and susceptible Trichostrongylus colubriformis.

Acetate, propionate, ethanol and propanol were the predominant end-products released during incubation of a thiabendazole resistant and a susceptible strain of Trichostrongylus colubriformis. The parasites in all the incubations appeared to be deficient in reducing equivalents if the end-products arose from the classical catabolic pathway through fumarate reductase (EC 1.3.1.6). Possible alternative pathways for accounting for redox balance, including beta-oxidation, the pentose phosphate pathway and amino acid metabolism were investigated. Palmitate was oxidised aerobically. Radiolabelled tricarboxylic acid cycle intermediates, citrate and alpha-ketoglutarate, were decarboxylated to 14CO2 indicating that at least a partial tricarboxylic acid cycle to succinyl-CoA via alpha-ketoglutarate operates both anaerobically and aerobically in T. colubriformis. These data and the pattern of end-products suggest the presence of two pathways to propanol and propionate either through fumarate reduction or alpha-ketoglutarate oxidation. T. colubriformis may apportion carbon flow through these pathways to maintain a stable redox ratio. Similar calculations on previously reported data indicate that both pathways may also operate in Haemonchus contortus. Exposure of resistant T. colubriformis to thiabendazole under anaerobic conditions caused an increased accumulation of end-products, especially propanol, in the incubation medium. The alpha-ketoglutarate pathway may lower the dependence of the parasite on the fumarate reductase route which is sensitive to thiabendazole. The operation of the alpha-ketoglutarate pathway, with propanol as an end-product, may provide a mechanism for regulating redox balance in trichostrongylidae.

1-Propanol↗

Comparative efficacy of levamisole, thiabendazole and fenbendazole against cattle gastrointestinal nematodes.

Efficacy of two older anthelmintics, levamisole and thiabendazole, was compared with a newer benzimidazole, fenbendazole, against naturally acquired gastrointestinal nematode infections in cattle superimposed with experimental infections of Bunostomum phlebotomum and Dictyocaulus viviparus. Twenty-four crossbred beef heifers of 7-9 months of age and 152 kg in average weight were randomly allocated to four groups of six calves. The cattle grazed on pastures contaminated with larvae of gastrointestinal nematodes and the lungworm for 2 months prior to Day 0. Treatment groups were as follows: Group 1--levamisole, topical at 10 mg kg-1; Group 2--thiabendazole paste at 110 mg kg-1; Group 3--fenbendazole paste at 10 mg kg-1; Group 4--untreated controls. All calves were necropsied for worm recovery between 8 and 10 days after treatment. Fecal egg/larval per gram counts at 18 and 42 h post-treatment indicated greatest reductions in Groups 1 and 2. By 7 days post-treatment, reduction in counts for all treated groups ranged from 99.1 to 100%, except for the 66.7% reduction of B. phlebotomum in Group 2. Seven nematode species were present in a sufficient number of untreated controls for valid efficacy assessment at necropsy. Efficacy of fenbendazole was 100% against all species, including Cooperia spp. L4 and immature (E5) D. viviparus. The overall efficacy of levamisole and thiabendazole was generally high (93.0-100% against Haemonchus placei adults, Cooperia punctata and C. spatulata adult males, Cooperia spp. adult females, Oesophogastomum radiatum, B. phlebotomum, and D. viviparus adults).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Gas chromatographic determination of thiabendazole in fruits as its methyl derivative.

Quantitative determination of thiabendazole, as its methyl derivative, has been achieved by gas-liquid chromatography with flame ionization detection and a column of 10% of DC-200 on Gas-Chrom Q at 240 degrees. Determination was possible with 10 to 200 ng per mul of the methylated reaction mixture. For determining thiabendazole in fruits, clean-up of the crude extracts by liquid-liquid partition allows satisfactory elimination of interference and permits determination in concentrations down to 0.1 ppm. The recovery of thiabendazole added to fruits at the 0.5-ppm level ranges from 90.3 to 94.9%. The methyl derivative was identified as N-methylthiabendazole by its elementary composition, by its melting-point, and by ultraviolet, infrared, mass and nuclear magnetic resonance spectroscopy.

Chromatography, Gas↗

Ion trap tandem mass spectrometric identification of thiabendazole phototransformation products on titanium dioxide.

The purpose of this study is to artificially produce degradation intermediates of thiabendazole, which could be reasonably similar to those really present in the environment. The formation of by-products from thiabendazole transformation has been evaluated by adopting irradiated titanium dioxide as a photocatalyst. Several species more hydrophilic than the thiabendazole have been identified and characterized by HPLC-multiple MS. A pattern of reactions accounting for the observed intermediates is proposed. Two different parallel pathways are operating (and through these pathways the transformation of the molecule proceeds) leading to several intermediate compounds. The main steps involved are: (1) the hydroxylation of the molecule on the aromatic ring with the formation of a species having [M+H]+ 218; a further oxidation leads to the ring-opening and to the formation of aldehydic and alcoholic structures ([M+H]+ 270, 268 and 152); and (2) the cleavage of a C-C bond and the formation of a species having [M+H]+ 119.

Chromatography, High Pressure Liquid↗