The ABCG2 protein in vitro transports the xenobiotic thiabendazole and increases the appearance of its residues in milk.

Thiabendazole (TBZ) is a broad-spectrum anthelmintic and fungicide used in humans, animals, and agricultural commodities. TBZ residues are present in crops and animal products, including milk, posing a risk to food safety and public health. ABCG2 is a membrane transporter which affects bioavailability and milk secretion of xenobiotics. Therefore, the aim of this work was to characterize the role of ABCG2 in the in vitro transport and secretion into milk of 5-hydroxythiabendazole (5OH-TBZ), the main TBZ metabolite. Using MDCK-II polarized cells transduced with several species variants of ABCG2, we first demonstrated that 5OH-TBZ is efficiently in vitro transported by ABCG2. Subsequently, using Abcg2 knockout mice, we demonstrated that 5OH-TBZ secretion into milk was affected by Abcg2, with a more than 2-fold higher milk concentration and milk to plasma ratio in wild-type mice compared to their Abcg2-/- counterpart.

Developmental defects induced by thiabendazole are mediated via apoptosis, oxidative stress and alteration in PI3K/Akt and MAPK pathways in zebrafish.

Thiabendazole, a benzimidazole fungicide, is widely used to prevent yield loss in agricultural land by inhibiting plant diseases derived from fungi. As thiabendazole has a stable benzimidazole ring structure, it remains in the environment for an extended period, and its toxic effects on non-target organisms have been reported, indicating the possibility that it could threaten public health. However, little research has been conducted to elucidate the comprehensive mechanisms of its developmental toxicity. Therefore, we used zebrafish, a representative toxicological model that can predict toxicity in aquatic organisms and mammals, to demonstrate the developmental toxicity of thiabendazole. Various morphological malformations were observed, including decreased body length, eye size, and increased heart and yolk sac edema. Apoptosis, reactive oxygen species (ROS) production, and inflammatory response were also triggered by thiabendazole exposure in zebrafish larvae. Furthermore, PI3K/Akt and MAPK signaling pathways important for appropriate organogenesis were significantly changed by thiabendazole. These results led to toxicity in various organs and a reduction in the expression of related genes, including cardiovascular toxicity, neurotoxicity, and hepatic and pancreatic toxicity, which were detected in flk1:eGFP, olig2:dsRED, and L-fabp:dsRed;elastase:GFP transgenic zebrafish models, respectively. Overall, this study partly determined the developmental toxicity of thiabendazole in zebrafish and provided evidence of the environmental hazards of this fungicide.

Recovery from spindle checkpoint-mediated arrest requires a novel Dnt1-dependent APC/C activation mechanism.

The activated spindle assembly checkpoint (SAC) potently inhibits the anaphase-promoting complex/cyclosome (APC/C) to ensure accurate chromosome segregation at anaphase. Early studies have recognized that the SAC should be silenced within minutes to enable rapid APC/C activation and synchronous segregation of chromosomes once all kinetochores are properly attached, but the underlying silencers are still being elucidated. Here, we report that the timely silencing of SAC in fission yeast requires dnt1+, which causes severe thiabendazole (TBZ) sensitivity and increased rate of lagging chromosomes when deleted. The absence of Dnt1 results in prolonged inhibitory binding of mitotic checkpoint complex (MCC) to APC/C and attenuated protein levels of Slp1Cdc20, consequently slows the degradation of cyclin B and securin, and eventually delays anaphase entry in cells released from SAC activation. Interestingly, Dnt1 physically associates with APC/C upon SAC activation. We propose that this association may fend off excessive and prolonged MCC binding to APC/C and help to maintain Slp1Cdc20 stability. This may allow a subset of APC/C to retain activity, which ensures rapid anaphase onset and mitotic exit once SAC is inactivated. Therefore, our study uncovered a new player in dictating the timing and efficacy of APC/C activation, which is actively required for maintaining cell viability upon recovery from the inhibition of APC/C by spindle checkpoint.

Nutritional inter-dependencies and a carbazole-dioxygenase are key elements of a bacterial consortium relying on a Sphingomonas for the degradation of the fungicide thiabendazole.

Thiabendazole (TBZ), is a persistent fungicide/anthelminthic and a serious environmental threat. We previously enriched a TBZ-degrading bacterial consortium and provided first evidence for a Sphingomonas involvement in TBZ transformation. Here, using a multi-omic approach combined with DNA-stable isotope probing (SIP) we verified the key degrading role of Sphingomonas and identify potential microbial interactions governing consortium functioning. SIP and amplicon sequencing analysis of the heavy and light DNA fraction of cultures grown on 13 C-labelled versus 12 C-TBZ showed that 66% of the 13 C-labelled TBZ was assimilated by Sphingomonas. Metagenomic analysis retrieved 18 metagenome-assembled genomes with the dominant belonging to Sphingomonas, Sinobacteriaceae, Bradyrhizobium, Filimonas and Hydrogenophaga. Meta-transcriptomics/-proteomics and non-target mass spectrometry suggested TBZ transformation by Sphingomonas via initial cleavage by a carbazole dioxygenase (car) to thiazole-4-carboxamidine (terminal compound) and catechol or a cleaved benzyl ring derivative, further transformed through an ortho-cleavage (cat) pathway. Microbial co-occurrence and gene expression networks suggested strong interactions between Sphingomonas and a Hydrogenophaga. The latter activated its cobalamin biosynthetic pathway and Sphingomonas its cobalamin salvage pathway to satisfy its B12 auxotrophy. Our findings indicate microbial interactions aligning with the 'black queen hypothesis' where Sphingomonas (detoxifier, B12 recipient) and Hydrogenophaga (B12 producer, enjoying detoxification) act as both helpers and beneficiaries.

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole.

BACKGROUND: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. METHODS: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10-9 M, 10-11 M, 10-13 M), pyrantel citrate (10-6 M, 10-8 M, 10-10 M), thiabendazole (10-5 M, 10-7 M, 10-9 M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system. RESULTS: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ - 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10-9 M of ivermectin. CONCLUSIONS: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.

The binding of orally dosed hydrophobic active pharmaceutical ingredients to casein micelles in milk.

Casein proteins (αS1-, αS2-, ß- and κ-casein) account for 80% of the total protein content in bovine milk and form casein micelles (average diameter = 130 nm, approximately 1015 micelles/mL). The affinity of native casein micelles with the 3 hydrophobic active pharmaceutical ingredients (API), meloxicam [351.4 g/mol; log P = 3.43; acid dissociation constant (pKa) = 4.08], flunixin (296.2 g/mol; log P = 4.1; pKa = 5.82), and thiabendazole (201.2 g/mol; log P = 2.92; pKa = 4.64), was evaluated in bovine milk collected from dosed Holstein cows. Native casein micelles were separated from raw bovine milk by mild techniques such as ultracentrifugation, diafiltration, isoelectric point precipitation (pH 4.6), and size exclusion chromatography. Acetonitrile extraction of hydrophobic API was then done, followed by quantification using HPLC-UV. For the API or metabolites meloxicam, 5-hyroxy flunixin and 5-hydroxy thiabendazole, 31 ± 3.90, 31 ± 1.3, and 28 ± 0.5% of the content in milk was associated with casein micelles, respectively. Less than ∼5.0% of the recovered hydrophobic API were found in the milk fat fraction, and the remaining ∼65% were associated with the whey/serum fraction. A separate in vitro study showed that 66 ± 6.4% of meloxicam, 29 ± 0.58% of flunixin, 34 ± 0.21% of the metabolite 5-hyroxy flunixin, 50 ± 4.5% of thiabendazole, and 33 ± 3.8% of metabolite 5-hydroxy thiabendazole was found partitioned into casein micelles. Our study supports the hypothesis that casein micelles are native carriers for hydrophobic compounds in bovine milk.

Characterization of the biodegradation, bioremediation and detoxification capacity of a bacterial consortium able to degrade the fungicide thiabendazole.

Thiabendazole (TBZ) is a persistent fungicide used in the post-harvest treatment of fruits. Its application results in the production of contaminated effluents which should be treated before their environmental discharge. In the absence of efficient treatment methods in place, biological systems based on microbial inocula with specialized degrading capacities against TBZ could be a feasible treatment approach. Only recently the first bacterial consortium able to rapidly transform TBZ was isolated. This study aimed to characterize its biodegradation, bioremediation and detoxification potential. The capacity of the consortium to mineralize 14C-benzyl-ring labelled TBZ was initially assessed. Subsequent tests evaluated its degradation capacity under various conditions (range of pH, temperatures and TBZ concentration levels) and relevant practical scenarios (simultaneous presence of other postharvest compounds) and its bioaugmentation potential in soils contaminated with increasing TBZ levels. Finally cytotoxicity assays explored its detoxification potential. The consortium effectively mineralized the benzoyl ring of the benzimidazole moiety of TBZ and degraded spillage level concentrations of the fungicide in aqueous cultures (750 mg L-1) and in soil (500 mg kg-1). It maintained its high degradation capacity in a wide range of pH (4.5-7.5) and temperatures (15-37 °C) and in the presence of other pesticides (ortho-phenylphenol and diphenylamine). Toxicity assays using the human liver cancer cell line HepG2 showed a progressive decrease in cytotoxicity, concomitantly with the biodegradation of TBZ, pointing to a detoxification process. Overall, the bacterial consortium showed high potential for future implementation in bioremediation and biodepuration applications.

Isolation of a bacterial consortium able to degrade the fungicide thiabendazole: the key role of a Sphingomonas phylotype.

Thiabendazole (TBZ) is a fungicide used in fruit-packaging plants. Its application leads to the production of wastewaters requiring detoxification. In the absence of efficient treatment methods, biological depuration of these effluents could be a viable alternative. However, nothing is known regarding the microbial degradation of the recalcitrant and toxic to aquatics TBZ. We report the isolation, via enrichment cultures from a polluted soil, of the first bacterial consortium able to rapidly degrade TBZ and use it as a carbon source. Repeated efforts using various culture-dependent approaches failed to isolate TBZ-degrading bacteria in axenic cultures. Denaturating gradient gel electrophoresis (DGGE) and cloning showed that the consortium was composed of α-, ß- and γ-Proteobacteria. Culture-independent methods including antibiotics-driven selection with DNA/RNA-DGGE, q-PCR and stable isotope probing (SIP)-DGGE identified a Sphingomonas phylotype (B13) as the key degrading member. Cross-feeding studies with structurally related chemicals showed that ring substituents of the benzimidazole moiety (thiazole or furan rings) favoured the cleavage of the imidazole moiety. LC-MS/MS analysis verified that TBZ degradation proceeds via cleavage of the imidazole moiety releasing thiazole-4-carboxamidine, which was not further transformed, and the benzoyl moiety, possibly as catechol, which was eventually consumed by the bacterial consortium as suggested by SIP-DGGE.

NHR-176 regulates cyp-35d1 to control hydroxylation-dependent metabolism of thiabendazole in Caenorhabditis elegans.

Knowledge of how drugs are metabolized and excreted is an essential component of understanding their fate within and among target and non-target organisms. Thiabendazole (TBZ) was the first benzimidazole (BZ) to be commercially available and remains one of the most important anthelmintic drugs for medical and veterinary use. We have characterized how Caenorhabditis elegans metabolizes and excretes TBZ. We have shown that TBZ directly binds to the nuclear hormone receptor (NHR)-176 and that this receptor is required for the induction by TBZ of the cytochrome P450 (CYP) encoded by cyp-35d1. Further, RNAi inhibition of cyp-35d1 in animals exposed to TBZ causes a reduction in the quantity of a hydroxylated TBZ metabolite and its glucose conjugate that is detected in C. elegans tissue by HPLC. This final metabolite is unique to nematodes and we also identify two P-glycoproteins (PGPs) necessary for its excretion. Finally, we have shown that inhibiting the metabolism we describe increases the susceptibility of C. elegans to TBZ in wild-type and in resistant genetic backgrounds.

Fate of thiabendazole through the treatment of a simulated agro-food industrial effluent by combined MBR/Fenton processes at µg/L scale.

This study has been carried out to assess the performance of a combined system consisting of a membrane bioreactor (MBR) followed by an advanced oxidation process (Fenton/Photo-Fenton) for removing the fungicide thiabendazole (TBZ) in a simulated agro-food industrial wastewater. Previous studies have shown the presence of TBZ in the effluent of an agro-food industry treated by activated sludge in a sequencing batch reactor (SBR), thus reinforcing the need for alternative treatments for removal. In this study, a simulated agro-food industry effluent was enriched with 100 µg L(-1) TBZ and treated by combined MBR/Fenton and MBR/solar photo-Fenton systems. Samples were directly injected into a highly sensitive liquid chromatography-triple quadrupole-linear ion trap-mass spectrometer (LC-QqLiT-MS/MS) analytical system to monitor the degradation of TBZ even at low concentration levels (ng L(-1)). Results showed that the biological treatment applied was not effective in TBZ degradation, which remained almost unaltered; although most dissolved organic matter was biodegraded effectively. Fenton and solar photo-Fenton, were assayed as tertiary treatments. The experiments were run without any pH adjustment by using an iron dosage strategy in the presence of excess hydrogen peroxide. Both treatments resulted in a total degradation of TBZ, obtaining more than 99% removal in both cases. To assure the total elimination of contaminants in the treated waters, transformation products (TPs) of TBZ generated during Fenton degradation experiments were identified and monitored by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS/MS). Up to four TPs could be identified. Two of them corresponded to mono-hydroxylated derivatives, typically generated under hydroxyl radicals driven processes. The other two corresponded with the hydrolysis of the TBZ molecule to yield benzoimidazole and thiazole-4-carboxamidine. All of them were also degraded during the treatment.

Thiabendazole uptake in shimeji, king oyster, and oyster mushrooms and its persistence in sterile and nonsterile substrates.

Thiabendazole in the substrates incurred from spraying and premixing was translocated to the pileus, stipe, and volva of selected mushrooms. The spraying on the substrates resulted in higher residues of thiabendazole in all three mushrooms than the premixing treatment. For premixing, in the five substrates, half-lives of thiabendazole were found to be 13.6 days for shimeji, 10.0 days for king oyster, 13.7 days for oyster, 19.1 days for sterilized substrate, and 8.4 days for nonsterilized substrate, respectively. For spraying, the longest and shortest half-lives were found to be 19.5 and 8.1 days for the nonsterilized and sterilized substrates, respectively. The residues of thiabendazole in three edible fungi were increased with the incubation days from 3 to 5 to 7. The residues of thiabendazole in king oyster were the highest among the three fungi while those in shimeji and oyster showed similar patterns.

Thiabendazole inhibits ubiquinone reduction activity of mitochondrial respiratory complex II via a water molecule mediated binding feature.

The mitochondrial respiratory complex II or succinate: ubiquinone oxidoreductase (SQR) is a key membrane complex in both the tricarboxylic acid cycle and aerobic respiration. Five disinfectant compounds were investigated with their potent inhibition effects on the ubiquinone reduction activity of the porcine mitochondrial SQR by enzymatic assay and crystallography. Crystal structure of the SQR bound with thiabendazole (TBZ) reveals a different inhibitor-binding feature at the ubiquinone binding site where a water molecule plays an important role. The obvious inhibitory effect of TBZ based on the biochemical data (IC(50) ~100 µmol/L) and the significant structure-based binding affinity calculation (~94 µmol/L) draw the suspicion of using TBZ as a good disinfectant compound for nematode infections treatment and fruit storage.

Development of competitive immunoassays to hydroxyl containing fungicide metabolites.

This paper describes the isolation of monoclonal antibodies and the development of competitive immunoassays to pesticide metabolites of the fungicides imazalil, carbendazim and thiabendazole. The metabolite specific hydroxyl residues were used as the reactive group with which to link the metabolite to the carrier proteins Keyhole Limpet Haemocyanin (KLH) and Bovine Serum Albumin (BSA). In each case immune responses in mice were raised and monoclonal antibodies were produced. Antibodies were developed into competitive ELISAs to the appropriate metabolite. The antibody raised to a metabolite of imazalil was optimised into a competitive ELISA format which had an assay IC50 of 7.5 µg/L and a limit of detection (LOD) of 1.1 µg/L. A single antibody isolated against the metabolite of carbendazim had assay IC50s of 3.2 and 2.7 µg/L for the metabolites of carbendazim and thiabendazole respectively with an LOD of 0.38 µg/L for both. These sensitive immunoassays may have application in the monitoring of human exposure to these fungicide residues either by occupational or non-occupational routes.

[Distribution and elimination of thiabendazole and its metabolite residue in laying hens].

OBJECTIVE: To explore the distribution and elimination of thiabendazole and its metabolite 5-hydrothiabendazole residues in the hens tissues including liver, muscle, heart, fat, as well as in eggs. METHODS: Laying hens were orally administred thiabendazole for 5 consecutive days (100mg per hen daily) and then the hens were sacrificed at the times of 1 day, 3 days, 5 days and 7 days after the end of treatment. Eggs, liver,muscle,fat and heart tissues were collected and homogenized. The samples were extracted by acetonitrile, further concentrated and purified by an Oasis MCX cartridge, and then the contents of thiabendazole and 5-hydrothiabendazole in tissue homogenates determinated by liquid chromatography electrospray ionisation tandem mass spectrometry (UPLC-ESI-MS/MS). RESULTS: The major residues in the tissue homogenates was 5-hydroxythiabendazole, with more higher concentrations than thiabendazole. Egg samples presented the large majority of both drug residues. For the tissue homogenates,the total concentrations of thiabendazole and 5-hydrothiabendazole residues followed the order of liver > heart > muscle > fat at 1 day after the treatment. The withdrawal period of thiabendazole for eggs was about 7 days. CONCLUSION: Distribution and elimination of thiabendazole and its metabolite residues in laying hens were primarily studied in this study.

In vitro metabolic activation of thiabendazole via 5-hydroxythiabendazole: identification of a glutathione conjugate of 5-hydroxythiabendazole.

Thiabendazole (TBZ) is a broad-spectrum antihelmintic used for treatment of parasitic infections in animals and humans and as an agricultural fungicide for postharvest treatment of fruits and vegetables. It is teratogenic and nephrotoxic in mice, and cases of hepatotoxicity have been observed in humans. Recent reports have demonstrated a correlation between 5-hydroxythiabendazole (5-OHTBZ) formation, a major metabolite of TBZ, and covalent binding of [(14)C]TBZ to hepatocytes, suggesting another pathway of activation of TBZ. Current in vitro studies were undertaken to probe the bioactivation of TBZ via 5-OHTBZ by cytochrome P450 (P450) and peroxidases and identify the reactive species by trapping with reduced glutathione (GSH). Microsomal incubation of TBZ or 5-OHTBZ supplemented with NADPH and GSH afforded a GSH adduct of 5-OHTBZ and was consistent with a bioactivation pathway that involved a P450-catalyzed two-electron oxidation of 5-OHTBZ to a quinone imine. The same adduct was detected in GSH-fortified incubations of 5-OHTBZ with peroxidases. The identity of the GSH conjugate suggested that the same reactive intermediate was formed by both these enzyme systems. Characterization of the conjugate by mass spectrometry and NMR revealed the addition of GSH at the 4-position of 5-OHTBZ. In addition, the formation of a dimer of 5-OHTBZ was discernible in peroxidase-mediated incubations. These results were consistent with a one-electron oxidation of 5-OHTBZ to a radical species that could undergo disproportionation or an additional one-electron oxidation to form a quinone imine. Overall, these studies suggest that 5-OHTBZ can also play a role in TBZ-induced toxicity via its bioactivation by P450 and peroxidases.

Artemisinin and thiabendazole are potent inhibitors of cytochrome P450 1A2 (CYP1A2) activity in humans.

OBJECTIVE: To investigate the likelihood of artemisinin and thiabendazole causing pharmacokinetic interactions involving cytochrome P450 (CYP1A2) in humans given their potent inhibitory effects on the isoform in vitro. METHODS: Ten healthy volunteers received caffeine (136.5 mg), and after a washout period of 48 h, the volunteers were given a caffeine tablet (136.5 mg) together with thiabendazole (500 mg). After an additional 14 days, the volunteers received caffeine together with artemisinin (500 mg). After each treatment, plasma was obtained up to 24 h post-dose. The plasma concentrations of the drugs were measured by HPLC with UV and MS detection. RESULTS: Using the ratio of paraxanthine to caffeine after 4 h as an indicator of CYP1A2 activity, thiabendazole and artemisinin inhibited 92 and 66%, respectively, of the enzyme activity in vivo. In addition, the pharmacokinetics of caffeine were altered in the presence of the drugs; increases in AUC(0-24) of 1.6-fold (P < 0.01) and 1.3-fold of caffeine in the presence of thiabendazole and artemisinin respectively were measured. The use of in vitro data to predict the effects of thiabendazole on the formation of paraxanthine yielded good results and underestimated the effects of artemisinin when total plasma concentrations were used. Corrections for protein binding resulted in underestimation of inhibitory effects on CYP1A2. CONCLUSIONS: Co-administration of thiabendazole or artemisinin with CYP1A2 substrates could result in clinically significant effects. Our results highlight the validity of in vitro data in predicting in vivo CYP inhibition. The formation of paraxanthine seems to be a better indicator of in vivo CYP1A2 activity than caffeine levels.

Synthesis, X-ray crystal structure, antimicrobial activity and photodynamic effects of some thiabendazole complexes.

An interesting series of metal complexes of thiabendazole (tbz) is synthesized and characterized by elemental analyses and spectroscopic studies. The crystal structure of the hydrogen bonded one dimensional Co(II) complex, namely [Co(tbz)(2)(NO(3))(H(2)O)](NO(3)) is solved by single crystal X-ray diffraction. The complex crystallizes in monoclinic space group P2(1)/a with unit cell parameters, a=14.366(2), b=11.459(4), c=15.942(3) A, beta=113.78(3) degrees and z=4. The unit cell packing reveals an extensive hydrogen bonding involving a water molecule, nitrate ligands and the protonated nitrogen atoms of the tbz ligands, resulting in a one dimensional hydrogen bonding pattern. The antimicrobial activity of the complexes against selected bacteria (Escherichia coli and Bacillus subtilis) and yeast (Aspergillus flavues) is estimated. The relationship between the enzymatic production of ROS and antimicrobial activity of the complexes is examined, and a good correlation between two factors is found. Photodynamic quantum yields of singlet oxygen production (RNO bleaching assay) and rate of superoxide generation (SOD inhibitable ferricytochrome c reduction assay and EPR spin trapping experiments using 5,5-dimethyl-1-pyrroline-N-oxide as spin trap) by the metal complexes have been studied.

Persistence of the fungicides thiabendazole, carbendazim and prochloraz-Mn in mushroom casing soil.

The persistence of the fungicides thiabendazole, carbendazim and prochloraz-Mn in mushroom casing soil was determined following their application at rates commonly used in the UK mushroom industry. Following drench applications, the concentration of all active ingredients was always higher in the top half of the casing soil layer than in with the bottom half. When carbendazim and prochloraz-Mn were applied using half the recommended volume of water per unit area, there was a tendency for carbendazim concentrations to be even higher in the top half of the casing soil, compared with the standard treatment, while concentrations of prochloraz-Mn were similar, irrespective of the volume of water used. Carbendazim and prochloraz-Mn concentrations in the top half of the casing layer decreased to < or = 13 mg kg(-1) by day 28/29, following different applications, whereas the thiabendazole concentration was consistently high during the course of the crop, being < or = 83 mg kg(-1) at day 31. Fungicides that do not persist at high concentrations in mushroom casing soil for the duration of the crop may not give good control of mushroom pathogens, particularly if the fungicide concentration falls to a level which is close to the EC50 value.

Release of protein-bound residues of thiabendazole from liver.

Tissue-bound residues of thiabendazole (TBZ), a veterinary anthelmintic and postharvest fungicide, are formed when this compound is incubated with rabbit hepatocytes or administered to mice or pigs. Several pretreatment steps were investigated for removing free TBZ and metabolites prior to the release of bound residues, and three procedures were evaluated for the release of bound residues from solvent-extracted rabbit hepatocytes: incubation under acidic conditions, enzymatic action using cystathionine beta-lyase, and Raney nickel desulfurization. Immunoaffinity chromatography utilizes monoclonal antibodies capable of binding TBZ or its 5-hydroxy metabolite enabled isolation of crossreactive residue fractions. Residues released from incurred pig liver and isolated by immunoaffinity included TBZ, as determined by HPLC with photodiode array detection. The methodology described should facilitate food safety assessments of TBZ.