Imazalil Research Articles

Factorial experimental design of imazalil-containing wastewater to be treated by Fenton-based processes

Abstract Fenton-based processes have been studied for the mineralization and detoxification of wastewaters from the postharvest of fruit, which were contaminated with the fungicide imazalil (IMZ). For this purpose, studies were carried out in a simulated agro-industrial wastewater. Statistical design of experiments was used to assess the influence of IMZ, Fe(II) and H2O2 concentration in the efficiency of the process. The most significant factor affecting the process was Fe(II). Higher mineralization was obtained with the photo-Fenton process when compared with the Fenton reaction. The optimized Fe(II) and H2O2 for the mineralization of 50 mg L−1 IMZ were 21.5 and 127.5 mg L−1, respectively. LC/MS/MS was used to identify the main products formed during the reaction and a degradation pathway was proposed. An operational cost of 2.06 €/m3 was estimated for the treatment of 10 m3 wastewater contaminated with IMZ.

Insights Into a Possible Influence on Gut Microbiota and Intestinal Barrier Function During Chronic Exposure of Mice to Imazalil.

The fungicide imazalil (IMZ) is widely used to prevent and treat fungal diseases in plants and animals. Here, male adult C57BL/6 mice were exposed to 0.1, 0.5, and 2.5 mg/kg body weight IMZ for 2, 5, or 15 weeks. The microbiota in cecal contents and feces changed during chronic IMZ exposure at phylum and genus levels. Sequencing of the V3-V4 region of the bacterial 16S rRNA gene revealed a significant change in the richness of microbiota in cecal contents and feces after exposure to 2.5 mg/kg IMZ for 15 weeks. Operational taxonomic unit (OTU) analysis indicated that 31.1% of cecal OTUs and 14.0% of fecal OTUs changed after IMZ exposure. In addition, chronic IMZ exposure also disturbed the intestinal barrier function of the mice, reducing mucus secretion, decreasing the expression of cystic fibrosis transmembrane conductance regulator (CFTR)-related genes in both the ileum and colon. Molecular docking analysis revealed that key hydrogen bonds were formed by nitrogen atoms of the imidazole bond with Val440 of CFTR and Ala697 of the SLC26 family. Our data suggested that gut microbiota and intestinal barrier were potential toxicological targets of IMZ.

Postharvest dip, drench and wax coating application of pyrimethanil on citrus fruit: Residue loading and green mould control

Residue loading of pyrimethanil (PYR) with application methods typically used in South African packhouses and green mould control was studied. PYR was applied curatively (after inoculation) and protectively (before inoculation) in dip, drench and wax coating treatments and fruit were inoculated with an imazalil (IMZ)-sensitive or an IMZ-resistant isolate of Penicillium digitatum, the causal agent of citrus green mould. The dip treatments consisted of PYR concentrations 0, 50, 100, 250, 500 and 1000 mg L−1; fruit were dipped for 60 s at 18 °C at pH 7. Residues loaded differed between fruit type and batch. At the registered concentration of 1000 mg L−1, an average of 1.96 mg kg−1 PYR (range 1.40–2.95 mg kg−1) was loaded. Increased residue levels generally resulted in improved control, but better curative than protective control was observed with effective residue levels of 0.27 and 0.91 mg kg−1 for 50% and 75% curative control of green mould, respectively. Effective residue levels for 50% protective control were 0.97–1.43 mg kg−1. Drench treatments consisted of exposure times of 30, 60 and 90 s with 1000 mg L−1 PYR. PYR residues on Valencia oranges were 2.46–4.22 mg kg−1 PYR, and on navel oranges were 2.09–7.64 mg kg−1. Green mould control on Valencia orange fruit was moderate (57.3–69.8% for curative treatment after 6 h) to relatively poor (4.9–14.6% control for curative treatments after 24 h and 18.1–26.4% for protective treatments). Control on navel oranges following these treatments was poor (0–7%). Drench applications with different combinations of PYR, IMZ, thiabendazole (TBZ), guazatine (GZT) and didecyl dimethyl ammonium chloride were applied to navel orange fruit with 60 s exposure time. In general, fungicide mixtures performed better than single fungicides (44.4–64.6% for mixtures vs. 24.3, 39.6 and 46.5% for PYR, GZT and TBZ respectively). Drench applications where fruit were inoculated with spore suspensions containing 106, 105, 104 or 103 spores mL−1 showed that infections from lower inoculum loads were better controlled (90.6–98.6% vs. 32.6–62.2% for 103vs. 106 spores mL−1). Wax with 2000 or 4000 mg L−1 PYR was applied at 0.6, 1.2 and 1.8 L wax ton−1 of fruit. Residues loaded on navel oranges were 1.75–6.33 mg kg−1, and on Valencia oranges were 1.05–9.91 mg kg−1. Green mould control on navel oranges was poor and were 0–12.2%. On Valencia oranges, higher levels of protective control (28.8–54.0%) than curative treatments (10.5–27.3%) was observed. The IMZ-resistant isolate was successfully controlled by all PYR treatments, in some cases better than the sensitive isolate. In conclusion, PYR provided very good curative control in dip applications, but gave poor results in drench and wax applications. Green mould control following PYR drench treatment improved on fruit inoculated with smaller spore loads, or when PYR was mixed with other fungicides.

Imazalil exposure induces gut microbiota dysbiosis and hepatic metabolism disorder in zebrafish

The fungicide imazalil (IMZ) is used extensively to preserve freshness, prevent decay and control fungal infections in fruits, vegetables or other plants. Recently, some studies have reported that the real in aquatic systems have reached very high levels. Here, male adult zebrafish were exposed to 100 and 1000μg/L IMZ for 1, 7, 21days, and the gut microbiota and hepatic metabolism were evaluated. Exposure to a high concentration of IMZ for 21days decreased mucin secretion in the gut. Sequencing of the V3–V4 region of the bacterial 16S rRNA gene revealed a significant increase in the diversity of gut microbiota in male zebrafish. At the phylum level, the composition of Proteobacteria and Bacteroidetes was decreased, while those Fusobacteria and Firmicutes increased in the gut after exposure to 1000μg/L IMZ for 21days. At the genus level, 29 species of microorganisms were significantly changed after IMZ exposure. Based on GC/MS metabolomics analysis, 101 metabolites were observably significantly altered in the 1000μg/L IMZ-treatment group. These changed metabolites were mainly associated with the pathway of glycolysis, amino acid metabolism, and lipid metabolism. In addition, the transcription of some genes related to glycolysis and lipid metabolism, including Aco, Cpt1, Acc1, Srebp1a and Fas, was decreased significantly in the liver of zebrafish when exposed to 100 and 1000μg/L IMZ for 7 or 21days. These results indicated that exposure to IMZ could cause gut microbiota dysbiosis and metabolic disorders in adult zebrafish.

Classification of imazalil resistance in an international collection of Penicillium digitatum isolates

A total of 213 Penicillium digitatum isolates with resistance to the fungicide imazalil (IMZ) originating from green moulded citrus fruit from several countries (South Africa, USA, Uruguay, Spain, Israel, Cyprus, Chile, Australia and Argentina) were tested for their Cyp51 IMZ-resistance group (R1 to R3) using a published multiplex PCR assay. As well, the level of resistance (measured as EC50 values) was determined. Isolates from the USA showed the highest resistance group diversity (isolates belonged to R1, R2 and R3 groups) and lowest EC50 values. The isolates from the other countries were all classified into the R3 group, except for Chile, which also contained R1 isolates. There was no correlation between the level of resistance of isolates and the resistance group genotype. In general, IMZ resistance was most commonly associated (88.3%) with the R3 group. This study is the first to investigate the distribution of IMZ resistance genotypes in P. digitatum populations from a broad international scale.

Identification and determination of chlorinated azoles in sludge using liquid chromatography quadrupole time-of-flight and triple quadrupole mass spectrometry platforms

Four antimycotic drugs (tioconazole, TCZ; sertaconazole, STZ; fenticonazole, FTZ and itraconazole, ITZ) and the fungicide imazalil (IMZ) are determined in sludge from sewage treatment plants (STPs) following a bottom-up analytical strategy. First, sludge extracts, obtained under different sample preparation conditions, were analyzed by liquid chromatography (LC) quadrupole time-of-flight (QTOF) mass spectrometry (MS). A non-target search strategy, combined with the use of the chlorine mass filter, permitted to detect several chlorinated pollutants including the above referred azoles, which either had not been previously reported (TCZ, STZ, FTZ and ITZ), or scarcely investigated (IMZ), in this environmental compartment. Then, the sample preparation procedure was validated using standards of these compounds and their sensitive and selective determination was performed by LC–MS/MS, based on a QqQ system. Under final working conditions, quantitative extraction yields were attained with negligible changes in ionization efficiencies between sample extracts and standards; therefore, the above compounds were quantified against authentic standard solutions, with absolute recoveries in the range from 75 to 124%, achieving a limit of quantification of 2ngg−1. Analysis of sludge from 10 municipal STPs demonstrated the ubiquity of the identified chlorinated azoles with average concentrations from 31ngg−1, for IMZ, to more than 200ngg−1, for ITZ.

A sequential treatment with sodium hypochlorite and a reduced dose of imazalil heated at 50 °C effectively control decay of individually film-wrapped lemons stored at 20 °C

Storage of individual seal-packaged citrus fruit at room temperature in China is a very common practice that requires a pre-storage treatment with a high concentration of an effective fungicide as imazalil (IMZ) to prevent decay.In this study, lemons were washed with NaOCl (200mgL−1) or not, treated with IMZ (50 or 1000mgL−1) at 20 or 50°C and individually wrapped with a 16-μm thick extensible polyvinylchloride film (Film A) or two heat shrinkable polyolefinic films, thick 15 (Film B) or 19-μm (FilmC).The sequential treatment with NaOCl and IMZ at 50mgL−1 at 50°C, was as effective as IMZ at 1000mgL−1 at 20°C in controlling Penicillium decay. Losses for decay in fruit wrapped with the two more permeable films (Film A and Film B) never exceeded 10%, while in those wrapped with Film C (the least permeable) peaked to 41%. All films reduced weight losses, which at the end of storage were 11% in fruit wrapped with Film A and below 4% in those wrapped with the other two films, while were 41% in unwrapped ones. After one week of storage, only 50% of unwrapped fruit were marketable whereas all wrapped fruit were still marketable after 8 weeks. Respiration as well as juice acetaldehyde and ethanol were slightly affected by the two more permeable films, while an abnormal production of CO2, acetaldehyde and ethanol occurred in those wrapped with film C. Changes in chemical parameters were relevant in fruit sealed with Film C and minor in those with Film A and Film B.Decay control and quality preservation of lemons stored at room temperature can be achieved for several weeks by a sequential treatment with NaOCl and a heated water emulsion of IMZ at 50mgL−1, when fruit are wrapped with plastic films highly permeable to gases.

Degradation alternatives for a commercial fungicide in water: biological, photo-Fenton, and coupled biological photo-Fenton processes.

Imazalil (IMZ) is a widely used fungicide for the post-harvest treatment of citrus, classified as "likely to be carcinogenic in humans" for EPA, that can be only partially removed by conventional biological treatment. Consequently, specific or combined processes should be applied to prevent its release to the environment. Biological treatment with adapted microorganism consortium, photo-Fenton, and coupled biological photo-Fenton processes were tested as alternatives for the purification of water containing high concentration of the fungicide and the coadjutants present in the commercial formulation. IMZ-resistant consortium with the capacity to degrade IMZ in the presence of a C-rich co-substrate was isolated from sludge coming from a fruit packaging company wastewater treatment plant. This consortium was adapted to resist and degrade the organics present in photo-Fenton-oxidized IMZ water solution. Bacteria colonies from the consortia were isolated and identified. The effect of H2O2 initial concentration and dosage on IMZ degradation rate, average oxidation state (AOS), organic acid concentration, oxidation, and mineralization percentage after photo-Fenton process was determined. The application of biological treatment to the oxidized solutions notably decreased the total organic carbon (TOC) in solution. The effect of the oxidation degree, limited by H2O2 concentration and dosage, on the percentage of mineralization obtained after the biological treatment was determined and explained in terms of changes in AOS. The concentration of H2O2 necessary to eliminate IMZ by photo-Fenton and to reduce TOC and chemical oxygen demand (COD) by biological treatment, in order to allow the release of the effluents to rivers with different flows, was estimated.

Simultaneous Determination of Seven Kinds of Fungicides in Citrus Fruits by Gas Chromatograghy/Mass Spectrometry.

A simple and accurate procedure was developed for the determination of seven fungicides, azoxystrobin (AZO), diphenyl (DP), fludioxonil (FLUDI), imazalil (IMZ), o-phenylphenol (OPP), pyrimethanil (PYRI) and thiabendazole (TBZ), in citrus fruits. The citrus fruit sample was extracted with acetonitrile and cleaned up with a graphite carbon/aminopropyl silanized silica gel solid-phase extraction cartridge using acetonitrile-toluene (3 : 1, v/v) as the eluent. Triphenylene was used as an internal standard (I.S.) at the concentration of 0.5 μg/mL. The sample solution was subjected to GC-MS utilizing the matrix-matched standard solution method. The recoveries of AZO, FLUDI, IMZ, OPP, PYRI and TBZ spiked in domestic citrus fruits (Satsuma mandarin) at the level of 0.01-10.0 μg/g were 72.8-104% and the limits of quantification were 0.01 μg/g. The recoveries of DP spiked in domestic citrus fruits at the level of 0.01-70.0 μg/g were 70.8-80.4% and the limit of quantification was 0.01 μg/g. The proposed method was applied to the determination of fungicides in citrus fruits purchased in various markets.

Oral imazalil exposure induces gut microbiota dysbiosis and colonic inflammation in mice

The fungicide imazalil (IMZ) is used extensively in vegetable and fruit plantations and as a post-harvest treatment to avoid rot. Here, we revealed that ingestion of 25, 50 and 100 mg IMZ kg−1 body weight for 28 d induced gut microbiota dysbiosis and colonic inflammation in mice. The relative abundance of Bacteroidetes, Firmicutes and Actinobacteria in the cecal contents decreased significantly after exposure to 100 mg kg−1 IMZ for 28 d. In feces, the relative abundance in Bacteroidetes, Firmicutes and Actinobacteria decreased significantly after being exposed to 100 mg kg−1 IMZ for 1, 14 and 7 d, respectively. High throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene revealed a significant reduction in the richness and diversity of microbiota in cecal contents and feces of IMZ-treated mice. Operational taxonomic units (OTUs) analysis identified 49.3% of OTUs changed in cecal contents, while 55.6% of OTUs changed in the feces after IMZ exposure. Overall, at the phylum level, the relative abundance of Firmicutes, Proteobacteria and Actinobacteria increased and that of Bacteroidetes decreased in IMZ-treated groups. At the genus level, the abundance of Lactobacillus and Bifidobacterium decreased while those of Deltaproteobacteria and Desulfovibrio increased in response to IMZ exposure. In addition, it was observed that IMZ exposure could induce colonic inflammation characterized by infiltration of inflammatory cells, elevated levels of lipocalin-2 (lcn-2) in the feces, and increased mRNA levels of Tnf-α, IL-1β, IL-22 and IFN-γ in the colon. Our findings strongly suggest that ingestion of IMZ has some risks to human health.

The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application

Imazalil (IMZ) is the most relied upon fungicide for use against citrus green mould, caused by Penicillium digitatum. In South Africa, the IMZ sulphate formulation is used as dip application in packhouses. Previous studies showed that IMZ efficacy and residue loading of this formulation is highly affected by pH in the application solution. This study investigated the effect of pH, exposure time and temperature on residue loading, green mould control and sporulation inhibition when citrus fruit were dipped in IMZ sulphate. Clementine mandarins, lemons and navel oranges were dipped in a 500mgL−1 solution of IMZ sulphate for 15, 45 or 90s, at a temperature of 23, 35 or 45°C, and at pH levels of 3 or 6. At pH 3, similar residues were loaded on fruit regardless of fruit type, temperature or exposure time (1.18–1.51mgkg−1). At pH 6, both temperature and exposure time influenced residue loading: at 23°C, higher residue levels were observed than at pH 3 (2.03–2.73mgkg−1, increasing with longer exposure time); at 35°C and 45°C, residue levels increased significantly with 15s, 30s and 45s exposure time (3.34, 5.11, 7.24mgkg−1 and 5.31, 9.65 and 13.10mgkg−1, respectively). Clementine mandarin fruit loaded higher residues at pH 6 (6.96mgkg−1) than lemons and navels (5.20 and 4.82mgkg−1, respectively). The MRL was frequently exceeded at longer exposure times. Green mould control was lower at pH 3 and was influenced by temperature and exposure time (92.9–97.4% and 42.8–45.1% for lemons and Clementine mandarins, respectively). Better control was observed at pH 6 regardless of temperature and exposure time (96.9–98.7% and 39.3–57.9% for lemons and Clementine mandarins, respectively). On navel oranges, increased exposure time led to increased green mould control (84.7–92.5%). Sporulation incidence was lower at pH 6 (12.5–28.8%, 19.1–39.2% and 4.6% for lemons, Clementine mandarins and navel oranges, respectively) than at pH 3 treatments (72.7–89.9%, 35.8–91.4% and 77.2% for lemons, Clementine mandarins and navel oranges, respectively). The results show that temperature, pH and exposure time are important parameters in dip application using IMZ sulphate formulation with significant effects on green mould control, more so than residue loading alone.

The fungicide imazalil induces developmental abnormalities and alters locomotor activity during early developmental stages in zebrafish

The fungicide imazalil (IMZ) is used extensively to protect vegetable fields, fruit plantations and post-harvest crops from rot. Likely due to its wide-spread use, IMZ is frequently detected in vegetable, fruit, soil and even surface water samples. Even though several previous studies have reported on the neurotoxicity of IMZ, its effects on the neurobehavior of zebrafish have received little attention to date. In this study, we show that the heartbeat and hatchability of zebrafish were significantly influenced by IMZ concentrations of 300 μg L−1 or higher. Moreover, in zebrafish larvae, locomotor behaviors such as average swimming speed and swimming distance were significantly decreased after exposure to 300 μg L−1 IMZ for 96 h, and acetylcholinesterase (AChE) expression and activity were consistently inhibited in IMZ-treated fish. Our results further suggest that IMZ could act as a neuroendocrine disruptor by decreasing the expression of neurotoxicity-related genes such as Glial fibrillary acidic protein (Gfap), Myelin basic protein (Mbp) and Sonic hedgehog a (Shha) during early developmental stages of zebrafish. In conclusion, we show that exposure to IMZ has the potential to induce developmental toxicity and locomotor behavior abnormalities during zebrafish development.

A crucial role of constitutive androstane receptor (CAR) in liver tumor development by imazalil in mice.

To clarify the major pathway of liver tumor development induced by imazalil (IMA), an imidazole fungicide, male constitutive androstane receptor (CAR)-knockout (CARKO) and wild-type (WT) mice were treated with IMA at 500 ppm in the diet up to 27 weeks after initiation by diethylnitrosamine. After 27 weeks of treatment, neither altered foci nor adenomas were significantly increased in CARKO mice, whereas both eosinophilic altered foci and adenomas were increased in WT mice. After 4 or 13 weeks of IMA treatment, liver hypertrophy was observed at the tumor-inducible dose without differences among genotypes or durations. Analysis of hepatic drug metabolite enzymes, performed after administration of multiple doses during a 1-week period, indicated that pregnane X receptor might be involved in liver hypertrophy because IMA markedly elevated Cyp3a11 and Cyp2b10 expression levels in a dose-dependent manner in both genotypes. Our results demonstrated that the CAR pathway was the main mechanism of liver tumor development induced by IMA. The carcinogenic pathway was different from that of liver hypertrophy.

Evaluation of sodium benzoate and other food additives for the control of citrus postharvest green and blue molds

The curative activity of the food additives dehydroacetic acid, dimethyl dicarbonate, ethylene diamine tetracetic acid, sodium acetate, and sodium benzoate (SB) was tested in in vivo preliminary screenings against green and blue molds on citrus fruit artificially inoculated 24h before with Penicillium digitatum and Penicillium italicum, respectively. SB was the most effective compound and it was further tested in trials simulating postharvest industrial applications. Dip treatments for 60s with 3% (w/v) SB heated above 50°C resulted in about 90% reduction of green and blue mold incidence on ‘Valencia’ oranges inoculated, treated, and incubated at 20°C and 90% RH for 7 days. This treatment was also effective on ‘Lanelate’ oranges, ‘Fino’ lemons and ‘Ortanique’ mandarins, but not on ‘Clemenules’ mandarins. Heated solutions combining SB with low doses (25 or 50μLL−1) of the fungicide imazalil (IMZ) were synergistic and greatly improved the efficacy of stand-alone treatments. On ‘Valencia’ oranges stored for 8 weeks at 5°C followed by 7 days of shelf-life at 20°C, this combination reduced the incidence of green and blue molds almost by 100%. It was found in additional trials to test the preventive activity that 3% SB dips at 50°C for 60s did not reduce green mold on ‘Valencia’ oranges treated, inoculated with P. digitatum 24h later, and incubated at 20°C for 7 days. It can be concluded from this work that heated SB aqueous solutions might be in the future an interesting nonpolluting disease control alternative for the commercialization of citrus in markets with zero tolerance to fungicide residues.

The potential of organic substrates based on mushroom substrate and straw to dissipate fungicides contained in effluents from the fruit-packaging industry – Is there a role for Pleurotus ostreatus?

Citrus fruit-packaging plants (FPP) produce large wastewater volumes with high loads of fungicides like ortho-phenylphenol (OPP) and imazalil (IMZ). No methods are in place for the treatment of those effluents and biobeds appear as a viable alternative. We employed a column study to investigate the potential of spent mushroom substrate (SMS) of Pleurotus ostreatus, either alone or in mixture with straw and soil plus a mixture of straw /soil to retain and dissipate IMZ and OPP. The role of P. ostreatus on fungicides dissipation was also investigated by studying in parallel the performance of fresh mushroom substrate of P. ostreatus (FMS) and measuring lignolytic enzymatic activity in the leachates. All substrates effectively reduced the leaching of OPP and IMZ which corresponded to 0.014–1.1% and 0.120–0.420% of their initial amounts respectively. Mass balance analysis revealed that FMS and SMS/Straw/Soil (50/25/25 by vol) offered the most efficient removal of OPP and IMZ from wastewaters respectively. Regardless of the substrate, OPP was restricted in the top 0–20cm of the columns and was bioavailable (extractable with water), compared to IMZ which was less bioavailable (extractable with acetonitrile) but diffused at deeper layers (20–50, 50–80cm) in the SMS- and Straw/Soil-columns. PLFAs showed that fungal abundance was significantly lower in the top layer of all substrates from where the highest pesticide amounts were recovered suggesting an inhibitory effect of fungicides on total fungi in the substrates tested. Our data suggest that biobeds packed with SMS-rich substrates could ensure the efficient removal of IMZ and OPP from wastewaters of citrus FPP.

Land Spreading of Wastewaters from the Fruit-Packaging Industry and Potential Effects on Soil Microbes: Effects of the Antioxidant Ethoxyquin and Its Metabolites on Ammonia Oxidizers.

Thiabendazole (TBZ), imazalil (IMZ), ortho-phenylphenol (OPP), diphenylamine (DPA), and ethoxyquin (EQ) are used in fruit-packaging plants (FPP) with the stipulation that wastewaters produced by their application would be depurated on site. However, no such treatment systems are currently in place, leading FPP to dispose of their effluents in agricultural land. We investigated the dissipation of those pesticides and their impact on soil microbes known to have a key role on ecosystem functioning. OPP and DPA showed limited persistence (50% dissipation time [DT50], 0.6 and 1.3 days) compared to TBZ and IMZ (DT50, 47.0 and 150.8 days). EQ was rapidly transformed to the short-lived quinone imine (QI) (major metabolite) and the more persistent 2,4-dimethyl-6-ethoxyquinoline (EQNL) (minor metabolite). EQ and OPP exerted significant inhibition of potential nitrification, with the effect of the former being more persistent. This was not reflected in the abundance (determined by quantitative PCR [qPCR]) of the amoA gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Considering the above discrepancy and the metabolic pattern of EQ, we further investigated the hypothesis that its metabolites and not only EQ were toxic to ammonia oxidizers. Potential nitrification, amoA gene abundance, and amoA gene transcripts of AOB and AOA showed that QI was probably responsible for the inhibition of nitrification. Our findings have serious ecological and practical implications for soil productivity and N conservation in agriculturally impacted ecosystems and stress the need to include metabolites and RNA-based methods when the soil microbial toxicity of pesticides is assessed.

LC-MS-MS Analysis of Urinary Biomarkers of Imazalil Following Experimental Exposures.

Imazalil (IMZ) is a fungicide used in the cultivation of vegetables, such as cucumbers, in green houses or post-harvest on fruit to avoid spoilage due to fungal growth. Agricultural workers can be occupationally exposed to IMZ and the general public indirectly by the diet. The purpose of this study was to develop and validate an LC–MS-MS method for the analysis of IMZ in human urine. The method used electrospray ionization and selected reaction monitoring in the positive mode. Excellent linearity was observed in the range 0.5–100 ng/mL. The limit of detection of the method was 0.2 ng/mL, and the limit of quantitation 0.8 ng/mL. The method showed good within-run, between-run and between-batch precision, with a coefficient of variation <15%. The method was applied to analyze urine samples obtained from two human volunteers following experimental oral and dermal exposure. The excretion of IMZ seemed to follow a two-compartment model and first-order kinetics. In the oral exposure, the elimination half-life of IMZ in the rapid excretion phase was 2.6 and 1.9 h for the female and the male volunteer, respectively. In the slower excretion phase, it was 7.6 and 13 h, respectively. In the dermal exposure, the excretion seemed to follow a single-compartment model and first-order kinetics. The elimination half-life was 10 and 6.6 h for the female and the male volunteer, respectively. Although the study is limited to two volunteers, some information on basic toxicokinetics and metabolism of IMZ in humans is presented.

Dissipation, metabolism and sorption of pesticides used in fruit-packaging plants: Towards an optimized depuration of their pesticide-contaminated agro-industrial effluents

Wastewaters from the fruit-packaging industry constitute a serious point source contamination with pesticides. In the absence of effective depuration methods, they are discharged in municipal wastewater treatment plants or spread to land. Modified biobeds could be an applicable solution for their treatment. We studied the dissipation of thiabendazole (TBZ), imazalil (IMZ), ortho-phenylphenol (OPP), diphenylamine (DPA) and ethoxyquin (EQ), used by the fruit-packaging industry, in anaerobically digested sewage sludge, liquid aerobic sewage sludge and in various organic substrates (biobeds packing materials) composed of soil, straw and spend mushroom substrate (SMS) in various volumetric ratios. Pesticide sorption was also determined. TBZ and IMZ showed higher persistence especially in the anaerobically digested sewage sludge (DT50=32.3–257.6d), in contrast to OPP and DPA which were rapidly dissipated especially in liquid aerobic sewage sludge (DT50=1.3–9.3d). EQ was rapidly oxidized mainly to quinone imine (QI) which did not persist and dimethyl ethoxyquinoline (EQNL, minor metabolite) which persisted for longer. Sterilization of liquid aerobic sewage sludge inhibited pesticide decay verifying the microbial nature of pesticide dissipation. Organic substrates rich in SMS showed the highest dissipation capacity with TBZ and IMZ DT50s of ca. 28d compared to DT50s of >50d in the other substrates. TBZ and IMZ showed the highest sorption affinity, whereas OPP and DPA were weakly sorbed. Our findings suggest that current disposal practices could not guarantee an efficient depuration of effluents from the fruit-packaging industry, whereas SMS-rich biobed organic substrates show efficient depuration of effluents from the fruit-packaging industry via accelerated dissipation even of recalcitrant fungicides.

Imazalil resistance in Penicillium digitatum and P. italicum causing citrus postharvest green and blue mould: Impact and options

Citrus green and blue mould, caused by Penicillium digitatum (PD) and Penicillium italicum (PI), respectively, are mostly controlled by means of postharvest fungicide applications. Currently, IMZ is regarded as the most effective fungicide in use. Effective IMZ concentrations that inhibit 50% (EC50) growth of nine PD and five PI isolates were assessed in vitro and the various isolates categorized according to their resistance (R) factors. Effective residue levels that provided 50% curative (ER50C) and protective (ER50P) control of these isolates were determined in vivo. All the PI isolates were sensitive, having EC50 values of 0.005–0.050μgmL−1. Three PD isolates were sensitive (0.027–0.038μgmL−1), while one resistant isolate was categorized as low resistant (R-factor of 19), one as moderately resistant (R-factor of 33.2), three as resistant (R-factor of 50–57.6) and one as highly resistant (R-factor of 70.7). Sensitive PD isolates had mean ER50C and ER50P values on Valencia orange fruit of 0.29 and 0.20μgg−1, and 0.33 and 0.32μgg−1 on navel fruit, respectively. ER50 values for resistant isolates did not always correlate with EC50 values and ranged from 1.22 to 4.56μgg−1 for ER50C and 1.00–6.62μgg−1 for ER50P values. ER50P values for resistant isolates could not be obtained on navel orange fruit, but ER50C values (1.42–1.65μgg−1) were similar to those obtained on Valencia fruit. The PI isolates all behaved similar to the sensitive PD isolates with ER50C and ER50P values on navel and Valencia fruit <0.38μgg−1. Alternative fungicides were assessed for the control of an IMZ sensitive, resistant and highly resistant PD isolate; these included sodium ortho-phenylpenate (SOPP), thiabendazole (TBZ), guazatine (GZT), imazalil (IMZ), pyrimethanil (PYR) and Philabuster® (PLB; a combination of IMZ and PYR), fludioxonil (FLU), azoxystrobin (AZO), Graduate®A+ (GRA; a combination of FLU and AZO) and propiconazole (PPZ). Multiple fungicide resistance was shown to IMZ, GZT, TBZ and PPZ in both resistant isolates. For the sensitive isolates, IMZ, SOPP, TBZ, GZT and PLB provided best curative control, while IMZ, GZT and PLB provided best protective control. For the IMZ-resistant isolates, SOPP, PYR and PLB gave the best curative control, while none of the fungicides provided adequate protective control.

Evaluation of the enantioselective binding of imazalil to human serum albumin by capillary electrophoresis

In this work, a methodology for the evaluation of enantioselective binding of imazalil (IMA) enantiomers to human serum albumin (HSA) that does not require the separation of free and bound to HSA fractions is developed. This methodology comprises the incubation of IMA-HSA designed mixtures for 30 min directly in the capillary electrophoresis system and the subsequent direct injection and chiral separation of IMA employing highly sulfated β-cyclodextrin as chiral selector and the complete filling technique. Two mathematical approaches were used to estimate apparent affinity constants (K1), protein binding and enantioselectivity (ES) for both enantiomers of IMA. Moderate enantioselective binding of IMA enantiomers to HSA (ES = 2.0) was shown by the 1:1 stoichiometry and log K1 values of 3.4 ± 0.4 and 3.1 ± 0.3 for the first and second eluted enantiomers, respectively.