Imazalil resulted in glucolipid metabolism disturbance and abnormal m6A RNA methylation in the liver of dam and offspring mice.

As a fungicide with the characteristics of high effectiveness, internal absorption and broad spectrum, imazalil is widely used to prevent and treat in fruits and vegetables. Here, pregnant C57BL/6 mice were exposed to imazalil at dietary levels of 0, 0.025‰, and 0.25‰ through drinking water during pregnancy and lactation. We then analyzed the phenotype, metabolome, and expression of related genes and proteins in the livers of mice. There was a marked decrease in the body and liver weights of male offspring mice after maternal imazalil exposure, while this effect on the dam and female offspring was slight. Metabolomics analyses revealed that imazalil significantly altered the metabolite composition of liver samples from both dams and offspring. The preliminary results of the analysis indicated that glucolipid metabolism was the pathway most significantly affected by imazalil. We performed a coabundance association analysis of metabolites with significant changes in the pathway of glycolipid metabolism, and IMZ altered the networks of both dams and offspring compared with the network in control mice, especially in male offspring. The hepatic triglyceride, non-esterified fatty acid and glucose levels were increased significantly in the dams but decreased significantly in male offspring after maternal imazalil exposure. Furthermore, the expression levels of genes associated with glycolipid metabolism and m6A RNA methylation were significantly affected by maternal intake of imazalil. Imazalil-induced glucolipid metabolism disturbance was highly correlated with m6A RNA methylation. In conclusion, maternal imazalil exposure resulted in glucolipid metabolism disturbance and abnormal m6A RNA methylation in the livers of dams and offspring mice. We expected that the information acquired in this study will provide novel evidence for understanding the effect of maternal imazalil exposure on potential health risks.

Investigations with Drugs and Pesticides Revealed New Species- and Substrate-Dependent Inhibition by Elacridar and Imazalil in Human and Mouse Organic Cation Transporter OCT2.

Multiple drugs are used to treat various indications as well as pesticides that are ingested unintentionally and enter the bloodstream. The residence time or bioavailability of these substances in circulation depends on several mechanisms, such as drug−drug interaction (DDI), drug−pesticide interaction, metabolizing enzymes and the hepatic and renal transport systems, involved in the elimination of the compounds from the body. One of these transporters is the Organic Cation Transporter 2 (OCT2) member of the solute carrier (SLC22) transporter family. OCT2 is highly expressed in the proximal tubule epithelial cells in human and mouse kidney, where it mediates the uptake of endogenous organic cations as well as numerous drugs and xenobiotics, and contributes to the first step of renal clearance. In this study, we examined OCT2 on two subjects: First, the transferability of data from mouse to human, since mice are initially examined in the development of new drugs to assess the renal excretion of organic cations. Second, to what extent the choice of substrate affects the properties of an inhibitor. For this purpose, the functional properties of hOCT2 and mOct2 were validated under the same experimental conditions with the known substrates metformin and 1-Methyl-4-phenylpyridinium iodide (MPP). While hOCT2 and mOct2 showed very low affinities for metformin with Km values of 3.9 mM and 3.5 mM, the affinity of hOCT2 and mOct2 for MPP (62 and 40 µM) was 64- and 89-fold higher, respectively. For our positive control inhibitor decynium22, we determined the following IC50 values for hOCT2 and mOct2: 2.2 and 2.6 µM for metformin uptake, and 16 and 6.9 µM for MPP uptake. A correlation analysis of the inhibitory effects of 13 drugs and 9 pesticides on hOCT2- and mOct2-mediated transport of metformin showed a correlation coefficient R2 of 0.88, indicating good interspecies correlation. Nevertheless, the bioenhancer elacridar and the fungicide imazalil showed species-dependent inhibitory potentials. Concentration-dependent inhibition of hOCT2- and mOct2-mediated metformin uptake by elacridar showed IC50 values of 20 µM and 1.9 µM and by imazalil 4.7 µM and 0.58 µM, respectively. In conclusion, although our data show comparable species-independent interactions for most compounds, there can be large species−specific differences in the interactions of individual compounds, which should be considered when extrapolating data from mice to humans. Furthermore, a comparison of the inhibitory potential of elacridar and imazalil on metformin uptake with that on MPP uptake reveals substrate-dependent differences in hOCT2 and mOct2 for both inhibitors. Therefore, it might be useful to test two different substrates in inhibition studies.

Molecular Physicochemical Properties of Selected Pesticides as Predictive Factors for Oxidative Stress and Apoptosis-Dependent Cell Death in Caco-2 and HepG2 Cells.

In this work, three pesticides of different physicochemical properties: glyphosate (GLY, herbicide), imidacloprid (IMD, insecticide), and imazalil (IMZ, fungicide), were selected to assess their cytotoxicity against Caco-2 and HepG2 cells. Cell viability was assessed by the Alamar Blue assay, after 24 and 48 h exposure to different concentrations, and IC50 values were calculated. The mechanisms underlying toxicity, namely cellular reactive oxygen species (ROS), glutathione (GSH) content, lipid peroxidation, loss of mitochondrial membrane potential (MMP), and apoptosis/necrosis induction were assessed by flow cytometry. Cytotoxic profiles were further correlated with the molecular physicochemical parameters of pesticides, namely: water solubility, partition coefficient in an n-octanol/water (Log Pow) system, topological polar surface area (TPSA), the number of hydrogen-bonds (donor/acceptor), and rotatable bonds. In vitro outputs resulted in the following toxicity level: IMZ (Caco-2: IC50 = 253.5 ± 3.37 µM, and HepG2: IC50 = 94 ± 12 µM) > IMD (Caco-2: IC50 > 1 mM and HepG2: IC50 = 624 ± 24 µM) > GLY (IC50 >>1 mM, both cell lines), after 24 h treatment, being toxicity time-dependent (lower IC50 values at 48 h). Toxicity is explained by oxidative stress, as IMZ induced a higher intracellular ROS increase and lipid peroxidation, followed by IMD, while GLY did not change these markers. However, the three pesticides induced loss of MMP in HepG2 cells while in Caco-2 cells only IMZ produced significant MMP loss. Increased ROS and loss of MMP promoted apoptosis in Caco-2 cells subjected to IMZ, and in HepG2 cells exposed to IMD and IMZ, as assessed by Annexin-V/PI. The toxicity profile of pesticides is directly correlated with their Log Pow, as affinity for the lipophilic environment favours interaction with cell membranes governs, and is inversely correlated with their TPSA; however, membrane permeation is favoured by lower TPSA. IMZ presents the best molecular properties for membrane interaction and cell permeation, i.e., higher Log Pow, lower TPSA and lower hydrogen-bond (H-bond) donor/acceptor correlating with its higher toxicity. In conclusion, molecular physicochemical factors such as Log Pow, TPSA, and H-bond are likely to be directly correlated with pesticide-induced toxicity, thus they are key factors to potentially predict the toxicity of other compounds.

Mixing postharvest fungicides and sanitizers results in unpredictable survival of microbes that affect cantaloupes.

Postharvest treatments with sanitizers and fungicides are applied to increase the quality, safety and shelf life of fresh produce including cantaloupes (also known as rockmelons). The primary role of sanitizers during cantaloupe washing is to prevent cross contamination of potentially pathogenic bacteria in washwater. Postharvest fungicide sprays or dips are employed to inhibit spoilage-causing fungi. While assessing the compatibility of these antimicrobials based on the measurement of active ingredients levels provides some indication of antimicrobial capacity, there is limited data on whether the interaction between these chemicals in wash water modifies their overall efficacy against relevant microorganisms. The aim of this research was to determine how chlorine- and peroxyacetic acid-based sanitizers interact with commercial guazatine- and imazalil-based fungicide formulations used on cantaloupes, and whether mixing these augments or suppresses anti-microbial activity against relevant human pathogens and spoilage fungi in wash water. The results were unpredictable: while most combinations were antimicrobial, the chlorine-based sanitizer when mixed with the guazatine-based fungicide had significantly reduced efficacy against pathogenic Salmonella spp. (~2.7 log) and the fungal spoilage organisms, Trichothecium roseum and Rhizopus stolonifera. Mixing the chlorine-based sanitizer with an imazalil-based fungicide produced a range of outcomes with antagonistic, indifferent and synergistic interactions observed for the fungal species tested. The peroxyacetic acid-based sanitizer led to indifferent interactions with the guazatine-based fungicide, while antagonism and synergy were observed when mixed with the imazalil-based fungicide. This study demonstrates that mixing postharvest agrichemicals used in the cantaloupe industry may increase the risk of microbial contamination and thereby potentially compromise food safety and quality.

Monitoring the behavior of imazalil and its metabolite in grapes, apples, and the processing of fruit wine at enantiomeric level.

BACKGROUND: Imazalil is widely used in agriculture, which may pose a threat to food safety. This study aimed to investigate the fate of imazalil and its main metabolite, R14821 (imazalil-M), in field grapes and apples, and in the processing of fruit wine at the enantiomeric level. RESULTS: Analysis method was established to determine imazalil and imazalil-M enantiomers in grape, apple, fruit wine and pomace. The method showed acceptable recoveries of 71.6-99.9% and precision with relative standard deviation of 0.3-11.7%. Processing factors (PFs) were 0.15-0.40 (for imazalil enantiomers) and <0.13-0.83 (for imazalil-M enantiomers) during the wine-making process. The PFs after individual steps including washing, peeling, fermentation, and clarification were all less than 1. No enantioselective dissipation of imazalil was found in grapes under field conditions with half-lives of 23.82-24.49 days. R-(-)-imazalil degraded slightly faster than S-(+)-imazalil in apples under field conditions with half-lives of 9.82-10.09 days. S-(+)-imazalil-M preferentially degraded in field grapes and apple. No significant enantioselectivity of imazalil and imazalil-M was observed during the wine-making process. The enantiomeric fraction (EF) values of imazalil were 0.484-0.511 and 0.509-0.522 in grape wine and cider, respectively. The EFs were 0.484-0.501(in grape wine) and 0.484-0.504 (in cider) for imazalil-M. CONCLUSION: The results showed that the wine-making process could reduce imazalil and imazalil-M residues in grapes and apples. The finding of non-enantioselectivity of imazalil during the processing of fruit wine was useful for accurate risk assessment for imazalil in raw and processing fruits. © 2021 Society of Chemical Industry.

Polyhexamethylene guanidine as a fungicide, disinfectant and wound protector in lemons challenged with Penicillium digitatum.

Citrus green mold, a postharvest disease caused by Penicillium digitatum, provokes important economic losses on lemon production. Here, the effectiveness of polyhexamethylene guanidine (PHMG) to inhibit P. digitatum growth and to control green mold on artificially infected lemons was evaluated. At sublethal concentrations, PHMG inhibited conidia germination and infectivity (5 mg L-1), and mycelial growth (50 mg L-1). Viability of conidia was completely suppressed by treatment with 500 mg L-1 PHMG. In this condition, membrane integrity loss, cell wall disruption and ultrastructural alterations were detected, as well as conidia distortion, deformation and collapse. In artificially inoculated lemons, a 30 s-immersion in 500 mg L-1 PHMG completely inhibited green mold. PHMG also exhibited a high disinfectant activity, even in the presence of 1% organic matter, with a better performance than the standard NaClO disinfectant. In addition, 500 mg L-1 PHMG protected wounds against infection. Taken together, our results indicate that PHMG is a promising fungicide for the postharvest control of green mold in lemon packinghouses.

Chronic exposure of mice to low doses of imazalil induces hepatotoxicity at the physiological, biochemical, and transcriptomic levels.

Imazalil (IMZ), which is a widely used fungicide, can accumulate in the body and threaten an animal's health. However, this fungicide has adverse effects on aquatic organisms and ultimately affects human health when it leaches into the environment. Our research tried to determine that if IMZ might cause liver damage and its potential to cause-related diseases. In this study, male adult C57BL/6 mice were exposed to 0.1, 0.5, or 2.5 mg/kg body weight IMZ in drinking water for 15 weeks. Then, we evaluated the liver damage at the physiological, biochemical, and transcriptome levels in mice after chronic IMZ exposure. We observed serious ballooning degeneration of hepatocytes in the IMZ-treated groups. And IMZ induced oxidative stress and caused the disorders of bile acid metabolism in mice. In addition, the transcriptome data showed that IMZ has substantial influence on several pathways, including metabolic pathways for drug metabolism, RNA transport, and bile secretion. We further confirmed that the mRNA expression of the key genes involved in oxidative stress and bile acid metabolism were changed of mice exposed to IMZ. Our data suggested that chronic IMZ exposure could induce hepatotoxicity in mice at the physiological, biochemical, and transcriptome levels.

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.

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 ß-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.

Role of aqueous Bryoria capillaris (Ach.) extract as a genoprotective agent on imazalil-induced genotoxicity in vitro.

In recent years, a number of studies have suggested that lichens might be the easily accessible sources of natural drugs that could be used as a possible food supplement. Extensive research is being carried out to explore the importance of lichen species, which are known to contain a variety of pharmacological active compounds. On the other hand, imazalil (IMA), a commonly used fungicide in both agricultural and clinical domains, is suspected to produce very serious toxic effects in vertebrates. In this context, the antigenotoxic effect of aqueous Bryoria capillaris (Ach.) extract (BCE) was studied against the genotoxic damage induced by IMA on cultured human lymphocytes using chromosomal aberrations (CA) and micronucleus (MN) as cytogenetic parameters. Human peripheral lymphocytes were treated in vitro with varying concentrations of BCE (5, 10, 25, 50 and 100 µg/mL), tested in combination with IMA (336 µg/mL). BCE alone was not genotoxic, and when combined with IMA treatment, it reduced the frequency of CAs and the rates of MN. A clear dose-dependent decrease in the genotoxic damage of IMA was observed, suggesting a genoprotective role of BCE. The results of the present study suggest that this plant extract per se do not have genotoxic potential, but can modulate the genotoxicity of IMA on peripheral human lymphocytes in vitro. In conclusion, our findings may have an important application in the protection of cultured human lymphocyte from the genetic damage and side effects induced by agricultural and medical chemicals that are hazardous to people.

Effects of maternal exposure to imazalil on behavioral development in F1-generation mice.

Female mice were exposed maternally to imazalil through diet to provide levels of 0 (control), 0.0006, 0.0018, and 0.0054% during gestation and lactation periods, and selected reproductive and neurobehavioral parameters were measured in F1 generation. There was no adverse effect of imazalil on litter size, litter weight, or sex ratio at birth. With respect to behavioral developmental parameters, surface righting on postnatal day 4 of male offspring was delayed significantly in a dose-related manner (p < 0.05). Regarding exploratory behavior in the F1 generation, movement time was significantly long (p = 0.0206) in the low-dose group of males at 8 weeks of age. Spontaneous behavior examination in males indicated that movement time increased but in females decreased in the low-dose groups in the F1 generation. The dose levels of imazalil in the present study produced some adverse effects in neurobehavioral parameters in mice.

Anti-genotoxic role of eicosapentaenoic acid against imazalil-induced DNA damage in vitro.

Eicosapentaenoic acid (EPA) is a polyunsaturated n-3 fatty acid and is essential to the health of mammals. Recent data show that EPA can act as anti-mutagenic agent. On the other hand, pesticides comprise a new and important class of environmental pollutants nowadays. Imazalil (IMA), a commonly used fungicide in both agricultural and clinical domains is suspected to produce very serious toxic effects in vertebrates. The present study investigated the anti-genotoxic effect of EPA against the genotoxic damage induced by IMA on cultured human lymphocytes using chromosomal aberration (CA) and micronucleus (MN) tests as cytogenetic endpoints. Peripheral blood cells were treated in vitro with varying concentrations of EPA (2.5, 5, 10, 20 and 40 µg/ml), tested in combination with IMA (336 µg/ml). Our results revealed that the rates of CAs and MNs in lymphocytes were significantly (p < 0.05) increased by IMA as compared to the controls. The results also showed that EPA alone was not genotoxic. Moreover, when combined with IMA treatment, EPA reduced the frequencies of CAs and MNs. A clear dose-dependent decrease in the genotoxic damage of IMA was observed, suggesting a genoprotective role of EPA. In conclusion, our data may have an important application for the protection of cultured human lymphocyte from the genetic damage and repercussions induced by agricultural and industrial chemicals hazardous in people.

Stereoselective behavior and residues of the imazalil during strawberry growth and strawberry wine production.

Imazalil is a chiral fungicide widely used to protect strawberries against gray mold, which may pose threats to food safety. This study aims to investigate the stereoselective behavior of imazalil during strawberry growth and strawberry wine production. A method was proposed and validated for the extraction and quantitative analysis of imazalil residues in strawberry, strawberry pomace, and strawberry wine by using ultra-high performance liquid chromatography-tandem mass spectrometry. The method exhibited mean recoveries ranging from 86.2% to 119.7% with relative standard deviations of 0.1-11.3%. The dissipation curve of imazalil during strawberry growth followed the first-order kinetic model with a half-life ranging from 6.5 to 7.1 days. Significant enantioselectivity of imazalil was observed in strawberry grown under field conditions and strawberry wine production process, with enantiomeric fraction values ranging from 0.51 (2 h) to 0.42 (27d) and from 0.48 (0d) to 0.52 (10d), respectively. (+)-imazalil was preferentially degraded in strawberry under field conditions, while (-)-imazalil was preferentially degraded during the fermentation process. The processing factor was lower than 1 for each procedure, indicating that the wine-making process can reduce imazalil residue in strawberry. These findings may facilitate a more accurate risk assessment of imazalil and provide important guidance for the safe and efficacious use of imazalil in agriculture.

Modification of the existing maximum residue levels for imazalil in courgettes, cucumbers and gherkins.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Certis Europe B.V. submitted a request to the competent national authority in the Netherlands to modify the existing maximum residue levels (MRLs) for the active substance imazalil in cucumbers, courgettes and gherkins. The data submitted in support of the request were found to be sufficient to derive an MRL proposal of 0.08 mg/kg for the whole group of cucurbits with edible peel. It is noted that the derived MRL is proposed to replace the existing tentative MRL of 0.1 mg/kg for courgettes, thus also addressing the data gap identified in the context of the MRL review. As regards to cucumbers and gherkins, it is noted that the MRL proposal derived in the current application is covered by the MRL of 0.5 mg/kg currently in place for these commodities. Nevertheless, it is also noted that the aforementioned MRLs are based on Codex maximum residue limits (CXLs) which were revoked in 2019 following the JMPR proposal. Should this proposal be implemented in the EU regulation in the future, the proposed MRL of 0.08 mg/kg as derived in the current application would be considered an appropriate fall-back option for the whole group of cucurbits with edible peel. Further risk management considerations are therefore required. Adequate analytical methods for enforcement are available to control the residues of imazalil on the commodities under consideration at the validated limit of quantification (LOQ) of 0.005 mg/kg (QuEChERS method based on LC-MS/MS) and 0.01 mg/kg (HPLC-MS/MS method). Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the use of imazalil according to the reported agricultural practices is unlikely to present a risk to consumer health. However, it should be noted that a high degree of uncertainty on the overall long-term exposure calculation remains due to the data gaps identified in the context of the MRL review on full toxicological assessment of the metabolite R014821, which is expected to occur following post-harvest uses of imazalil, and animal metabolites FK-772 and FK-284.

Earthworms synergize with indigenous soil functional microorganisms to accelerate the preferential degradation of the highly toxic S-enantiomer of the fungicide imazalil in soil.

The roles of soil and earthworm gut microorganisms in the degradation of the chiral fungicide imazalil (IMA) enantiomers were systemically studied in soil-earthworm systems. S-IMA degraded slower than R-IMA in soil without earthworms. After the addition of earthworms, S-IMA degraded faster than R-IMA. Methylibium was the potential degradative bacterium likely related to the preferential degradation of R-IMA in soil. However, the addition of earthworms significantly decreased the relative abundance of Methylibium, especially in R-IMA-treated soil. Meanwhile, a new potential degradative bacterium Aeromonas first appeared in soil-earthworm systems. Compared with enantiomer-treated soil, the relative abundance of indigenous soil bacterium Kaistobacter significantly boomed in enantiomer-treated soil with earthworms. Interestingly, Kaistobacter in the earthworm gut also obviously increased after exposure to enantiomers, particularly in S-IMA-treated soil, which was associated with the significant increase in Kaistobacter in soil. More importantly, the relative abundances of Aeromonas and Kaistobacter in S-IMA-treated soil were obviously higher than those in R-IMA-treated soil after the addition of earthworms. Moreover, these two potential degradative bacteria were also potential bacterial hosts of the biodegradation genes p450 and bph. Collectively, gut microorganisms are important helpers in soil pollution remediation by participating in the preferential degradation of S-IMA mediated by indigenous soil microorganisms.

Deciphering the degradation characteristics of the fungicides imazalil and penflufen and their effects on soil bacterial community composition, assembly, and functional profiles.

The adsorption-desorption and degradation characteristics of two widely applied fungicides, imazalil and penflufen, and the responses of soil bacterial diversity, structure, function, and interaction after long-term exposure were systemically studied in eight different soils. The adsorption ability of imazalil in soil was significantly higher than that of penflufen. Both imazalil and penflufen degraded slowly in most soils following the order: imazalil > penflufen, with soil pH, silt, and clay content being the potential major influencing factors. Both imazalil and penflufen obviously inhibited the soil microbial functional diversity, altered the soil bacterial community and decreased its diversity. Although exposure to low and high concentrations of imazalil and penflufen strengthened the interactions among the soil bacterial communities, the functional diversity of the co-occurrence network tended to be simple at high concentrations, especially in penflufen treatment. Both imazalil and penflufen markedly disturbed soil nitrogen cycling, especially penflufen seriously inhibited most nitrogen cycling processes, such as nitrogen fixation and nitrification. Meanwhile, sixteen and ten potential degradative bacteria of imazalil and penflufen, respectively, were found in soils, including Kaistobacter and Lysobacter. Collectively, the long-term application of imazalil and penflufen could cause residual accumulation in soils and subsequently result in serious negative effects on soil ecology.

Encapsulation of Imazalil in HKUST-1 with Versatile Antimicrobial Activity.

Based on high surface areas, adjustable porosity and microbicide activity, metal-organic frameworks (MOFs) HKUST-1 are widely used as drug release carriers for their slow degradation characteristics under slightly acidic conditions. In this work, porous HKUST-1 was reacted rapidly by cholinium salt (as the deprotonation agent and template) in an aqueous solution at room temperature. A novel antimicrobial system based on an imazalil encapsulated metal organic framework (imazalil IL-3@HKUST-1) was established. Imazalil IL-3@HKUST-1 could achieve synergism in inhibiting pathogenic fungi and bacteria. Moreover, six days after treatment, the slow and constant release of imazalil from imazalil IL@HKUST-1 exhibited better sustainability and microbicidal activity than imazalil. We believe that the method may provide a new strategy for related plant diseases caused by bacteria or fungi.

Neurotoxicity Assessment of Four Different Pesticides Using In Vitro Enzymatic Inhibition Assays.

Pesticides affect different organs and tissues according to their bioavailability, chemical properties and further molecular interactions. In animal models exposed to several classes of pesticides, neurotoxic effects have been described, including the reduction of acetylcholinesterase activity in tissue homogenates. However, in homogenates, the reduction in enzymatic activity may also result from lower enzymatic expression and not only from enzymatic inhibition. Thus, in this work, we aimed to investigate the neurotoxic potential of four distinct pesticides: glyphosate (herbicide), imazalil (fungicide), imidacloprid (neonicotinoid insecticide) and lambda-cyhalothrin (pyrethroid insecticide), by assessing their inhibitory effect on the activity of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase, by using direct in vitro enzymatic inhibition methods. All pesticides dose-dependently inhibited AChE activity, with an inhibition of 11 ± 2% for glyphosate, 48 ± 2% for imidacloprid, 49 ± 3% for imazalil and 50 ± 3% for lambda-cyhalothrin, at 1 mM. Only imazalil inhibited BChE. Imazalil induced dose-dependent inhibition of BChE with identical pattern as that observed for AChE; however, for lower concentrations (up to 500 µM), imazalil showed higher specificity for AChE, and for higher concentrations, the same specificity was found. Imazalil, at 1 mM, inhibited the activity of BChE by 49 ± 1%. None of the pesticides, up to 1 mM, inhibited tyrosinase activity. In conclusion, the herbicide glyphosate shows specificity for AChE but low inhibitory capacity, the insecticides imidacloprid and λ-cyhalothrin present selective AChE inhibition, while the fungicide IMZ is a broad-spectrum cholinesterase inhibitor capable of inhibiting AChE and BChE in an equal manner. Among these pesticides, the insecticides and the fungicide are the ones with higher neurotoxic potential.

Cross-generational effects of maternal exposure to imazalil on anaerobic components and carnitine absorption associated with OCTN2 expression in mice.

Imazalil (IMZ) is a fungicide recommended by the Chinese ministry of agriculture. However, recent study was observed high level of IMZ by dietary exposure in pregnant women. To determine the cross-generational effects, C57BL/6 mice were exposed to IMZ at dietary levels of 0, 0.025‰, and 0.25‰ during the gestation and lactation periods. Then, we assessed the changes in growth phenotypes, carnitine levels, and gut microbiota in F0, F1 or F2 generations. The growth phenotypes of dams didn't observe significant difference, but there were significant changes in the offspring. Plasma samples revealed low levels of free carnitine (C0), long-chain acyl-carnitines and total carnitine. In particular, C0 may be regarded as relatively potential, specific markers by maternal IMZ exposure. Caco2 cell culture and animal experiment confirmed IMZ affected carnitine absorption through the organic cation transporter type-2 (OCTN2) protein encoded by solute carrier family 22A member 5 (SLC22A5) gene in colon. Maternal IMZ exposure also had a greater effect on gut microbiota in offspring, especially anaerobic bacteria, which positively correlated with C0 and acyl-carnitines. These results suggested that maternal IMZ exposure affected carnitine absorption through OCTN2 protein, which led to the decline of anaerobic bacteria and unbalanced intestinal homeostasis.

Isolation, characterization and industrial application of a Cladosporium herbarum fungal strain able to degrade the fungicide imazalil.

Imazalil (IMZ) is an imidazole fungicide commonly used by fruit-packaging plants (FPPs) to control fungal infections during storage. Its application leads to the production of pesticide-contaminated wastewaters, which, according to the European Commission, need to be treated on site. Considering the lack of efficient treatment methods, biodepuration systems inoculated with tailored-made inocula specialized on the removal of such persistent fungicides appear as an appropriate solution. However, nothing is known about the biodegradation of IMZ. We aimed to isolate and characterize microorganisms able to degrade the recalcitrant fungicide IMZ and eventually to test their removal efficiency under near practical bioengineering conditions. Enrichment cultures from a soil receiving regular discharges of effluents from a FPP, led to the isolation of a Cladosporium herbarum strain, which showed no pathogenicity on fruits, a trait essential for its biotechnological exploitation in FPPs. The fungus was able to degrade up to 100 mg L-1 of IMZ. However, its degrading capacity and growth was reduced at increasing IMZ concentrations in a dose-dependent manner, suggesting the involvement of a detoxification rather than an energy-gain mechanism in the dissipation of IMZ. The isolate could tolerate and gradually degrade the fungicides fludioxonil (FLD) and thiabendazole (TBZ), also used in FPPs and expected to coincide alongside IMZ in FPP effluents. The capacity of the isolate to remove IMZ in a practical context was evaluated in a benchtop immobilized-cell bioreactor fed with artificial IMZ-contaminated wastewater (200 mg L-1). The fungal strain established in the reactor, completely dominated the fungal community and effectively removed >96% of IMZ. The bioreactor also supported a diverse bacterial community composed of Sphingomonadales, Burkholderiales and Pseudomonadales. Our study reports the isolation of the first IMZ-degrading microorganism with high efficiency to remove IMZ from agro-industrial effluents under bioengineering conditions.