Environmental Hot Spots and Resistance-Associated Application Practices for Azole-Resistant Aspergillus fumigatus, Denmark, 2020-2023.

Azole-resistant Aspergillus fumigatus (ARAf) fungi have been found inconsistently in the environment in Denmark since 2010. During 2018-2020, nationwide surveillance of clinical A. fumigatus fungi reported environmental TR34/L98H or TR46/Y121F/T289A resistance mutations in 3.6% of isolates, prompting environmental sampling for ARAf and azole fungicides and investigation for selection of ARAf in field and microcosmos experiments. ARAf was ubiquitous (20% of 366 samples; 16% TR34/L98H- and 4% TR46/Y121F/T289A-related mechanisms), constituting 4.2% of 4,538 A. fumigatus isolates. The highest proportions were in flower- and compost-related samples but were not correlated with azole-fungicide application concentrations. Genotyping showed clustering of tandem repeat-related ARAf and overlaps with clinical isolates in Denmark. A. fumigatus fungi grew poorly in the field experiment with no postapplication change in ARAf proportions. However, in microcosmos experiments, a sustained complete (tebuconazole) or partial (prothioconazole) inhibition against wild-type A. fumigatus but not ARAf indicated that, under some conditions, azole fungicides may favor growth of ARAf in soil.

Aquatic and sediment ecotoxicity data of difenoconazole and its potential environmental risks in ponds bordering rice paddies.

Difenoconazole has a widespread agricultural use to control fungal diseases in crops, including rice. In edge-of-field surface waters the residues of this lipophilic fungicide may be toxic to both pelagic and benthic organisms. To allow an effect assessment we mined the regulatory and open literature for aquatic toxicity data. Since published sediment toxicity data were scarce we conducted 28 d sediment-spiked toxicity test with 8 species of benthic macroinvertebrates. Ecotoxicological threshold levels for effects were assessed by applying the species sensitivity distribution approach. Based on short-term L(E)C50's for aquatic organisms from water-only tests an acute Hazardous Concentration to 5% of the species (HC5) of 100 µg difenoconazole/L was obtained, while the HC5 based on chronic NOEC values was a factor of 104 lower (0.96 µg difenoconazole/L). For benthic macroinvertebrates the chronic HC5, based on 28d-L(E)C10 values, was 0.82 mg difenoconazole/kg dry weight sediment. To allow a risk assessment for water- and sediment-dwelling organisms, exposure concentrations were predicted for the water and sediment compartment of an edge-of-field pond bordering rice paddies treated with difenoconazole using the Chinese Top-Rice modelling approach, the Chinese Nanchang exposure scenario and the Equilibrium Partitioning theory. It appeared that in the vast majority of the 20 climate years simulated, potential risks to aquatic and sediment organisms cannot be excluded. Although the HC5 values based on laboratory toxicity data provide one line of evidence only, our evaluation suggests population- and community-level effects on these organisms due to chronic risks in particular.

Toxicity interactions of azole fungicide mixtures on Chlorella pyrenoidosa.

It is acknowledged that azole fungicides may release into the environment and pose potential toxic risks. The combined toxicity interactions of azole fungicide mixtures, however, are still not fully understood. The combined toxicities and its toxic interactions of 225 binary mixtures and 126 multi-component mixtures on Chlorella pyrenoidosa were performed in this study. The results demonstrated that the negative logarithm 50% effect concentration (pEC50 ) of 10 azole fungicides to Chlorella pyrenoidosa at 96 h ranged from 4.23 (triadimefon) to 7.22 (ketoconazole), while the pEC50 values of the 351 mixtures ranged from 3.91 to 7.44. The high toxicities were found for the mixtures containing epoxiconazole. According to the results of the model deviation ratio (MDR) calculated from the concentration addition (MDRCA ), 243 out of 351 (69.23%) mixtures presented additive effect at the 10% effect, while the 23.08% and 7.69% of mixtures presented synergistic and antagonistic effects, respectively. At the 30% effect, 47.29%, 29.34%, and 23.36% of mixtures presented additive effects, synergism, and antagonism, respectively. At the 50% effect, 44.16%, 34.76%, and 21.08% of mixtures presented additive effects, synergism, and antagonism, respectively. Thus, the toxicity interactions at low concentration (10% effect) were dominated by additive effect (69.23%), whereas 55.84% of mixtures induced synergism and antagonism at high concentration (50% effect). Climbazole and imazalil were the most frequency of components presented in the additive mixtures. Epoxiconazole was the key component induced the synergistic effects, while clotrimazole was the key component in the antagonistic mixtures.

Species-specific differences in the inhibition of 11ß-hydroxysteroid dehydrogenase 2 by itraconazole and posaconazole.

11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) converts active 11ß-hydroxyglucocorticoids to their inactive 11-keto forms, thereby preventing inappropriate mineralocorticoid receptor activation by glucocorticoids. Disruption of 11ß-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypokalemia, hypernatremia and hypertension. Recently, the azole antifungals itraconazole and posaconazole were identified to potently inhibit human 11ß-HSD2, and several case studies described patients with acquired AME. To begin to understand why this adverse drug effect was missed during preclinical investigations, the inhibitory potential of itraconazole, its main metabolite hydroxyitraconazole (OHI) and posaconazole against 11ß-HSD2 from human and three commonly used experimental animals was assessed. Whilst human 11ß-HSD2 was potently inhibited by all three compounds (IC50 values in the nanomolar range), the rat enzyme was moderately inhibited (1.5- to 6-fold higher IC50 values compared to human), and mouse and zebrafish 11ß-HSD2 were very weakly inhibited (IC50 values above 7 µM). Sequence alignment and application of newly generated homology models for human and mouse 11ß-HSD2 revealed significant differences in the C-terminal region and the substrate binding pocket. Exchange of the C-terminus and substitution of residues Leu170,Ile172 in mouse 11ß-HSD2 by the corresponding residues His170,Glu172 of the human enzyme resulted in a gain of sensitivity to itraconazole and posaconazole, resembling human 11ß-HSD2. The results provide an explanation for the observed species-specific 11ß-HSD2 inhibition by the studied azole antifungals. The obtained structure-activity relationship information should facilitate future assessments of 11ß-HSD2 inhibitors and aid choosing adequate animal models for efficacy and safety studies.

Azole-resistant Aspergillus fumigatus isolates carrying TR34/L98H mutations in Taiwan.

Cumulative evidence described the emergence and geographical expansion of azole-resistant A. fumigatus associated with azole treatment failure. To investigate the status of azole resistance in A. fumigatus in Taiwan, we studied 38 A. fumigatus clinical isolates cultivated from 31 patients at two teaching hospitals from 2011 to 2014. Three isolates obtained from respiratory samples of two azole-naïve patients with pulmonary aspergillosis were found to display multi-azole resistance and cross resistance to agricultural azole fungicides, and all carried TR34/L98H mutations in cyp51A gene. The prevalence rates of azole resistance were 7.9% and 6.5% based on isolates and patients respectively. A phylogenetic analysis suggested genetic diversity of the TR34/L98H isolates in Taiwan, including a unique genotype distinct from strains outside Taiwan. The result underlines the emergence of such isolates in Taiwan as well, emphasising the importance of further surveillance for azole-resistant A. fumigatus and implementation of strategies that prevent fungicide-driven resistance selection.

Toxic interactions at the physiological and biochemical levels of green algae under stress of mixtures of three azole fungicides.

Assessing the interactions between environmental pollutants and these mixtures is of paramount significance in understanding their negative effects on aquatic ecosystems. However, existing research often lacks comprehensive investigations into the physiological and biochemical mechanisms underlying these interactions. This study aimed to reveal the toxic mechanisms of cyproconazole (CYP), imazalil (IMA), and prochloraz (PRO) and corresponding these mixtures on Auxenochlorella pyrenoidosa by analyzing the interactions at physiological and biochemical levels. Higher concentrations of CYP, IMA, and PRO and these mixtures resulted in a reduction in chlorophyll (Chl) content and increased total protein (TP) suppression, and malondialdehyde (MDA) content exhibited a negative correlation with algal growth. The activity of catalase (CAT) and superoxide dismutase (SOD) decreased with increasing azole fungicides and their mixture concentrations, correlating positively with growth inhibition. Azole fungicides induced dose-dependent apoptosis in A. pyrenoidosa, with higher apoptosis rates indicative of greater pollutant toxicity. The results revealed concentration-dependent toxicity effects, with antagonistic interactions at low concentrations and synergistic effects at high concentrations within the CYP-IMA mixtures. These interactions were closely linked to the interactions observed in Chl-a, carotenoid (Car), CAT, and cellular apoptosis. The antagonistic effects of CYP-PRO mixtures on A. pyrenoidosa growth inhibition can be attributed to the antagonism observed in Chl-a, Chl-b, Car, TP, CAT, SOD, and cellular apoptosis. This study emphasized the importance of gaining a comprehensive understanding of the physiological and biochemical interactions within algal cells, which may help understand the potential mechanism of toxic interaction.

Early life stage exposure to fenbuconazole causes multigenerational cardiac developmental defects in zebrafish and potential reasons.

With the increasing use of triazole fungicides in agriculture, triazole pesticides have aroused great concern about their toxicity and ecological risk. The current study investigated the impairments of embryonic exposure to fenbuconazole (FBZ) on cardiac transgenerational toxicity and related mechanisms. The fertilized eggs were exposed to 5, 50 and 500 ng/L FBZ for 72 h, and the larvae were then raised to adulthood in clean water. The adult fish were mated with unexposed fish to produce maternal and paternal F1 and F2 embryos, respectively. The results showed that increased arrhythmia were observed in F0, F1 and F2 larvae. Transcriptome sequencing indicated that the pathway of adrenergic signaling in cardiomyocytes was enriched in F0 and F2 larvae. In both F0 and F1 adult zebrafish hearts, ADRB2 protein expression decreased, and transcription of genes related to cardiac development and Ca2+ homeostasis was downregulated. These alterations might cause cardiac developmental defects. Significantly decreased protein levels of H3K9Ac and H3K14Ac might be linked with the downregulation in transcription of cardiac development genes. Protein‒protein interaction analysis exhibited that the pathway affecting the heart was well inherited in the paternal line. These results provide new ideas for the analysis and prevention of congenital heart disease.

Toxic mechanism of three azole fungicides and their mixture to green alga Chlorella pyrenoidosa.

Currently, few studies have investigated the joint toxicity mechanism of azole fungicides at different exposure times and mixed at the relevant environmental concentrations. In this study, three common azole fungicides, namely, myclobutanil (MYC), propiconazole (PRO), and tebuconazole (TCZ), were used in studying the toxic mechanisms of a single substance and its ternary mixture exposed to ambient concentrations of Chlorella pyrenoidosa. Superoxide dismutase (SOD), catalase (CAT), chlorophyll a (Chla), and total protein (TP), were used as physiological indexes. Results showed that three azole fungicides and ternary mixture presented obvious time-dependent toxicities at high concentrations. MYC induced a hormetic effect on algal growth, whereas PRO and TCZ inhibit algal growth in the entire range of the tested concentrations. The toxicities of the three azole fungicides at 7 days followed the order PRO > TCZ > MYC. Three azole fungicides and their ternary mixture induced different levels of SOD and CAT activities in algae at high concentrations. The ternary mixture showed additive effects after 4 and 7 days exposure, but no effect was observed at actual environmental concentrations. The toxic mechanisms may be related to the continuous accumulation of reactive oxygen species, which not only affected protein structures and compositions but also damaged thylakoid membranes, hindered the synthesis of proteins and chlorophyll a, and eventually inhibited algal growth. These findings increase the understanding of the ecotoxicity of azole fungicides and use of azole fungicides in agricultural production.

Metabolomics reveals the reproductive abnormality in female zebrafish exposed to environmentally relevant levels of climbazole.

Climbazole (CBZ) ubiquitously detected in the aquatic environment may disrupt fish reproductive function. Thus far, the previous study has focused on its transcriptional impact of steroidogenesis-related genes on zebrafish, but the underlying toxic mechanism still needs further investigation at the metabolic level. In this study, adult zebrafish were chronically exposed to CBZ at concentrations of 0.1 (corresponding to the real concentration in surface water), 10, and 1000 µg/L and evaluated for reproductive function by egg production, with subsequent ovarian tissue samples taken for histology, metabolomics, and other biochemical analysis. After 28 days' exposure, fecundity was significantly decreased in all exposure groups, with the inhibition of oocytes in varying developmental stages to a certain degree. The decrease in retinoic acid and sex hormones, down-regulated genes important in steroidogenesis, and increase in oxidized/reduced glutathione ratio and occurrence of apoptotic cells were observed in zebrafish ovaries following exposure to CBZ even at environmentally realistic concentrations, suggesting that alternations in steroidogenesis and oxidative stress can play significant roles in CBZ-triggered reproductive toxicity. Besides, mass spectrometry imaging analysis validated the results from metabolomics analysis. Our findings provide novel perspectives for unveiling the mechanism of reproductive dysfunction by CBZ and highlight its risk to fish reproduction.

Effects of Azole Fungicides on Secreted Metabolomes of Botrytis cinerea.

Botrytis cinerea, gray mold, is one of the most notorious phytopathgens, causing serious economic loss in the agricultural industry. The phytotoxic effects are mainly derived from secreted virulence proteins and terpenoid-type secondary metabolites. Azole fungicides are commonly used to manage the disease. However, their biochemical effects other than sterol biosynthesis were not documented, especially toxic secreted metabolites. In this study, six azole fungicides were treated with in vitro and in vivo conditions. Comprehensive profiles of primary and secondary metabolites in culture media were evaluated to assess the fungal metabolomes under pesticide-stressed conditions. The results indicated that extensive metabolic differentiation was induced by azole fungicides. Epoxiconazole clearly reduced the extracellular phytotoxin concentrations, while the level of indole-3-acetic acid was increased. In addition, significant differentiation of primary metabolism could be deduced from secreted metabolite profiles, including the tricarboxylic acid cycle and aromatic amino acid catabolism. Cellular lipid profiles, including fatty acids and sterol, have been altered drastically by azoles, which indicate extensive changes of cellular lipid metabolism. These system-wide metabolic alterations resulted in reduced plant damages, proven by the in vivo assay with tomato. Overall, azole fungicides induced significant changes of endo- and exometabolomes and could reduce the fungal infection. The experimental results will provide a more detailed understanding of physiological changes of phytopathogens under pesticide treatment and information for new pesticide development.

The synergistic potential of azole fungicides does not directly correlate to the inhibition of cytochrome P450 activity in aquatic invertebrates.

The ability of azole fungicides to inhibit cytochrome P450 dependent metabolism is proposed to be the main mechanism for their synergizing effect on pyrethroid insecticide toxicity in aquatic invertebrates. This study investigates the correlation between inhibition strength and synergistic potential of azole fungicides in the crustacean Daphnia magna and the insect larvae Chironomus riparius. Inhibition strength was measured in vivo toward the cytochrome P450 catalysed conversion of 7-ethoxycoumarin to 7-hydroxycoumarin (ECOD). Synergistic potentials were determined as the ratio between predicted and observed toxicity of mixtures based on the model of concentration addition (CA) and independent action (IA). Azoles (n = 9-11) enhanced the toxicity of α-cypermethrin in D. magna (Synergy ratios CA: 0.8 - 16; IA: 1.1 - 22) and inhibited cytochrome P450 activity by different degrees (IC50: 0.0023 - 36 µM for D. magna and 0.08 - 24 µM for C. riparius). Inhibition strengths were strongly correlated in the two organisms (r: 0.937 p: 0.019 for triazoles and r: 0.903 p: 0.097 for imidazoles). Lipophilicity governed the inhibition strength of triazoles in both species (r > 0.9, p < 0.05). No correlation was observed between inhibition strengths and synergistic potentials. Several reasons for the apparent lack of correlation were discussed.

Can the inhibition of cytochrome P450 in aquatic invertebrates due to azole fungicides be estimated with in silico and in vitro models and extrapolated between species?

Azole fungicides, designed to halt fungal growth by specific inhibition of fungal cytochrome P450 (CYP51), inhibit cytochrome P450s involved in the metabolism of xenobiotics in several non-target organisms thus raising environmental concern. The present study investigates the degree by which inhibition strengths of azoles toward cytochrome P450 in rat liver, the insect Chironomus riparius larvae and the snail Lymnaea stagnalis can be extrapolated from estimated in silico affinities. Azoles' affinities toward human cytochrome P450 isoforms involved in xenobiotic metabolism (CYP3A4, CYP2C9 and CYP2D6) as well as fungal CYP51 were estimated with a ligand-protein docking model based on the ChemScore scoring function. Estimated affinities toward the selected enzymatic structures correlated strongly with measured inhibition strengths in rat liver (ChemScore vs. logIC50 among cytochrome P450 isoforms: -0.662 < r < -0.891, n = 17 azoles), while weaker correlations were found for C. riparius larvae (-0.167 < r < -0.733, n = 9) and L. stagnalis (-0.084 < r < -0.648, n = 8). Inhibition strengths toward C. riparius and rat liver activities were found to be highly correlated to each other (r: 0.857) while no significant relationship was found between either of the species and L. stagnalis. The inhibition of cytochrome P450 due to azole fungicides could be estimated in vitro and to a lesser extent in silico for C. riparius but not for L. stagnalis, possibly due to different enzymatic susceptibility toward azole inhibition among the species.

Synergism between macrolide antibiotics and the azole fungicide ketoconazole in growth inhibition testing of the green alga Pseudokirchneriella subcapitata.

Macrolide antibiotics and azole fungicides are detected widely in the aquatic environment as a result of their increased use in humans and animal livestock disease and their incomplete removal by wastewater treatment plants. In most cases, ecotoxicological tests are performed by using individual chemical substances, but because of the coexistence of a number of chemicals in the environment, organisms are exposed to many chemicals simultaneously. Therefore, it is important to evaluate effects of chemical interactions, adding to potential hazards of individual chemical. Here, we investigated the synergetic effects of combined chemicals (the azole fungicide ketoconazole and either of two macrolide antibiotics, erythromycin and clarithromycin) in growth inhibition testing using Pseudokirchneriella subcapitata according to OECD Test guideline 201. Combination index plots, isobolograms, and curve-shift analyses revealed that the combination of macrolide antibiotic and ketoconazole at various ratios resulted in strong synergism that enhanced growth inhibition of P. subcapitata, suggesting the necessity of investigating potential hazard of combined chemicals for regulatory purposes.

Analysis of azole fungicides in fish muscle tissues: Multi-factor optimization and application to environmental samples.

Azole fungicides have been reported to be accumulated in fish tissue. In this study, a sensitive and robust method using high-performance liquid chromatography-tandem mass spectrometry combined with ultrasonic extraction, solid-liquid clean-up, liquid-liquid extraction and solid-phase extraction (SPE) for enrichment and purification have been proposed for determination of azole fungicides in fish muscle samples. According to the results of non-statistical analysis and statistical analysis, ethyl acetate, primary secondary amine (PSA) and mixed-mode cation exchange cartridge (MCX) were confirmed as the best extraction solvent, clean-up sorbent and SPE cartridge, respectively. The satisfied recoveries (81.7-104%) and matrix effects (-6.34-7.16%), both corrected by internal standards, were performed in various species of fish muscle matrices. Method quantification limits of all azoles were in the range of 0.07-2.83ng/g. This optimized method was successfully applied for determination of the target analytes in muscle samples of field fish from Beijiang River and its tributaries. Three azole fungicides including climbazole, clotrimazole and carbendazim were detected at ppb levels in fish muscle tissues. Therefore, this analytical method is practical and suitable for further clarifying the contamination profiles of azole fungicides in wild fish species.

Multimedia fate modeling and risk assessment of a commonly used azole fungicide climbazole at the river basin scale in China.

Climbazole is an antidandruff active ingredient commonly used in personal care products, but little is known about its environmental fate. The aim of this study was to evaluate the fate of climbazole in water, sediment, soil and air compartments of the whole China by using a level III multimedia fugacity model. The usage of climbazole was calculated to be 345 t in the whole China according to the market research data, and after wastewater treatment a total emission of 245 t was discharged into the receiving environment with approximately 93% into the water compartment and 7% into the soil compartment. The developed fugacity model was successfully applied to estimate the contamination levels and mass inventories of climbazole in various environmental compartments of the river basins in China. The predicted environmental concentration ranges of climbazole were: 0.20-367 ng/L in water, and 0.009-25.2 ng/g dry weight in sediment. The highest concentration was mainly found in Haihe River basin and the lowest was in basins of Tibet and Xinjiang regions. The mass inventory of climbazole in the whole China was estimated to be 294 t, with 6.79% in water, 83.7% in sediment, 9.49% in soil, and 0.002% in air. Preliminary risk assessment showed high risks in sediment posed by climbazole in 2 out of 58 basins in China. The medium risks in water and sediment were mostly concentrated in north China. To the best of our knowledge, it is the first report on the emissions and multimedia fate of climbazole in the river basins of the whole China.