Fungicidal

Azoxystrobin (AZ) is a broad-spectrum synthetic fungicide widely used in agriculture globally. However, there are concerns about its fate and effects in the environment. It is reportedly transformed into azoxystrobin acid as a major metabolite by environmental microorganisms. Bacillus licheniformis strain TAB7 is used as a compost deodorant in commercial compost and has been found to degrade some phenolic and agrochemicals compounds. In this article, we report its ability to degrade azoxystrobin by novel degradation pathway. Biotransformation analysis followed by identification by electrospray ionization-mass spectrometry (MS), high-resolution MS, and nuclear magnetic resonance spectroscopy identified methyl (E)-3-amino-2-(2-((6-(2-cyanophenoxy)pyrimidin-4-yl)oxy)phenyl)acrylate, or (E)-azoxystrobin amine in short, and (Z) isomers of AZ and azoxystrobin amine as the metabolites of (E)-AZ by TAB7. Bioassay testing using Magnaporthe oryzae showed that although 40 μg/mL of (E)-AZ inhibited 59.5 ± 3.5% of the electron transfer activity between mitochondrial Complexes I and III in M. oryzae, the same concentration of (E)-azoxystrobin amine inhibited only 36.7 ± 15.1% of the activity, and a concentration of 80 μg/mL was needed for an inhibition rate of 56.8 ± 7.4%, suggesting that (E)-azoxystrobin amine is less toxic than the parent compound. To our knowledge, this is the first study identifying azoxystrobin amine as a less-toxic metabolite from bacterial AZ degradation and reporting on the enzymatic isomerization of (E)-AZ to (Z)-AZ, to some extent, by TAB7. Although the fate of AZ in the soil microcosm supplemented with TAB7 will be needed, our findings broaden our knowledge of possible AZ biotransformation products. [1]

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The effects of the fungicides azoxystrobin (a strobilurin) and epoxiconazole (a sterol biosynthesis inhibitor) on phyllosphere fungi, senescence and yield were studied in winter wheat in field trials free of visible disease and under controlled environmental conditions. In two field trials, treatments with each of the two fungicides prolonged green leaf area retention and increased yield compared with untreated control plots. Azoxystrobin maintained green leaf area for longer than epoxiconazole and, in one trial, treatments with azoxystrobin gave a greater yield response than epoxiconazole. Mycelial growth on leaf surfaces, mainly originating from saprophytic fungi, was reduced by each of the fungicides. Papilla formation and hypersensitive reactions, almost exclusively against infection attempts by Mycosphaerella spp. (most probably M. graminicola), occurred with high frequency in the leaves. These defence reactions presumably incurred a significant energy cost, accelerating plant senescence. Fewer defence reactions were recorded in azoxystrobin-treated leaves than in epoxiconazole-treated and untreated leaves. Inoculation in a glasshouse experiment with the saprophytic fungi Alternaria alternata and Cladosporium macrocarpum accelerated wheat senescence. Control of the saprophytes by azoxystrobin or epoxiconazole treatments caused a delay in the accelerated senescence, but without significant increase in above-ground biomass and yield. Neither fungicide influenced senescence, above-ground biomass or yield in noninoculated wheat plants. In growth chamber experiments azoxystrobin inhibited spore germination and mycelial growth of A. alternata and C. macrocarpum. Epoxiconazole had little inhibitory effect on spore germination, but strongly inhibited mycelial growth of both saprophytes. Both fungicides reduced A. alternata-induced papilla formation in wheat leaves, with epoxiconazole being more effective. Inoculation with either of the two saprophytes did not significantly increase wheat leaf respiration, in contrast to inoculation with the nonhost pathogen Erysiphe graminis f.sp. hordei. Treatment with azoxystrobin did not affect this latter increase in respiration whereas it was reduced by epoxiconazole treatment. It is proposed that the greater inhibition of infection attempts from Mycosphaerella spp. by azoxystrobin, compared with epoxiconazole, may account for the greater yield given by azoxystrobin in field plots [2].

Azoxystrobin is a widely used systemic fungicide in the Northeast of China, but it is unclear how azoxystrobin impacts the soil microbiome. Thus, we studied the impact of azoxystrobin on the microbial community structure and function in Chinese Spodosols. Field soil that had never been exposed to azoxystrobin was amended in laboratory batch experiments with 0 mg kg-1, 2 mg kg-1, 25 mg kg-1 and 50 mg kg-1 of azoxystrobin. Four soil enzymes (urease, invertase, phosphatase and catalase) were monitored to assess the impact of azoxystrobin on carbon, nitrogen and phosphorus cycling as well as the microbial activity. The results show that the urease, invertase, and phosphatase (hydrolytic enzymes) activities was inhibited by as little as 2 mg kg−1 of azoxystrobin after 35 days, whereas the catalase (oxidoreductase enzyme) activity was promoted by the same concentration in most cases. Biolog Ecoplate analysis indicated that the utilization of different carbon sources was inhibited by azoxystrobin. 16S rRNA sequencing showed the bacterial operational taxonomic unit richness and the Shannon index decreased with increasing azoxystrobin concentration. Relative abundances of Sphingomonas decreased while Amycolatopsis and Sphingomonas increased by addition of increasing levels of azoxystrobin. In conclusion, azoxystrobin application to Spodosols leads to reduced urease, invertase, and phosphatase activities, which can impact nutrient cycling and carbon utilization. Furthermore, azoxystrobin application changed the microbiome community structure.[3]

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Pesticides are continually entering the soil ecosystem because of safety assurance of high-yield food in agricultural intensification. It is highly urgent to evaluate their effects on the soil biota. This study characterized the dose-dependent changes in the gut bacterial and fungal community of Enchytraeus crypticus after oral exposure to an environmental dose of the fungicide azoxystrobin (AZ; 0.5, 1, and 10 mg/L) for 21 days. AZ not only induced the growth opportunistic pathogens and reduced the relative abundance of beneficial bacteria in the E. crypticus gut, but also destroyed the stability of the gut microecology of E. crypticus. Meanwhile, the dose-dependent effects of AZ were observed on the number and normalized abundance of antibiotic resistance genes (ARGs; copies/bacterial cell), and trace dose of AZ (> 0 and < 0.085 μg/individual) might enrich the ARG numbers in the gut of E. crypticus. Moreover, we used structural equation modeling to speculate that apart from mobile genetic elements and the bacterial community, the microbial interaction of E. crypticus gut might be another key contributor that drived the emergence and dissemination of ARGs. This study provides new perspectives in assessing the gut health of soil fauna under pesticide pollution in intensive agricultural production [4].

Quantitative analysis of Azoxystrobin/AZ degradation [1]
Luria broth (LB, Sambrook and Russell, 2001) agar plates were prepared by dissolving the following in 1 L of Milli-Q water: 10 g tryptone, 10 g NaCl, 5 g yeast extract, and 16 g agar. The media was then sterilized by autoclaving at 121 °C for 20 min. TAB7 cells in glycerol stock were streaked on LB agar plates and incubated overnight at 30 °C. A single colony was then inoculated into a test tube containing LB (5 mL) and incubated overnight at 30 °C with shaking (300 strokes/min). Cells were harvested via centrifugation at 5000×g for 5 min using sterile centrifuge tubes, re-suspended in fresh LB (5 mL) supplemented with 100 ppm AZ previously dissolved in DMSO (stock solution: 2000 ppm). The bacterial cultures were then incubated at 30 °C with shaking (300 strokes/min) for up to 60 days. For the negative control, AZ was added to sterile TAB7-free LB and incubated under the same conditions. At 10-day intervals, liquid culture tubes (triplicates) were sacrificed and an internal standard (100 ppm isoprothiolane dissolved in DMSO) was added to each sample before two rounds of extraction with ethyl acetate, using 2.5 mL of ethyl acetate each time. After extraction, the solvent was completely evaporated using a centrifugal evaporator, and the resultant residue was re-dissolved in methanol, filtered through 0.22-μm PTFE filters, and injected (30 μL) into a Hitachi Elite LaChrom L2455 HPLC system equipped with a diode array detector, autosampler injector, thermostat column compartment, and a PEGASIL-B octadecyl silica (ODS) analytical column (4.6 mm i.d. × 250 mm length). The mobile phase was acetonitrile:water (70:30, v/v) with a flow rate of 1 mL/min, and the column temperature during the analysis was maintained at 40 °C. The detection wavelength was set at 235 nm.

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Preparation of Magnaporthe oryzae mitochondrial fraction [1]
The fungicidal activity of (E)-azoxystrobin amine and AzoxystrobinAZ against rice blast fungus M. oryzae Ina 86–137 (Japanese race 007.0) was tested by collecting the mitochondrial membrane fraction of the fungus and measuring the inhibition of NADH:cytochrome c oxidoreductase activity (Complexes I and III, Motoba et al., 1988). First, the mitochondrial fraction from 5-day-old M. oryzae grown in YPS liquid medium (5 g/L yeast extract, 10 g/L soluble starch, 20 g/L sucrose) was extracted by freezing the cells with liquid nitrogen and grinding them in lysis buffer (1 M sorbitol, 50 mM sodium citrate, pH 5.8). The resulting liquid was centrifuged at 1000×g and 4 °C for 5 min to remove the debris, followed by another round of centrifugation at 15,000×g and 4 °C for 15 min to precipitate the mitochondrial fraction. The precipitate was suspended in complex measurement solution (0.25 M sucrose, 1 mM dithiothreitol, 0.1 mM EDTA, 3 mM Tris-HCl, pH 7.4) and used for the NADH:cytochrome c assay immediately. For protein measurement, the fraction was treated with 8 M urea for 30 min at room temperature before the protein was quantified using a BioRad Protein Assay kit.

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Complex I and III activity measurement [1]
First, 40–50 μg of fresh mitochondrial extract was suspended in 2 mL of complex measurement solution. Then, azoxystrobin amine or Azoxystrobin/AZ, 0.8 mM KCN, and 30 μM salicylhydroxamic acid (SHAM) were added to the assay mixture, followed by 100 μM cytochrome c, and the change in absorbance at 548 nm was measured for 200 s. After allowing the absorbance to stabilize for approximately 50 s, 25 μM NADH was injected into the cuvette and cytochrome c reducing activity was measured as the absorbance increased. The absorption measurement was done using a UV–Vis spectrophotometer at 25 °C. Antimycin A (35 μg/mL) was used as the positive control. The slope of the absorbance in dAbs/min was considered to indicate transfer efficiency and used to compare the inhibitory activity of the compound against that of the negative control.

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Fungicide treatments [2]
Treatments in both experiments comprised of Azoxystrobin, epoxiconazole or untreated, 12 pots of each. In each tent there were 12 pots (four of each treatment) fully randomized within the tent. The experimental sprayings were performed using a tracksprayer with a Teejet 8002 EVS tip nozzle delivering 200 L ha−1. The fungicides were applied at the manufacturers' recommended field rates: ‘Amistar’ (azoxystrobin 250 g ha−1) and ‘Opus’ (epoxiconazole 125 g ha−1) in a three-spray programme at GS 31–32, GS 37–39 and GS 45–51 (as determined when > 50% of the plants reached the target growth stages).
‘Amistar’ (azoxystrobin 250 g ha−1) or ‘Opus’ (epoxiconazole 125 g ha−1) was applied in 200 L ha−1 spray volume with hand-held spray booms in a three-spray programme at GS 31, GS 39 and GS 59. The three plots in each block were treated with Azoxystrobin, epoxiconazole or left untreated and were randomized within each block. In Suffolk, the trial consisted of 18 blocks with plots each of 6 × 2·5 m. In Berkshire, the trial was divided into nine blocks with plots 12 × 2·5 m in size.

Azoxystrobin/AZ (C22H17N3O5; CAS 131860-33-8; white powder; 95% purity) was dissolved in methanol/water (1:5) and a 1 g/L solution was made to prepare 0.5, 1, and 10 mg azoxystrobin /kg dry oat (environmentally relevant concentration) as the following treatment groups: AZ0.5, AZ1, and AZ10 (Zhang et al.,

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Measurement of the soil antibiotic background value and the Azoxystrobin/AZ residue of E. crypticus [4]
To characterize the background value of antibiotics in the used soil suspension, a Shimadzu liquid chromatograph coupled with ABI 3200 triple-quadruple tandem mass spectrometry was used to detect 19 types of antibiotics according to a previously published protocol (Table S3) (Hong et al., 2018). At the end of the 21-day Azoxystrobin/AZ exposure, six adult E. crypticus were used to determine the AZ residue levels using solid-phase extraction-high-performance liquid chromatography according to a previously described method (Zhang et al., 2019a, Zhang et al., 2019b).

Absorption, Distribution and Excretion
Eight male and female rats were orally administered 1 mg/kg body weight of unlabeled azoxystrobin daily for 14 consecutive days, followed by a single oral dose of 1 mg/kg body weight of 14C-pyrimidinyl-labeled azoxystrobin. Following repeated administration, approximately 89.1% and 86.5% of the administered dose were excreted in feces within 7 days in males and females, respectively, and approximately 12.5% and 17.0% of the administered dose were excreted in urine within 7 days, respectively. Excretion of radioactive material was rapid in both males and females, with over 96% of the radioactive material eliminated within the first 48 hours. Approximately 0.62% and 0.39% of the administered dose were detected in the cadavers of males and tissues of females within 7 days post-administration. The highest concentration of azoxystrobin-derived radioactive material was observed in the kidneys after repeated administration (<0.04 μg equivalent/g in both males and females). The concentrations detected in the liver were 0.02 μg equivalents/g in males and 0.01 μg equivalents/g in females. At the end of the experiment, the total radioactivity concentration in the blood of both male and female rats was 0.01 μg equivalents/g. In the toxicokinetic studies, male and female Alpk:APfSD rats were divided into groups (n=5-8 per group) and administered a single dose of 1 or 100 mg/kg body weight of azoxystrobin (99% purity) by gavage, or repeated 14 times daily at a dose of 1 mg/kg body weight. Bile duct cannulation rats were administered a single dose of 100 mg/kg body weight of azoxystrobin by gavage to assess bile metabolites. The solvent was polyethylene glycol (PEG 600), and the dose was 4 mL/kg body weight. The treated rats were housed in stainless steel metabolic cages for 7 days. Urine was collected 6 hours after administration, and urine and feces were collected at 12, 24, 36, and 48 hours after administration, and every 24 hours thereafter, up to day 7 after administration. At each collection, the cages were rinsed with water, and the rinsing fluid was collected along with the urine. At the end of the study, the cages were thoroughly rinsed with ethanol/water (1:1 v/v) and retained for radiochemical analysis. Carbon dioxide and volatile substances were collected. After 7 days, various organs and tissues were removed for radioactive analysis. …For rats receiving a single low dose (1 mg/kg body weight), the total radioactive excretion (urine, feces, and rinsing fluid) in male and female rats over 7 days was 93.75% and 91.44%, respectively. The majority (>85%) of urinary and fecal excretion occurred within the first 36 hours after administration. In these rats, approximately 83.2% and 72.6% of the administered dose were excreted in feces in males and females over 7 days, and approximately 10.2% and 17.9% of the administered dose were excreted in urine, respectively. Approximately 0.34% and 0.31% of the administered dose were detected in the cadavers and tissues of male and female rats within 7 days after administration. For rats receiving this dose (1 mg/kg body weight), the tissues with the highest concentrations of radiolabeled substances were the liver (mean 0.009 μg equivalent/g for both male and female rats) and the kidney (0.027 μg equivalent/g for male rats and 0.023 μg equivalent/g for female rats). At the end of the experiment, the total radioactivity concentration in the blood of both male and female rats was 0.004 μg equivalent/g. Less than 0.6% of the administered dose was recovered in exhaled breath. For rats receiving a single higher dose (100 mg/kg body weight), the total radioactivity excretion rates (urine, feces, and cage cleaning fluid) within 7 days were 98.29% and 97.22% for male and female rats, respectively. The majority (>82%) of urinary and fecal excretion occurred within the first 48 hours after administration. At this dose, approximately 89.37% and 84.53% of the administered dose were excreted in feces in male and female rats, respectively, and approximately 8.54% and 11.54% were excreted in urine, respectively, within 7 days. Approximately 0.33% of the administered dose was detected in the cadavers and tissues of male and female rats within 7 days after administration. At this higher dose, the tissues with the highest concentrations of radiolabeled substances were the kidneys (male: 1.373 μg Equivalent/g; female: 1.118 μg Equivalent/g) and the liver (male: 0.812 μg Equivalent/g; female: 0.714 μg Equivalent/g). At the end of the experiment, the total radioactive concentrations in the blood of male and female rats were 0.389 μg Equivalent/g and 0.379 μg Equivalent/g, respectively. This study also investigated the excretion and tissue distribution of radioactive materials within 48 hours following a single oral administration of 1 mg/kg body weight of azoxystrobin to male and female rats. Rats treated with the drug were placed in metabolic cages to collect urine, feces, exhaled gases, and volatile substances. One male and one female rat were sacrificed 24 and 48 hours after administration, respectively. Each rat received radiolabeled azoxystrobin, and the administration site was marked. The carcasses of each rat were frozen and sectioned for whole-body X-ray examination. Within 48 hours, male and female rats excreted approximately 89% and 86% of the (14)C-pyrimidinyl-labeled azoxystrobin, respectively, in urine and feces. Most of the radioactive material was excreted in feces, with less than 17% in urine. Male and female rats treated with (14)C-phenylacrylate-labeled azoxystrobin excreted approximately 80% and 97% of the administered dose, respectively, within 48 hours. Most of the radioactive material was excreted in feces, with less than 21% in urine. After 48 hours, male and female rats excreted approximately 0.01% of the administered dose as carbon dioxide traps and volatile metabolites, respectively. Male and female rats treated with 14C-cyanophenyl-labeled azoxystrobin excreted approximately 95% and 98% of the administered dose, respectively, within 48 hours. Most of the radioactive material was excreted in feces, with <16% radioactive material in urine. After 48 hours, small amounts of radioactive material were excreted in both male and female rats as carbon dioxide (<0.3%) and volatile metabolites (0.01%). Whole-body autoradiography showed similar distribution of all radiolabeled substances in male and female rats. After 24 hours, most radiolabeled substances were present in the digestive tract, moderate amounts in the kidneys, and small amounts in the liver. Whole-body autoradiography showed a significant decrease in radioactivity 48 hours after administration. These results indicate no significant differences in excretion rate, route of excretion, or tissue distribution among the three different labeled azoxystrobin formulations. No sex-related differences in excretion were found. The small differences in excretion volume are primarily due to the small number of rats used in this study. There were no significant differences in the amount of radioactivity recovered in exhaled breath and volatile substances among the three radiolabeled substances and between different sexes. Based on the results of this study, subsequent studies on excretion and tissue retention were conducted using only pyrimidinyl-labeled azoxystrobin.

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Metabolism/Metabolites
... (14)C-cyanophenyl-labeled azoxystrobin was administered to cannulated and uncannulated rats at a dose of 100 mg/kg body weight. Urine, fecal, and bile samples were collected for 72 hours. This study aimed to reassess certain plant and goat metabolites that had not been previously identified in rats and to further elucidate the metabolic pathways of azoxystrobin in rats. Three metabolites previously detected in plants or goats were also identified. Compound 13 (2-hydroxybenzonitrile) is a product of diphenyl ether bond cleavage and was detected in bile and urine as a glucuronide conjugate at concentrations up to 1.8% of the administered dose. Compound 20 ((2-(6-(2-cyanophenoxy)pyrimidin-4-yloxy)phenyl)acetic acid) was also detected in bile and urine at concentrations up to 1.3% of the administered dose. Compound 35 (2-(2-(6-(2-cyanophenoxy)pyrimidin-4-yloxy)phenyl)glycolic acid) was detected in urine, feces, and bile at concentrations up to 0.6%. Compounds 24 (methyl 2-(2-(6-(2-cyanophenoxy)pyrimidin-4-yloxy)phenyl)glycolate and 30 (2-(6-(2-cyanophenoxy)pyrimidin-4-yloxy)benzoic acid) were not detected.
Bile duct cannulated rats were administered radiolabeled pyrimidin, cyanophenyl, or phenyl acrylate ring-based azoxystrobin via gavage at a dose of 100 mg/kg body weight. Comparison of excretion rate, pathway, and metabolite profile showed (consistent with previous findings) no significant differences in metabolism among the three different labeled forms, indicating extremely low ether bond cleavage between aromatic rings. Therefore, we designed a metabolite identification experiment in which 14C-pyrimidinyl-labeled azoxystrobin was administered by gavage to bile-tubed rats. Feces, bile, and cage flushing fluid were collected from bile-tubed rats at 6, 12, 24, 36, and 48 hours and stored at -20°C. Bile, fecal, and urine samples were collected and mixed between 0 and 48 hours. Male and female samples were collected separately. In single-dose (high or low dose) and continuous 14-day administration experiments, urine and feces were collected within 168 hours post-administration for quantitative metabolite analysis. A portion of the bile samples was enzymatically hydrolyzed overnight at 37°C and pH 5.6 with cholylglycine hydrolase (30 units/mL). Metabolites were identified using a variety of analytical techniques, including thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), proton nuclear magnetic resonance (NMR), and mass spectrometry (MS). Based on data from bile excretion following a single administration of 100 mg/kg body weight of 14C-pyrimidinyl, 14C-phenylacrylate, or 14C-cyanophenyl-labeled azoxystrobin to rats, 48 hours later, male rats excreted 74.4% of the pyrimidinyl-labeled radioactive material in their bile, while female rats excreted 80.7%; male rats excreted 56.6% of the cyanophenyl-labeled radioactive material in their bile, while female rats excreted 62.5%; and male rats excreted 64.4% of the cyanophenyl-labeled radioactive material in their bile, while female rats excreted 63.6%. Quantitative analysis showed no significant difference in bile excretion between male and female rats. The study found that azoxystrobin is extensively metabolized in rats. Fifteen metabolites were detected in feces and identified. Seven other metabolites were detected but not identified. The levels of all unidentified metabolites did not exceed 4.9% of the administered dose. Quantitative data on various metabolites in feces, urine, and bile of rats given a single dose of 100 mg/kg body weight of azoxystrobin… The quality balance of the metabolite identification studies indicated that although excretion studies showed an overall recovery rate of 91.75–103.99% (of which 72.6–89.3% was found in feces), a considerable proportion of administered radiolabeled substances (45.6–73.6%) remained unexplained. The proportion of unexplained radiolabeled substances was particularly significant in the single low-dose group and the repeated low-dose group. The study authors noted that the differences in recovery rates may be due to the following reasons: During metabolite identification, feces were extracted using acetonitrile, which allowed the parent compound to be separated when it was present in the feces (i.e., in the higher-dose group). For the groups receiving a single low dose or repeated low doses (where the parent compound content was extremely low), most of the radiolabeled substances in the feces were bound to polar metabolites, which were not present in the acetonitrile extract. Therefore, the concentration of radiolabeled substances in the extract was very low. For the higher-dose group, more parent compound was not absorbed, resulting in more of the parent compound being separated into the acetonitrile extract. Glucuronide conjugates (metabolite V) were the most common metabolite in bile from both males (29.3%) and females (27.4%). Metabolite I (the parent compound) was not detected in bile. Other bile metabolites accounted for 0.9% to 9.0% of the administered dose, respectively. In bile duct cannulated rats, approximately 15.1% and 13.6% of the radioactivity in the feces of male and female rats, respectively, was metabolite I (the parent compound). The parent compound was not detected in the urine of either male or female rats with bile duct cannulation. The major metabolite in the urine of bile duct cannulated rats was an unidentified metabolite 2, accounting for approximately 1.8% and 2.0% of the administered dose in males and females, respectively. No evidence was found that metabolism was dose-dependent, but sex differences in biotransformation were observed, with females producing more metabolites than males. Biotransformation was not dose-dependent. The study authors considered absorption to be dose-related. Oral absorption at a dose of 1 mg/kg body weight was nearly complete (100%) because the parent compound was not detected. Oral absorption at higher doses (100 mg/kg body weight) was estimated to be approximately 74–81% because approximately 19–26% of the parent compound was detected. However, due to low recovery rates after extraction, especially at lower doses, it is difficult to estimate the true oral absorption rate. ...There are two main metabolic pathways: one is hydrolysis to generate methoxyic acid, which then undergoes glucuronide conjugation to generate metabolite V; the other is the conjugation of the cyanobenzene ring with glutathione, followed by further metabolism through a series of intermediates (VI, VII, and VIII) to generate mercaptouric acid metabolite IX. Azoxystrobin also exhibits hydroxylation at positions 8 and 10 of the cyanobenzene ring, followed by glucuronide conjugation (metabolites II, III, IVa, and IVb). Several minor metabolic pathways involving the acrylate moiety ultimately generate metabolites XIII and XIV. Three metabolites (X, XII, and XV) generated through ether bond cleavage were also identified.
The metabolic pathways of the drug were determined in male and female rats after a single oral dose of 1 and 100 mg·kg⁻¹ of [(14)C]-methyl-(E)-2-[2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl]-3-methoxyacrylate (azoxyl), respectively, and in surgically prepared rats with bile duct cannulation after a single oral dose of 100 mg·kg⁻¹. 2. Azoxyl has extensive metabolism, producing at least 15 metabolites. There are sex differences, with females producing more metabolites than males. 3. The two main metabolic pathways are the hydrolysis of methoxyic acid followed by conjugation with glucuronic acid, and the further metabolism of cyanophenyl rings with glutathione to thiouric acid. In addition, there are several minor metabolic pathways.
Organic nitriles are converted into cyanide ions in the liver by cytochrome P450 enzymes. Cyanide ions are rapidly absorbed and distributed throughout the body. Cyanide ions are primarily metabolized to thiocyanate by thiocyanate oxidase or 3-mercaptopyruvate-thiotransferase. Cyanide metabolites are excreted in urine. (L96)

Toxicity Summary
Organic nitriles can decompose into cyanide ions both in vivo and in vitro. Therefore, the main toxic mechanism of organic nitriles is the production of toxic cyanide ions, or hydrogen cyanide. Cyanide ions are inhibitors of cytochrome c oxidase in the fourth electron transport chain complex (located on the mitochondrial membrane of eukaryotic cells). It forms a complex with the ferric atom in this enzyme. The binding of cyanide ions to this cytochrome prevents electrons from being transferred from cytochrome c oxidase to oxygen. As a result, the electron transport chain is disrupted, and the cell can no longer perform aerobic respiration to produce ATP for energy. Tissues that rely primarily on aerobic respiration, such as the central nervous system and the heart, are particularly susceptible to this. Cyanide can also exert some toxic effects by binding to catalase, glutathione peroxidase, methemoglobin, hydrocobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, succinate dehydrogenase, and copper/zinc superoxide dismutase. Cyanide binds to the iron ions in methemoglobin to form inactive methemoglobin cyanide. (L97)

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Toxicity Data
LC50 (Rats)> 4670 mg/m3

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Non-human Toxicity Values
LD50 (Rats, Oral)> 5000 mg/kg
LD50 (Rats, Dermal)> 2000 mg/kg

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Toxicity Data
LC50 (Rats)> 4670 mg/m3

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Antidote and First Aid Measures
/SRP:/ Immediately take first aid measures: Ensure adequate decontamination has been performed. If the patient stops breathing, begin artificial respiration immediately, preferably using a ventilator on demand, bag-valve-mask, or simple breathing mask, and follow the training instructions. Perform cardiopulmonary resuscitation if necessary. Immediately flush contaminated eyes with running water. Do not induce vomiting. If vomiting occurs, lean the patient forward or place them in the left lateral decubitus position (head down if possible) to maintain an open airway and prevent aspiration. Keep the patient calm and maintain normal body temperature. Seek immediate medical attention. /Class A and Class B Poisoning/
/SRP:/ Basic Treatment: Establish a patent airway (using oropharyngeal or nasopharyngeal airways if necessary). Suction if necessary. Observe for signs of respiratory failure and provide assisted ventilation if necessary. Administer oxygen via a non-invasive ventilation mask at a flow rate of 10 to 15 liters per minute. Monitor for pulmonary edema and treat as necessary… Monitor for shock and treat as necessary… Anticipate seizures and treat as necessary… If eyes are contaminated, flush with water immediately. During transport, continuously flush each eye with 0.9% saline… Do not use emetics. In case of ingestion, rinse mouth and dilute with 5 mL/kg to 200 mL of water, provided the patient is able to swallow, has a strong gag reflex, and does not drool… After decontamination, cover skin burns with a dry, sterile dressing… /Class A and Class B Poisons/
/SRP:/ Advanced Treatment: For patients with altered mental status, severe pulmonary edema, or severe respiratory distress, consider oropharyngeal or nasopharyngeal endotracheal intubation to control the airway. Positive pressure ventilation with a bag-valve-mask may be effective. Consider medical treatment for pulmonary edema… Consider the use of a beta-agonist (such as salbutamol) for severe bronchospasm… Monitor heart rhythm and treat arrhythmias if necessary… Start intravenous infusion of 5% glucose solution /SRP: "Keep patent", minimum flow rate/. If signs of hypovolemia appear, use 0.9% normal saline (NS) or lactated Ringer's solution. Administer fluids with caution in cases of hypotension with signs of hypovolemia. Watch for signs of fluid overdose… Use diazepam or lorazepam to treat seizures… Use promecaine hydrochloride to assist eye irrigation… /Toxins A and B/

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Human Toxicity Excerpt
/Genetic Toxicity/In vitro human lymphocyte chromosome aberration assay: At moderate to severe cytotoxic doses (i.e., a reduction of ≥16-70% in mitotic cells), the assay showed positive chromosome aberration induction (5-50 ug/mL +S9) regardless of the presence of S9 activation.

[1]. Azoxystrobin amine: A novel azoxystrobin degradation product from Bacillus licheniformis strain TAB7. Chemosphere. 2021 Jun;273:129663.

[2]. Fungicidal effects of azoxystrobin and epoxiconazole on phyllosphere fungi, senescence and yield of winter wheat. Plant Pathology, 50: 190-205.

[3]. Fungicide azoxystrobin induced changes on the soil microbiome. Applied Soil Ecology, 2020, 145: 103343.

[4]. Oral azoxystrobin driving the dynamic change in resistome by disturbing the stability of the gut microbiota of Enchytraeus crypticus. J Hazard Mater. 2022 Feb 5;423(Pt B):127252.

Azoxystrobin is an aryloxypyrimidine compound with a 4,6-diphenoxypyrimidine skeleton. One benzene ring has a cyano group substituted at the C-2 position, while the other benzene ring has a 2-methoxy-1-(methoxycarbonyl)vinyl group substituted at the C-2 position. It inhibits mitochondrial respiration by blocking electron transfer between cytochrome b and c1, and is therefore widely used as an agricultural fungicide. Azoxystrobin can act as an inhibitor of the mitochondrial cytochrome bc1 complex, an exogenous substance, an environmental pollutant, an antifungal pesticide, and a quinone exogenous inhibitor. It is a nitrile, aryloxypyrimidine, acrylate, enol ether, methyl ester, and methoxyacrylate antifungal agent. Azoxystrobin is a methoxyacrylate analog and also a methoxyacrylate fungicide. Azoxystrobin (trade name: Anmidar, Syngenta) is a commonly used fungicide in agriculture. Azoxystrobin has the broadest known spectrum of activity among antifungal agents. This substance is used as an active ingredient to protect plants and fruits and vegetables from fungal diseases. Azoxystrobin binds very tightly to the Qo site of mitochondrial electron transport chain complex III, ultimately preventing ATP production. Azoxystrobin is widely used in agricultural production, especially in wheat cultivation.

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Mechanism of Action
Method of Action: A fungicide with protective, eradicative, systemic, and endophytic properties. It strongly inhibits spore germination and, in addition to inhibiting mycelial growth, also has antispore activity. Its mechanism of action is to inhibit mitochondrial respiration by blocking electron transport between cytochrome b and cytochrome c1.
Controls pathogenic strains resistant to 14 demethylase inhibitors, benzamides, dicarboxamides, or benzimidazoles.

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This study shows that Bacillus licheniformis TAB7 strain can convert (E)-AZ to (E)-3-amino-2-(2-((6-(2-cyanophenoxy)pyrimidin-4-yl)oxy)phenyl)acrylate (abbreviated as (E)-azoxystrobinamine) and its (Z)-isomer. In addition, this study reports a new AZ degradation/conversion product for the first time. (Z)-AZ may be an enzymatic product of AZ degradation, indicating that isomerases are involved in the conversion process. The results of this study enrich the list of known AZ degradation metabolites. Although it is not yet clear whether TAB7 produces the same metabolites in environments with low AZ concentrations, the discovery of these novel degradation products suggests the need for further investigation into the potential environmental impact and fate of these products, which will be explored in future studies. [1]

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The field trials conducted in this study showed that even when less than 1% of the leaf area was visibly affected by disease, there were extensive fungal-plant interactions on fungicide-treated wheat leaves that could only be observed under a microscope. The high frequency of defense responses against fungal infections is likely to have an adverse effect on the final yield due to the associated energy consumption. Therefore, the reduction of defense responses by azoxystrobin treatment compared to cyclooxygenazole treatment may partially explain why the green leaf retention and yield of azoxystrobin-treated plots were better than those of cyclooxygenazole-treated plots. The results of this study present an example of fungal control that can only be observed under a microscope, and its potential impact on crop yield may be attributed to other factors. [2]

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This study investigated the effects of azoxystrobin on microbial diversity and carbon source utilization in gray soils. The activities of three hydrolases (urease, invertase, and phosphatase) decreased, while the activity of catalase increased. Soil moisture content (AWCD) and utilization of six types of carbon sources both decreased. Overall, enzyme activity decreased with increasing azoxystrobin concentration. These results indicate that azoxystrobin and its metabolites inhibit microorganisms essential for soil carbon, nitrogen, and phosphorus cycling. Compared with the control group and azoxystrobin group 1, the soil bacterial community changes in the high-concentration azoxystrobin treatment groups (AZO 2 and AZO 3) were more significant. Therefore, the concentration of fungicide applied to the soil is an important consideration when considering the effects of soil microbiome. Although azoxystrobin is considered to have low toxicity, these results suggest that long-term application of azoxystrobin can affect soil bacterial communities, thereby inhibiting important soil nutrient cycling and thus affecting farmland productivity. However, it must be noted that this study mainly focused on gray soils, so the effects on other soils may vary depending on soil microbial communities and other soil parameters. Future research should focus on the effects of pyraclostrobin on soil nutrients and determine its safe dosage to ensure long-term soil health and productivity. [3]

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This study shows that oral pyraclostrobin severely interferes with the structure and composition of bacteria and fungi in the gut of Cryptosporidium, induces the growth of opportunistic pathogens, reduces the relative abundance of beneficial bacteria, and disrupts the stability of the Cryptosporidium gut microbiota. The study reveals the threat of environmental doses of pyraclostrobin to the gut health of Cryptosporidium. Meanwhile, AZ showed a dose-dependent effect on the normalized abundance and number of antibiotic resistance genes (ARGs) by altering the gut microbiota of E. crypticus. The results also showed that trace amounts of AZ (>0 and <0.085 μg/individual) may enrich ARGs in the gut of E. crypticus. Furthermore, scanning electron microscopy (SEM) results showed that bacterial/fungal (B/F) abundance was significantly correlated with ARG abundance (copy number/bacterial cell), indicating that the interaction between bacteria and fungi in the gut of Escherichia coli may be a key factor leading to changes in the number and abundance (copy number/bacterial cell) of ARGs. These findings provide a new perspective for assessing the gut health of soil animals and provide a theoretical basis for guiding the use of pesticides in intensive agricultural production. [4]

C22H17N3O5

403.39

403.12

C, 65.50; H, 4.25; N, 10.42; O, 19.83

143130-94-3

Azoxystrobin;131860-33-8

6537969

Typically exists as solids at room temperature

1.3±0.1 g/cm3

581.3±50.0 °C at 760 mmHg

305.3±30.1 °C

0.0±1.6 mmHg at 25°C

1.626

5.13

8

8

30

646

0

O(C1C=C(N=CN=1)OC1C=CC=CC=1C#N)C1C=CC=CC=1/C(=C/OC)/C(=O)OC

(Z)-Azoxystrobin; methyl (Z)-2-[2-[6-(2-cyanophenoxy)pyrimidin-4-yl]oxyphenyl]-3-methoxyprop-2-enoate; methyl (Z)-2-(2-(6-(2-cyanophenoxy)pyrimidin-4-yl)oxyphenyl)-3-methoxyprop-2-enoate; 893-412-2; (Z)-Azoxystrobin; 143130-94-3; (Z)-Methyl 2-(2-((6-(2-cyanophenoxy)pyrimidin-4-yl)oxy)phenyl)-3-methoxyacrylate; MMV021057; Z-Azoxystrobin;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)