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 given 14 consecutive daily oral doses of unlabelled azoxystrobin at 1 mg/kg bw followed by a single oral dose of (14)C-pyrimidinyl-labelled azoxystrobin at 1 mg/kg bw. For the repeated doses, about 89.1% and 86.5% of the administered dose was excreted in the feces of the males and females rats within 7 days, respectively, and about 12.5% and 17.0% of the administered dose was excreted in the urine of the males and females rats within 7 days, respectively. In males and females, excretion of radioactivity was rapid, with > 96% being excreted during the first 48 hr. Approximately 0.62% and 0.39% of the administered dose was found in the carcass and tissues within 7 days after dosing in male and female rats, respectively. For the repeated dose, the highest concentrations of azoxystrobin-derived radioactivity were found in the kidneys (males and females, < 0.04 ug equivalents/g). The concentrations found in the liver were 0.02 and 0.01 ug equivalents/g for males and females, respectively. At termination, the total concentration of radioactivity in blood was 0.01 ug equivalents/g for males and females.
In toxicokinetic studies, groups of male and female Alpk:APfSD rats (five to eight per group, depending on experiment) were given azoxystrobin (purity, 99%) with or without pyrimidinyl label as a single dose at 1 or 100 mg/kg bw by gavage or as 14 repeated doses of 1 mg/kg bw per day. Biliary metabolites were assessed using rats with cannulated bile ducts given a single dose at 100 mg/kg bw by gavage. The vehicle was polyethylene glycol (PEG 600) at 4 mL/kg bw. Treated rats were housed in stainless steel metabolism cages for 7 days. Urine was collected at 6 hr, and urine and feces were collected separately at 12, 24, 36, 48 h and at 24 hr intervals until 7 days after dosing. At each collection, cages were rinsed with water and cage-washing collected together with the urine. At the end of the study, cages were thoroughly rinsed with ethanol/water (1:1 v/v) and retained for radiochemical analysis. Carbon dioxide and volatiles were trapped. After 7 days, various organs and tissues were removed and analyzed for radioactivity. ... For rats receiving a single lower dose (1 mg/kg bw), total excretion of radioactivity (urine, feces, and cage wash) was 93.75% and 91.44% for males and females, respectively over the 7 days. Most (> 85%) of the urinary and fecal excretion took place during the first 36 hr after dosing. In these rats, about 83.2% and 72.6% of the administered dose was excreted in the feces of males and females within 7 days, respectively, and about 10.2% and 17.9% of the administered dose was excreted in the urine of the males and females within 7 days, respectively. Approximately 0.34% and 0.31% of the administered dose was found in the carcass and tissues within 7 days after dosing in males and females, respectively. For rats at this dose (1 mg/kg bw), the highest concentrations of radiolabel were found in the liver (mean for males and females, 0.009 ug equivalents/g) and in the kidneys (males, 0.027 ug equivalents/g; and females, 0.023 ug equivalents/g). At termination, the total concentration of radioactivity in blood was 0.004 ug equivalents/g for males and females. Less than 0.6% of the administered dose was recovered in the expired. For rats receiving the single higher dose (100 mg/kg bw), total excretion of radioactivity (urine, feces, and cage wash) was 98.29% and 97.22% for males and females, respectively, over the 7 days. Most (> 82%) of the urinary and fecal excretion took place during the first 48 hr after dosing. At this dose, about 89.37% and 84.53% of the administered dose was excreted in the feces of the males and females within 7 days, respectively, and about 8.54% and 11.54% of the administered dose was excreted in the urine of the males and females within 7 days, respectively. Approximately 0.33% and 0.33% of the administered dose was found in the carcass and tissues within 7 days after dosing in males and females rats, respectively. At this higher dose, the highest concentrations of radiolabel were found in the kidneys (males, 1.373 ug equivalents/g; and females, 1.118 ug equivalents/g) and in the liver (males, 0.812 ug equivalents/g; and females, 0.714 ug equivalents/g). At termination, the total concentration of radioactivity in blood was 0.389 ug equivalents/g for males and 0.379 ug equivalents/g for females
The excretion and tissue distribution of radioactivity was investigated for 48 h in male and female rats given a single dose of azoxystrobin at 1 mg/kg bw by gavage. Treated rats were housed in metabolism cages to facilitate the collection of urine, feces, exhaled air and volatiles. One male and one female rat receiving azoxystrobin radiolabelled in each position were killed at 24 hr and 48 hr after dosing. Each carcass was frozen and sectioned in preparation for whole-body radiography. About 89% and 86% of the administered dose of (14)C-pyrimidinyl-labelled azoxystrobin was excreted within 48 hr in the urine and feces of male and female rats, respectively. Most of the radioactivity was excreted in the feces, with < 17% in the urine. The male and female rats treated with (14)C-phenylacrylate-labelled azoxystrobin excreted about 80% and 97% of the administered dose within 48 hr, respectively. Most of the radioactivity was excreted via the feces with < 21% in the urine. At 48 hr, males and females, excreted approximately 0.01% of the administered dose as carbon dioxide trap and approximately 0.01% as volatile metabolites. The male and female rats treated with (14)C-cyanophenyl- labelled azoxystrobin excreted about 95% and 98% of the administered dose within 48 hr, respectively. Most of the radioactivity was excreted via the feces, with < 16% in the urine. At 48 hr, males and females excreted small amounts of radioactivity as carbon dioxide (< 0.3%) and as volatile metabolites (0.01%). For all radiolabels, the distribution of radioactivity was similar in males and females, as shown by whole-body autoradiography. At 24 hr, most of the radiolabel was present in the alimentary canal, moderate amounts in the kidneys and small amounts in the liver. Forty-eight hours after dosing, the whole-body autoradiography results showed a marked reduction in radioactivity. The results of these studies indicated that there were no significant differences between the rates and routes of excretion or tissue distribution of azoxystrobin labelled in one of three positions. No sex-related difference in excretion profile was evident. Minor differences in excretion were primarily due to the small numbers of rats used in the study. No significant differences in the amount of radioactivity recovered in the exhaled air and as volatiles were observed between the three radiolabels or between sexes. On the basis of the results of this study, other studies of excretion and tissue retention were conducted using only pyrimidinyl-labelled azoxystrobin.

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Metabolism / Metabolites
... (14)C-Cyanophenyl-labelled azoxystrobin was given to bile duct cannulated and non-cannulated rats at a dose of 100 mg/kg bw. Samples of urine, feces and bile were collected for up to 72 hr. The purpose of this study was to reevaluate certain plant and goat metabolites that were previously not identified in rats and further elucidate the metabolic pathway of azoxystrobin in rats. Three further metabolites, previously detected in either plants or goats, were identified. Compound 13 (2-hydroxybenzonitrile), resulting from cleavage of the diphenyl ether link, was detected in the bile and urine as the glucoronide conjugate at a concentration of up to 1.8% of the administered dose. Compound 20 ((2-(6-(2-cyanophenoxy) pyrimidin-4-yloxy) phenyl)acetic acid) was also detected in the bile and urine at a concentration of up to 1.3%. Compound 35 (2-(2-(6-(2-cyanophenoxy) pyrimidin-4-yloxy) phenyl)glycolic acid) was detected in the urine, feces and bile at a concentration of 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 given azoxystrobin radiolabelled in either the pyrimidinyl, cyanophenyl or phenylacrylate rings at 100 mg/kg bw by gavage. Comparison of the rates and routes of excretion and the profile of the metabolites showed (as previously) that there were no significant differences in the metabolism of the three differently labelled forms, thus indicating that there was minimal cleavage of the ether linkages between the aromatic rings. Experiments designed to identify metabolites were therefore conducted in bile-duct cannulated rats given (14)C-pyrimidinyl labelled azoxystrobin by gavage. In the bile-duct cannulated rats, excreta, bile, and cage wash were collected at 6, 12, 24, 36, and 48 hr and stored at -20 °C. Samples of bile, feces and urine were collected between 0 hr and 48 hr and pooled. Samples for males and females were separated. Urine and feces were collected at up to 168 hr after dosing from rats given the single dose (higher or lower) and from rats receiving repeated doses for 14 days, and were used for quantification of metabolites. Some bile samples were enzymatically digested using cholylglycine hydrolase at 30 units/mL, pH 5.6 at 37 °C overnight. Metabolites were identified using various analytical techniques, such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), proton nuclear magnetic resonance spectroscopy (NMR) and mass spectrophotometry (MS). On the basis of biliary excretion data for rats given a single dose of either (14)C-pyrimidinyl-, (14)C-phenylacrylate-, or (14)C-cyanophenyl-labelled azoxystrobin at 100 mg/kg bw, 74.4% (males) and 80.7% (females) of the pyrimidinyl-derived radioactivity was excreted in the bile after 48 hr. For the cyanophenyl-derived radioactivity, 56.6% and 62.5% was excreted in the bile of males and females, respectively. For the phenylacrylate-derived radioactivity, 64.4% (males) and 63.6% (females) was excreted in the bile. Quantitatively, there were no significant differences in biliary excretion between males and females. Azoxystrobin was found to undergo extensive metabolism in rats. A total of 15 metabolites were detected in the excreta and subsequently identified. Seven additional metabolites were detected but not identified. None of the unidentified metabolites represented more than 4.9% of the administered dose. The quantitative data for the various metabolites in the faeces, urine and bile of rats receiving a single dose of azoxystrobin at 100 mg/kg bw ... . The mass balance for the study of metabolite identification indicated that a substantial percentage of the administered radiolabel (45.6-73.6%) was unaccounted for, although the studies of excretion showed total recovery of 91.75-103.99%, with 72.6-89.3% being in the feces. The percentage of unaccounted-for radiolabel was especially notable in the groups receiving a single lower dose and a repeated lower dose. The study authors indicated that the variable efficiency in recovery could be explained by the fact that, for metabolite identification, feces were extracted with acetonitrile which allowed partitioning of the parent compound when it was present in the faeces (i.e. rats receiving the higher dose). For the groups receiving a single lower dose or repeated lower dose (where quantities of the parent compound were minimal), most of the faecal radiolabel was associated with polar metabolites that would not be present in the acetonitrile extract. The resulting concentration of radiolabel in the extract would, therefore, be very low. For the group receiving the higher dose, greater amounts of parent compound were left unabsorbed, thereby resulting in greater amounts of parent compound available for partitioning into the acetonitrile extract. The glucuronide conjugate (metabolite V) was the most prevalent biliary metabolite in both males (29.3%) and females (27.4%). Metabolite I (parent compound) was not detected in the bile. Each of the other biliary metabolites accounted for between 0.9% and 9.0% of the administered dose. In the bile-duct cannulated rats, about 15.1% and 13.6% of the faecal radioactivity was metabolite I (parent compound) in male and female rats, respectively. No parent compound was detected in the urine of bile-duct cannulated male and female rats. The predominant metabolite in the urine of the bile-duct cannulated rats was unidentified metabolite 2, which accounted for about 1.8% and 2.0% of the administered dose in male and female rats, respectively. There was no evidence for a dose-influencing metabolism, but a sex-specific difference in biotransformation was observed, with females producing more metabolites than did males. Biotransformation was unaffected by dose. The study authors suggested that absorption was dose-dependent. The oral absorption at 1 mg/kg bw was nearly complete (100%) since no parent compound was detected. The oral absorption at the higher dose (100 mg/kg bw) was estimated to be approximately 74-81% since about 19-26% of the parent compound was detected. However, it is difficult to estimate the true oral absorption value owing to poor recoveries after extraction, especially at the lower dose. ... There were two principal metabolic pathway: hydrolysis to the methoxyacid, followed by glucuronide conjugation to give metabolite V; and glutathione conjugation of the cyanophenyl ring followed by further metabolism via a number of intermediates (VI, VII, and VIII) to the mercapturic acid metabolite IX. Azoxystrobin was also hydroxylated at the 8 and 10 positions on the cyanophenyl ring followed by glucuronide conjugation (metabolites II, III, IVa and IVb). There were several minor pathways involving the acrylate moiety, resulting in formation of the metabolite XIII and XIV. Three metabolites (X, XII, and XV) arising via the cleavage of the ether linkages were identified.
The metabolic fate of [(14)C]-methyl-(E)-2-[2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl]-3-methoxyacrylate (azoxystrobin) was determined in the male and female rat following a single oral dose of 1 and 100 mg x kg(-1) and in surgically prepared, bile duct-cannulated rats following a single oral dose of 100 mg x kg(-1). 2. Azoxystrobin was extensively metabolized with at least 15 metabolites. There was a sex difference, with females producing more metabolites than males. 3. The two principal metabolic pathways were hydrolysis of the methoxyacid followed by glucuronic acid conjugation and glutathione conjugation of the cyanophenyl ring followed by further metabolism leading to the mercapturic acid. There were also several other minor pathways.
Organic nitriles are converted into cyanide ions through the action of cytochrome P450 enzymes in the liver. Cyanide is rapidly absorbed and distributed throughout the body. Cyanide is mainly metabolized into thiocyanate by either rhodanese or 3-mercaptopyruvate sulfur transferase. Cyanide metabolites are excreted in the urine. (L96)

Toxicity Summary
Organic nitriles decompose into cyanide ions both in vivo and in vitro. Consequently the primary mechanism of toxicity for organic nitriles is their production of toxic cyanide ions or hydrogen cyanide. Cyanide is an inhibitor of cytochrome c oxidase in the fourth complex of the electron transport chain (found in the membrane of the mitochondria of eukaryotic cells). It complexes with the ferric iron atom in this enzyme. The binding of cyanide to this cytochrome prevents transport of electrons from cytochrome c oxidase to oxygen. As a result, the electron transport chain is disrupted and the cell can no longer aerobically produce ATP for energy. Tissues that mainly depend on aerobic respiration, such as the central nervous system and the heart, are particularly affected. Cyanide is also known produce some of its toxic effects by binding to catalase, glutathione peroxidase, methemoglobin, hydroxocobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, succinic dehydrogenase, and Cu/Zn superoxide dismutase. Cyanide binds to the ferric ion of methemoglobin to form inactive cyanmethemoglobin. (L97)

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Toxicity Data
LC50 (rat) > 4670 mg/m3

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Non-Human Toxicity Values
LD50 Rat oral >5000 mg/kg
LD50 Rat percutaneous >2000 mg/kg

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Toxicity Data
LC50 (rat) > 4670 mg/m3

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Antidote and Emergency Treatment
/SRP:/ Immediate first aid: Ensure that adequate decontamination has been carried out. If patient is not breathing, start artificial respiration, preferably with a demand valve resuscitator, bag-valve-mask device, or pocket mask, as trained. Perform CPR if necessary. Immediately flush contaminated eyes with gently flowing water. Do not induce vomiting. If vomiting occurs, lean patient forward or place on the left side (head-down position, if possible) to maintain an open airway and prevent aspiration. Keep patient quiet and maintain normal body temperature. Obtain medical attention. /Poisons A and B/

/SRP:/ Basic treatment: Establish a patent airway (oropharyngeal or nasopharyngeal airway, if needed). Suction if necessary. Watch for signs of respiratory insufficiency and assist ventilations if needed. Administer oxygen by nonrebreather mask at 10 to 15 L/min. Monitor for pulmonary edema and treat if necessary ... . Monitor for shock and treat if necessary ... . Anticipate seizures and treat if necessary ... . For eye contamination, flush eyes immediately with water. Irrigate each eye continuously with 0.9% saline (NS) during transport ... . Do not use emetics. For ingestion, rinse mouth and administer 5 mL/kg up to 200 mL of water for dilution if the patient can swallow, has a strong gag reflex, and does not drool ... . Cover skin burns with dry sterile dressings after decontamination ... . /Poisons A and B/

/SRP:/ Advanced treatment: Consider orotracheal or nasotracheal intubation for airway control in the patient who is unconscious, has severe pulmonary edema, or is in severe respiratory distress. Positive-pressure ventilation techniques with a bag valve mask device may be beneficial. Consider drug therapy for pulmonary edema ... . Consider administering a beta agonist such as albuterol for severe bronchospasm ... . Monitor cardiac rhythm and treat arrhythmias as necessary ... . Start IV administration of D5W /SRP: "To keep open", minimal flow rate/. Use 0.9% saline (NS) or lactated Ringer's if signs of hypovolemia are present. For hypotension with signs of hypovolemia, administer fluid cautiously. Watch for signs of fluid overload ... . Treat seizures with diazepam or lorazepam ... . Use proparacaine hydrochloride to assist eye irrigation ... . /Poisons A and B/

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Human Toxicity Excerpts
/GENOTOXICITY/ In vitro chromosome aberrations in human lymphocytes assay: The test was positive for the induction of chromosomal aberrations in both the presence and absence of S9 activation at doses (5-50 ug/mL +S9) that were moderately to severely cytotoxic (ie, > or = 16-70% reductions in mitotic cells, respectively).

[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 having a 4,6-diphenoxypyrimidine skeleton in which one of the phenyl rings is cyano-substituted at C-2 and the other carries a 2-methoxy-1-(methoxycarbonyl)vinyl substituent, also at C-2. An inhibitor of mitochondrial respiration by blocking electron transfer between cytochromes b and c1, it is used widely as a fungicide in agriculture. It has a role as a mitochondrial cytochrome-bc1 complex inhibitor, a xenobiotic, an environmental contaminant, an antifungal agrochemical and a quinone outside inhibitor. It is a nitrile, an aryloxypyrimidine, an enoate ester, an enol ether, a methyl ester and a methoxyacrylate strobilurin antifungal agent.
Azoxystrobin is a methoxyacrylate analog and a strobilurin fungicide.
Azoxystrobin (brand name Amistar, Syngenta) is a fungicide commonly used in agriculture. Azoxystrobin possesses the broadest spectrum of activity of all known antifungals. The substance is used as an active agent protecting plants and fruit/vegetables from fungal diseases. Azoxystrobin binds very tightly to the Qo site of Complex III of the mitochondrial electron transport chain, thereby ultimately preventing the generation of ATP. Azoxystrobin is widely used in farming, particularly in wheat farming.

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Mechanism of Action
Mode of action: fungicide with protectant, eradicant, translaminar & systemic properties. Powerfully inhibits spore germination &, in addition to its ability to inhibit mycelial growth, also shows antisporulant activity. Acts by inhibiting mitochondrial respiration by blocking electron transfer between cytochrome b & cytochrome c1. Controls pathogenic strains resistant to the 14 demethylase inhibitors, phenylamides, dicarboxamides or benzimidazoles.

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In this work, we showed that B. licheniformis strain TAB7 can transform (E)-AZ into methyl (E)-3-amino-2-(2-((6-(2-cyanophenoxy)pyrimidin-4-yl)oxy)phenyl)acrylate ((E)-azoxystrobin amine in short) and its (Z)-isomer. Additionally, novel AZ degradation/transformation products are reported for the first time. (Z)-AZ is possibly an enzymatic reaction product of AZ degradation, highlighting the involvement of isomerases in the transformation process. The results from this study add to the list of known AZ degradation metabolites. Although it is still unknown whether TAB7 can produce the same metabolites in environments where AZ is present in lower concentrations, the discovery of these novel degradation products indicates that further studies on the potential effects and fates of these products in the environment are necessary and will be addressed in future studies. [1]

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The field trials conducted in this study showed that extensive fungal–plant interactions detectable only by microscopy do occur on fungicide-treated wheat leaves, even though only < 1% leaf area was visibly diseased. The high frequency of defence reactions against attempted fungal infection makes it highly probable that the associated energy expenditure can adversely influence the final yield. The reduction of defence reactions obtained with azoxystrobin treatments, particularly compared with treatment with epoxiconazole, can therefore be part of the explanation for the superior green leaf conservation and yield recorded for azoxystrobin-treated plots compared with epoxiconazole-treated plots. The present results demonstrate an example of fungal control, observable only by microscopy, with a probable influence on yield that otherwise might have been attributed to other factors. [2]

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In this study, we investigated the effect of azoxystrobin on the microbial diversity and the utilization of carbon sources in Spodosols. The activities of three hydrolytic enzymes (urease, invertase and phosphatase) decreased and catalase activity increased. The AWCD values and the utilization of six classes of carbon sources decreased. In general, as azoxystrobin concentrations increased, the enzyme activity decreased. These results suggest that microbes essential to cycling soil C, N, and P were inhibited by azoxystrobin and its metabolites. The bacterial communities of soil treated with higher azoxystrobin concentration (AZO 2 and AZO 3) changed much more than control and AZO 1. Thus, the concentration of fungicide applied to soils is important to consider when considering soil microbiome impacts. Although azoxystrobin is considered to have low toxicity, these findings suggest that persistent azoxystrobin application can impact bacterial soil communities, which may inhibit important soil nutrient cycling to sustain productive croplands. However, it must be noted that this study focused on Spodosols, thus impacts to other soils may vary depending on microbial communities present and various other soil parameters. Future work should focus on quantifying soil nutrient impacts and determining safe doses of azoxystrobin to ensure long term soil health and productivity. [3]

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This study showed that oral exposure of AZ severely interferes with the structure and composition of bacteria and fungi in the gut of E. crypticus, inducing the growth of opportunistic pathogens and reducing the relative abundance of beneficial bacteria as well as destroying the stability of the gut microecology of E. crypticus. It is revealed that the environmental dose of AZ threatened the gut healthy of E. crypticus. Meanwhile, AZ presented a dose-dependent effect on the normalized abundance and number of ARGs by changing the gut microbiota of E. crypticus. The result also concluded that the trace dose of AZ (> 0 and < 0.085 μg/individual) might enrich the ARGs in the gut of E. crypticus. Moreover, SEM showed that the B/F richness significantly correlated with the ARG abundance (copies/bacterial cell), suggesting that the interaction of bacteria and fungi in the gut of E. crypticus may be a key contributor to the shifts in the number and abundance of ARGs (copies/bacterial cell). These findings provided new perspectives for assessing the gut health of soil fauna and 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

Typically exists as solids at room temperature

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.)