Antifungal

Azoxystrobin and Picoxystrobin are two primary strobilurin fungicides used worldwide. This study was conducted to test their effects on embryonic development and the activity of several enzyme in the zebrafish (Danio rerio). After fish eggs were separately exposed to azoxystrobin and picoxystrobin from 24 to 144 h post fertilization (hpf), the mortality, hatching, and teratogenetic rates were measured. Additionally, effects of azoxystrobin and picoxystrobin on activities of three important antioxidant enzymes [catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD)] and two primary detoxification enzymes [carboxylesterase (CarE) and glutathione S-transferase (GST)] and malondialdehyde (MDA) content in zebrafish larvae (96 h) and livers of adult zebrafish of both sexes were also assessed for potential toxicity mechanisms. Based on the embryonic development test results, the mortality, hatching, and teratogenetic rates of eggs treated with azoxystrobin and Picoxystrobin all showed significant dose- and time-dependent effects, and the 144-h LC50 values of azoxystrobin and picoxystrobin were 1174.9 and 213.8 μg L−1, respectively. In the larval zebrafish (96 h) test, activities of CAT, POD, CarE, and GST and MDA content in azoxystrobin and picoxystrobin-treated zebrafish larvae increased significantly with concentrations of the pesticides compared with those in the control. We further revealed that azoxystrobin and picoxystrobin exposure both caused significant oxidative stress in adult fish livers and the changes differed between the sexes. Our results indicated that picoxystrobin led to higher embryonic development toxicity and oxidative stress than azoxystrobin in zebrafish and the male zebrafish liver had stronger ability to detoxify than that of the females. [1]

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Strobilurins is the most widely used class of fungicides, but is reported highly toxic to some aquatic organisms. In this study, zebrafish embryos were exposed to a range concentrations of three strobilurins (pyraclostrobin, trifloxystrobin and Picoxystrobin) for 96 h post-fertilization (hpf) to assess their aquatic toxicity. The 96-h LC50 values of pyraclostrobin, trifloxystrobin and Picoxystrobin to embryos were 61, 55, 86 μg/L, respectively. A series of symptoms were observed in developmental embryos during acute exposure, including decreased heartbeat, hatching inhibition, growth regression, and morphological deformities. Moreover, the three fungicides induced oxidative stress in embryos through increasing reactive oxygen species (ROS) and malonaldehyde (MDA) contents, inhibiting superoxide dismutase (SOD) activity and glutathione (GSH) content as well as differently changing catalase (CAT) activity and mRNA levels of genes related to antioxidant system (Mn-sod, Cu/Zn-sod, Cat, Nrf2, Ucp2 and Bcl2). In addition, exposure to the three strobilurins resulted in significant upregulation of IFN and CC-chem as well as differently changed expressions of TNFa, IL-1b, C1C and IL-8, which related to the innate immune system, suggesting that these fungicides caused immunotoxicity during zebrafish embryo development. The different response of enzymes and genes in embryos exposed to the three fungicides might be the cause that leads to the difference of their toxicity. This work made a comparison of the toxicity of three strobilurins to zebrafish embryos on multi-levels and would provide a better understanding of the toxic effects of strobilurins on aquatic organisms[2].

Enzyme activities in the adult zebrafish liver study [1]
Glass beakers of 20 L with 120 zebrafish (2 months old; male and female ratio, 1:1) and 15 L of test solution per breaker were used for the adult zebrafish study. Test concentrations were the same as in 2.4. (larval zebrafish study). Exposure studies were also repeated three times, and the solution in each breaker was also renewed every 24 h to maintain a relatively constant test chemical concentration and water quality. Then, 30 zebrafish per treatment were sampled for enzyme activity assays at 7, 14, 21 and 28 d. The zebrafish livers were isolated for enzyme activity (CAT, SOD, POD, CarE, and GST) and MDA content assays according to the same method as in 2.4. (larval zebrafish study).

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Chemical analyses [1]
Water samples of exposure media (10 mL per replicate) for azoxystrobin (150, 300, 500, 1000, 1500, 2000 μg L−1) or Picoxystrobin (15, 25, 50, 100, 200, 400 μg L−1) in the embryonic development test were collected at 0 and 24 h before the renewal. The actual concentrations of azoxystrobin or Picoxystrobin were both determined by ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS). The final geometric mean concentrations were used for the LC50 calculation in the embryonic development test. Briefly, a 5 mL water sample for azoxystrobin or picoxystrobin was added to a 10 mL centrifuge tube, followed by the addition of 5 mL of acetonitrile; the solution was shocked for 30 s, and then 2 g of NaCl and 3 g of MgSO4 were added. The solution was shocked for 1 min and then centrifuged at 4000 rpm for 5 min, and finally, the upper liquid was filtered through a 0.22 μm micropore membrane for Agilent 7890A UPLC/MS/MS analysis equipped with an ACQUITYUPLC®BEHC181.7 μm column and Electro-Spray Ionization (ESI) at multiple reaction monitoring (MRM) mode. Each sample was repeated three times for the validation of nominal concentrations.

Embryonic development test [1]
\nEmbryonic development toxicity was tested according to OECD Guideline 210 (OECD, 2013) with some modifications. A group of twenty fertilized eggs at 3 hpf were exposed to each test concentration in a standard 24-well plate (one egg and 2 mL of solution per well), and the spare four wells were treated as controls (the system water). For the LC50, embryos were exposed to azoxystrobin at nominal concentrations of 0, 150, 300, 500, 1000, 1500, and 2000 μg L−1 or Picoxystrobin at nominal concentrations of 0, 15, 25, 50, 100, 200, and 400 μg L−1. An additional group of 20 embryos were exposed to the solvent acetone solutions on a separate 24-well plate, which served as a solvent control. A positive control at the fixed concentration of 4 mg L−1 3,4-dichloroaniline was performed with each egg batch used for testing. Exposure studies were repeated three times, and the exposure solution in each well was renewed every 24 h to maintain a relatively constant test chemical concentration. The plates were placed in an incubator at 26 ± 1 °C. The mortality, hatching rates, and teratogenetic rates of the embryos were checked under an Olympus BX63 microscope at 24, 48, 72, 96 and 144 hpf.\n

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\n\nEnzyme activitiesin the larval zebrafish study [1]
\nA standard 6-well plate with 30 fertilized eggs at 3 hpf and 10 mL of test solution per well was used for the larval zebrafish study. Test concentrations were based on preliminary LC50 results of the adult acute toxicity test (data not given), and the highest dose was set at the previous LC50/6. The test solutions were a series of concentrations of azoxystrobin (nominal concentrations of 0, 0.25, 2.5, 25, and 250 μg L−1) and Picoxystrobin (nominal concentrations of 0, 0.02, 0.2, 2, and 20 μg L−1). Exposure studies were repeated three times and the solution in each well was also renewed every 24 h to maintain a relatively constant test chemical concentration and water quality. The plates were placed in an incubator at 26 ± 1 °C for 96 h, and the dead eggs or larval zebrafish were removed promptly during the exposure. Then, the treated larval zebrafish were used for enzyme activity (CAT, POD, CarE, and GST) and MDA content assays according to the manufacturer's recommendations. The activities of CAT, POD, CarE, and GST are expressed as U mg−1 based on protein content. The MDA content is expressed as nmol mg−1.\n

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\n\n\n\t\nExposure for embryos acute toxicity [2]
\nAcute-toxicity test of zebrafish embryo was conducted according to the OECD Draft Proposal-Fish Embryo Toxicity (FET) Test (OECD, 2013) and a previously proposed method (Fraysse et al., 2006). Embryos at 2 hpf were randomly distributed in 24-well culture plates (2 mL solution and 1 embryo per well) for exposure to the test solutions (pyraclostrobin: 30.0, 37.5, 47.0, 58.6, 73.0 μg/L; Trifloxystrobin: 30.0, 37.5, 47.0, 58.6, 73.0 μg/L; Picoxystrobin: 60, 69, 79, 91, 105 μg/L) for 96 h. Test concentrations were designed based on pre-experiment data (data not shown). Reconstituted water was used to prepare all test solutions, which was also served as blank control. Solvent control was arranged containing the same acetone and Tween-80 contents as that in the test solutions with the highest concentrations of each fungicide. In each 24-well plate, 20 wells contained test solution, and 4 wells contained reconstituted water as the internal control. Each concentration and control replicated three times (per plate as one replicate) and contained 60 embryos. All tested 24-well plates were placed in an incubator (27 ± 1 °C; 14:10 h light/dark photoperiod). The plates were covered with transparent lids to prevent evaporation. The exposure solution was renewed every 24 h to keep the appropriate concentration of fungicides and water quality. Dead individuals were immediately removed during exposure. Morphological development and abnormalities were checked daily and recorded using an inverted microscope. The heartbeat rates were measured by counting the number of heartbeat of surviving zebrafish embryos/larvae at 72 hpf in a 20 s period using a microscope. Hatching rate of embryos was calculated as a percentage of the hatched eggs at 72 hpf. The body length of 96 hpf larvae was measured by using Aigo GE-5.\n

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\n\nExposure for enzyme activity and gene expression tests [2]
\nEmbryos at 2 hpf were randomly transferred into test solutions (pyraclostrobin: 0, 10, 20, 40 μg/L; Trifloxystrobin: 0, 10, 20, 40 μg/L; Picoxystrobin: 0, 15, 30, 60 μg/L) in 1 L beakers. The concentrations were selected based on the results of acute toxicity and some reported environmental concentrations. The lowest concentration was about 1/6 of the 96 h-LC50 value and lower than that detected in paddy water in China (Cao et al., 2015; Guo et al., 2016); the highest concentration was about 2/3 of the 96 h-LC50 value and had adverse effects on embryos. Each beaker contained 500 mL of exposure solution and 200 embryos, and there were 3 beakers in each concentration treatment. The external conditions during exposure, including the temperature, humidity and light cycle, were the same as that in the acute toxicity test. The exposure solution was renewed every 24 h to keep the appropriate concentration of fungicides and water quality. At 96 hpf, embryos (120 for antioxidant index measurement; 30 for RNA extraction) from each replicate were collected and washed twice with reconstituted water. The embryo samples were stored at −80 °C for further study.

Acute toxicity of three fungicides to zebrafish embryos[2]
The results of the acute toxicity test on embryos showed that the three methoxyacrylate fungicides were highly toxic to zebrafish embryos. According to the 96-hour LC50 value, trifluoperazon was the most toxic to embryos (55 μg/L), followed by Picoxystrobin (61 μg/L) and Picoxystrobin (86 μg/L) (Table 1).

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Effects of three methoxyacrylate fungicides on embryonic development[2]
The results showed that in embryos fertilized after 72 hours, treatment with the three fungicides significantly inhibited the embryonic heartbeat. In the treatment groups with Picoxystrobin and trifluoperazon at a concentration of 58.6 μg/L and Picoxystrobin at a concentration of 91 μg/L, the 20-second heartbeat rate of the embryos decreased to 44.89%, 41.50% and 44.11% of the control group, respectively (Figure 1A). Meanwhile, exposure to Picoxystrobin and trifluralin at concentrations higher than 58.6 μg/L, and Picoxystrobin at a concentration of 69 μg/L, all resulted in a significant decrease in embryo hatching rate after 72 hours. No hatching was observed in the trifluralin treatment group at a concentration of 73.0 μg/L (Fig. 1B). At 96 hours post-fertilization (hpf), the body length of hatched larvae in all three fungicide treatment groups was significantly reduced in a dose-dependent manner (Fig. 1C).

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Teratogenic effects of three methoxyacrylate fungicides[2] on embryos
Picoxystrobin, trifluralin, and Picoxystrobin induce morphological abnormalities during embryonic development, including growth retardation, pericardial edema, yolk sac edema, yolk sac malformation, and pigmentation defects (Fig. 2A). After exposure to these three methoxyacrylate fungicides, the cumulative malformation rate increased significantly in a dose-dependent manner (Fig. 2B). At 96 hours (96 hpf) after fertilization, no deformed individuals were observed in the control group, while the deformity rate of the treatment groups treated with Picoxystrobin 73.0 μg/L, trifluoperazon 58.6 and 73.0 μg/L, and Picoxystrobin 91 and 105 μg/L all reached 100%.

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Effects of the three fungicides on embryonic ROS and MDA content [2]
The results showed that the highest concentrations of the three fungicides could significantly induce an increase in ROS content in the embryos. Compared with the control group, the ROS content of the Picoxystrobin, trifluoperazon, and Picoxystrobin treatment groups increased by 1.28 times, 1.63 times, and 1.49 times, respectively (Figure 3A). Compared with the control group, all concentrations of trifluoperazon significantly induced the MDA level of zebrafish embryos, increasing by 2.98 times, 3.60 times, and 3.97 times, respectively. Meanwhile, treatments with Picoxystrobin at 20 and 40 μg/L, and Picoxystrobin at 30 and 60 μg/L increased the MDA level of embryos by 1.42 times, 2.82 times, 1.66 times, and 2.64 times, respectively, while the MDA level in the Picoxystrobin at 10 μg/L and Picoxystrobin at 15 μg/L did not change significantly (Figure 3B).

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Effects of the three fungicides on embryonic enzyme activity [2]
The results showed that all three methoxyacrylate fungicides significantly reduced SOD activity (Figure 4A) and GSH content (Figure 4C) in embryos. In the 40 μg/L Picoxystrobin and trifluralin, 30 μg/L Picoxystrobin and 60 μg/L Picoxystrobin treatment groups, the relative SOD level of embryos was significantly reduced, only 0.77 times, 0.63 times, 0.69 times, and 0.49 times that of the control group, respectively. The GSH content in the Picoxystrobin treatment group decreased in a dose-dependent manner, and the GSH content was also decreased in the 40 μg/L trifluoperazon and 60 μg/L Picoxystrobin treatment groups. 40 μg/L Picoxystrobin and trifluoperazon significantly reduced the activity of CAT, while 15, 30 and 60 μg/L Picoxystrobin significantly induced the activity of CAT, with the activity increasing by 1.85 times, 1.96 times and 1.48 times respectively compared with the control group (Figure 4B).

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Effects of the three fungicides on embryonic gene expression [2]
Changes in the expression level of oxidative stress-related genes [2]
All concentrations of trifluoperazon and 15 and 30 μg/L Picoxystrobin significantly induced the mRNA expression of Mn-sod, while Picoxystrobin had no inducing effect (Figure 5A). Picoxystrobin at a concentration of 10 μg/L induced Cu/Zn-sod transcription, but significantly inhibited it at concentrations of 20 and 40 μg/L (Fig. 5B). Both 40 μg/L Picoxystrobin and trifluoperazon significantly reduced Cat mRNA levels, by only 0.67-fold and 0.57-fold, respectively, compared to the control group (Fig. 5C). Nrf2 mRNA expression levels were decreased in all Picoxystrobin and 60 μg/L Picoxystrobin treatments, but increased in the 20 and 40 μg/L trifluoperazon treatments (Fig. 5D). Compared to the control group, 10 μg/L, 40 μg/L, and 60 μg/L Picoxystrobin significantly induced Ucp2 mRNA levels, increasing them by 1.46-fold, 1.50-fold, and 1.38-fold, respectively (Fig. 5E). 20 μg/L, 40 μg/L Picoxystrobin, and 60 μg/L Picoxystrobin significantly inhibited Bcl2 transcription, but no significant changes were observed in the trifluoperazine treatment group (Fig. 5F).

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Changes in expression levels of immune system-related genes[2]
In all trifluoperazine treatment groups, the mRNA expression level of TNFα in embryos was significantly reduced, while no significant changes were observed in the Picoxystrobin and Picoxystrobin treatment groups (Fig. 6A). At Picoxystrobin concentrations of 20 and 40 μg/L, the transcription level of IL-1β was significantly inhibited; while 40 μg/L trifluoperazine and 15 μg/L Picoxystrobin significantly induced the expression of IL-1β in zebrafish embryos, upregulating by 1.53-fold and 3.68-fold, respectively (Fig. 6B). Compared with the control group, the expression levels of IFN were upregulated by 4.66-fold, 2.38-fold, and 2.21-fold, respectively, after treatment with the highest concentrations of Picoxystrobin, trifluoperazine, and Picoxystrobin (Fig. 6C). CC-chem transcription levels were induced by 20 and 40 μg/L Picoxystrobin, 40 μg/L trifluoperazine, and 60 μg/L Picoxystrobin, increasing by 1.73-fold, 3.48-fold, 1.87-fold, and 1.78-fold, respectively (Fig. 6D). 10 and 20 μg/L trifluoperazine significantly inhibited C1C mRNA expression, while 40 μg/L Picoxystrobin increased C1C mRNA levels by 1.62-fold compared to the control group (Fig. 6E). IL-8 mRNA levels were significantly reduced in zebrafish embryos under all concentrations of trifluoperazine treatment (Fig. 6F).

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29111t Picoxystrobin fungicide t Aquatic plants t Blue-green algae Anabaena flos-aquaet N.R. t96 hrt EC50 t>t3000 extreme test tPPB
29115t Picoxystrobin R403814 degrading agent t fungicide t Fish Fathead minnow tPimephales promelast 0.59 gt96 hrt LC50 t>t10 (extreme test) tPPM
29116t Picoxystrobin fungicide t Mallard duck t Anas platyrhynchost 10 Dt8 DtLC50 t>t5200 tPPM
29117t Picoxystrobin fungicide t Avest white quail Colinus virginianus t22 weeks t14 DtLD50t>t2250tMGK
29118tPicoxystrobin R408509Degrading agenttFungicidetFishestPimephalespromelast0.59gt96hrtLC50t>t10(Extreme Test)tPPM

[1]. Effects of two strobilurins (azoxystrobin and picoxystrobin) on embryonic development and enzyme activities in juveniles and adult fish livers of zebrafish (Danio rerio). Chemosphere. 2018 Sep;207:573-580.
[2]. Developmental toxicity, oxidative stress and immunotoxicity induced by three strobilurins (pyraclostrobin, trifloxystrobin and picoxystrobin) in zebrafish embryos . Chemosphere, 2018, 207: 781-790.

Picoxystrobin is an acrylate, specifically the methyl ester of (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]prop-2-enoic acid. It is a cereal fungicide used to control various diseases, including brown rust, brown spot, powdery mildew, and net blotch. It is a mitochondrial cytochrome bc1 complex inhibitor and antifungal pesticide. It is an aromatic ether, acrylate, enol ether, organofluorine compound, pyridine compound, and also a methoxyacrylate antifungal agent. In studies on liver enzyme activity in adult zebrafish, Picoxystrobin treatment significantly activated glutathione S-transferase (GST) activity in the livers of both male and female zebrafish in the early stages, while GST activity decreased in the later stages, attributed to reduced substrate or competitive inhibition (Egaas et al., 1999). Except for the female zebrafish treated with 0.2 μg L⁻¹, CarE enzyme activity was significantly inhibited in all treatment groups at day 7 (P < 0.01), but significantly activated after day 7. Due to excessive early production of reactive oxygen species (ROS), malondialdehyde (MDA) levels were significantly higher than in the control group, followed by significant activation of superoxide dismutase (SOD) activity. Throughout the experiment, SOD activity in the livers of female zebrafish was significantly inhibited by the lowest dose (0.02 μg L⁻¹) of Picoxystrobin on day 14, subsequently returning to control levels, but was significantly activated by the highest dose (20 μg L⁻¹) of Picoxystrobin; while SOD activity in the livers of male zebrafish was inhibited later. Catalase (CAT) and peroxidase (POD) activities in the livers of both male and female zebrafish were significantly activated from day 14 to day 28, indicating that Picoxystrobin caused sustained oxidative damage. The results showed that oxidative damage was more severe in female zebrafish than in male zebrafish because the liver of male zebrafish had a stronger detoxification capacity for Picoxystrobin than that of female zebrafish. In summary, both azoxystrobin and Picoxystrobin exhibited developmental toxicity and oxidative stress in the livers of zebrafish larvae and adults. The liver of male zebrafish had a stronger detoxification capacity for either azoxystrobin or Picoxystrobin than that of female zebrafish. Due to the relatively high environmental concentration of azoxystrobin in runoff water, it may pose a potential risk to Chinese zebrafish larvae. Although there are currently no reports on the residual environmental concentration of Picoxystrobin in natural water bodies, Picoxystrobin should also be given more attention because it has higher toxicity and oxidative stress effects on zebrafish embryonic development than azoxystrobin. [1] In summary, our results indicate that Picoxystrobin, trifluralin and Picoxystrobin exhibit high acute toxicity to zebrafish embryos. Embryos exposed to these three fungicides all exhibited decreased heart rate, hatching inhibition, growth regression, and morphological deformities, and these symptoms were concentration-dependent. The potential mechanism of this developmental toxicity may be partly related to the abnormal generation of reactive oxygen species (ROS), increased malondialdehyde (MDA) content, changes in antioxidant enzyme activity, and changes in the mRNA levels of genes related to oxidative stress and the immune system. These different changes in parameters may be the reason for the differences in toxicity among the three fungicides. Since the target of methoxyacrylate fungicides for fungal action is mitochondrial complex III, future research is needed on the effects of these fungicides on fish mitochondria to fully understand the toxic mechanisms of methoxyacrylate fungicides on aquatic organisms. [2]

C18H16F3NO4

367.32

367.103

C, 58.86; H, 4.39; F, 15.52; N, 3.81; O, 17.42

117428-22-5

Picoxystrobin-d3

11285653

Typically exists as solid at room temperature

1.3±0.1 g/cm3

453.1±45.0 °C at 760 mmHg

75°

227.9±28.7 °C

0.0±1.1 mmHg at 25°C

1.522

4.48

0

8

7

26

495

0

CO/C=C(\C1=CC=CC=C1COC2=CC=CC(=N2)C(F)(F)F)/C(=O)OC

IBSNKSODLGJUMQ-SDNWHVSQSA-N

InChI=1S/C18H16F3NO4/c1-24-11-14(17(23)25-2)13-7-4-3-6-12(13)10-26-16-9-5-8-15(22-16)18(19,20)21/h3-9,11H,10H2,1-2H3/b14-11+

methyl (E)-3-methoxy-2-[2-[[6-(trifluoromethyl)pyridin-2-yl]oxymethyl]phenyl]prop-2-enoate

Picoxystrobin; 117428-22-5; Picoxystrobin [ISO]; UNII-62DH7GEL1P; 62DH7GEL1P; DTXSID9047542; CHEBI:83197; PICOXYSTROBIN [MI];

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)

DMSO : 100 mg/mL (272.24 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.81 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.81 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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