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]
Embryonic 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.

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Enzyme activitiesin the larval zebrafish study [1]
A 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.

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Exposure for embryos acute toxicity [2]
Acute-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.

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Exposure for enzyme activity and gene expression tests [2]
Embryos 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 embryo acute toxicity test showed that the three strobilurin fungicides were highly toxic to zebrafish embryos. According to the 96-h LC50 values, trifloxystrobin was the most toxic one to embryos (55 μg/L), followed by pyraclostrobin (61 μg/L) and Picoxystrobin (86 μg/L) (Table 1).

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Effects of three strobilurins on embryonic development [2]
The results showed that the heartbeat of embryos at 72 hpf was significantly inhibited in all three fungicides treatments. The 20s heartbeat rate of embryos in 58.6 μg/L pyraclostrobin and trifloxystrobin, and 91 μg/L Picoxystrobin treatments dropped to 44.89%, 41.50% and 44.11% of their controls, respectively (Fig. 1A). Simultaneously, exposure to concentrations higher than 58.6 μg/L pyraclostrobin and trifloxystrobin, as well as 69 μg/L picoxystrobin resulted in significant decrease in hatching rate of embryos at 72 hpf. No hatching was observed in 73.0 μg/L trifloxystrobin treated group (Fig. 1B). At 96 hpf, the body length of hatched larvae in all three fungicides treatments were obviously reduced in a dose-dependent manner (Fig. 1C).

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Teratogenic effects on embryos caused by three strobilurins [2]
Pyraclostrobin, trifloxystrobin and Picoxystrobin induced morphological abnormalities during the embryonic development, including growth retardation, pericardial edema, yolk sac edema, yolk sac deformity and pigmentation defect (Fig. 2A). The percentage of cumulative malformation following exposure to the three strobilurins significantly increased in a dose-dependent manner (Fig. 2B). At 96 hpf, no malformed individual was observed in control group, while the malformation rate reached 100% in 73.0 μg/L pyraclostrobin, 58.6 and 73.0 μg/L trifloxystrobin, 91 and 105 μg/L picoxystrobin treated groups.

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Effect of three fungicides on ROS and MDA contents of embryos [2]
The results showed that ROS content in embryos was obviously induced by the highest concentrations of all three fungicides, with 1.28-, 1.63- and 1.49-fold increase in pyraclostrobin, trifloxystrobin and Picoxystrobin treated groups, respectively, in comparison with that of the control group (Fig. 3A). All concentrations of trifloxystrobin significantly induced MDA levels of zebrafish embryos, with 2.98-, 3.60- and 3.97-fold increase, respectively, when compared with that of control groups. Simultaneously, the MDA levels of embryos were elevated of 1.42-, 2.82-, 1.66- and 2.64-fold by 20 and 40 μg/L pyraclostrobin, 30 and 60 μg/L picoxystrobin, respectively, but were not significantly changed in 10 μg/L pyraclostrobin and 15 μg/L picoxystrobin treated groups (Fig. 3B).

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Effect of three fungicides on enzyme activity of embryos [2]
The results showed that the three strobilurin fungicides caused an apparent decrease of SOD activity (Fig. 4A) and GSH content (Fig. 4C) in embryos. The relative SOD level of embryos in 40 μg/L pyraclostrobin and trifloxystrobin, 30 and 60 μg/L Picoxystrobin treatments significantly reduced, which were only 0.77-, 0.63-, 0.69-, and 0.49-fold of the control group, respectively. The GSH content decreased in a dose-dependent manner in pyraclostrobin treatment group, and reduced GSH content was also observed in 40 μg/L trifloxystrobin and 60 μg/L picoxystrobin treated groups. The activity of CAT was significantly reduced by 40 μg/L pyraclostrobin and trifloxystrobin, but obviously induced by picoxystrobin at 15, 30 and 60 μg/L, with 1.85-, 1.96- and 1.48-fold increase, respectively, when compared with that of the control (Fig. 4B).

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Effect of three fungicides on gene expression of embryos [2]
Altered expression level of genes related to oxidative stress [2]
The mRNA expression of Mn-sod was obviously induced by all concentrations of trifloxystrobin and 15, 30 μg/L Picoxystrobin, but not by pyraclostrobin (Fig. 5A). The transcription of Cu/Zn-sod was induced by pyraclostrobin at concentration of 10 μg/L, but significantly inhibited at 20 and 40 μg/L (Fig. 5B). The mRNA level of Cat was markedly reduced by 40 μg/L pyraclostrobin and trifloxystrobin, which were only 0.67- and 0.57-fold of the control group, respectively (Fig. 5C). The mRNA expression level of Nrf2 decreased in all concentrations of pyraclostrobin and 60 μg/L picoxystrobin groups, while it increased in 20 and 40 μg/L trifloxystrobin treatments (Fig. 5D). The mRNA level of Ucp2 was significantly induced by 10 and 40 μg/L pyraclostrobin as well as 60 μg/L picoxystrobin, with 1.46-, 1.50- and 1.38-fold increase, respectively, when compared with that of the control (Fig. 5E). The transcription of Bcl2 was obviously inhibited by 20 and 40 μg/L pyraclostrobin and 60 μg/L picoxystrobin, but no significant alteration was observed in trifloxystrobin treated groups (Fig. 5F).

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Altered expression level of genes related to immune system [2]
The mRNA expression level of TNFα in embryos significantly decreased in all concentrations of trifloxystrobin, while no apparent change was observed in pyraclostrobin and Picoxystrobin treated groups (Fig. 6A). Pyraclostrobin at 20 and 40 μg/L obviously inhibited the transcription level of IL-1b, whereas 40 μg/L trifloxystrobin and 15 μg/L picoxystrobin markedly induced IL-1b expression in zebrafish embryos, with 1.53- and 3.68-fold increase, respectively (Fig. 6B). The expression levels of IFN in the highest concentrations of pyraclostrobin, trifloxystrobin and picoxystrobin were upregulated 4.66-, 2.38- and 2.21-fold, respectively, relative to the control group (Fig. 6C). The transcription level of CC-chem were induced by 20 and 40 μg/L pyraclostrobin, 40 μg/L trifloxystrobin and 60 μg/L picoxystrobin, with 1.73-,3.48-,1.87-, and 1.78-fold increase, respectively (Fig. 6D). Trifloxystrobin at 10 and 20 μg/L obviously inhibited the mRNA expression of C1C, while 40 μg/L pyraclostrobin induced C1C mRNA level by 1.62-fold when compared with that of the control group (Fig. 6E). Significant decrease of mRNA level of IL-8 was observed in zebrafish embryos at all concentrations of trifloxystrobin (Fig. 6F).

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29111 Picoxystrobin Fungicide Aquatic Plant Blue-green algae Anabaena flos-aquae N.R. 96 hr EC50 > 3000 limit test PPB
29115 Picoxystrobin R403814 degradate Fungicide Fishes Fathead minnow Pimephales promelas 0.59 g 96 hr LC50 > 10 (limt test) PPM
29116 Picoxystrobin Fungicide Aves Mallard duck Anas platyrhynchos 10 D 8 D LC50 > 5200 PPM
29117 Picoxystrobin Fungicide Aves Bobwhite quail Colinus virginianus 22 wk 14 D LD50 > 2250 MGK
29118 Picoxystrobin R408509 degradate Fungicide Fishes Fathead minnow Pimephales promelas 0.59 g 96 hr LC50 > 10 (limt test) PPM

[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 enoate ester that is the methyl ester of (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]prop-2-enoic acid. A cereal fungicide used to control a wide range of diseases including brown rust, tan spot, powdery mildew and net blotch. It has a role as a mitochondrial cytochrome-bc1 complex inhibitor and an antifungal agrochemical. It is an aromatic ether, an enoate ester, an enol ether, an organofluorine compound, a member of pyridines and a methoxyacrylate strobilurin antifungal agent.

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In the study of adult zebrafish liver enzyme activities, picoxystrobin treatments significantly activated GST enzyme activities of the male and female zebrafish livers at the early stage, whereas GST enzyme activity decreased at the later stage, which was attributed to the reduction of reaction substrate or competitive inhibition (Egaas et al., 1999). In all treated groups except for the 0.2 μg L−1-treated female group, CarE enzyme activity was significantly inhibited (P < 0.01) at 7 d but was significantly activated after 7 d. The MDA contents were significantly higher than those in the control because of the excess production of ROS at the early stage, and then the SOD enzyme activity was significantly activated. In the process of the entire test, the SOD enzyme activity of female zebrafish liver was significantly inhibited by the lowest dose (0.02 μg L−1) of picoxystrobin at 14 d, then recovered to the control level but was significantly activated by the highest dose (20 μg L−1) of picoxystrobin, whereas the SOD enzyme activity of male zebrafish liver was inhibited during the later period. The significantly activated CAT and POD enzyme activities of the male and female zebrafish livers from 14 to 28 d showed that picoxystrobin produced persistent oxidation damage. The results showed that oxidation damage would be more serious for the female than the male zebrafish, because the ability of male zebrafish livers to detoxify picoxystrobin was stronger than that of female zebrafish livers.
In summary, both azoxystrobin and picoxystrobin generated developmental toxicityand induced oxidative stress in larval zebrafish and adult zebrafish livers. Male zebrafish liver had a stronger ability to detoxify azoxystrobin or picoxystrobin than that of female zebrafish liver. Azoxystrobin would be a potential risk for larval zebrafish in China because of the relatively high environmental concentrations in runoff water. Picoxystrobin should also receive more attention because this fungicide led to higher embryonic development toxicity and oxidative stress than azoxystrobin in zebrafish, despite the fact that no report on the environmental concentrations of picoxystrobin residuals in natural waters is currently available. [1]

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In summary, our results demonstrated that pyraclostrobin, trifloxystrobin and picoxystrobin exhibited high level of acute toxicity to zebrafish embryos. Embryos exposed to the three fungicides showed decreased heart rate, hatching inhibition, growth regression, and morphological deformities in a concentration-dependent manner. The underlying mechanisms of this developmental toxicity might be partly related to the abnormal generation of ROS, increase of MDA content, change of antioxidant enzymes activities and mRNA levels of genes related to oxidative stress and immune system. The different changes of these parameters might be responsible for the toxicity difference between the three fungicides. As the molecule target of strobilurins on fungus is mitochondria complex Ⅲ, the influence of these fungicides on fish mitochondria is needed in future study to fully understand the toxic mechanisms of strobilurin 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.)