Picoxystrobin   中文名称: 啶氧菌酯

Antifungal

嘧菌酯和啶氧菌酯是全球广泛使用的两种甲氧基丙烯酸酯类杀菌剂。本研究通过斑马鱼（Danio rerio）模型评估其对胚胎发育及多种酶活性的影响。将鱼卵分别暴露于两种杀菌剂（受精后24至144小时，hpf），测定死亡率、孵化率和畸形率；同时检测96 hpf幼鱼及成年雌雄鱼肝脏中三种抗氧化酶[过氧化氢酶（CAT）、超氧化物歧化酶（SOD）和过氧化物酶（POD）]、两种解毒酶[羧酸酯酶（CarE）和谷胱甘肽S-转移酶（GST）]的活性以及丙二醛（MDA）含量以探究毒性机制。胚胎发育实验显示，嘧菌酯和啶氧菌酯处理的鱼卵死亡率、孵化率及畸形率均呈现显著剂量-时间依赖性，144小时半数致死浓度（LC50）分别为1174.9和213.8 μg·L−1。在96 hpf幼鱼实验中，与对照组相比，暴露组CAT、POD、CarE和GST活性及MDA含量均随农药浓度显著升高。进一步研究发现，两种杀菌剂均导致成年鱼肝脏显著氧化应激且存在性别差异。结果表明啶氧菌酯比嘧菌酯对斑马鱼具有更强的胚胎发育毒性和氧化应激效应，而雄性斑马鱼肝脏解毒能力优于雌性。[1]

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甲氧基丙烯酸酯类是目前使用最广泛的杀菌剂，但对部分水生生物具有高毒性。本研究将斑马鱼胚胎暴露于三种杀菌剂（吡唑醚菌酯、肟菌酯和啶氧菌酯）不同浓度（受精后96小时内），评估其水生毒性。三种杀菌剂对胚胎的96小时LC50值分别为61、55和86 μg/L。急性暴露期间观察到胚胎出现心跳减缓、孵化抑制、生长迟滞及形态畸形等症状。此外，这些杀菌剂通过增加活性氧（ROS）和MDA含量、抑制SOD活性与GSH含量，并差异调节CAT活性及抗氧化系统相关基因（Mn-sod、Cu/Zn-sod、Cat、Nrf2、Ucp2和Bcl2）的mRNA水平诱导氧化应激。暴露还导致先天免疫相关因子IFN和CC-chem显著上调，并差异影响TNFa、IL-1b、C1C和IL-8的表达，表明其可引发胚胎发育期免疫毒性。三种杀菌剂引发的酶活性和基因表达差异响应可能是其毒性差异的原因。本研究通过多层面比较揭示了甲氧基丙烯酸酯类对水生生物的毒性效应。[2]

成年斑马鱼肝脏酶活性研究 [1]
采用20 L玻璃烧杯（每杯盛装15 L测试溶液并放养120尾2月龄斑马鱼，雌雄比1:1）进行成年鱼实验。测试浓度与2.4节（幼鱼研究）相同。暴露实验重复三次，每24小时更换溶液以维持化学物质浓度和水质稳定。分别于7、14、21和28天每组取样30尾鱼，参照2.4节方法分离肝脏测定CAT、SOD、POD、CarE和GST酶活性及MDA含量。

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化学分析 [1]
胚胎发育实验中，于每次换液前0和24小时分别采集暴露介质水样（每组10 mL），包括嘧菌酯（150、300、500、1000、1500、2000 μg·L−1）和啶氧菌酯（15、25、50、100、200、400 μg·L−1）组。采用超高效液相色谱-串联质谱（UPLC/MS/MS）测定实际浓度，以几何平均浓度计算LC50值。具体步骤：取5 mL水样加入10 mL离心管，加5 mL乙腈震荡30秒，再加入2 g NaCl和3 g MgSO4震荡1分钟，4000 rpm离心5分钟后上清液经0.22 μm微孔膜过滤，使用配备ACQUITYUPLC®BEHC181.7 μm色谱柱的Agilent 7890A UPLC/MS/MS系统，在电喷雾电离（ESI）和多反应监测（MRM）模式下进行分析。每个样品重复测定三次以验证标称浓度。

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

57.65

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.81 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.81 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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