Some standard content:
ICS 65. 100. 10
National Standard of the People's Republic of China
GB/T 19567.3---2004
Bacillus thuringiemsis wettable powder
Bacillus thuringgiensis wettable powder2004-06-22Promulgated
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaAdministrative Committee of Standardization of the People's Republic of China
Implementation on 2004-12-01
GB/T19567.3—2004
Bacillus thuringiemsis (B.) is the most widely used microbial insecticide in recent years. Its main insecticidal component is the toxin protein in the parasporal body. Among them, the relative molecular weight of the toxin protein toxic to lepidopteran pests detected in this standard is 130kDa. In the bioassay, beet armyworm is used to detect the toxicity of Bacillus thuringiensis wettable powders with characteristics against the genus Spodoptera of the order Spodoptera, and diamondback moth and cotton bollworm are used to detect the toxicity of Bacillus thuringiensis wettable powders with activity against other scale insect pests. This standard is formulated based on the relevant materials such as the Bacillus thuringiensis industry and enterprise standards previously formulated in my country, combined with the actual situation in my country. This standard makes specific requirements and provisions for the requirements, test methods, sampling, packaging, transportation, etc. of Bacillus thuringiensis wettable powders, thereby providing a unified technical basis for the production of Bacillus thuringiensis. Appendix A of this standard is an informative appendix, and Appendix B is a normative appendix. This standard was proposed by the Ministry of Agriculture of the People's Republic of China. The drafting units of this standard are the Pesticide Control Institute of the Ministry of Agriculture, the National Engineering Research Center for Microbial Pesticides of Huazhong Agricultural University, and the Hubei Provincial Biological Pesticide Engineering Research Center.
The main drafters of this standard are Jiang Hui, Yu Ziniu, Chen Shouwen, Gong Kaimei, Wang Xiaojun, Wu Xinping and Gu Baogen. This standard is interpreted by the Pesticide Testing Institute of the Ministry of Agriculture. Scope
Bacillus thuringiensis wettable powder
GB/T 19567.3--2004
This standard specifies the requirements, test methods, and signs, labels, packaging, storage and transportation of Bacillus thuringiensis wettable powder. This standard applies to Bacillus thuringiensis wettable powder made from Bacillus thuringiensis raw powder and adjuvants for the control of lepidopteran pests.
2 Normative references
The clauses in the following documents become the clauses of this standard through reference in this standard. For any dated referenced document, all subsequent amendments (excluding outdated content) or revisions are not applicable to this standard. However, parties to an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For any referenced document without an H date, the latest version shall apply to this standard. GB/T1250 Method for expressing and determining limit values GB/T1600---2001 Method for determining moisture content in liquid pesticides GB/T1601 Method for determining pH value of pesticides
GB/T1604 Acceptance rules for commercial pesticides
GB/T1605:2001 Sampling method for commercial pesticides GB3796 General test for pesticide packaging
GB/T54512001 Method for determining wettability of pesticide wettable powders GB/T16150--1995 Method for determining fineness of pesticide powders and wettable powders 3 Requirements
3.1 Appearance: Off-white or brown loose powder, without lumps. 3.2 The thuringia bud runner shade-avoidable powder should meet the requirements of Table 1 Table 1 Bacillus thuringiensis wettable powder control items Index items
Toxic protein (13Gke)/(%)
Toxicity titer (P.1., H.α., S. e.)/(IU/mg) pH value
Water/(%)
Suspension rate (active ingredient)/()
Mixing time/min
Fineness (75μm)/(%)
6. C-~7. 5
Note: P, 2.Ha and Se are the abbreviations of Pluteltex.ytastelta, Heliathtsarmigera and Spodupteraeriga, respectively.
CB/T 19567.3--2004
4 Test method
Unless otherwise specified, the reagents used in this method are all analytically pure and the solutions described are all water bath solutions. 4.1 Sampling
The "Sampling of Solid Preparations" in 2001 is carried out, and the sampling packages are determined by the random number table method. The final sampling is not in accordance with B/T16052
The minimum amount is 100g:
4.2 Toxin protein content
The content is determined by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel image processing method. 4.2.1 Method summary
The traceable powder of Bacillus thuringiensis is treated with an alkaline solution to degrade it into toxin protein. Then, by S10SPAGE, the toxin protein is separated from other impurities according to the difference in molecular weight of the protein pairs. After that, the protein zone area is scanned by electrophoresis image and quantified.
4.2.2 Instruments and equipment
a) Electrophoresis apparatus;
b) Vertical electrophoresis bottle (1.5mm concave grooved rubber frame), gel plate area 170mm×170mm1.5mm, 20-hole sample slot frame);
c) Electrophoresis gel imaging system:
d) Centrifuge: 10 000t/min
e) Analysis time: accurate to 0.0001.
4.2.3 Reagents and solutions
4.2.3.1 Ammonium persulfate (APS).
4.2.3.2 Sodium dodecyl sulfate (SDS), 4.2.3.3 Tetramethylethylenediamine (TFMEID). 4.2.3.4 Sodium oxide.
4.2.3.5 30% acrylamide gel mother solution: weigh 0.8 g of 30% acrylamide + methylene bisacrylamide (original name: methyl bisacrylamide), add it to 100 ml of distilled water, filter, and store in a dark place for later use. 4.2.3.6 Separation buffer: weigh 18.17% of trimethylol hexadecane (1-1S) and 0.4% of DS in distilled water, adjust pH to 8 with concentrated hydrochloric acid, and dilute to 100 mL with distilled water: 4.2.3.7 Concentration gel buffer: weigh 6.06% of 1RIS and 0.4% of SS in distilled water, adjust pH to 16.8 with concentrated hydrochloric acid, and dilute to 100 mL with distilled water.
4.2.3.8 Electrode buffer: weigh 3.036 g of Tris, 14.42 g of glycine, 1 μg of SDS, dissolve in water and make up to 1000 mL. 4.2.3.9 3× sample solution: 1 mol/L, pH 6.8 Tris-HCl 18.75 ml., SDS 6 g, glycerol 30 ml., mercaptoethanol 15 mL, a little phenol blue, make up to 100 mL with distilled water. 4.2.3.10 Fixing solution: weigh 500 mL of 95% ethanol, 160 mL of ice ethyl, make up to 1000 mL with distilled water. 4.2.3.11 Staining Weigh 1 g of Coomassie Brilliant Blue (CBB) R-250, add 250 mL of 95% ethanol, 80 mL of glacial acetic acid, and distilled water to make up to 1000 mL, dissolve and use: 4.2.3.12 Decolorizing solution: Take 250 ml of 95% ethanol and 80 ml of glacial acetic acid. Make up to 1000 ml with distilled water. 4.2.3.13 Toxin protein standard sample: original powder with a toxin strong white (relative molecular weight protein is 130 kDa) content of 8.0%. 4.2.4 Sample treatment Weigh 20.0 mg of standard sample and test sample (accurate to 0.1 mg), transfer to a 1.5 ml centrifuge tube, add 1. ml of water to fully float. Take 100 μL and add it to another 1.5mL centrifuge tube. Add 25 μL of 0.5 mol/L sodium hydroxide solution (so that the final concentration of sodium hydroxide solution is 0.1 mol/L). Let stand for about 5 min. Then add 75 μL of 3× sample diluent. Make the final volume 200 μL. Boil in 100℃ boiling water for 6 min. Centrifuge (2 gates r/mia) for 0 min and take the upper supernatant for electrophoresis. 4.2.5 SDS-PAGE separation of toxin protein
4.2.5.1 Preparation of 7.5% acrylamide gel using discontinuous buffer system. The gel preparation method is shown in Appendix A (informative appendix). 4.2.5.2 Sample loading
GB/T 19567.3--2004
Take the above standard sample solution.1 layer of clear solution, load 6, 8, 10, 12, 14 μL (toxin protein content is about 3g~7μg) in the loading hole of polyacrylamide gel respectively as standard curve; then take a fixed volume of sample solution 1 layer of clear solution (toxin protein content is about 5g), add it to the loading hole, inject electrode buffer and then turn on the power. 4.2.5.3 Electrophoresis
The initial voltage of electrophoresis is controlled at about 190V. After the sample enters the separation gel, increase the voltage to 170V. Continue electrophoresis. When the indicator front reaches about 1cm from the bottom, stop electrophoresis, take out the gel plate, and soak it in 7.5% (volume fraction) acetic acid for 30min. 4.2.5.4 Staining
Remove the separation gel part and stain it with Coomassie brilliant vegetable (CFB) R-250 staining solution overnight. 4.2.5.5 Decolorization
Pour off the staining solution, wash the gel with rinse solution first, then add decolorization solution, keep decolorizing at 37°C, change the decolorization solution several times, until the background disappears to
4.2.6 Determination
After decolorization, the protein band with a molecular weight of 130kLa can be clearly seen, and the gel is analyzed by gel imaging system. The percentage of bovine serum albumin in the sample (x) is calculated according to formula (1). X
X #X 100
M—the amount of protein calculated by the standard protein regression curve; c—the concentration of the sample,
V-the amount of sample added,
n—the dilution multiple of the sample.
4.2.7 Allowable difference
Take the arithmetic mean as the determination result. The relative deviation of the results of two parallel determinations is less than or equal to 5%. 4.3 Determination of group potency
Perform according to Appendix B (Normative Appendix).
4. 4 Determination of pH value
Perform according to (GB/T1601).
4. 5 Determination of fineness
Perform according to 2.2 of G3/T16150-1995. 4.6 Determination of water content
Perform according to the "azeotropic distillation method" in GB/T160G2001. 4.7 Determination of buoyancy rate
4.7.1 Instruments and reagents
a) Measuring cylinder: 250 mL, tool:
b) Liquid pipette:
) Stopwatch;
d) Erlenmeyer flask: 500 mL.100 ml.;
e) Constant temperature water bath:
) Standard hard water: Preparation method is carried out according to the standard hard water preparation method in GB/T5151. -1)
GB/T19567.3—2004
4.7.2 Operation steps
Weigh the wettable powder sample at 20℃.0 (accurate to 0.2), put it into a triangular bottle with glass beads, add 100mL of standard hard water, shake it left and right for 60 seconds, transfer all the floating water into a 250mL true stopper volume, and dilute it to 250mL with standard hard water.
Put the volume into a 30℃ 1 ℃: In a constant temperature water bath, when the suspension reaches 30℃, cover the cylinder, pick up the cylinder and gently shake the sediment, then regularly shake it up and down 305 with the center of the cylinder as the center to make a uniform suspension. Put the cylinder back in the constant temperature water bath, open the lid, move the arm for 30 minutes, and use the suction method to extract 9/10 of the upper part of the cylinder (complete within 15 minutes). During the extraction process, the pipette should descend along the cylinder wall with the surface, and do not stir the lower sediment. According to the method in the normative appendix (B), the toxicity potency of the wettable powder and the remaining 25mL suspension in the cylinder is determined. 4. 7.3 Calculation
The floating rate (Y) of the wettable powder is calculated according to formula (2): Y11.1×CQ×100%
Wherein;
C is the toxicity titer of the wettable powder to be tested;
Q is the toxicity titer of the 25 mL suspension left at the bottom of the measuring tube. 4.7.4 Allowable difference
The difference between the results of two repeated determinations should not exceed 10%. 4.8 Determination of mixing time
It shall be determined according to the "azeotropic distillation method\" in GB/T1600-2001. 5 Inspection rules
It shall comply with the relevant provisions of GB/F1601. The limit shall be handled according to GB/T1250. 6 Marking, labeling, packaging, storage and transportation
6.1 The product packaging shall comply with the provisions of GB3796, and the standard number used and the test insects used for the test shall be indicated. 6.2 Coagulable powders are mainly packaged in plastic bags and sealed. 6.3 When storing, avoid sunlight and pressure, and place in a cool and dry place. 6.4 When transporting, be careful to handle with care to prevent damage. 6.5 Warranty period: Under normal storage and transportation conditions, the quality guarantee period of wettable powder is two years from the month of production. The toxicity titer and toxin protein content of the product shall not be lower than the 3.2 index when it leaves the factory. Within two years, the toxicity titer and toxin protein content of the product shall not be lower than 70% of the 3.2 index. A.1 Plate preparation
Appendix 4
(Informative Appendix)
Preparation of electrophoresis gel
GB/T 19567.3--2004
This experiment uses a double vertical electrophoresis tank. The specific operation depends on the laboratory conditions. The basic operation is to select two glass plates of the same size according to the size of the electrophoresis plate, where the end of the plate has a groove of 2cm to 3cm high. After the two glass plates are cleaned and dried, put a "gap strip" (plastic strip or adhesive strip can be used, and its width and thickness are determined according to needs) on both sides of the glass plate without grooves, then put the glass plate with grooves on it, and fix the two glass plates with clips, so that a certain gap is formed between the two glass plates. The lower end of the gap should be closed to prevent the glue from leaking out. Generally, it is sealed with adhesive tape, and the adhesive tape is torn off after the glue solidifies; or it can be sealed with 1% to 1.5% concentration of agar. The method is: add electrode buffer or distilled water to agar, and heat it in a boiling water bath to dissolve. Put the glass plate device with gaps vertically in a small groove with a height of 3cm and a width of 3cm, which is wider and longer than the glass plate (the commercial electrophoresis tank has a matching device), and pour the dissolved agar glue into the small groove. After cooling, take it out, and the lower end of the glass plate device is sealed, and polyacrylamide glue can be poured. A.2 Preparation of separation gel
Take out the gel-making reagent from the refrigerator and balance it to room temperature. Prepare separation gel according to the formula in Table A.1. The concentration of separation gel in this experiment is 7.5%. Prepare the gel solution according to the formula in Table A.1, mix well, and quickly inject it into the gap between the two glass plates until the gel surface is about 3 cm away from the groove of the glass plate. Then gently add 1 cm high distilled water on the gel surface. When adding distilled water, usually add it slowly along the glass plate without disturbing the gel surface. Place the gel plate vertically at room temperature for about 1 hour to allow it to condense. At this time, a very clear interface can be seen between the gel and the distilled water. Then pour off the distilled water on the gel surface.
A. 3 Preparation of concentrated gel
The method is determined according to the actual situation. The preparation method is prepared according to the formula in Table A.1. Mix the glue according to the formula in Table A.1, take a small amount and pour it into the gap between the glass plates, rinse and separate the gel surface, then pour it out, and pour the remaining glue into the gap between the glass plates. Make the glue liquid level flush with the groove of the glass plate, and place it at room temperature for 20min~-30min. The concentrated glue can be condensed. After solidification, slowly take out the "comb". When taking it out, be careful not to break the gel holes. After taking out the "comb", add distilled water to the gel holes formed to rinse the uncondensed terpenes, pour out the distilled water in the holes, and then add electrode buffer. Place the glass plate filled with gel vertically on the electrophoresis tank. The glass plate with grooves is close to the electrophoresis tank to form a liquid bottle. Grab the electrode buffer into it so that it contacts the buffer in the gel holes. Add electrode buffer to the reservoir at the lower end of the electrophoresis tank. Table A.1 Formula of SDS-PAGE gel
30% terpenes mother solution
Separation gel buffer
Concentration gel buffer
Double-water
10% ammonium persulfate (APS)
Tetramethyl Ethyl Ether (TEMED)
Separation gel
O. li tnt.
tv, 1 mL
Concentrated gel
t.. s ml.
0. 02 tr.
GB/T 19567.32004
Appendix B
(Normative Appendix)
Determination of toxicity
B. 1 Method for determination of toxicity I
I. Method for determination using Plutella xylestella as test insect B.1. 1 Reagents or materials
Standard substance: CS·1995, Hb200001U/mg. Plutella xylestella larvae. Edible rapeseed oil.
Yeast powder: for industrial use.
Vitamin C: for medical use, analytical grade.
Agar:Gel strength is greater than 300 g/cm. Potassium dihydrogen phosphate: analytically pure.
Polysorbate-80: viscosity 3.5×101m/s5.5X10-*m2/s. Leaf powder: Brassica napus leaves, dried at 80℃, crushed, and passed through 80 mesh sieve. Yan sugar, analytically pure.
Cellulose powder CF-11.
Potassium hydroxide: analytically pure.
Sodium chloride: analytically pure.
15% paraben: methyl paraben (chemically pure) dissolved in % alcohol, 10% formaldehyde solution: formaldehyde (analytical pure) dissolved in distilled water. Casein solution: 2g of casein (BR biological reagent), add 2mL of 0.001mol/L potassium hydroxide and 8mL of distilled water, sterilize. Phosphate buffer: 8.5 g sodium chloride, 6.0 g potassium dihydrogen phosphate, 30 g potassium dihydrogen phosphate, 0.1 mL polysorbate-80 solution, 1 000 mL distilled water.
B. 1.2 Instruments and equipment
Ground-mouthed Erlenmeyer flask: 250 ml with stopper.
Analytical balance: accurate to 0.1 tg.
Electric stirrer: stepless speed regulation, 100 r/min~1000 r/min. Medical scalpel.
Vibrator,
Microwave oven or electric stove.
Water bath.
Insect tube: 9 cm×2.5 cm.
Small beaker: 50 ml
Large beaker: 500 ml.
Test tube: 18 mmX180 mm
Glass beads: 5 mm in diameter.
Pipette: 10 mL, 5 mL, 2 mL, 1 mL. B, 1. 3 Determination steps
B. 1. 3. 1 Preparation of infection fluid
.1.3, 1.1 Standard
GB/T 19567.3--2004
Use an analytical balance to accurately weigh 100.mg~150.0wg (accurate to 0.1g) of the standard. Place it in a 250ml ground-mouth two-corner bottle containing 10 glass beads. Add 100 mL of phosphate buffer, soak for 10 min, shake in a vibrator for 3 min to obtain a standard stock solution with a concentration of 1 mg/mL (the stock solution can be stored in a refrigerator at 4°C for 10 min), then dilute the standard stock solution into six dilution solutions with concentrations of 1.000, 0.500, 0.250, 0.125, 0.0625, 0.0313 mg/ml. B.1.3.1.2 Wettable powder samples
Weigh the sample with the toxicity titer equivalent to the standard (accurate to 0.2 mg), add 100 mL of phosphate buffer, and then prepare the sample infection solution according to the preparation method of the standard.
For samples with too high or too low toxicity titers, a preliminary test should be conducted with three degrees with large differences before the determination to estimate the range of the lethal concentration (LCse value), and the dilution concentration should be designed accordingly. B. 1. 3.2 Preparation of infected feed
What is the feed formula: 0.5g vitamin C, 10ml cheese solution, 3.0g vegetable leaf powder, 1.5g yeast powder, 1.0g cellulose powder, 2.0g agar powder, 6.0g sucrose, 0.2ml seed oil, 0.5mL 10% formaldehyde solution, 1.0mL 15% nipakine, 100ml distilled water. Add sucrose, yeast powder, casein solution and agar powder into 90 IIL distilled water and mix well. Stir and boil to dissolve agar completely, add nipakine and stir. Mix the other ingredients into a paste with the remaining 10mL distilled water, and when the agar cools to about 75℃, mix it thoroughly, stir with a spoon, and keep it warm in a 55℃ water bath for later use. Take 7 50ml beakers, write labels, preheat in a 55℃ water bath, add 1mL of infection solution of corresponding concentration to each beaker, and use the flushing solution as a blank control. Add 1mL of dissolved infection feed to each beaker, stir with an electric stirrer for 20s to fully mix the infection wave and feed in each beaker. Cover the beaker quietly, wait for cooling and solidification, and cut the infected feed into 1cm×1cm feed blocks with a medical scalpel. Take 4 blocks of each concentration and put them in 4 insect tubes, put one block in each tube, and write labels. B.1.3.3 Infection
Randomly take the insect tubes with feed placed. Put 10 third-instar larvae of diamondback moth in each tube, 4 tubes for each concentration, plug them with cotton plugs, write labels, and raise them under the same inter-raising conditions.
B. 1. 4 Result inspection and calculation
48h after infection, check the death of the test insects. The standard for judging dead insects is to gently touch the insect body with a fine swab. Those without any reaction are considered dead.
Calculate the mortality rate of the test insects at each concentration of the standard and sample, and check the Abbott table or calculate the corrected mortality rate (x). The blank control mortality rate needs to be corrected if it is below 10%. If it is higher than 10%, the test result is invalid. The corrected mortality rate is calculated according to formula (B.1):
X, =9×100%
Where:
T——death rate after treatment with the agent;
C——death rate after blank control
Convert the infection concentrations into logarithmic values, convert the corrected mortality rate into the probability of death value, use the least squares method to calculate the LCso value of the standard and the IL.Cu value of the sample to be tested, and calculate the toxicity titer (X) of the sample to be tested. The toxicity titer is calculated according to formula (B.2):
Wu Zhong:
S-LC value of standard,
P-titer of standard;
Y·LCx value of sample.
(B, 2)
GB/T 19567.3--2004
B.1.5 Permissible difference
The toxicity assay method allows relative deviation, but the maximum relative deviation of the results of three repeated measurements of each sample shall not exceed 20%. The mortality rate caused by each concentration of the toxicity assay preparation should be between 10% and 90%, with at least two concentrations above and below the 50% mortality rate. B.2 Toxicity titer determination method - determination method using cotton bollworm (Heliothisarmigera) as test insect B.2.1 Reagents and materials
Standard; CS-1995, Hab, 20000IU/mg Cotton bollworm larvae: Heliathisarmigera. Soybean powder: Soybeans are fried and ground and passed through a 6-mesh sieve. Dayou powder: Pass through a 60-day sieve.
Yeast powder: Industrial use.
36% acetic acid solution, acetic acid (chemically pure), dissolved in distilled water, sodium benzoate: analytically pure.
Formaldehyde: analytically pure.
Vitamin C, medical use, analytically pure.
Agar powder: Gel strength greater than 300g/cm. Phosphate buffer: Same as B.1.1.
B.2.2 Apparatus and equipment
Analytical balance: accurate to 0.1 mg
Electric stirrer: stepless speed regulation, 1001/min-~6000x/tmin. Microwave oven or electric stove,
Oscillator.
Water bath.
Tissue culture plate: 24 wells.
Porcelain plate, 30cm×20cm.
Quick-necked conical flask: 250mL, with stopper.
Large beaker: 1000mL,
Small beaker, 50mL.
Test tube: 18mm×180mm,
Glass beads: 5mm in diameter
Syringe, 50mL,
Specimen cylinder.
Constant temperature incubator,
B,2. 3 Determination steps
B.2.3.1 Feed preparation
Feed formula: 12g yeast powder, 24% yellow powder, 1.5g vitamin C, 0.42g sodium benzoate: 3.9mL 36% acetic acid, 300ml distilled water..
Put soybean powder, yeast powder, vitamin C, sodium benzoate and 36% acetic acid into a large beaker, and moisten with 100mL boiling water. Add the remaining 200 ml of distilled water to the agar powder and boil it in the microwave oven to make the agar completely melt. Take it out and cool it to 70℃, mix it with other ingredients, stir it in an electric stirrer at high speed for 1 minute, quickly move it to a 60℃ water bath and cover it to keep it warm. B.2.3.2 Preparation of infection solution
Weigh 100.011g 150.0mg (accurate to 0.1mg) of wettable powder sample into a ground-mouth cold conical flask containing glass beads, add 100ml of phosphate buffer, soak for 10min, and oscillate on a vibrator for 1tmin to form the mother solution. Weigh 150.0mg GB/T 19567.3--2004
300.0mg of standard product (accurate to 0.1 machine), prepare the mother solution as above, dilute the sample and standard stock solution with phosphate buffer in a certain multiple, dilute each sample and standard solution to at least 5 concentrations, and set buffer as control, draw 3mL of infection solution of each concentration into a 50mL beaker for use. Draw 3mL of phosphate buffer for control. B.2.3.3 Mixing and subpackaging of feed and infection concentration Use a syringe to draw 27tnL of feed, inject into the beaker containing the sample or standard infection solution, stir with an electric stirrer for 0.5min, and quickly pour into each small hole on the tissue culture plate [the amount poured does not need to be the same, and the bottom of the hole is the recommended amount]. Solidify and set aside. B. 2. 3, 4 Infection
At room temperature of 26℃~30℃, shake the newly hatched larvae (within 12h after hatching) that have not been fed into a specimen tank with a diameter of 20cm and wait for several minutes. Select healthy larvae at the top of the tank as test worms, and gently move them into the small holes of the tissue plate with infected feed with a brush, with 1 larva in each hole. Put 48 larvae in each concentration and blank control, cover with plastic sheet, then stack the tissue culture plates, tie them tightly with rubber bands, and place them upright in a 30℃ constant temperature incubator for 72h. B. 2.4 Result inspection and statistical analysis
Check the number of dead and live worms with the naked eye or a magnifying glass. Touch the worm body with a thin stick, and the one with no reaction is considered dead, and the mortality rate is calculated. If there is death in the control, the corrected mortality rate can be calculated by looking up the Abbott correction value table or using formula (B.1). The control mortality rate does not need to be corrected if it is below 6%, but needs to be corrected if it is between 6% and 15%. If it is greater than 15%, the test is invalid. Convert the concentration into logarithmic value, convert the mortality rate or corrected mortality rate into probability value, use the least square method to calculate the LCse value of the standard and sample respectively, and calculate the toxicity titer according to formula (B.2): B.2.5 Allowable integer
The allowable relative deviation requirements of the toxicity determination method are the same as those in B.1.5. B.3 Toxicity titer determination method - determination method using Spodoptera exigua as test insect B.3.1 Materials and reagents
Standard: CS-2002, Hra, 20000IU/mg. Beet armyworm larvae: Spodoptera exigua. Soybean powder: stir-fry soybeans, crush them and pass through a 60-day sieve. Yeast powder (200 days): for industrial use.
Vitamin A: medical use, analytical grade.
15% paraben: methyl paraben (chemically pure) in 95% alcohol. 10% formaldehyde: formaldehyde (analytical pure) dissolved in distilled water. Agar: gel strength greater than 300 g/crn, distilled water.
B.3.2 Instruments
Analytical balance: accuracy 0.1 mg.
Electric stirrer: stepless speed regulation, 100r/min～6000r/min. Microwave oven or electric stove.
Vibrator.
Water bath.
Tissue culture plate: 24 wells.
Porcelain plate: 30 cm×20 cm.
H-shaped triangular flask: 250 mL, with stopper.
Large beaker: 1000ml..
Small beaker: 50mL
Test tube: 18mm×180mm.
Glass beads: 5mm in diameter
GB/T 19567.3---2004
Syringe: 50 tnl.,
Brown cylinder.
Constant temperature incubator.
B, 3. 3 Determination steps
B, 3. 3. 1 Material formula
32g soybean powder (60 days), 16g alcohol powder (200 days), 6g agar, 2g of paraben C, 6.7mL of 15% nipaquinone, 4mL of 10% formaldehyde, 400ml of water
Put soybean powder, yeast powder, vitamin C, nipaquinone and 10% formaldehyde in a large beaker, add 150ml of water, mix and set aside. Add the remaining 250mL of water to agar, heat to boiling in a microwave oven to completely dissolve the agar, take out and cool to 70℃. Mix with other ingredients, stir at high speed in an electric stirrer for 1min, quickly move to a 60℃ water bath pot and cover to keep warm. 3.3.2 Preparation of infection solution
Weigh 150.0mg~300.0mg (accurate to 0.1mg) of Cb2002 standard, place it in a 250mL triangular bottle with glass beads, add 100ml of phosphine buffer, soak for 10min, and oscillate on a vortex shaker for 1min to prepare the standard infection mother solution. Weigh 100.00mg~50.00mg of wettable powder sample, place it in a 250mL triangular bottle with glass beads, add 100ml of phosphate buffer, soak for 10min, and oscillate on a vortex shaker for 1min to prepare the sample infection mother solution. Dilute the standard and sample mother solutions into a certain concentration gradient by the two-cycle dilution method. Each sample is diluted at least 5 concentration gradients. 3mL of infection solution of each concentration is drawn into a 50mL small beaker for use. 3mL of phosphate buffer is drawn for control. B.3.3.3 Mixing and subpackaging of feed and infection solution Use a syringe to draw 27mL of the feed and inject it into the beaker containing the sample or standard product infection solution. Stir with an electric stirrer for 0.5min and quickly pour it into the small holes of the tissue culture plate (the amount poured does not need to be uniform, and the bottom of the hole should be covered). Solidify and set aside. B.3.3.4 Infection with insects
At room temperature of 26℃~-30℃, put the newly hatched larvae (within 12h after hatching) that have been fed into a specimen cylinder with a diameter of 30cm, wait for a few minutes, select the healthy larvae that crawl up to the mouth of the cylinder as the test insects, and use a brush to gently move them into the small holes of the tissue plate with infected feed, with 100 larvae in each hole. Put 48 larvae for each concentration and blank control, cover them with plastic sheets, then stack the tissue culture plates one by one, tie them tightly with rubber bands, and place them vertically in a 25℃ incubator for 72h. R.3.4 Result inspection and statistical analysis
Check the number of dead and live worms with the naked eye or a magnifying glass. Touch the worm body with a thin stick. If there is no reaction at all, the worm is dead. Calculate the mortality rate. If there is death in the control, check the Abbatt correction value table or calculate the corrected mortality rate according to formula (B.1). No correction is required for the control mortality rate below 6%, correction is required between 6% and 15%, and the test is invalid if it is greater than 15%. Convert the concentration into a logarithmic value, the mortality rate or the corrected mortality rate into a rate value, use the least squares method or a calculator with statistical functions to calculate the LC of the standard and sample respectively, and calculate the toxicity titer according to formula (B.2). B.3.5 Allowable relative deviation requirements for the toxicity determination method are the same as those in B.1.5. 4 Detection of suspected samples with other effective or active ingredients For some suspected samples that meet the analytical indicators in 3.2 of the main text, the toxicity contribution rate of the cytosolic mixture in the sample can be determined to determine whether the sample is adulterated with other active ingredients.
B.4.1 Separation of different components in wettable powders Dilute the wettable powder with acetone to a solution of 10 mg/nl, centrifuge at 5000 r/min for 10 min at 4°C, resuspend the precipitated part with an equal volume of acetone, centrifuge again, wash by centrifugation for 3 times, and retain the precipitated component; suspend the precipitated component with an equal volume of distilled water, centrifuge and retain the precipitated component, centrifuge and wash for 3 times, and retain the precipitated component. The precipitated component is the cytosolic mixture in the wettable powder. B.4.2 Determination of toxicity titer
According to the method in B.1, B.2 or B.3, the precipitated components (cytosolic mixture) of unit mass of wettable powder and condensable powder are respectively subjected to toxicity titer determination.1) Calculate the corrected mortality rate. If the control mortality rate is below 6%, no correction is required. If it is between 6% and 15%, correction is required. If it is greater than 15%, the test is invalid. Convert the concentration into a logarithmic value, convert the mortality rate or the corrected mortality rate into a probability value, use the least square method to calculate the LCse value of the standard and sample respectively, and calculate the toxicity titer according to formula (B.2): B.2.5 Allowable integer
The allowable relative deviation requirements for the toxicity determination method are the same as those in B.1.5. B.3 Toxicity titer determination method - determination method using Spodoptera exigua as test insect B.3.1 Materials and reagents
Standard: CS-2002, Hra, 20000 IU/mg. Beet armyworm larvae: Spodoptera exigua. Soybean powder: stir-fry soybeans, crush them and pass through a 60-day sieve. Yeast powder (200 days): for industrial use.
Vitamin C: medical use, analytical grade.
15% paraben: methyl paraben (chemically pure) in 95% alcohol. 10% formaldehyde: formaldehyde (analytical pure) dissolved in distilled water. Agar: gel strength greater than 300 g/crn, distilled water.
B.3.2 Instruments
Analytical balance: accuracy 0.1 mg.
Electric stirrer: stepless speed regulation, 100r/min～6000r/min. Microwave oven or electric stove.
Vibrator.
Water bath.
Tissue culture plate: 24 wells.
Porcelain plate: 30 cm×20 cm.
H-shaped triangular flask: 250 mL, with stopper.
Large beaker: 1000ml..
Small beaker: 50mL
Test tube: 18mm×180mm.
Glass beads: 5mm in diameter
GB/T 19567.3---2004
Syringe: 50 tnl.,
Brown cylinder.
Constant temperature incubator.
B, 3. 3 Determination steps
B, 3. 3. 1 Material formula
32g soybean powder (60 days), 16g alcohol powder (200 days), 6g agar, 2g of paraben C, 6.7mL of 15% nipaquinone, 4mL of 10% formaldehyde, 400ml of water
Put soybean powder, yeast powder, vitamin C, nipaquinone and 10% formaldehyde in a large beaker, add 150ml of water, mix and set aside. Add the remaining 250mL of water to agar, heat to boiling in a microwave oven to completely dissolve the agar, take out and cool to 70℃. Mix with other ingredients, stir at high speed in an electric stirrer for 1min, quickly move to a 60℃ water bath pot and cover to keep warm. 3.3.2 Preparation of infection solution
Weigh 150.0mg~300.0mg (accurate to 0.1mg) of Cb2002 standard, place it in a 250mL triangular bottle with glass beads, add 100ml of phosphine buffer, soak for 10min, and oscillate on a vortex shaker for 1min to prepare the standard infection mother solution. Weigh 100.00mg~50.00mg of wettable powder sample, place it in a 250mL triangular bottle with glass beads, add 100ml of phosphate buffer, soak for 10min, and oscillate on a vortex shaker for 1min to prepare the sample infection mother solution. Dilute the standard and sample mother solutions into a certain concentration gradient by the two-cycle dilution method. Each sample is diluted at least 5 concentration gradients. 3mL of infection solution of each concentration is drawn into a 50mL small beaker for use. 3mL of phosphate buffer is drawn for control. B.3.3.3 Mixing and subpackaging of feed and infection solution Use a syringe to draw 27mL of the feed and inject it into the beaker containing the sample or standard product infection solution. Stir with an electric stirrer for 0.5min and quickly pour it into the small holes of the tissue culture plate (the amount poured does not need to be uniform, and the bottom of the hole should be covered). Solidify and set aside. B.3.3.4 Infection with insects
At room temperature of 26℃~-30℃, put the newly hatched larvae (within 12h after hatching) that have been fed into a specimen cylinder with a diameter of 30cm, wait for a few minutes, select the healthy larvae that crawl up to the mouth of the cylinder as the test insects, and use a brush to gently move them into the small holes of the tissue plate with infected feed, with 100 larvae in each hole. Put 48 larvae for each concentration and blank control, cover them with plastic sheets, then stack the tissue culture plates one by one, tie them tightly with rubber bands, and place them vertically in a 25℃ incubator for 72h. R.3.4 Result inspection and statistical analysis
Check the number of dead and live worms with the naked eye or a magnifying glass. Touch the worm body with a thin stick. If there is no reaction at all, the worm is dead. Calculate the mortality rate. If there is death in the control, check the Abbatt correction value table or calculate the corrected mortality rate according to formula (B.1). No correction is required for the control mortality rate below 6%, correction is required between 6% and 15%, and the test is invalid if it is greater than 15%. Convert the concentration into a logarithmic value, the mortality rate or the corrected mortality rate into a rate value, use the least squares method or a calculator with statistical functions to calculate the LC of the standard and sample respectively, and calculate the toxicity titer according to formula (B.2). B.3.5 Allowable relative deviation requirements for the toxicity determination method are the same as those in B.1.5. 4 Detection of suspected samples with other effective or active ingredients For some suspected samples that meet the analytical indicators in 3.2 of the main text, the toxicity contribution rate of the cytosolic mixture in the sample can be determined to determine whether the sample is adulterated with other active ingredients.
B.4.1 Separation of different components in wettable powders Dilute the wettable powder with acetone to a solution of 10 mg/nl, centrifuge at 5000 r/min for 10 min at 4°C, resuspend the precipitated part with an equal volume of acetone, centrifuge again, wash by centrifugation for 3 times, and retain the precipitated component; suspend the precipitated component with an equal volume of distilled water, centrifuge and retain the precipitated component, centrifuge and wash for 3 times, and retain the precipitated component. The precipitated component is the cytosolic mixture in the wettable powder. B.4.2 Determination of toxicity titer
According to the method in B.1, B.2 or B.3, the precipitated components (cytosolic mixture) of unit mass of wettable powder and condensable powder are respectively subjected to toxicity titer determination.1) Calculate the corrected mortality rate. If the control mortality rate is below 6%, no correction is required. If it is between 6% and 15%, correction is required. If it is greater than 15%, the test is invalid. Convert the concentration into a logarithmic value, convert the mortality rate or the corrected mortality rate into a probability value, use the least square method to calculate the LCse value of the standard and sample respectively, and calculate the toxicity titer according to formula (B.2): B.2.5 Allowable integer
The allowable relative deviation requirements for the toxicity determination method are the same as those in B.1.5. B.3 Toxicity titer determination method - determination method using Spodoptera exigua as test insect B.3.1 Materials and reagents
Standard: CS-2002, Hra, 20000 IU/mg. Beet armyworm larvae: Spodoptera exigua. Soybean powder: stir-fry soybeans, crush them and pass through a 60-day sieve. Yeast powder (200 days): for industrial use.
Vitamin C: medical use, analytical grade.
15% paraben: methyl paraben (chemically pure) in 95% alcohol. 10% formaldehyde: formaldehyde (analytical pure) dissolved in distilled water. Agar: gel strength greater than 300 g/crn, distilled water.
B.3.2 Instruments
Analytical balance: accuracy 0.1 mg.
Electric stirrer: stepless speed regulation, 100r/min～6000r/min. Microwave oven or electric stove.
Vibrator.
Water bath.
Tissue culture plate: 24 wells.
Porcelain plate: 30 cm×20 cm.
H-shaped triangular flask: 250 mL, with stopper.
Large beaker: 1000ml..
Small beaker: 50mL
Test tube: 18mm×180mm.
Glass beads: 5mm in diameter
GB/T 19567.3---2004
Syringe: 50 tnl.,
Brown cylinder.
Constant temperature incubator.
B, 3. 3 Determination steps
B, 3. 3. 1 Material formula
32g soybean powder (60 days), 16g alcohol powder (200 days), 6g agar, 2g of paraben C, 6.7mL of 15% nipaquinone, 4mL of 10% formaldehyde, 400ml of waterwww.bzxz.net
Put soybean powder, yeast powder, vitamin C, nipaquinone and 10% formaldehyde in a large beaker, add 150ml of water, mix and set aside. Add the remaining 250mL of water to agar, heat to boiling in a microwave oven to completely dissolve the agar, take out and cool to 70℃. Mix with other ingredients, stir at high speed in an electric stirrer for 1min, quickly move to a 60℃ water bath pot and cover to keep warm. 3.3.2 Preparation of infection solution
Weigh 150.0mg~300.0mg (accurate to 0.1mg) of Cb2002 standard, place it in a 250mL triangular bottle with glass beads, add 100ml of phosphine buffer, soak for 10min, and oscillate on a vortex shaker for 1min to prepare the standard infection mother solution. Weigh 100.00mg~50.00mg of wettable powder sample, place it in a 250mL triangular bottle with glass beads, add 100ml of phosphate buffer, soak for 10min, and oscillate on a vortex shaker for 1min to prepare the sample infection mother solution. Dilute the standard and sample mother solutions into a certain concentration gradient by the two-cycle dilution method. Each sample is diluted at least 5 concentration gradients. 3mL of infection solution of each concentration is drawn into a 50mL small beaker for use. 3mL of phosphate buffer is drawn for control. B.3.3.3 Mixing and subpackaging of feed and infection solution Use a syringe to draw 27mL of the feed and inject it into the beaker containing the sample or standard product infection solution. Stir with an electric stirrer for 0.5min and quickly pour it into the small holes of the tissue culture plate (the amount poured does not need to be uniform, and the bottom of the hole should be covered). Solidify and set aside. B.3.3.4 Infection with insects
At room temperature of 26℃~-30℃, put the newly hatched larvae (within 12h after hatching) that have been fed into a specimen cylinder with a diameter of 30cm, wait for a few minutes, select the healthy larvae that crawl up to the mouth of the cylinder as the test insects, and use a brush to gently move them into the small holes of the tissue plate with infected feed, with 100 larvae in each hole. Put 48 larvae for each concentration and blank control, cover them with plastic sheets, then stack the tissue culture plates one by one, tie them tightly with rubber bands, and place them vertically in a 25℃ incubator for 72h. R.3.4 Result inspection and statistical analysis
Check the number of dead and live worms with the naked eye or a magnifying glass. Touch the worm body with a thin stick. If there is no reaction at all, the worm is dead. Calculate the mortality rate. If there is death in the control, check the Abbatt correction value table or calculate the corrected mortality rate according to formula (B.1). No correction is required for the control mortality rate below 6%, correction is required between 6% and 15%, and the test is invalid if it is greater than 15%. Convert the concentration into a logarithmic value, the mortality rate or the corrected mortality rate into a rate value, use the least squares method or a calculator with statistical functions to calculate the LC of the standard and sample respectively, and calculate the toxicity titer according to formula (B.2). B.3.5 Allowable relative deviation requirements for the toxicity determination method are the same as those in B.1.5. 4 Detection of suspected samples with other effective or active ingredients For some suspected samples that meet the analytical indicators in 3.2 of the main text, the toxicity contribution rate of the cytosolic mixture in the sample can be determined to determine whether the sample is adulterated with other active ingredients.
B.4.1 Separation of different components in wettable powders Dilute the wettable powder with acetone to a solution of 10 mg/nl, centrifuge at 5000 r/min for 10 min at 4°C, resuspend the precipitated part with an equal volume of acetone, centrifuge again, wash by centrifugation for 3 times, and retain the precipitated component; suspend the precipitated component with an equal volume of distilled water, centrifuge and retain the precipitated component, centrifuge and wash for 3 times, and retain the precipitated component. The precipitated component is the cytosolic mixture in the wettable powder. B.4.2 Determination of toxicity titer
According to the method in B.1, B.2 or B.3, the precipitated components (cytosolic mixture) of unit mass of wettable powder and condensable powder are respectively subjected to toxicity titer determination.100mg wettable powder sample, place it in a 250mL triangular flask with glass beads, add 100ml phosphate buffer, soak for 10min, and oscillate on a vortex shaker for 1min to prepare the sample infection stock solution. Dilute the standard product and sample stock solution to a certain concentration gradient by two-cycle dilution method. Each sample is diluted to at least 5 concentration gradients. 3mL of each concentration infection solution is pipetted into a 50mL small beaker for standby use. 3mL phosphate buffer is pipetted for control. B.3.3.3 Mixing and subpackaging of feed and infection solution Pipet 27mL of feed into the beaker containing the sample or standard product infection solution, stir with an electric stirrer for 0.5min, and quickly pour into each small hole of the tissue culture plate (the amount poured does not need to be consistent, and the bottom of the hole is covered), and solidify for standby use. B.3.3.4 Infection with insects
At room temperature of 26℃~-30℃, place the newly hatched larvae (within 12 hours after hatching) that have been fed into a specimen tank with a diameter of 30cm, wait for a few minutes, select the healthy larvae that have climbed up the tank mouth as test worms, and gently move them into the small holes of the tissue plate with infected feed with a brush, with 1 worm in each hole. Put 48 worms in each concentration and blank control, cover with plastic sheets, then stack the tissue culture plates one by one, tie them tightly with rubber bands, and place them vertically in a 25℃ incubator for 72 hours. R.3.4 Result inspection and statistical analysis
Check the number of dead and live worms with the naked eye or a magnifying glass, touch the worm body with a thin stick, and the worms that have no reaction at all are dead worms, and calculate the snake mortality rate. If there is death in the control, check the Abbatt correction value table or calculate the corrected mortality rate according to formula (B.1). The control mortality rate does not need to be corrected if it is below 6%, but needs to be corrected if it is between 6% and 15%, and the test is invalid if it is greater than 15%. Convert the concentration into a logarithmic value, the mortality rate or the corrected mortality rate into a rate value, and use the least square method or a calculator with statistical functions to calculate the LC of the standard and sample respectively, and calculate the toxicity titer according to formula (B.2). B.3.5 The allowable relative deviation requirements for the tolerance determination method are the same as those in B.1.5. 4 Detection of suspected samples mixed with other effective or active ingredients For some suspected samples that meet the analytical indicators in 3.2 of the main text, the toxicity contribution rate of the cytosolic mixture in the sample can be determined to determine whether the sample is mixed with other active ingredients.
B.4.1 Separation of different components in wettable powders Dilute the wettable powder with acetone to a solution of 10 mg/nl, centrifuge at 5000 r/min for 10 min at 4°C, resuspend the precipitated part with an equal volume of acetone, centrifuge again, wash by centrifugation for 3 times, and retain the precipitated component; suspend the precipitated component with an equal volume of distilled water, centrifuge and retain the precipitated component, centrifuge and wash for 3 times, and retain the precipitated component. The precipitated component is the cytosolic mixture in the wettable powder. B.4.2 Determination of toxicity titer
According to the method in B.1, B.2 or B.3, the precipitated components (cytosolic mixture) of unit mass of wettable powder and condensable powder are respectively subjected to toxicity titer determination.100mg wettable powder sample, place it in a 250mL triangular flask with glass beads, add 100ml phosphate buffer, soak for 10min, and oscillate on a vortex shaker for 1min to prepare the sample infection stock solution. Dilute the standard product and sample stock solution to a certain concentration gradient by two-cycle dilution method. Each sample is diluted to at least 5 concentration gradients. 3mL of each concentration infection solution is pipetted into a 50mL small beaker for standby use. 3mL phosphate buffer is pipetted for control. B.3.3.3 Mixing and subpackaging of feed and infection solution Pipet 27mL of feed into the beaker containing the sample or standard product infection solution, stir with an electric stirrer for 0.5min, and quickly pour into each small hole of the tissue culture plate (the amount poured does not need to be consistent, and the bottom of the hole is covered), and solidify for standby use. B.3.3.4 Infection with insects
At room temperature of 26℃~-30℃, place the newly hatched larvae (within 12 hours after hatching) that have been fed into a specimen tank with a diameter of 30cm, wait for a few minutes, select the healthy larvae that have climbed up the tank mouth as test worms, and gently move them into the small holes of the tissue plate with infected feed with a brush, with 1 worm in each hole. Put 48 worms in each concentration and blank control, cover with plastic sheets, then stack the tissue culture plates one by one, tie them tightly with rubber bands, and place them vertically in a 25℃ incubator for 72 hours. R.3.4 Result inspection and statistical analysis
Check the number of dead and live worms with the naked eye or a magnifying glass, touch the worm body with a thin stick, and the worms that have no reaction at all are dead worms, and calculate the snake mortality rate. If there is death in the control, check the Abbatt correction value table or calculate the corrected mortality rate according to formula (B.1). The control mortality rate does not need to be corrected if it is below 6%, but needs to be corrected if it is between 6% and 15%, and the test is invalid if it is greater than 15%. Convert the concentration into a logarithmic value, the mortality rate or the corrected mortality rate into a rate value, and use the least square method or a calculator with statistical functions to calculate the LC of the standard and sample respectively, and calculate the toxicity titer according to formula (B.2). B.3.5 The allowable relative deviation requirements for the tolerance determination method are the same as those in B.1.5. 4 Detection of suspected samples mixed with other effective or active ingredients For some suspected samples that meet the analytical indicators in 3.2 of the main text, the toxicity contribution rate of the cytosolic mixture in the sample can be determined to determine whether the sample is mixed with other active ingredients.
B.4.1 Separation of different components in wettable powders Dilute the wettable powder with acetone to a solution of 10 mg/nl, centrifuge at 5000 r/min for 10 min at 4°C, resuspend the precipitated part with an equal volume of acetone, centrifuge again, wash by centrifugation for 3 times, and retain the precipitated component; suspend the precipitated component with an equal volume of distilled water, centrifuge and retain the precipitated component, centrifuge and wash for 3 times, and retain the precipitated component. The precipitated component is the cytosolic mixture in the wettable powder. B.4.2 Determination of toxicity titer
According to the method in B.1, B.2 or B.3, the precipitated components (cytosolic mixture) of unit mass of wettable powder and condensable powder are respectively subjected to toxicity titer determination.
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