Some standard content:
HG3616—1999
This standard is formulated based on the relevant materials such as the Bacillus thuringiensis enterprise standards formulated in my country in the past, combined with the actual situation in my country. This standard makes specific requirements and provisions on the requirements, test methods, sampling, packaging, transportation, etc. of Bacillus thuringiensis raw powder, thereby providing a unified technical basis for the production of Bacillus thuringiensis. Appendix A of this standard is a suggestive appendix, and Appendix B is a standard appendix. This standard was proposed by the Technical Supervision Department of the former Ministry of Chemical Industry of the People's Republic of China. This standard is under the jurisdiction of the Shenyang Chemical Industry Research Institute of the Ministry of Chemical Industry. The main drafting unit of this standard is the Department of Applied Chemistry of China Agricultural University. The participating drafting units of this standard are: Hubei Biopesticide Engineering Research and Development Center, Jinan Kerbel Bioengineering Co., Ltd. The main drafters of this standard are: Liu Fengmao, Wang Kaimei, Qian Chuanfan, Zhong Liansheng, Zhao Xinxin, and Wang Qiwen. 1156
Chemical Industry Standard of the People's Republic of China
Bacillus thuringiensis original powder
Bacillus thuringiensis technical HG3616—1999
Bacillus thuringiensis (Bt) is currently the most widely used microbial insecticide. Its main insecticide component is the toxin protein in the parasporal crystal, among which the relative molecular mass of the protein toxic to Lepidoptera is 130,000. Scope
This standard specifies the requirements, test methods, and marking, labeling, packaging, storage and transportation of Bacillus thuringiensis original powder. This standard is applicable to Bacillus thuringiensis original powder for the prevention and control of Lepidoptera pests. 2 Referenced standards
The provisions contained in the following standards constitute the provisions of this standard through reference in this standard. When this standard is published, the versions shown are valid. All standards will be revised, and parties using this standard should explore the possibility of using the latest versions of the following standards. GB/T1250—1989 Method for expressing and determining limit values GB/T1600--1979 (1989) Method for determining moisture content of pesticides GB/T1601—1993 Method for determining pH value of pesticides GB/T1604—1995 Acceptance rules for commercial pesticides GB/T1605--1979 (1989) Method for sampling commercial pesticides GB3796--1983 General rules for pesticide packaging
GB/T16150—1995 Method for determining fineness of pesticide powders and wettable powders 3 Requirements
3.1 Appearance: off-white to brown powder.
3.2 Bacillus thuringiensis raw powder should meet the requirements of Table 1. Table 1 Index of control items for Bacillus thuringiensis powder
Toxin protein, %
Toxicity titer ([Px IU /mgHa IU /mg])pH value
Water content, %
Fineness, 45 μm, %
First-class product
Qualified product
Note: Px and Ha are the abbreviations of Plutella xylostella and Heliothis armigera, respectively. Approved by the State Administration of Petroleum and Chemical Industry on June 16, 1999 and implemented on June 1, 2000
4. Test method
HG 3616--1999
Unless otherwise specified, all reagents used in this method are analytically pure and all solutions are aqueous solutions. 4.1 Sampling
The sampling is carried out in accordance with the "raw powder sampling" in GB/T1605--1979 (1989). The sampling packages are determined by the random number table method. The final sampling volume should be no less than 100g.
4.2 Identification test
When there is doubt about the evaluation of product quality by bioassay, the following methods can be used for identification. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method is used to determine whether the relative molecular mass of the effective toxin protein is 130,000, and at the same time determine whether its content meets the requirements of the indicators in 3.2. 4.3 Toxin protein content determination method
The toxin protein content can be determined by the SDS-PAGE-scanning method and the SDS-PAGE-elution colorimetric method. The precision and accuracy of the two methods are similar. The former is determined as the arbitration method due to its higher degree of automation. 4.3.1 SDS-PAGE-scanning method (arbitration method) 4.3.1.1 Method summary
Treat the Bacillus thuringiensis powder and spore crystals with alkaline solution to degrade them into toxin proteins. Then, use SDS-PAGE to separate the toxin proteins from other impurities according to the difference in relative molecular weight of the proteins. Then, use a thin layer scanner or electrophoresis image scanner to scan the protein band area for quantitative analysis.
4.3.1.2 Instruments and equipment
Electrophoresis instrument.
Sandwich-type vertical electrophoresis tank (1.5mm concave grooved rubber mold frame), gel plate area 145mm×100mm (1.5mm, 12-hole sample tank mold).
High-speed thin layer chromatography scanner or electrophoresis image scanner. Centrifuge: 10000r/min.
Analytical balance: accurate to 0.0001g.
4.3.1.3 Reagents and solutions
Ammonium persulfate (AP).
Sodium dialkyl sulfate (SDS).
Tetramethylethylenediamine (TEMED).
Sodium hydroxide.
30% acrylamide: weigh 30g acrylamide and 0.8g methylenebisacrylamide (formerly known as methylenebisacrylamide), dissolve in 100mL distilled water, filter, and store in a dark place at 4℃ for later use. 1mol/I., pH8.8 tris(hydroxymethylaminomethane)-HCl buffer: weigh 30.25g tris(hydroxymethylaminomethane) and dissolve in distilled water, adjust to pH8.8 with concentrated hydrochloric acid, and dilute to 250mL with distilled water. 1mol/L, pH6.8 tris(hydroxymethylaminomethane)-HCl buffer: weigh 12.10g tris(hydroxymethylaminomethane) and dissolve it in distilled water, adjust to pH6.8 with concentrated hydrochloric acid, and dilute to 100mL with distilled water. Electrode buffer: weigh 3.03g tris(hydroxymethylaminomethane), 14.42g glycine, 1g sodium dodecyl sulfate, dissolve in water and dilute to 1 000mL.
3× sample diluent: 18.75mL 1mol/L, pH6.8 tris(hydroxymethylaminomethane)-HCl, 6g sodium dodecyl sulfate, 30mL glycerol, 15mL mercaptoethanol, a little bromophenol blue, dilute to 100mL with distilled water. Staining solution: weigh 1g Coomassie Brilliant Blue (CBB) R-250, add 450mL methanol, 100mL glacial acetic acid, 450mL distilled water, dissolve and filter before use.
Decolorizing solution: Measure 100mL of methanol and 35mL of glacial acetic acid, and make up to 1000mL with distilled water. 1158
HG 3616- 1999
Rinsing solution: Measure 30mL of anhydrous ethanol, 10mL of glacial acetic acid, and 60mL of distilled water, mix well and use. Toxin protein standard sample: Original powder with a toxin protein (relative molecular weight of 130,000) content of 9.3%. 4.3.1.4 Sample treatment
Weigh 20mg of each standard sample and sample (accurate to 0.1mg), transfer to a 5mL centrifuge tube, and add 2mL of water to fully suspend. Then add 0.45mL of 0.55mol/L sodium hydroxide solution (so that the final concentration of sodium hydroxide solution is 0.1mol/L), let it stand for about 5 minutes, then add 1.30mL of 3× sample diluent to make the final volume 3.75mL, boil in 100℃ boiling water for 6 minutes, centrifuge (2000r/min) for 10 minutes, and take the upper clear liquid for electrophoresis. 4.3.1.5 SDS-PAGE separation of toxin protein
a) Preparation of 8%~10% acrylamide gel
Use a discontinuous buffer system. The gel preparation method is shown in Appendix A (Suggested Appendix). b) Sample loading
Take the supernatant of the above standard sample solution and load 6, 8, 10, 12, and 14 μL (the toxin protein content is about 3-7 ug) in the sample wells of the polyacrylamide gel respectively as the standard curve, and then take a fixed volume of the supernatant of the sample solution (the toxin protein content is about 5 ug); add it to the sample well, inject the electrode buffer, and turn on the power. c) Electrophoresis
The initial voltage of electrophoresis is controlled at about 100V. After the sample enters the separation gel, increase the voltage to 120V and continue electrophoresis. When the indicator front reaches about 1 cm from the bottom, stop electrophoresis, take out the gel plate, and place it at 7.Soak in 5% (volume percentage) acetic acid for 30 minutes. d) Staining
Remove the separation gel part and stain it with Coomassie Brilliant Blue (CBB) R-250 staining solution overnight. e) Decolorization
Pour off the staining solution, wash the gel with rinse solution first, then add decolorizing solution, heat at 37℃ to decolorize it, and change the decolorizing solution several times until the background is clear.
4.3.1.6 Determination
After decolorization of the gel plate, the 130,000 protein band can be clearly seen. Scan the band with a high-speed thin layer chromatography scanner or electrophoresis image scanner at a scanning wavelength of 600nm.
The percentage content (X) of toxin protein in the sample is calculated according to formula (1). mz×100
·Where: ml—the amount of toxin protein in the sample obtained from the standard curve, μg, -2mL the mass of the sample in the diluent, mg; m2
V,--—the final volume of the sample, mL (3.75mL); V
the volume of the sample injected into the gel loading hole, μL. 4.3.1.7 Allowable difference
Take its arithmetic mean as the determination result. The relative deviation of the results of two parallel determinations is less than or equal to 8%. 4.3.2SDS-PAGE-elution colorimetric method
4.3.2.1 Method summary
Treat the Bacillus thuringiensis powder-associated spore crystals with alkaline solution to degrade them into the original toxin disorder, and then separate the toxin protein from other impurities through SDS-PAGE according to the difference in the relative molecular mass of the protein, and then cut the gel, elute, and measure the absorbance. 4.3.2.2 Apparatus and equipment
Spectrophotometer.
Others are the same as 4.3.1.2.
4.3.2.3 Reagents and solutions
Others are the same as 4.3.1.3.
4.3.2.4 Sample treatment
Same as 4.3.1.4.
4.3.2.5 Separation of toxin protein by SDS-PAGE
a) Preparation of 8%~～10% polyacrylamide gel is the same as 4.3.1.5a).
b) Sample loading
HG 3616 - 1999
Take the supernatant of the above standard solution and load 15, 20, 30, 40, 50μL (the toxin protein content is about 7.5-25μg) in the loading wells respectively as the standard curve. Then take a certain volume of the supernatant of the sample solution (the toxin protein content is about 15μg), add it to the loading wells, inject the electrode buffer, and turn on the power. c) Electrophoresis
Same as 4.3.1.5c).
d) Staining
Same as 4.3.1.5d).
e) Decolorization
Same as 4.3.1.5e).
4.3.2.6 Determination
Use a scalpel to scrape the test zone, put it into a glass test tube, add 3.0.mL of 25% pyridine (volume fraction), shake at 37°C to elute the Cox Brilliant Blue (CBB) R-250 adsorbed by the toxin protein, and after equilibrium, use a spectrophotometer to measure the absorbance of the bath solution at 605nm with 25% pyridine as a reference, and calculate the toxin protein content using formula (1). 4.3.2.7 Allowable difference
Take its arithmetic mean as the determination result. The relative deviation of the results of two parallel determinations is less than or equal to 8%. 4.4 Determination of toxicity
Perform according to Appendix B (Appendix to the standard).
4.5 Determination of pH value
Perform according to GB/T1601.
4.6 Determination of finenessbZxz.net
Determine in accordance with 2.2 of GB/T16150-1995. 4.7 Determination of moisture
Determine in accordance with the "azeotropic distillation method" in GB/T1600-1979 (1989). 5 Inspection rules
Should comply with the relevant provisions of GB/T1604. The limit value shall be handled in accordance with GB/T1250. 6 Marking, labeling, packaging, and shipping
6.1 Product packaging shall comply with the provisions of GB3796, and the standard number used shall be indicated. 6.2 The raw powder is mainly packaged in plastic bags and sealed. 6.3 When storing, it should be strictly protected from sunlight and pressure, and placed in a cool and dry place. 6.4 When transporting, be careful to handle with care to prevent damage. 6.5 Guarantee period: Under normal storage and transportation conditions, the quality guarantee period of the raw powder is two years from the date 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 product toxicity titer and toxin protein content shall not be lower than 60% of the 3.2 index. 1160
A1 Plate making
HG3616--1999
Appendix A
(Suggested appendix)
Preparation of electrophoresis gel
This experiment uses plate electrophoresis. The specific operation depends on the laboratory conditions. The basic operation is generally based on the height of the electrophoresis tank, select two glass plates of the same size, one of which has a groove of 2 to 3 cm high at one end. After the two glass plates are washed and dried, put a "gap strip" on both sides of the glass plate without a groove (plastic strips or rubber strips are both acceptable, and their width and thickness are determined according to needs). 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 injected glue from leaking out. Generally, it is sealed with adhesive tape. After the injected glue solidifies, the adhesive tape can be torn off; or it can be sealed with 1% to 1.5% agar. The method is: add electrode buffer or distilled water to the agar, heat it in a boiling water bath to dissolve, and place the glass plate device with the gap vertically in a small groove 3cm high and 3cm wide, 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 while it is hot. After cooling, take out the glass plate device, and the lower end is sealed, and the polyacrylamide glue can be poured. A2 Preparation of separation gel
Take out the gel-making reagent from the refrigerator and balance it to room temperature. Prepare the separation gel according to Table A1. The concentration of the separation gel in this test is 10%. After the glue is prepared and mixed, quickly inject it into the gap between the two glass plates until the glue surface is about 3.5 cm away from the groove of the glass plate. Then gently spread 1 cm high distilled water on the glue surface. When adding distilled water, usually add it slowly along the glass plate, do not disturb the glue surface. Place the glue plate vertically at room temperature for about 20 to 30 minutes to solidify it. At this time, a very clear interface can be seen between the gel and the steamed filling water. Then suck out the distilled water on the glue surface.
A3 Preparation of concentrated glue
The amount to be used depends on the actual situation, and the preparation method is according to Table A1. Table A1 SDS-PAGE gel formula
Preparation solution
30% acrylamide
1 mol/L, pH8.8 Tris(hydroxymethyl)aminomethane-HCl1 mol/L, pH6.8 Tris(hydroxymethyl)aminomethane-HClDistilled water
10% sodium dodecyl sulfate
10% ammonium persulfate
Tetramethylethylenediamine
Separation gel
Concentrated gel
20 μL
Mix the above solutions, pour a small amount into the gap between the glass plates, rinse the surface of the separation gel, and then pour it out. Then pour the remaining gel into the gap between the glass plates, make the gel surface flush with the concave part of the glass plate, and then insert the "comb" and leave it at room temperature for 20 to 30 minutes, and the concentrated gel can be condensed. After condensation, slowly take out the comb, and be careful not to break the gel holes when taking it out. After removing the comb, add distilled water to the formed gel holes to rinse the uncoagulated acrylamide, etc., pour out the distilled water in the holes, and then add electrode buffer. Fix the glass plate filled with gel vertically on the electrophoresis tank, and the glass plate with grooves is close to the electrophoresis tank to form a reservoir. Add electrode buffer to 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. 1161
B1 Method for determination of toxicity titer 1
B1.1 Reagents or materials
HG 3616—1999
Appendix B
(Standard Appendix)
Determination of toxicity titer
Determination method using Plutella xylostella as test insect (arbitration method) Standard: CS-95, Hsab, titer 20 000IU/mg. Plutella xylostella, larvae of diamondback moth. Edible rapeseed oil.
Yeast powder: industrial use.
Vitamin C: medical use, analytical grade.
Agar powder: gel strength greater than 300g/cm2. Dipotassium hydrogen phosphate: analytical grade.
Polysorbate-80: viscosity 3.5×10-45.5×10-m2/s. Leaf powder: Brassica napus leaves, dried at 80℃, ground, and passed through 80 mesh sieve. Sucrose: analytical grade.
Cellulose powder CF-11.
Potassium hydroxide: analytical grade.
Sodium chloride: analytical grade.
15% paraben: methyl paraben (chemically pure) dissolved in 95% ethanol. 10% formaldehyde solution: formaldehyde (analytical grade) dissolved in distilled water. Casein solution: 2g casein (BR biological reagent) plus 2mL 0.001mol/L potassium hydroxide, 8mL distilled water, sterilized. Phosphate buffer: 8.5g sodium chloride, 6.0g dipotassium hydrogen phosphate, 3.0g potassium dihydrogen phosphate; 0.1mL polysorbate-80 solution; 1000mL distilled water.
B1.2 Instruments and equipment
Ground-mouth triangular flask: 250mL.
Analytical balance: accurate to 0.1mg.
Electric stirrer: stepless speed regulation, 1006000r/min. Medical scalpel.
Oscillator.
Water bath.
Insect tube: 9cm×2.5cm.
Beaker: 50mL.
Test tube: 18 mmX180 mm.
Glass beads: $5mm.
Pipette: 10 mL.
B1. 3 Determination steps
B1.3.1 Preparation of infection fluid
a) Standard
Use an analytical balance to accurately weigh 100.0~150.0mg of the standard (accurate to 0.1mg), and put it into a 250mL 1162
HG 3616--1999
ground-mouth Erlenmeyer flask containing 10 glass beads. Add 100mL of phosphate buffer, soak for 10min, and shake on an oscillator for 30min. Obtain a standard stock solution with a concentration of about 1mg/mL (the stock solution can be stored in a 4℃ refrigerator for 10 days). Then dilute the standard stock solution into six diluted infection solutions with concentrations of 1.000, 0.500, 0.250, 0.125, 0.0625, and 0.0313mg/mL. b) Wettable powder samples
Weigh an appropriate amount of sample (accurate to 0.2 mg) equivalent to the toxicity of the standard product, add 100 mL of phosphate buffer, and then prepare the sample infection solution according to the preparation method of the standard product.
c) Suspension samples
Oscillate the sample for 20 minutes and shake it thoroughly. Pipette 10.00 mL of the sample and add it to a ground-mouth triangular flask containing 90 mL of sterile distilled water. Wash it three times and shake it thoroughly to obtain a mother solution containing 100 μL/mL. Dilute the mother solution into six dilutions with contents of 5.000, 2.500, 1.250, 0.625, 0.313 and 0.156 μL/mL respectively. For some samples with too high or too low potency, it is necessary to conduct preliminary tests with three concentrations with large differences before determination to estimate the range of LCs0 value (median lethal concentration) and design the dilution concentration accordingly. B1.3.2 Preparation of infected feed
Feed formula: 0.5g vitamin C, 1.0 mL casein solution, 3.0g vegetable leaf powder, 1.5g yeast powder, 1.0g vitamin powder, 2.0g agar powder, 6.0g sucrose, 0.2mL rapeseed oil, 0.5mL 10% formaldehyde solution, 1.0mL 15% paraben, 100mL distilled water. Add sucrose, yeast powder, casein solution, and agar powder into 90mL distilled water and mix well. Stir and boil to melt agar completely, add paraben, and stir. Mix other ingredients into a paste with the remaining 10mL distilled water. When agar cools to about 75℃, mix it thoroughly, stir, and place it in a 55℃ water bath to keep warm for later use. Take 7 50mL beakers, write labels, and preheat in a 55℃ water bath; add 1mL of the corresponding concentration of infection solution to each beaker, and use the buffer solution as a blank control. Add 9mL of melted infection feed to each beaker; stir with an electric stirrer for 20s to fully mix the infection solution and feed in each beaker. : Let the beaker stand, wait for it to cool and solidify, and use a medical scalpel to cut the infected feed into 1 cm×1 cm feed blocks. Take 4 feed blocks of each concentration and put them in 4 insect tubes, one block in each tube, and write labels. B1.3.3 Infection
Randomly take the insect tubes with feed placed, and put 10 third-instar larvae of Plutella xylostella in each tube. Put 4 tubes of each concentration, plug them with cotton plugs, write labels, and raise them under the same feeding conditions.
B1.4 Result inspection and calculation
Check the death of the test insects 48 hours after infection. The standard for judging dead insects is to touch the insect body lightly with a thin swab, and those without any reaction are considered dead.
Calculate the mortality rate of the tested insects at each concentration of the standard and sample, and calculate the corrected mortality rate (X,) by looking up the Abbott table or using formula (B1). If the control mortality rate is below 10%, correction is required, and if it is greater than 10%, the test result is invalid. X
In the formula: T—treatment mortality rate;
C——control mortality rate.
·(B1)
Convert each concentration of the infection fluid into a logarithmic value, convert the corrected mortality rate into a mortality probability value, use the least squares method to calculate the LCso value of the standard and the LCso value of the sample to be tested, and then calculate the toxicity titer (Xz) [PxIU/mg(μL) or Ha IU/mg(μL)] of the sample to be tested according to formula (B2):
Where: S——LCso value of the standard;
P——titer of the standard,
Y——LCso value of the sample.
B1.5 Allowable difference
（B2）
HG3616—1999
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 the concentration of the toxicity assay preparation should be between 10% and 90%, and there should be at least two concentrations above and below the 50% mortality rate. B2 Method for determination of toxicity titer II -
B2.1 Reagents or materials
Method for determination using cotton bollworm (Heliothis armigera) as test insect Standard: CS-95, Hab, titer 20000IU/mg. Cotton bollworm larvae; Heliothis armigera. Soybean powder: roasted soybeans, grind and pass through a 60-mesh sieve. Barley flour: pass through a 60-mesh sieve.
Yeast powder: for 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/cm2. Phosphate buffer: same as B1.1.
B2.2 Instruments and equipment
Analytical balance: accurate to 0.1mg.
Electric stirrer: stepless speed regulation, 100～6000r/min. Microwave oven or electric stove.
Oscillator.
Water bath.
Tissue culture plate: 24 holes.
Sugar porcelain plate: 30cm×20cm.
Ground-mouth triangle flask: 250mL, with stopper.
Large beaker: 1000mL.
Small beaker: 50mL.
Test tube: 18 mmX180 mm.
Glass beads: 5 mm.
Syringe: 50mL.
Specimen cylinder.
Constant temperature incubator.
B2.3 Determination steps
B2.3.1 Feed preparation
Feed formula: 12g yeast powder, 24g soybean 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 in a large beaker, add 100mL distilled water to moisten, add the remaining 200mL distilled water to the agar powder, heat in a microwave oven until boiling to melt the agar completely, 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 and cover to keep warm. B2.3.2 Preparation of infection solution
Weigh 100.0~150.0mg wettable powder sample into a ground-mouth stoppered flask with glass beads, add 100mL phosphate buffer, soak for 10min, and shake on an oscillator for 1min to form a mother solution. Or after fully shaking the suspension sample, draw 1.00mL into a ground-mouth stoppered flask with glass beads, add 99mL phosphate buffer, soak for 10min, and shake on an oscillator for 1min to form a mother solution. Weigh 150.0~300.0mg standard (accurate to 0.1164
HG3616—1999
on an analytical balance.1mg), prepare the mother solution as above. Dilute the sample and standard stock solutions with phosphate buffer in a certain multiple, dilute each sample and standard at least 5 times, and set a buffer solution as a control. Pipette 3mL of infection solution of each concentration into a 50mL beaker for standby use, and pipette 3mL of phosphate buffer for the control. B2.3.3 Mixing and subpackaging of feed and infection solution Pipette 27mL of feed into the beaker containing the sample or standard infection solution, stir at high speed with an electric stirrer for 0.5min, and quickly pour into the small holes on the tissue culture plate (the amount poured does not need to be consistent, and the bottom of the hole should be covered), and solidify for standby use. B2.3.4 Infection
At room temperature of 26.～30℃, shake the newly hatched larvae (within 12 hours after hatching) that have not been fed into a specimen cylinder with a diameter of 20cm, wait for a few minutes, select healthy larvae on the upper end of the cylinder as test worms, and gently move them into the small holes of the tissue plate with infected feed with a brush, one worm in each hole. Put 48 worms in each concentration and blank control, cover with plastic sheet, then stack the tissue culture plates one by one, tie them tightly with rubber bands, and place them upright in a 30℃ constant temperature incubator for 72 hours. B2.4 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 mortality rate. If there is death in the control, you can check the Abbott correction value table or calculate the corrected mortality rate according to formula (B1). The control mortality rate does not need to be corrected if it is below 6%, but needs to be corrected between 6% and 15%, and the determination is invalid if it is greater than 15%. The concentration is converted into a logarithmic value, and the mortality rate or corrected mortality rate is converted into a probability value. The LCso of the standard and sample are calculated using the least squares method, and the toxicity titer is calculated according to formula (B2). B2.5 Allowable difference
The allowable relative deviation requirements for the toxicity determination method are the same as B1,5. 1165
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