GB/T 15440-1995 Specification for sample pretreatment for the detection of toxicity of organic pollutants in the environment
Some standard content:
National Standard of the People's Republic of China
Guidelines for preparing samples for genetoxicity testing of organic pollutants in environment
GB/T15440-1995
This specification specifies the technical requirements for sample pretreatment for genetic toxicity testing of organic pollutants in environment. This specification is divided into five parts,
Part 1 Pretreatment of atmospheric inhalable particulate matter samples Part 2 Pretreatment of surface water and wastewater samples Part 3 Pretreatment of non-aqueous liquid waste samples Part 4 Pretreatment of soil and sediment samples Part 5 Pretreatment of solid waste samples
The definitions given in the specification are limited to use within this specification and are not universal. This specification does not provide a comprehensive description of the quality control and operational safety of sample pretreatment, but only explains specific issues. Other issues should comply with the relevant principles of qualified laboratory standards. The quality control principles of this specification are all based on the premise that the genetic toxicity detection system itself is positive. This specification does not provide a separate record form. The record of the sample pretreatment process should comply with the principles of conventional analysis and testing. Part I
Pretreatment of atmospheric inhalable particulate matter samples
1 Scope of application
This specification applies to non-volatile organic matter in atmospheric inhalable particulate matter, and does not apply to gaseous and semi-gaseous organic matter in atmospheric inhalable particulate matter.
2 Reference standards
GB6921 Determination of atmospheric dust concentration
GB6682 Laboratory water specifications and experimental methods: 3 Definitions
Inhalable particulate matter: Particulate matter that can be suspended in the air for a long time, with an aerodynamic equivalent diameter of 10m, and can enter the human respiratory tract.
4 Instruments and equipment
4.1 Sampling system: Meet the requirements of GB6921. 4.2 Ultrafine glass fiber filter membrane: filtration efficiency not less than 99.99% Approved by the State Environmental Protection Agency on March 25, 1995, and implemented on August 1, 1995
4.3 Soxhlet extractor: 500mL volume.
4.4 Ultrasonic cleaner: 250W power.
4.5 Separator.
4.6 Rotary extractor.
4.7 KD concentrator.
4.8 Steel cylinder.
4.9 Pressure reducing valve.
4.1D high-purity nitrogen.
GB/T15440--1995
4.11 General laboratory equipment and glassware. To avoid interference from other organic substances, all glassware III and equipment that directly contacts the sample must be cleaned and rinsed with pure water and pure organic solvents. 5 Test
5.1 Pure water: water that meets the first-level water standard in GB 6682 Laboratory Water Specifications, that is, conductivity ≤0.01 uS/cm (25℃), absorbance ≤0.001 (254nm, 1cm optical path), and silicon dioxide content ≤0.01mg/L. It can be made by using deionized water (adding a small amount of KMnO.) through sub-gravimetric heat increase in all-glassware.
5.2 Solvents: The grade must not be lower than analytical grade, and all should be redistilled in glass containers before use. 5.2. 1 Dichloromethane.
5.2.2 Dimethyl sulfoxide (DMSO).
5.3 Sodium hydroxide: c(NaOH)-1 mol/L. 5.4 Hydrochloric acid: r(HCI)-1 mol/L.
5.5 Anhydrous sodium sulfate.
6 Sample collection
According to the method of GB 6921, a certain amount of atmospheric inhalable particulate matter shall be collected, and the flow rate and sampling time shall be recorded. Before sampling, the filter membrane shall be burned at 500℃ for half an hour and then weighed. After sampling, the folded filter membrane shall be wrapped with sulfuric acid paper, put into a black paper bag, transported at 4℃ and weighed after balancing under the weighing conditions before sampling for at least 24 hours. The weighed filter membrane shall be put into a plastic bag and stored at a temperature below -20℃ and away from light. Sample preparation shall be carried out as soon as possible after sampling.
The sampling time shall not exceed 24 hours. If sufficient dust cannot be collected during this period, two or more samplers may be set up at the same sampling site and the dust samples obtained shall be combined into one sample. 7 Sample preparation
7.1 Separation and extraction of organic matter
Soxhlet extraction and ultrasonic extraction can be used. If necessary, different organic components can be extracted. 7.1.1 Soxhlet extraction
Fold the sample filter membrane into a roll and put it into the extractor. Use 100 mL of difluoromethane at the boiling point of the solvent (40℃) for 2 times, each time for 16 hours (reflux 5-10 times per hour), and combine the two extracts after cooling to room temperature. Use a microporous all-glass frit funnel with a pore size of ≤0.5 mm to filter the extract into a round-bottom flask. 7.1.2 Ultrasonic extraction
Cut the filter membrane into 1 cm small pieces, put it into a glass test tube with a polytetrafluoroethylene liner in the bottle stopper, and extract it with dichloromethane in an ultrasonic cleaner for 2 times, each time for 10 minutes. Use a microporous all-glass frit funnel with a pore size of ≤0.5 mm to filter the extract into a round-bottom flask. 7.1.3 Extraction of different organic components
7.1.3.1 Place the extract filtrate obtained by Soxhlet extraction or ultrasonic extraction into a separatory bucket and extract three times with 1 mol/L hydrochloric acid, each time using about 50~75 mL (pH of the extract is 1~2).
GB/T15440—1995
7.1.3.2 Alkaline component: Combine the aqueous phases obtained from the three extractions in 7.1.3.1, adjust the pH to 11~12 with 1 mol/L sodium hydroxide, extract three times with dichloromethane, each time using about 50~100 mL, combine the organic phases from the three extractions, and remove water through anhydrous sodium sulfate. This is the alkaline component.
7.1.3.3 Neutral component: Combine the organic phases extracted three times in 7.1.3.1, and extract three times with 1 mol/L sodium hydroxide, each time using about 50~75mL (the pH of the extract is 11~12). Combine the organic phases extracted three times, and remove water by passing through anhydrous sodium sulfate. This is the neutral component.
7.1.3.4 Acidic component: Combine the aqueous phases extracted three times in 7.1.3.3, and adjust the pH to 1~2 with 1 mol/L hydrochloric acid. Extract three times with dihydromethane, each time using about 100mL. Combine the organic phases extracted three times, and remove water by passing through anhydrous sodium sulfate. This is the acidic component.
Note that during the extraction process, if the emulsion layer between the aqueous phase and the organic phase exceeds 1/3 of the organic phase, physical methods such as centrifugation should be used for separation.
7.2 Concentration of extracts
When the volume of the extracts obtained in 7.1.1 and 7.1.2 and the extracts of each organic component obtained in 7.1.3 is less than 1L, they can be concentrated using a KD concentrator. If the volume of the extract is greater than 1L, use a rotary evaporator to reduce the extract to nearly 100mL and condense it using a KD concentrator. Keep 50% of the condensed extract for gravimetric and chemical analysis, and continue to concentrate the other 50% using a KD concentrator to 1mL. 7.3 Solvent replacement
Dry the concentrated extract using a steady stream of nitrogen in a 40°C water bath. Add an appropriate amount (about 1 mL) of DMSO or other solvents required for genetic toxicity testing to dissolve the extract and dilute to an appropriate concentration for rapid toxicology testing. The extract should be stored at -20°C away from light and tested within 3 weeks from the date of extraction. 8 Expression of sample calculation results
8.1 The total amount of gas collected is expressed in m.
8.2 The total amount of inhalable particulate matter collected is expressed in mg. 8.3 The amount of extracted organic matter is expressed in mg.
9 Quality Control
9.1 Set up on-site sampling and solvent control, that is, place blank filter membranes on site during sampling (pay attention to prevent contamination). In addition to not participating in the incoming samples, the blank filter membranes must go through the entire process from membrane preparation to genetic toxicity testing. If the genetic toxicity test of such a control is positive, the sampling process and preparation system should be checked separately to find out the reasons and repeat the control test until it is qualified. 9.2 Set up a positive control, that is, drop the control substance that will definitely produce a positive result in the genetic test to be conducted on the blank filter membrane, and let it go through the whole process of sample preparation. If the result of genetic toxicology test is negative, the preparation system should be checked to find out the reason, and the positive control test should be repeated until it is qualified.
10 Safety
Since the toxicity and carcinogenicity involved in sample preparation are not completely clear, it should be treated as a substance with potential health hazards, and the operator should keep the contact to a minimum.
Part II
11 Scope of application
Pretreatment of surface water and wastewater samples
This specification applies to non-volatile organic compounds in surface water and wastewater, but not to volatile organic compounds. 12 Definition
Surface water. This specification includes river water, river water, lake water, pond water, etc. 515
GB/T154401995
Wastewater, including industrial wastewater and domestic sewage in this specification. Non-aqueous phase; in this specification, it is a liquid phase separated from the water medium, and the main component is not water. Sedimented solid phase, in this specification, it is a solid phase or semi-solid phase separated from the spot water sample that has been left to stand for 24 hours. Instruments and equipment
13.1 Sampling bottle: a brown, screw-mouthed, wide-mouthed glass bottle with a polytetrafluoroethylene liner on the bottle cap, or a rubber stopper with an aluminum foil lining under non-corrosive conditions.
13.2 Separating funnel.
13.3 Water storage container: a glass or wall porcelain container with a lower opening. 13.4 Glass resin column, not less than 10cm high, with a column diameter to column length ratio of 14 to 110. 13.5 Infusion pump.
13.6~13.11 are the same as 4.6~4.11 respectively. 14 Reagents
14.1 Pure water: the same as \5.1.
14.2 Sodium hydroxide: c(NaOH)=10mol/L, 14.3 1-1(V/V) sulfuric acid
14.4 Sodium hydroxide c(NaOH)=1mol/L. 14.5 Hydrochloric acid tc(HCL)=1 mol/L
14.6 Solvents: the grade shall not be lower than analytical grade, and all shall be redistilled in glass containers before use. 14.6.1 Dichloromethane.
14.6.2 Methanol.
14.6.3 Acetone.
14.6.4 Hexane.
14.6.5 Dimethyl sulfoxide (DMSO).
14.7 XAD-2 resin or equivalent macroporous resin. The resin should be rinsed with pure water and methanol, and then extracted with methanol, dichloromethane, hexane, and acetone in a Soxhlet extractor for 8 hours to remove organic matter. The purified resin is immersed in methanol and refrigerated in a refrigerator for later use. 15 Sample collection
Manually collect an appropriate amount of sample and put it into a sampling bottle. The sample should completely fill the container, seal it, and return it to the laboratory within 2 hours for processing as soon as possible.
16 Sample preparation
16.1 Separation and extraction of organic matter
16.1.1 Sample pretreatment before separation and extraction of organic matter Place the sampling bottle at 4℃ for 24h to separate the non-aqueous liquid phase, aqueous phase and sedimented solid phase. Treat the non-aqueous liquid phase according to the sample preparation specifications for non-aqueous liquid waste; treat the sedimented solid phase according to the sample preparation specifications for solid waste. Treat the aqueous phase as follows: surface water with a suspended matter weight of less than 5% can be directly subjected to macroporous resin extraction of organic matter; surface water and wastewater with a suspended matter weight of more than 5% can be subjected to liquid-liquid extraction of organic matter. 16.1.2 Liquid-liquid extraction of organic matter
Take 2 portions of 1500 mL of water sample and divide them into 2 200 nml separatory funnels. Use 10 mol/1.5 sodium hydroxide to adjust the pH to 4. Adjust to 11. Add 150 mL of dichloromethane to each separatory funnel and shake for 2 minutes, paying attention to degassing. Let it stand for at least 10 minutes to separate the organic phase from the aqueous phase and separate the organic phase. Then extract twice with 100 mL of dichloromethane each. Combine the three extracts in a 1,000 mL flask. If the emulsion layer between the organic phase and the aqueous phase is more than 1/3 of the solvent layer, physical methods such as centrifugation can be used to separate the two phases. Use 1 * 1 acid solution to separate the water 516
GB/T 15440—1995
The pH of the phase is adjusted to below 2. Solvent extraction is performed three times with 150mL, 100mL, and 100mL of dichloromethane. The organic phases extracted three times are also combined into a 1000mL flask.
16.1.3 Macroporous resin separation and extraction of organic matter. The purified resin is loaded into the resin column together with acetone. The upper and lower wheels of the resin are respectively sealed and covered with glass wool. The water container is connected to the resin column. Drain the acetone in the column and wash the column 3 times with pure water. Allow the water sample to flow through the resin column at a flow rate of 1 to 2 times the column volume/min. The amount of water sample should not exceed 2000 times the column volume. Use vacuum to separate the purified resin and acetone. Pump out the remaining water in the column, then use 4 to 8 column volumes of 85115 hexane:acetone and 8 column volumes of methylene chloride to wash out the organic matter three times. The bubbling time is 10 minutes each time. Then drip slowly and collect the eluate in a flask. 16.1.4 If necessary, extract different organic components from the extract of 16.1.2 and the eluate of 16.1.3 according to 7.1.3. 16.2. Concentration of extract
Concentrate the extract or eluate obtained in 16.1.2, 16.1.3, and 16.1.4 according to the method described in 7.2. 16.3 Solvent replacement
With 7. 3 Same.
17 Expression of sample volume calculation results
17.1 The volume of water phase obtained by sample pretreatment or the volume of water sample flowing through the resin column is expressed in L. 17.2 The weight of organic extract is expressed in mg. 18 Quality control
18.1 Set up resin and solvent controls, that is, use pure water instead of water sample to complete the whole process of sample preparation. Use the obtained concentrate for genetic toxicity test. If it is positive, check the resin and solvent system respectively, find out the cause, and repeat the test until it is qualified. 18.2 Set up positive samples.
18.3 Set up positive controls, see 9.2.
19 Safety
Same as 10.
Part III Pretreatment of non-aqueous liquid waste samples 20 Scope of application
This specification applies to volatile and non-volatile organic matter in non-aqueous liquid waste. 21 Specification
Non-aqueous liquid waste: In this specification, it refers to liquids whose main component is not water, including water-soluble and non-water-soluble liquids and mixtures of certain liquids. The solid content in the liquid should be less than 5%. 22 Instruments and equipment
22.1 Sampling bottle 1000~1500mL, screw-mouth glass bottle with polytetrafluoroethylene liner on the bottle cap. 22.2 Ultrasonic cleaner.
22.3 Separating funnel
22.4 Same as the instruments and equipment listed in 4.6~4.10. 22.5-~General laboratory equipment and glassware, the treatment method is the same as 4.11. 23 Reagents
23.1 Pure water, the same as 5.1.
GB/T.15440-1995
23.2 Solvents: The grade shall not be lower than analytical grade, and all shall be redistilled in glass containers before use. 23-2.1 Dichloromethane.
23.2.2 Dimethyl sulfoxide (DMSO).
23-3 Sodium hydroxide: c(NaOH)=1 mol/L. 23.4 Hydrochloric acid: c(HCI)=1 mol/L.
23:5 Anhydrous sodium sulfate.
24 Sample collection
Randomly collect a certain amount of samples in a glass container. The sample should fill the container and be sent to the laboratory within 2 hours! Place the sample at room temperature for 1 to 2 hours before use and weigh it. Sample preparation should be carried out as soon as possible after sampling. 25 Sample preparation
25.1 Volatile non-aqueous liquid waste that does not require component extraction does not need to be subjected to organic matter extraction and can be directly used for mutagenicity detection by experimental means such as the desiccator method.
25.2 Non-volatile non-aqueous wastes that do not require component extraction do not require organic matter extraction. Instead, a certain amount of the test solution needs to be weighed and dissolved or suspended in a certain amount of DMSO or other solvents required for genetic toxicity testing. After ultrasonic treatment at room temperature for 5 minutes, genetic toxicology testing can be performed.
25.3.: Extraction of different organic components: If genetic toxicity testing requires samples to be extracted by component, proceed as follows. First, weigh nearly 5g of sample, put it into a 250mL conical flask, add 100mL of dichloromethane, and stir the flask to dissolve the sample. If it is not complete, use a separatory funnel to separate the layers (the liquid phase that is insoluble in dichloromethane can be directly used for genetic toxicology testing.) The subsequent procedures are the same as 7.1.3.
25.4 Concentration of the extract: Concentrate the components obtained in 25.3 according to the method described in 7.2. 25.5 Solvent replacement: Same as 7.3.
26Expression of the calculation results of the number of items
26.1 The total amount of sample is in g.
26-225.4 The weight of the concentrated sample is in g. 26.3 The amount of each organic component extracted is in mz. 27 Quality Control
27.1 Set up a solvent control, that is, do not use the sample. Only use the required solvent to complete the entire sample preparation process. If the genetic toxicity test result of the obtained concentrate is positive, the solvent should be replaced and the solvent blank test should be repeated until it is qualified. 27.2 Set up parallel samples.
27.3 Set up a positive control, see 9.2.
28 Safety
Similar to 10.
Chapter 4 Pretreatment of Soil and Sediment Samples 29 Scope of Application
This specification applies to non-volatile organic matter in ten soils and sediments. 518
30 Definition
.GB/T 15440—1995
Soil: In this specification, it refers to the material with particle diameter less than 2 mm, which can support plant growth and is combined into one. Sediment: In this specification, it refers to the soil-like material that is deposited and residual in the aquatic environment. 31 Instruments and equipment
31.1 Sampling bottle: brown, wide-mouthed, screw-mouthed glass bottle with a polytetrafluoroethylene liner. 31.2 Sampling spoon or shovel.
31.3 Soxhlet extractor.
31.4 Grinder.
31.5 Ultrasonic cleaner: 500W power. 31.6 Separating funnel.
31.7 Instruments and equipment listed in 4.6~4,10.
31.8 General laboratory equipment and glassware, the treatment method is the same as 4.11. 32 Reagents
32.1 Pure water, same as 5.1.
32.2 Solvents: The grade shall not be lower than analytical grade, and all shall be redistilled in glass containers before use. 32.2.1 Dichloromethane.
32.2.2 Dimethyl sulfoxide (DMSO).
32.3 Anhydrous sodium sulfate
32.4 Sodium hydroxide: c(NaOH)=1 mol/L. 32.5 Hydrochloric acid c(HCl)=1 mol/L.
33 Sample collection
Put the sample into the sampling bottle, and the sample should fill the container. If collecting underwater samples, use water samples to wash away the surface covering. Sediment samples should be covered with water samples before screwing on the bottle cap. Samples should be stored and transported at 4℃. If more than 48 hours, they should be stored at -10℃. Generally, the sampling volume is about 500g in the case of oil or other organic pollution. The sampling volume of soil with less serious pollution or high water content should be 2~3kg. When sampling, attention should be paid to the representativeness of the sample to the sampling site. 5~10 samples should be collected from sampling points in different directions within the sampling site, and mixed and evenly mixed into one sample. Sample preparation should be carried out as soon as possible after sample collection. 34 Sample preparation
34.1 Separation and extraction of organic matter
34.1.1 Sample pretreatment before separation and extraction of organic matter Air dry the sample, crush the sample pellets, and sieve through 40~60 mesh. The sampling is divided by quartering until a suitable sample is selected. Two samples should be retained, one of which is used to measure the water content. The sediment sample is centrifuged to remove the water in the sample, and the sample weight should be measured before and after the water is removed. Then, another sample is retained to measure the water content as with the soil.
34.1.2 Soxhlet extractionwwW.bzxz.Net
Organic matter in the sample can be extracted by Soxhlet extraction. Mix 10 g soil sediment sample with 10 g anhydrous sodium sulfate and place in a Soxhlet extractor. Use 300 mL difluoromethane to extract for 16 h at the boiling point of the solvent (reflux 5 to 10 times per hour). Let the extract cool, filter, and then pass it through an anhydrous sodium sulfate column to remove moisture. 34.1.3 Grinding extraction
Grinding extraction can also be used.
GB/T15440—1995
Mix 25 soil or sediment samples with 25 g anhydrous sodium sulfate and place in a grinding jar. Add about 150 mL of dichloromethane, tighten the grinder cover, grind for 30 minutes, and pour out the solvent. Repeat the extraction with 75 mL of dichloromethane twice, or until the extract is colorless. Combine the extracts, filter, and pass them through an anhydrous sodium sulfate column to remove moisture. 34.1.4 Ultrasonic extraction
Ultrasonic extraction can be used for samples that are not suitable for Soxhlet extraction or grinding extraction. Mix 25 g of soil or sediment sample with 50 g of anhydrous sodium sulfate and place in a 250 mL beaker. Extract twice with 150 mL of difluoromethane in an ultrasonicator or until the extract is colorless. Combine the extracts, filter, and pass them through an anhydrous sodium sulfate column to remove water. 34.1.5 Extraction of different organic components
If necessary, extract different organic components as follows. First, dissolve 10 g of the extract obtained by the above methods in 30 mL of dichloromethane and place in a separatory funnel. The subsequent procedures are the same as 7.1.3. 34.2 Concentration of extract
Concentrate the extract obtained in 34.1 as described in 7.2. 34.3 Solvent replacement
Same as 7.3.
35 Display of sample quantity calculation results
35.1 The total sample quantity is expressed in g.
35.2 The dry weight of soil sample is the difference between the total soil sample quantity and the water content, expressed in &; the dry weight of sediment sample is the difference between the total sediment sample quantity and the weight and water content of water sample, expressed in. 35.3 The amount of each organic matter and each organic component extracted is measured in mg. 36 Quality control
36.1 Set up solvent control, the same as 27.1. 36.2 Set up parallel samples.
36.3 Set up positive control, see 9.2.
37 Safety
The same as 10.
Chapter V Pretreatment of Solid Waste Samples
38 Applicable Scope
This specification applies to non-volatile organic matter in solid waste. 39 Definition
Solid waste: In this specification, it refers to waste with a water content of less than 50%, composed of particles with a certain hardness and adhesion, or substances that remain non-liquid or gaseous at 25°C. 40 Instruments and equipment
40-1 Sample container: Brown, large-mouthed, screw-mouthed glass bottle with a mulberry polytetrafluoroethylene liner on the bottle cap. If the sample is corrosive to glass, a polytetrafluoroethylene container can be used.
40.2 Sampling spoon or shovel.
40.3 Soxhlet extractor.
40.4 Grinder.
40.5 Separating funnel.
40.6 Instruments and equipment listed in 4.6~4.10.
41 Reagents
Same as 32.
42 Sample collection
GB/T 15440—1995
Use a sampling spoon or shovel to collect an appropriate amount of sample into the sample container. The sampling operation should be carried out under weak light conditions. The sample should be stored and transported at 4°C or below. Generally, the sample preparation should be carried out immediately after sampling. 43 Sample preparation
43.1 Methods for removing liquid phase and reducing sample particle size Use the following appropriate physical methods to remove the liquid phase in the sample and reduce the sample particle size. Let it stand at 4°C for 24 hours to separate the liquid phase from the solid phase; a.
Oily or sticky waste can be treated with anhydrous sodium sulfate or silica gel, b.
Grind and sieve through 40~～60 mesh;
d. Oily or sticky waste can be frozen and then ground to reduce its particle size. 43.2 Separation and extraction of organic matter
43.2.1 Soxhlet extraction
Mix 10 g of solid sample with 10 g of anhydrous sodium sulfate, put it into a Soxhlet extractor, and extract it with 300 mL of difluoromethane for 16 h. Let the extract pass through anhydrous sodium sulfate to remove water.
43.2.2 Grinding extraction
Mix 10 g of solid sample with 10 g of anhydrous sodium sulfate, put it into a grinder, and add 300 mL of difluoromethane. Grind for 30 s, rest for 5 min, and grind for another 15 s. Repeat the above operation. After grinding, filter the extract with filter paper and pass it through anhydrous sodium sulfate to remove water. 43.2.3 Extraction of different organic components:
Same as 7.1.3.
43.3 Concentration of extracts
Concentrate the extracts obtained in 43.2.1, 43.2.2 and 43.2.3 as described in 7.2. 43.4 Solvent replacement
Same as 7.3.
44 Presentation of sample calculation results
44. 1 Total sample volume in g.
44.2 Amount of extracted organic matter and each organic component in mg. 45 Quality control
Set up solvent control, same as 27.1.
45.2 Set up replicates.
45.3 Set up positive control, see 9.2.
Safety
Same as 10.
Additional Notes:
GB/T 15440—1995
This standard was proposed by the Science and Technology Standards Division of the State Environmental Protection Administration. This standard was drafted by the Beijing Environmental Protection Science Research Institute and the Chinese Academy of Environmental Sciences. The main drafters of this standard are Wang Jing and Yan Leisheng. This standard is interpreted by the State Environmental Protection Administration. 522
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