Some standard content:
National Standard of the People's Republic of China
Standard methods for bioassay of daphnia magna straus
GB/T16125-1995
This standard refers in part to the international standard IS06341:1982 "Water quality - Determination of inhibition of activity of Daphnia magna straus (Crustacea, Cladocera)" (UDC617.777:576.097.2) and the American Standard Method "Standard Methods for the Examination of Water and Wastewater" 804B. Bioassay procedures for Daphnia (Daphnia).
1 Subject content and scope of application
This standard specifies the survival and movement effects (acute test) and growth and reproduction test methods (chronic effects) in the bioassay technology of Daphnia magna straus.
This standard is applicable to the evaluation of the toxicity of soluble chemical substances; the evaluation of the comprehensive toxicity of industrial wastewater and industrial solid waste overflow; the evaluation of wastewater treatment effects; and also applicable to the toxicity evaluation of surface water, groundwater and sediments in water. 2 Principle of the method
2.1 Determine the initial concentration ECs value, which refers to the concentration that makes 50% of the large Daphnia exposed to the test solution lose their ability to move within 24h and 48h under the conditions specified in this standard, and indicate the lowest concentration that makes all experimental Daphnia lose their ability to move, and the highest concentration that cannot make any experimental Daphnia lose their ability to move. 2.2 Determine the initial concentration LCs value, which is the concentration that makes 50% of the large Daphnia exposed to the test solution stop beating within 24h and 48h under the conditions specified in this standard, and indicate the lowest concentration that makes all large Daphnia die and the highest concentration that does not cause death. 2.3 Determine the growth curve. The growth rate of the experimental Daphnia at different concentrations is different, and its growth curve is also different. Compare with the growth curve of the control group to judge its growth effect.
2.4 Determination of reproductive output. Under the same conditions, the number of young Daphnia born per litter is different due to the toxicity of different concentrations of experimental solution. Compare with the control group to determine its toxicity. 3 Reagents and materials
3.1 Test organisms
Use a large Daphnia (Daphnia magnastraus) 62Dm pure strain biological strain, which is the same age and mother. ECso and LCs tests use 1- to 3-year-old Daphnia, 10 per container, parallel 2-3 groups: Reproduction and growth tests use newly born young water eggs, 5-6 Daphnia per concentration group.
3.2 Dilution water
Natural water (surface water or groundwater) and tap water (natural water) that are not polluted by toxic substances can be used, with a pH value of 7-8.6, dissolved oxygen of more than 4 mg/L, or more than 60% of the saturated oxygen in the air, and a water hardness of 250±22 mg/L (expressed as CaCO.). Artificially prepared water that meets the above conditions can also be used.
3.3 Instruments
3.3.1 General laboratory glass instruments.
Approved by the State Administration of Technical Supervision on December 15, 1995 and implemented on July 1, 1996
3.3.2 Dissolved oxygen measuring instrument.
3.3.3 Stereo microscope.
3.3.4 pH meter.
GB/T16125-1995
3.3.5 Test container: glass crystal III or beaker. 3.4 Preparation of samples and experimentsbzxz.net
3.4.1 The samples are required to be representative. The sampling bottle should be filled with water. The collected wastewater samples are required to be tested for toxicity within 6 hours, otherwise they must be refrigerated.
3.4.2 Preparation of test solution
3.4.2.1 Preparation of stock solution
Dissolve a known amount of the substance to be tested in deionized water, first prepare a high-concentration experimental stock solution, and continuously dilute it by multiples of 10 to different concentrations, expressed in mg/1. When preparing a high-concentration stock solution for insoluble substances, a small amount of organic solvent (such as acetone) can be added to assist dissolution, but the final concentration of the organic solvent in each experimental solution must not exceed 0.5 mg/L, and a solvent control must be added at the same time. 3.4.2.2 The original water sample of industrial wastewater is the experimental stock solution, which is taken as 100%, and is prepared into various concentrations according to percentage (percentage) with dilution water. 3.4.2.3 Industrial solid waste. First grind it, add deionized water at a solid-liquid ratio of 1:10, shake it well and soak it for 24 hours, filter it with filter paper, and the dissolved part is the leaching stock solution (100%) of the industrial solid waste to be tested, and then prepare various concentrations with dilution water according to percentage. 4 Experimental steps
4.1 Large water egg EC5o and LCs experiment
4.1.1 Preliminary test
Determine the concentration range of the formal test, and require to know the concentration at which all the test algae survive (or have no effect) and the concentration at which all the test algae die (or do not move), to determine whether the sample to be tested is stable and whether the experimental water is healthy. 4.1.2 Formal test
The LCso result is obtained from the number of dead test water fleas in each concentration. The ECso result is obtained from the number of inactive test water fleas in each concentration. At the beginning and end of the experiment, the pH value, water temperature and dissolved oxygen (DO) of the experimental solution were measured. For unstable experimental solutions, the measures of regular replacement were adopted, and the experimental water eggs were not fed during the experiment. 4.2 Reproduction and growth experiment of large water fleas (chronic effect) 4.2.1 Preliminary test
48hLCso test was carried out. The concentration that did not cause the death of water fleas was obtained by 48hLCs. ×0.1, and 2 to 3 growth and reproduction test concentrations were selected within the range of the survival safety concentration of water eggs. 4.2.2 Formal test
According to the concentration selected in the preliminary test, the experimental solutions of various concentrations were prepared, and 4 to 6 groups were paralleled. Each group had a control. The experiment was carried out under normal laboratory lighting to prevent direct sunlight. The pH and DO were measured at the beginning and end of the experiment, and the temperature of the whole experiment was recorded with the highest and lowest thermometers. During the experiment, the experimental young water fleas were taken out with a pipette at a fixed time every 24 hours, and were moved into the corresponding Scenedesmus algae liquid tanks to feed for 1 hour, and then moved back to the medium tank, and the poisoning solution was replaced immediately.
4.3 Determination of sensitivity of Daphnia
The 24hECs of Daphnia magna determined by analytically pure potassium dichromate was below 2mg/L, indicating that the sensitivity of Daphnia magna in this experiment was qualified. 4.4 Data processing and experimental results
4.4.1EC and LC5 test
Use semi-logarithmic paper or probability paper to obtain ECs and LCs values, or use computer LCs program to process data. The experimental results are expressed in mg/L or percentage, and there should be no abnormalities in the control group in the experiment. 4.4.2 Reproduction and growth test
Use the observation time as the horizontal axis and the average body length as the vertical axis to draw the growth curve of each concentration, compare it with the growth curve of the control group, and judge the effective concentration and the ineffective concentration. Count the total number of young Daphnia in each test solution, and obtain the total reproductive amount and reproductive quantity (average number of young Daphnia per litter), 600
GB/T16125-1995
compare whether there is a significant difference with the control group. There should be no abnormality in the control group during the experiment. 5
Test report
When this standard method is clearly adopted, it also includes: a.
Concentration.
The source, species name, product name, number, age, domestication time, health status, etc. of the large Daphnia used in the experiment; the name, source, physical and chemical properties, sampling time and storage of the measured substance, as well as the preparation of the experimental solution; experimental methods, conditions, dilution water, etc.;
data processing, and 24h, 48h ECso, LCs results. Growth curve and reproductive output, poisoning symptoms, 0% and 100% impact 601
GB/T16125---1995
Appendix A
Cultivation and reproduction technology of large water fleas
(Supplement)
A1 In a 1-2L round glass tank or beaker, add 500mL~1000mL of culture water (tap water with natural aeration for more than three days or uncontaminated filtered surface water), add 10~20 large water fleas, and feed fresh Scenedesmus algae solution to make the algae concentration of (6~8)×105/mL the best. Cultivate under natural light conditions indoors, avoid direct sunlight, culture temperature 15~25℃, pH value 7.5±0.5, dissolved oxygen above 2mg/L, and replace the culture medium 1~2 times a week. A2 Isolation of experimental juvenile Daphnia
Select 10 to 20 Daphnia with high egg-bearing capacity, feed them with sufficient bait, filter out the juvenile Daphnia with a 1mm pore size 24 hours before the experiment, and perform a second sieving 6 to 12 hours before the experiment. The newly born juvenile Daphnia obtained are juvenile Daphnia with a birth size of 6 to 24l. Continue to culture this Daphnia for two days to obtain experimental water eggs that are 72 hours old. A3 Cultivation of Scenedesmus
Use artificially cultured Scenedesmus algae liquid to feed large Daphnia, and use the following inorganic salts as the nutrient solution for Scenedesmus algae species. Ammonium sulfate (NH4SO4)
Magnesium sulfate (MgSO4·7H4SO4)
0),
sodium bicarbonate (NaHCO,),
potassium dihydrogen phosphate (KHPO,),
calcium chloride (CaCl,·2H2O),
deionized water
1000mL
Scenes algae can be cultured in a large triangular flask at 15-25℃ water temperature and 49W sunlight (8h~16h), stirring 5-6 times a day. The Scenedesmus algae cultured with the above formula is expanded with dilution water, inoculated at 1:1 (algae solution to dilution water ratio) or 1:2, and urea is added once every 3 days. The final concentration of urea does not exceed 10mg/L. This Scenedesmus algae solution can be used to feed large Daphnia directly. Additional remarks:
This standard is proposed by the Ministry of Health of the People's Republic of China. This standard is drafted by the Institute of Environmental Health and Sanitary Engineering, Chinese Academy of Preventive Medicine. The main drafters of this standard are: Xiu Ruiqin, Gao Shirong, Xu Yongxiang, Ren Gaiying, Guo Qi. This standard is entrusted by the Ministry of Health to the Institute of Environmental Health Monitoring, Chinese Academy of Preventive Medicine, which is responsible for the interpretation.
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