CN105651986A - Method for determining toxicity of pesticide granule by using daphnia magna - Google Patents
Method for determining toxicity of pesticide granule by using daphnia magna Download PDFInfo
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- CN105651986A CN105651986A CN201511008512.3A CN201511008512A CN105651986A CN 105651986 A CN105651986 A CN 105651986A CN 201511008512 A CN201511008512 A CN 201511008512A CN 105651986 A CN105651986 A CN 105651986A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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Abstract
The invention provides a method for determining the toxicity of a pesticide granule by using daphnia magna. The method comprises the following steps: dissolving the pesticide granule to prepare a pesticide stock solution; mixing the pesticide stock solution with an Elendt M4 culture solution to prepare a mixed solution with gradient concentrations, continuously culturing for 48h with the mixed solution as domestic water of the daphnia magna, and recording the biological effect parameters of the daphnia magna and the physical and chemical parameters of the mixed solution every 24h in the culture process; and calculating the median inhibitory concentration EC50 of the pesticide to the daphnia magna according to the biological effect parameters of the daphnia magna and the physical and chemical parameters of the pesticide stock solution, and judging the toxicity of the pesticide granule. The method has the advantages of simple operation, strong practicality, fast detection speed and high accuracy.
Description
Technical field
The present invention relates to cultural technique field, a kind of method particularly relating to large-scale mensuration granular pesticide toxicity.
Background technology
The large-scale advantage as test species is a lot of, is easily managed as small, greatly reduces cultured space and management cost, can reduce for examination thing consumption and waste liquid amount in process of the test accordingly; Egg laying amount is big, and a tail raun can lay eggs weekly 1��2 time, and each egg laying amount is 100��300 pieces; Large-scale embryo is transparent, it is simple to observe its early development form. Both at home and abroad all using large-scale as the model organism of environment toxicological test in chemicals, agriculture chemical registration, be widely used in the fields such as life sciences, toxicology, environmental science and agricultural sciences.
At present, the widely used large-scale research carrying out Pesticide environment safety evaluatio, but adopt the large-scale research as granular pesticide toxicity screening model less, lack practicality and the reliability of relevant data assessment models. Therefore it provides a kind of method of easy operation, practical large-scale screening granular pesticide toxicity is even more important.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, it is provided that a kind of method of large-scale mensuration granular pesticide toxicity, has the advantages such as easy and simple to handle, practical, detection speed is fast, degree of accuracy height.
For solving above-mentioned technical problem, it is provided that a kind of method of large-scale mensuration granular pesticide toxicity, comprise the following steps:
(1) granular pesticide is dissolved, make pesticide storing solution;
(2) by described pesticide storing solution and ElendtM4Culture fluid is hybridly prepared into the mixed liquor of gradient concentration, by described mixed liquor as large-scale domestic water, continues to cultivate 48h, in incubation, records the physical and chemical parameter of large-scale biological effect parameter and described mixed liquor when 24h;
(3) pesticide is calculated to half-inhibition concentration EC according to the physical and chemical parameter of large-scale biological effect parameter and pesticide storing solution50, it is judged that the toxicity of granular pesticide.
Above-mentioned method, it is preferred that described step (1) particularly as follows: by described granular pesticide, with the mixed solution of acetone and tween 80 dissolve, ultrasonic, be filtrated to get described pesticide storing solution.
Above-mentioned method, it is preferred that in the mixed solution of described acetone and tween 80, the mass concentration��0.100g/L of tween 80, the mass concentration��0.100mL/L of described acetone.
Above-mentioned method, it is preferred that large-scale for derive from same maternal parthenogenetic reproduction 3 generation more than described in described step (2), the date of birth non-the first-born health children less than 24h.
Above-mentioned method, it is preferred that in the process that described step (2) carries out, described large-scale not feeding.
Above-mentioned method, it is preferred that ElendtM described in described step (2)4Culture fluid is the ElendtM of air aeration pump more than Air Exposure 24h4Culture fluid, described ElendtM4The pH of culture fluid is 6.0��9.0, and hardness is 140��250mg/L, dissolved oxygen >=3mg/L.
Above-mentioned method, it is preferred that in described step (2), described biological effect parameter is large-scale suppression ratio; Described physical and chemical parameter is the water temperature of mixed liquor, pH value, dissolved oxygen content. Judging that the standard of described large-scale suppression is as gently shaking beaker, not moving about in 15s is considered as movable suppression, but allows the faint swing of appendage.
Above-mentioned method, it is preferred that in described step (2), described incubation control water temperature be 18 DEG C��22 DEG C, illumination every day 16h, dissolved oxygen concentration be not less than 3.0mg/L.
Above-mentioned method, it is preferred that the criterion of the toxicity of described granular pesticide is: EC50> 10.0mga.i./L represents low toxicity, 1.00mga.i./L < EC50�� 10.0mga.i./L represents poisoning, 0.100mga.i./L < EC50�� 1.00mga.i./L represents high poison, EC50�� 0.100mga.i./L represents severe toxicity.
Above-mentioned method, it is preferred that the method for described large-scale mensuration granular pesticide toxicity is additionally provided with blank group and solvent control group; Described blank group is with ElendtM4Culture fluid is large-scale domestic water; Described solvent control group is with the mixed solution of acetone and tween 80 for described large-scale domestic water; Described blank group and the medium-and-large-sized suppressed rate of solvent control group not can exceed that 10% proves reliable experiment result.
In solvent control group, the content of acetone and tween 80 should be test group and makes the maximum of consumption, and the mass concentration at tween 80 answers��0.100g/L, and the mass concentration of described acetone answers��0.100mL/L.
Compared with prior art, it is an advantage of the current invention that:
(1) method that the invention provides the large-scale screening granular pesticide toxicity of a kind of easy and simple to handle, practical model organism; And granular pesticide effective ingredient is fully extracted, evaluate it to large-scale toxicity, there is speed fast, the advantage that degree of accuracy is high.
Detailed description of the invention
Below in conjunction with concrete preferred embodiment, the invention will be further described, but protection domain not thereby limiting the invention.
Embodiment
The material adopted in following example and instrument are commercially available.
Embodiment 1:
(1) preparation of pesticide storing solution:
Weighing 10% fosthiazate granule 1.3889g, use 50.00mL acetone solution, be then settled in 100mL volumetric flask with 0.1% tween 80 aqueous solution, after 2h sonic oscillation, discard solid particle, take supernatant and cross 0.4 ��m of moisture film, obtaining concentration is 2.50 �� 103The finely dispersed solution of mga.i./L.
The solution distilled water pipetting 1.000mL is settled in 100mL volumetric flask, is configured to the pesticide storing solution that concentration is 25.0mga.i./L.
(2) preparation of experimental liquid:
ElendtM4The preparation of culture fluid: by ElendtM4Culture fluid carries out air aeration pump more than Air Exposure 24h, detects ElendtM4The pH of culture fluid is 7.15, and hardness is 189mg/L, and dissolved oxygen is 7.82mg/L.
Experimental group: the pesticide storing solution pipetted successively in 0.300mL, 0.600mL, 1.200mL, 2.400mL, 4.800mL step (1) uses ElendtM respectively4Culture fluid is settled in 250mL volumetric flask, and obtaining concentration is 3.00 �� 10-2mga.i./L��6.00��10-2The experimental liquid of mga.i./L, 0.120mga.i./L, 0.240mga.i./L, 0.480mga.i./L.
Blank group (CK1): with ElendtM4Culture fluid is as experimental liquid.
Solvent control group (CK2): pipette 0.024mL acetone and 0.024mL0.1% tween 80 aqueous solution to 250ml volumetric flask, and use ElendtM4Culture fluid constant volume.
(3) contamination
Selecting and derive from same maternal parthenogenetic reproduction 4 generation, date of birth, the non-the first-born health children of 6h��24h, joins in the experimental liquid of above-mentioned steps (2) at random. 5, every beaker, cultivates, incubation control water temperature be 20 DEG C, cultivate 16h, dark culturing 8h under illumination every day, dissolved oxygen concentration is not less than 3.0mg/L. Large-scale not feeding in contamination process.
After contamination starts, measure and record the temperature of experimental liquid, pH value, dissolved oxygen amount etc. in beaker in 24h, 48h. After contamination, 24h, 48h observe and record large-scale suppression number. Judging that the standard of described large-scale suppression is as gently shaking beaker, not moving about in 15s is considered as movable suppression, but allows the faint swing of appendage.
Table 1 is that 10% fosthiazate granule is to large-scale acute toxicity motility inhibition test data record result. Table 2 is the water temperature of each concentration group during test, pH value, the testing result of dissolved oxygen.
Table 1:10% fosthiazate granule is to large-scale acute toxicity test data logger
In processing procedure, large-scale occurring in that travelling positive, spins in original place, and the biological inhibition effect such as appendage and the faint swing of feeler, the concentration more high time is more of a specified duration, and symptom also can be more serious.
Table 2: the testing result table of the water temperature of domestic water, pH value, dissolved oxygen in water vat
The Ph of experimental group 2 to 4, temperature, dissolved oxygen content etc. are all in the scope that experimental group 1 and experimental group 5 form.
(4) data process
Adopt statistic software SPSS 12.0, with the logarithm value of concentration for the treatment of for independent variable x, with the probit value of (correction) mortality rate for dependent variable y, set up virulence regression equation. Calculate 10% fosthiazate granule to large-scale inhibition concentration EC50With 95% confidence limit, concrete result of calculation is referring to table 3.
Table 3:10% fosthiazate granule is to large-scale the acute toxicity tests table
(5) evaluation of result
According to EC50Value judges pesticide poison level. Toxicity grading standard is: low toxicity (EC50> 10.0mga.i./L), poisoning (1.00mga.i./L < EC50�� 10.0mga.i./L), high poison (0.100mga.i./L < EC50�� 1.00mga.i./L), severe toxicity (EC50 ��0.100mga.i./L)��
Under this experimental condition, the acute movable toxic grade suppressed of class is " high poison " by 10% fosthiazate granule.
Embodiment 2:
(1) preparation of pesticide storing solution:
Weigh 5% chlopyrifos granule 0.5000g, use 50.00mL acetone solution, be then settled in 100mL volumetric flask with 0.1% tween 80 aqueous solution, after 2h sonic oscillation, discarding solid particle, take supernatant and cross 0.4 ��m of moisture film, obtaining concentration is the finely dispersed solution of 250mga.i./L.
Pipette the above-mentioned solution ElendtM of 0.100mL4Culture fluid is settled in 100mL volumetric flask, is configured to the pesticide storing solution that concentration is 0.250mga.i./L.
(2) preparation of experimental liquid:
ElendtM4The preparation of culture fluid: by ElendtM4Culture fluid carries out air aeration pump more than Air Exposure 24h, detects ElendtM4The pH of culture fluid is 7.15, and hardness is 189mg/L, dissolved oxygen 7.82mg/L.
Experimental group: the pesticide storing solution pipetted successively in 0.500mL, 1.000mL, 2.000mL, 4.000mL, 8.00mL step (1) uses ElendtM respectively4Culture fluid is settled in 250mL volumetric flask, and obtaining concentration is 5.00 �� 10-4mga.i./L��1.00��10-3mga.i./L��2.00��10-3mga.i./L��4.00��10-3mga.i./L��8.00��10-3The test medicinal liquid of mga.i./L.
Blank group (CK1): with ElendtM4Culture fluid is as experimental liquid.
Solvent control group (CK2): pipette 0.004mL acetone and 0.004mL0.1% tween 80 aqueous solution to 250ml volumetric flask, and use ElendtM4Culture fluid constant volume.
(3) contamination
Selecting and derive from same maternal parthenogenetic reproduction 5 generation, date of birth, the non-the first-born health children of 6h��24h, joins in the experimental liquid of above-mentioned steps (2) at random. 5, every beaker, cultivates, incubation control water temperature be 20 DEG C, cultivate 16h, dark culturing 8h under illumination every day, dissolved oxygen concentration is not less than 3.0mg/L. Large-scale not feeding in contamination process.
After contamination starts, measure and record the temperature of experimental liquid, pH value, dissolved oxygen amount etc. in beaker in 24h, 48h. After contamination, 24h, 48h observe and record large-scale suppression number. Judging that the standard of described large-scale suppression is as gently shaking beaker, not moving about in 15s is considered as movable suppression, but allows the faint swing of appendage.
Table 4 be 5% chlopyrifos granule to large-scale to large-scale acute toxicity motility inhibition test data record result. Table 5 is the water temperature of each concentration group during test, pH value, the testing result of dissolved oxygen.
Table 4:5% chlopyrifos granule is to large-scale acute toxicity test data logger
In processing procedure, large-scale occurring in that travelling positive, spins in original place, and the biological inhibition effect such as appendage and the faint swing of feeler, the concentration more high time is more of a specified duration, and symptom also can be more serious.
Table 5: the testing result table of the water temperature of domestic water, pH value, dissolved oxygen in water vat
The Ph of experimental group 2 to 4, temperature, dissolved oxygen content etc. are all in the scope that experimental group 1 and experimental group 5 form.
(4) data process
Adopt statistic software SPSS 12.0, with the logarithm value of concentration for the treatment of for independent variable x, with the probit value of (correction) mortality rate for dependent variable y, set up virulence regression equation. Calculate 5% chlopyrifos granule to large-scale inhibition concentration EC50With 95% confidence limit, concrete result of calculation is referring to table 6.
Table 6:5% chlopyrifos granule is to large-scale the acute toxicity tests table
(5) evaluation of result
According to EC50Value judges pesticide poison level. Toxicity grading standard is: low toxicity (EC50> 10.0mga.i./L), poisoning (1.00mga.i./L < EC50�� 10.0mga.i./L), high poison (0.100mga.i./L < EC50�� 1.00mga.i./L), severe toxicity (EC50��0.100mga.i./L)��
Under this experimental condition, the acute movable toxic grade suppressed of class is " severe toxicity " by 5% chlopyrifos granule.
The above, be only presently preferred embodiments of the present invention, and the present invention not does any pro forma restriction. Although the present invention discloses as above with preferred embodiment, but is not limited to the present invention. Any those of ordinary skill in the art, when without departing from the spirit of the present invention and technical scheme, all may utilize the method for the disclosure above and technology contents and technical solution of the present invention is made many possible variations and modification, or be revised as the Equivalent embodiments of equivalent variations. Therefore, every content without departing from technical solution of the present invention, to any simple modification made for any of the above embodiments, equivalent replacement, equivalence change and modify according to the technical spirit of the present invention, all still fall within the scope of technical solution of the present invention protection.
Claims (10)
1. the method for one kind large-scale mensuration granular pesticide toxicity, it is characterised in that comprise the following steps:
(1) granular pesticide is dissolved, make pesticide storing solution;
(2) by described pesticide storing solution and ElendtM4Culture fluid is hybridly prepared into the mixed liquor of gradient concentration, by described mixed liquor as large-scale domestic water, continues to cultivate 48h, in incubation, records the physical and chemical parameter of large-scale biological effect parameter and described mixed liquor when 24h;
(3) pesticide is calculated to half-inhibition concentration EC according to the physical and chemical parameter of large-scale biological effect parameter and pesticide storing solution50, it is judged that the toxicity of granular pesticide.
2. method according to claim 1, it is characterised in that described step (1) particularly as follows: by described granular pesticide, with the mixed solution of acetone and tween 80 dissolve, ultrasonic, be filtrated to get described pesticide storing solution.
3. method according to claim 2, it is characterised in that in the mixed solution of described acetone and tween 80, the mass concentration��0.100g/L of tween 80, the mass concentration��0.100mL/L of described acetone.
4. method according to claim 1, it is characterised in that large-scale for derive from same maternal parthenogenetic reproduction 3 generation more than described in described step (2), the date of birth non-the first-born health children less than 24h.
5. method according to claim 1, it is characterised in that in the process that described step (2) carries out, described large-scale not feeding.
6. method according to claim 1, it is characterised in that ElendtM described in described step (2)4Culture fluid is the ElendtM of air aeration pump more than Air Exposure 24h4Culture fluid, described ElendtM4The pH of culture fluid is 6.0��9.0, and hardness is 140��250mg/L, dissolved oxygen >=3mg/L.
7. method according to any one of claim 1 to 6, it is characterised in that in described step (2), described biological effect parameter is large-scale suppression ratio; Described physical and chemical parameter is the water temperature of mixed liquor, pH value, dissolved oxygen content.
8. method according to any one of claim 1 to 6, it is characterised in that in described step (2), described incubation control water temperature be 18 DEG C��22 DEG C, illumination every day 16h, dissolved oxygen concentration be not less than 3.0mg/L.
9. method according to any one of claim 1 to 6, it is characterised in that the criterion of the toxicity of described granular pesticide is: EC50> 10.0mga.i./L represents low toxicity, 1.00mga.i./L < EC50�� 10.0mga.i./L represents poisoning, 0.100mga.i./L < EC50�� 1.00mga.i./L represents high poison, EC50�� 0.100mga.i./L represents severe toxicity.
10. method according to any one of claim 1 to 6, it is characterised in that the method for described large-scale mensuration granular pesticide toxicity is additionally provided with blank group and solvent control group; Described blank group is with ElendtM4Culture fluid is large-scale domestic water; Described solvent control group is with the mixed solution of acetone and tween 80 for described large-scale domestic water; Described blank group and the medium-and-large-sized suppressed rate of solvent control group not can exceed that 10% proves reliable experiment result.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110988323A (en) * | 2019-12-10 | 2020-04-10 | 东南大学 | Method for evaluating toxicity of total sediments by utilizing daphnia magna |
| CN114878761A (en) * | 2022-04-25 | 2022-08-09 | 贵阳市花溪区农业农村局 | Method for rapidly screening organophosphorus pesticide residues in strawberries by utilizing daphnia magna and application of method |
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| CN1432538A (en) * | 2002-01-18 | 2003-07-30 | 何文辉 | Method of cultivating algivorous insects to tackle blue-green alga pollution in water |
| CN103983727A (en) * | 2014-05-29 | 2014-08-13 | 南京大学 | Method for identifying key toxic substances in pesticide wastewater by taking daphnia magna toxicity as guide |
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2015
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1432538A (en) * | 2002-01-18 | 2003-07-30 | 何文辉 | Method of cultivating algivorous insects to tackle blue-green alga pollution in water |
| CN103983727A (en) * | 2014-05-29 | 2014-08-13 | 南京大学 | Method for identifying key toxic substances in pesticide wastewater by taking daphnia magna toxicity as guide |
Non-Patent Citations (2)
| Title |
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| 宋伟华等: "农药助剂和实验室常用有机溶剂对大型潘的室内急性毒性研究", 《农药科学与管理》 * |
| 蒋红云等: "4 种新烟碱类杀虫剂对大型溞的急性活动抑制", 《农药科学与管理》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110988323A (en) * | 2019-12-10 | 2020-04-10 | 东南大学 | Method for evaluating toxicity of total sediments by utilizing daphnia magna |
| CN114878761A (en) * | 2022-04-25 | 2022-08-09 | 贵阳市花溪区农业农村局 | Method for rapidly screening organophosphorus pesticide residues in strawberries by utilizing daphnia magna and application of method |
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