CN105651983A - Method for determining toxicity of pesticide water dispersible granule by using zebra fish - Google Patents
Method for determining toxicity of pesticide water dispersible granule by using zebra fish Download PDFInfo
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- CN105651983A CN105651983A CN201511006190.9A CN201511006190A CN105651983A CN 105651983 A CN105651983 A CN 105651983A CN 201511006190 A CN201511006190 A CN 201511006190A CN 105651983 A CN105651983 A CN 105651983A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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 water dispersible granule by using zebra fish. The method comprises the following steps: dissolving the pesticide water dispersible granule to prepare a pesticide stock solution; mixing the pesticide stock solution with water to prepare a mixed solution with gradient concentrations, continuously culturing for 96h with the mixed solution as domestic water of the zebra fish, and recording the biological effect parameters of the zebra fish and the physical and chemical parameters of the mixed solution every 24h in the culture process; and calculating the median lethal concentration LC50 value of the pesticide to the zebra fish according to the biological effect parameters of the zebra fish, and judging the toxicity of the pesticide water dispersible 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, particularly relate to a kind of method that zebra fish measures pesticide water dispersible granules toxicity.
Background technology
Zebra fish (Brachydoniorerio) belongs to the short red Buddhist nun fish of Chordata spoke fin net-rope Li Xingmu carp section and belongs to, there is easy raising, the feature such as egg laying amount is big, sexual maturation cycle is short, individual little Yi operation, the similarity height of zebra fish gene and Human genome, has important scientific research meaning simultaneously. Therefore, zebra fish has become model animals conventional in scientific research, is widely used in the fields such as life science, toxicology, environmental science and agricultural sciences. Zebrafish embryo and juvenile fish are often used to acute, sub-chronic, chronic toxicity test research in novel chemical substance registration, and zebra fish adult fish is normally used for agriculture chemical registration acute toxicity test research.
At present, extensively adopted zebra fish to carry out the research of Pesticide environment safety evaluation, but adopted zebra fish less as the research of pesticide water dispersible granules toxicity screening model, lacked practicality and the reliability of relevant data assessment models. Therefore, it is provided that a kind of method of easy operation, practical zebra fish screening pesticide water dispersible granules toxicity is especially important.
Summary of the invention
The technical problem to be solved in the present invention overcomes the deficiencies in the prior art, it is provided that a kind of zebra fish measures the method for pesticide water dispersible granules toxicity, has easy and simple to handle, practical, detection speed fast, the advantages such as tolerance range height.
For solving the problems of the technologies described above, it provides a kind of zebra fish measures the method for pesticide water dispersible granules toxicity, comprises the following steps:
(1) pesticide water dispersible granules is dissolved, make agricultural chemicals storing solution;
(2) described agricultural chemicals storing solution and water are hybridly prepared into the mixed solution of gradient concentration, by the domestic water of described mixed solution as zebra fish, continue to cultivate 96h, in culturing process, when 24h, record the biological effect parameter of zebra fish and the physical and chemical parameter of described mixed solution;
(3) agricultural chemicals is calculated to the median lethal concentration LC of zebra fish according to the biological effect parameter of zebra fish50Value, judges the toxicity of pesticide water dispersible granules.
Above-mentioned method, it is preferable that, described step (1) is specially: be added to the water by described agricultural chemicals dispersible granule, carries out stirring or having children outside the state plan obtaining described agricultural chemicals storing solution.
Above-mentioned method, it is preferable that, the selection body length of zebra fish described in described step (2) is the zebra fish of 1��3cm, bouncing.
Above-mentioned method, it is preferable that, before adopting zebra fish to measure pesticide water dispersible granules toxicity in 24h, described zebra fish not feeding, in the process that described step (2) carries out, described zebra fish not feeding.
Above-mentioned method, it is preferable that, water described in described step (2) is water aeration, and described aeration water hardness is 10��240mg/L, pH is 6.0��8.5, and dissolved oxygen content is higher than the 80% of air saturation value.
Above-mentioned method, it is preferable that, in described step (2), described biological effect parameter is mortality ratio and the toxicity symptom of fish; Described physical and chemical parameter is the water temperature of mixed solution, pH value, dissolved oxygen content.
Above-mentioned method, it is preferable that, in described step (2), described culturing process control water temperature is 21��25 DEG C, illumination every day 12��16h, dissolved oxygen content be higher than the 60% of air saturation value.
Above-mentioned method, it is preferable that, the judging criterion of the toxicity of described pesticide water dispersible granules is: LC50> 10.0mga.i./L represents low poison, 1.00mga.i./L < LC50�� 10.0mga.i./L represents poisoning, 0.100mga.i./L < LC50�� 1.00mga.i./L represents high poison, LC50�� 0.100mga.i./L represents severe toxicity.
Above-mentioned method, it is preferable that, the method that described a kind of zebra fish measures agricultural chemicals water dispersant toxicity is also provided with blank group; Described blank group take water aeration as the domestic water of zebra fish. In described blank group, the mortality ratio of zebra fish is 0 proves reliable experiment result.
Compared with prior art, it is an advantage of the current invention that:
(1) the present invention provides the method for a kind of easy and simple to handle, practical model organism zebra fish screening pesticide water dispersible granules toxicity, pesticide water dispersible granules effective constituent is fully dissolved out, evaluate it to the toxicity of zebra fish, there is speed fast, the advantage that tolerance range is high.
(2) the present invention provides the method for a kind of model organism zebra fish screening pesticide water dispersible granules toxicity, is exposed in the pesticide solution by zebra fish juvenile fish, by the toxicity symptom of observation and comparison zebra fish and mortality ratio, judges the toxicity of agricultural chemicals. Mortality ratio is finally pass through LC50Characterizing, and toxicity symptom can as auxiliary judgment measure, accuracy is higher.
Embodiment
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 examples and instrument are commercially available. The water aeration adopted in following examples is the tap water adopting air aeration pump more than Air Exposure 24h.
Embodiment 1:
(1) preparation of agricultural chemicals storing solution:
Taking 50% boscalid amine water dispersible granules 1.2500g, dissolve with distilled water and be settled in 25mL volumetric flask, obtaining concentration is 2.50 �� 104The storing solution of mga.i./L.
(2) preparation of domestic water:
Getting the square glass cylinder of 7 10L, every cylinder injects the water aeration of 5L. The water temperature of this water aeration is 23.7 DEG C, and pH value is 7.62, and water hardness is that 16.2mg/L is (with CaCO3Meter), dissolved oxygen is the 99.6% of air saturation value.
Experimental group: the agricultural chemicals storing solution being equipped with in the square glass cylinder of water aeration respectively to 5 to inject 3.000mL, 3.600mL, 4.320mL, 5.18mL, 6.22mL step (1) obtains the zebra fish domestic water that concentration is respectively 15.0mga.i./L, 18.0mga.i./L, 21.6mga.i./L, 25.9mga.i./L, 31.1mga.i./L.
Blank group (CK): using water aeration as the domestic water of zebra fish.
(3) contaminate
Select the wild-type AB strain zebra fish juvenile fish that body length is 1��3cm (average body length 2.13 �� 0.25cm), put in square glass cylinder at random, each cylinder adds 10 tail fishes and cultivates, illumination every day 12h. Zebra fish is 24h not feeding before contamination, also not feeding in contamination process.
After contamination starts, measuring in 24h, 48h, 72h and 96h and record the temperature of experimental liquid in glass jar, pH value, dissolved oxygen amount etc., concrete detected result is in table 2.
After contamination, 6h, 24h, 48h, 72h and 96h observe and record toxicity symptom and the death toll of zebra fish. At once fish of checkmating when finding that fish is dead is pulled out. The standard judging zebra fish death is that fish does not have visible to move, as the gill instigate, to touch afterbody reactionless. Judge that the poisoning standard of zebra fish is: side trip occurred in zebra fish, cylinder bottom or the health obviously biological phenomenon such as bending of lying on one's side.
Table 1 is that 50% boscalid amine water dispersible granules is to the statistics of mortality ratio in zebra fish acute toxicity test data. Table 2 is the water temperature of each concentration group between trial period, pH value, the detected result of dissolved oxygen.
Table 1:50% boscalid amine water dispersible granules is to zebra fish acute toxicity test data recorder
Meanwhile, in table 1, experimental group 1��5, cultivates 6��96h, and side trip has occurred in zebra fish, cylinder bottom of lying on one's side, the health obviously biological effect such as bending, dead.
Table 2: the detected result table of the water temperature of domestic water, pH value, dissolved oxygen in water vat
(4) data processing
Adopt statistic software SPSS 12.0, taking the logarithmic value of concentration for the treatment of as independent variable(s) x, taking the probit value of (correction) mortality ratio as dependent variable y, set up virulence regression equation. Calculate 50% boscalid amine water dispersible granules to the median lethal concentration LC of zebra fish50With 95% fiducial limit, concrete calculation result is see table 3.
Table 3:50% boscalid amine water dispersible granules is to zebra fish the acute toxicity tests table
(5) result evaluation
According to LC50Value judges agricultural chemicals poison level. Toxicity grading standard is: low poison (LC50> 10.0mga.i./L), poisoning (1.00mga.i./L < LC50�� 10.0mga.i./L), high poison (0.100mga.i./L < LC50�� 1.00mga.i./L), severe toxicity (LC50��0.100mga.i./L)��
Under this test conditions, 50% boscalid amine water dispersible granules is to the LC of zebra fish acute toxicity50(96h) being 22.7mga.i./L, be greater than 10.0mga.i./L, toxic grade is " low poison ".
Embodiment 2:
(1) preparation of agricultural chemicals storing solution:
Taking 30% indoxacarb water dispersible granule 0.8333g, dissolve with distilled water and be settled in 50mL volumetric flask, obtaining concentration is 5.00 �� 103The storing solution of mga.i./L.
(2) preparation of domestic water:
Getting the square glass cylinder of 7 10L, every cylinder injects the water aeration of 5L. The water temperature of this water aeration is 22.1 DEG C, and pH value is 7.65, and water hardness is that 26.1mg/L is (with CaCO3Meter), dissolved oxygen is the 96.7% of air saturation value.
Experimental group: the agricultural chemicals storing solution being equipped with in the square glass cylinder of water aeration respectively to 5 to inject 2.000mL, 3.000mL, 4.500mL, 6.75mL, 10.10mL step (1) obtains the zebra fish domestic water that concentration is respectively 2.00mga.i./L, 3.00mga.i./L, 4.50mga.i./L, 6.75mga.i./L, 10.1mga.i./L.
Blank group (CK): using water aeration as the domestic water of zebra fish.
(3) contaminate
Select the wild-type AB strain zebra fish juvenile fish that body length is 1��3cm (average body length 2.24 �� 0.18cm), allow at random in square glass cylinder, each cylinder adds 10 tail fishes and cultivates, illumination every day 12h.Zebra fish is 24h not feeding before contamination, also not feeding in contamination process.
After contamination starts, measuring in 24h, 48h, 72h and 96h and record the temperature of experimental liquid in glass jar, pH value, dissolved oxygen amount etc., detected result is in table 5.
After contamination, 6h, 24h, 48h, 72h and 96h observe and record toxicity symptom and the death toll of zebra fish. At once fish of checkmating when finding that fish is dead is pulled out. The standard judging zebra fish death is that fish does not have visible to move, as the gill instigate, to touch afterbody reactionless. Judge that the poisoning standard of zebra fish is: side trip occurred in zebra fish, cylinder bottom or the health obviously biological phenomenon such as bending of lying on one's side.
Table 4 is that 30% indoxacarb water dispersible granule is to the statistics of mortality ratio in zebra fish acute toxicity test data. Table 5 is the water temperature of each concentration group between trial period, pH value, the detected result of dissolved oxygen.
Table 4:30% indoxacarb water dispersible granule is to zebra fish acute toxicity test data recorder
Meanwhile, in table 4 experimental group 1��5, cultivating 6��96h, has there is side trip in zebra fish, cylinder bottom of lying on one's side, the health obviously biological effect such as bending, dead.
Table 5: the detected result table of the water temperature of domestic water, pH value, dissolved oxygen in water vat
(4) data processing
Adopt statistic software SPSS 12.0, taking the logarithmic value of concentration for the treatment of as independent variable(s) x, taking the probit value of (correction) mortality ratio as dependent variable y, set up virulence regression equation. Calculate 30% indoxacarb water dispersible granule to the median lethal concentration LC of zebra fish50With 95% fiducial limit, concrete calculation result is see table 3.
Table 6:30% indoxacarb water dispersible granule is to zebra fish the acute toxicity tests table
(5) result evaluation
According to LC50Value judges agricultural chemicals poison level. Toxicity grading standard is: low poison (LC50> 10.0mga.i./L), poisoning (1.00mga.i./L < LC50�� 10.0mga.i./L), high poison (0.100mga.i./L < LC50�� 1.00mga.i./L), severe toxicity (LC50��0.100mga.i./L)��
Under this test conditions, 30% indoxacarb water dispersible granule is to the LC of zebra fish acute toxicity50(96h) being 4.31mga.i./L, be between 1.00��10.0mga.i./L, toxic grade is " poisoning ".
The above is only the better embodiment of the present invention, and the present invention not does any restriction in form. Although the present invention discloses as above with better embodiment, but and it is not used to limit the present invention. Any those of ordinary skill in the art, when not departing from spirit and the technical scheme of the present invention, all can utilize the Method and Technology content of above-mentioned announcement that technical solution of the present invention is made many possible variations and modification, or be revised as the equivalent embodiment of equivalent variations. Therefore, every content not departing from technical solution of the present invention, to any simple modification made for any of the above embodiments, equivalent replacement, equivalence change and modifies according to the technical spirit of the present invention, all still belongs in the scope of technical solution of the present invention protection.
Claims (9)
1. the method for a zebra fish mensuration pesticide water dispersible granules toxicity, it is characterised in that, comprise the following steps:
(1) pesticide water dispersible granules is dissolved in water, make agricultural chemicals storing solution;
(2) described agricultural chemicals storing solution and water are hybridly prepared into the mixed solution of gradient concentration, by the domestic water of described mixed solution as zebra fish, continue to cultivate 96h, in culturing process, when 24h, record the biological effect parameter of zebra fish and the physical and chemical parameter of described mixed solution;
(3) agricultural chemicals is calculated to the median lethal concentration LC of zebra fish according to the biological effect parameter of zebra fish50Value, judges the toxicity of pesticide water dispersible granules.
2. method according to claim 1, it is characterised in that, described step (1) is specially: be added to the water by described pesticide water dispersible granules, carries out stirring or ultrasonic obtains described agricultural chemicals storing solution.
3. method according to claim 1, it is characterised in that, the selection body length of zebra fish described in described step (2) is the zebra fish of 1��3cm, bouncing.
4. method according to claim 1, it is characterised in that, before adopting zebra fish to measure pesticide water dispersible granules toxicity in 24h, described zebra fish not feeding, in the process that described step (2) carries out, described zebra fish not feeding.
5. method according to claim 1, it is characterised in that, water described in described step (2) is water aeration, and described aeration water hardness is 10��240mg/L, pH is 6.0��8.5, and dissolved oxygen content is higher than the 80% of air saturation value.
6. method according to any one of claim 1 to 5, it is characterised in that, in described step (2), described biological effect parameter is mortality ratio and the toxicity symptom of fish; Described physical and chemical parameter is the water temperature of mixed solution, pH value, dissolved oxygen content.
7. method according to any one of claim 1 to 5, it is characterised in that, in described step (2), described culturing process control water temperature is 21��25 DEG C, illumination every day 12��16h, dissolved oxygen content be higher than the 60% of air saturation value.
8. method according to any one of claim 1 to 5, it is characterised in that, the judging criterion of the toxicity of described pesticide water dispersible granules is: LC50> 10.0mga.i./L represents low poison, 1.00mga.i./L < LC50�� 10.0mga.i./L represents poisoning, 0.100mga.i./L < LC50�� 1.00mga.i./L represents high poison, LC50�� 0.100mga.i./L represents severe toxicity.
9. method according to any one of claim 1 to 5, it is characterised in that, the method that described zebra fish measures pesticide water dispersible granules toxicity is also provided with blank group; Described blank group take water aeration as the domestic water of zebra fish; There are not the fish phenomena of mortality in described blank group and then prove reliable experiment result.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106950360A (en) * | 2017-03-23 | 2017-07-14 | 广东中科英海科技有限公司 | A kind of toxotest method of agricultural chemicals |
| CN107228925A (en) * | 2017-05-21 | 2017-10-03 | 中山大学 | A kind of method of the potential teratogenesis of utilization zebrafish embryo Fast Evaluation Chinese patent drug |
| CN110402893A (en) * | 2019-06-20 | 2019-11-05 | 中山大学 | A kind of preparation and its application of Nrf2 gene delection zebra fish mutant |
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| CN103301479B (en) * | 2013-05-07 | 2014-12-31 | 南京工业大学 | Method for evaluating acute toxicity of drug by using model animal zebra fish fry |
| CN104931326A (en) * | 2015-07-03 | 2015-09-23 | 南开大学 | Method for extracting zebra fish metabolite and application thereof |
| CN105115951A (en) * | 2015-08-10 | 2015-12-02 | 山东省科学院生物研究所 | Method for rapidly evaluating damage effect on hepatic function of zebra fishes by compounds |
| CN105044317A (en) * | 2015-08-26 | 2015-11-11 | 广东省微生物研究所 | Method for predicating embryotoxicity of non-steroidal anti-inflammatory drug type novel pollutants on early-phase life stage of zebra fish |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106950360A (en) * | 2017-03-23 | 2017-07-14 | 广东中科英海科技有限公司 | A kind of toxotest method of agricultural chemicals |
| CN107228925A (en) * | 2017-05-21 | 2017-10-03 | 中山大学 | A kind of method of the potential teratogenesis of utilization zebrafish embryo Fast Evaluation Chinese patent drug |
| CN110402893A (en) * | 2019-06-20 | 2019-11-05 | 中山大学 | A kind of preparation and its application of Nrf2 gene delection zebra fish mutant |
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Application publication date: 20160608 |