CN101982072A - Method for testing toxicity of environmental estrogen on whitebait embryonic development - Google Patents
Method for testing toxicity of environmental estrogen on whitebait embryonic development Download PDFInfo
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Abstract
The invention discloses a method for testing toxicity of environmental estrogen on whitebait embryonic development. The method of the invention utilizes the sensitivity of embryo to the simulation of environmental pollutant or chemical matter to perform a test under semi-static state on whitebait embryo which is exposed in standard diluted aqueous solution of subject having a series of concentrations. The test lasts about 30 days, begins as exposing a live embryo at the blastula stage in the aqueous solution of subject, and ends with hatching all embryos in a control group and an exposed group. The toxicity endpoint contains death, monstrosity and delayed hatching. The method comprises the steps of confirming the toxicity of the subject on the whitebait embryonic development by observing the toxicity endpoint of the exposed group and comparing with the control group; researching on the influence of two natural estrogens E2 and EE2 on the whitebait embryo-yolk sac stage so as to confirm the toxic dose-effect relationship of the natural estrogens to the whitebait embryonic development, and evaluating the potential risk of this type of fish exposed under the estrogen with environmental concentration.
Description
Technical field
The present invention relates to a kind of toxotest method, be specially environmental estrogens, belong to the fish acute toxicity field tests big silverfish embryonic development toxotest method.
Background technology
In the last thirty years, exogenous compounds in the environment---environmental estrogens (Environmental estrogens) can cause human and wildlife grows and the Health cost of proliferation, caused people's great attention, the environmental estrogens harm of having reported at present comprises: semen minimizing, wild fish sexual development disorder (comprising hermaphroditism, milter feminisation etc.), the minimizing of amphibian (as alligator, toad) population etc.Environmental estrogens is a class important in the incretion interferent, they or belong to natural estrogen (promptly drain and enter environment) by the endogenous oestrogenic hormone of organism, the synthesis of chemicals that perhaps belongs to synthetic oestrogenic hormone and have estrogen active, the function and the behavior of endogenous estrogen in the energy analogue body after it enters organism, cause the class estrogen response, thereby hormonal balance, reproduction, growth and the behavior etc. of organism are caused negative effect.Studies confirm that, had hundreds and thousands of kinds of compounds in the environment at present with different oestrogenic hormone vigor.Wherein, several frequently seen environmental estrogens and oestrogenic hormone vigor thereof are as shown in table 1 with the source;
* EEq (17 β-E
2Equivalent): the oestrogenic hormone vigor equivalent that is converted into 17 beta estradiols.
Oestrogenic hormone vigor equivalent is big more, and the internal secretion disturbing effect potential to organism is just strong more, therefore, and E
2And EE
2Be two kinds of environmental estrogens of greatest concern, their chemical structural formula and physicochemical property see Table 2.The former is the strongest a kind of of oestrogenic hormone vigor in the natural estrogen, and the value of being often used as is 1 oestrogenic hormone equivalent, to weigh the oestrogenic hormone vigor of other compounds.E
2Form by human or animal's secretion, mainly enter natural environment by ight soil and urine.It is reported E in the ight soil of cow head drainage every day
2Content reaches 170 ~ 1230 μ g/kg, adult female E
2Excretion can reach 82 ~ 695 μ g/ days, and period of gestation women E
2Excretion can reach at ordinary times 1000 times.The latter is the oestrogenic hormone of synthetic, mainly as the controversies in hormone replacement in the elderly medication of contraceptive for female and treatment syndrome in climacteric;
Since half a century, because the rise of global industrialized acceleration and intensification livestock and poultry farm is discharged into the E in the environment
2And EE
2Amount significantly increases.At present, E
2And EE
2Be reported in the various surrounding mediums and had (seeing Table 3).Though traditional sewage treatment plant (STP) is to E
2And EE
2Certain removal effect (oestrogenic hormone vigor reduce by 80 ~ 97%) is arranged, but have report to confirm, if organism lives in the (E that contains ng/L trace level in the receiving water body of STP water outlet for a long time
2Or EE
2), can bring out body and produce negative effect.Because E
2And EE
2Have higher Log Kow value, they can exist in the organism adipose tissue for a long time, therefore can bring bigger threat to health by the biological magnification of food chain;
* mud dry weight.
No matter environmental estrogens is to enter surface water by the STP discharging, still the release action by bed mud enters water body once more, the water environment always important tax of environmental estrogens is deposited medium, therefore, fish (as zebra fish, plan carp, blue or green Medaka, rainbow trout etc.) can be used as the important tests biology of environmental estrogens ecological risk assessment.Fish acute toxicity test (Fish Acute Toxicity Test) is the necessary component of present chemicals eco-toxicity test, yet test Mesichthyes lethality rate is unique test terminal point, and the morality of this test and science just are being subjected to increasing query.Therefore, some method of testings that substitute obtain researchers' exploitation and checking, for example fish cell or cell-line testing in vitro method, fish embryo method of testing etc., wherein (Fish Embryo Toxicity, FET) test is an alternative method of extensively being approved also potentialization at present to fish embryo toxicity.
People such as Lange have reported the acute toxicity that utilizes zebrafish embryo to test ten kinds of specific compounds as test carrier in nineteen ninety-five, result of study is found, these ten kinds of compounds can cause that zebrafish embryo grows obvious damage, and directing terminal comprises that the embryo is solidified, the growth of the quantity of the growth of primitive gut, muscle segment, organ, heartbeat, painted etc.Thereafter, zebrafish embryo is tested (
Danio rerioEmbryo test, DarT) result compares with the toxotest result who utilizes rainbow trout cell-line RTG-2 as test carrier, find that the DarT test is more sensitiveer than adult fish or RTG-2 cell acute toxic test, thereby recommend the alternative method of DarT test as the adult fish acute toxic test.Subsequently in order to verify the reliability of this method, several government tests chamber, Europe has been carried out the DarT test of compound and urban sewage simultaneously, and the result compared, find that the DarT test result not only has good correlation (R with adult fish acute toxic test result
2=0.84), and between different laboratory not the significance difference (
p0.05).2005, Braunbeck etc. provide data acknowledgement except that zebra fish other some the test fingerlings, as fathead minnow (
Pimephales promelas) and Japanese blue or green Medaka (
Oryzias latipes), its body early embryo stage also can be used as the FET test.From 2005, FET tested by the daily toxotest project of German mandatory requirement as the total water outlet of STP, and the total water outlet zebrafish embryo test of STP subsequently also is adopted as international standard.2006, OECD issued the draft of FET test as the chemicals test event.
The FET test has three big advantages: (1) short (wherein the DarT test only needs 48 hours) consuming time; (2) data are true and reliable; (3) observable toxicity terminal point is more and sensitive, comprises deadly, semilethal, teratogenesis etc.Wherein, last point is more suitable for the poisonous effect of testing the compound (as environmental estrogens) that does not have lethal effect under ambient concentration usually.The test fingerling of FET test recommendation only limits to zebra fish and Japanese blue or green Medaka in the world at present, but these two fingerlings are not this soil species of China, and in China's nature water environment, there are not these two species to exist, therefore, choose a kind of China distinctive, have extensive representational fingerling and carry out FET test, thereby estrogenic chemicals in China's water body environment is carried out the ecological risk assessment, have important theory and be worth and realistic meaning.And big silverfish (
Protosalanx hyalocraniusAbbott) just in time meet this research demand.
Current, domestic the research of big silverfish also is confined to its biological property, propagation and implantation technique, do not utilize big silverfish embryo to carry out the FET testing research as yet.This paper utilizes the susceptibility of big silverfish embryo stage to chemical substance, two kinds of natural estrogen (E of research
2And EE
2) to the influence in big silverfish embryo-yolk sac stage, determining their dosage-effect relations, and estimate the potential risk that this fingerling is exposed to the existence oestrogenic hormone environmental level under to big silverfish embryonic development toxicity.This paper uses big silverfish as test organism both at home and abroad first, carries out oestrogenic hormone embryotoxicity Study on Effect.
Summary of the invention
The objective of the invention is to be exposed to the potential risk that exists under the oestrogenic hormone environmental level to test this fingerling in order to provide a kind of environmental estrogens to big silverfish embryonic development toxotest method.
Purpose of the present invention can be achieved through the following technical solutions.
Environmental estrogens is to big silverfish embryonic development toxotest method, and concrete steps are as follows:
1) E2 that at first takes by weighing 1 mg is dissolved in the 500 ml standard dilution waters, is mixed with the E2 storing solution of 2 mg/L, reagent bottle is sealed with film be placed on 2 ℃ of refrigerators and keep in Dark Place, and the EE2 storing solution of preparation 2 mg/L that use the same method is also preserved;
When 2) testing, take out above-mentioned storing solution, wait to return to room temperature and fully shake up after, is 1,0.2,0.04 mg/L with the standard dilution water respectively at compound concentration in the glass culture dish, 8, the E2 of 1.6 μ g/L and 10,0.05 mg/L and the exposure solution of EE2, and respectively pipette 20 ml and put into culture dish; Blank is 20 ml standard dilution waters, and other gets Yangtze River Water (filtering through absorbent cotton) 20 ml as the contrast of natural water environment, more than expose liquid and contrast liquid all be provided with 3 groups parallel;
3) the random choose live embryo that is in blastula stage places and carries out viewing test under the inverted microscope, in being equipped with the culture dish that exposes liquid and contrast liquid, each puts into 10 embryos, then the embryo is placed in the constant incubator and cultivate, under 2-10 ℃ of condition, keep constant temperature, unglazed photograph, test duration 30 days, use the terminal point of inverted microscope observation test every day, and record;
4) use SPSS 13.0 for Windows softwares, to big silverfish embryo under the exposure concentrations with blank under survival, g and D carry out significance analysis.
Described standard dilution water, adopt analytically pure chemicals and full glass distilled water or deionized water (electrical conductivity≤10 μ S/cm), getting each 25 ml of calcium chloride solution, Adlerika, sodium bicarbonate solution and 4 kinds of solution of sodium chloride solution is mixed, be diluted to 1 L, thinned water needs to store for future use then through aeration (be not less than saturation of the air value 90%) till the oxygen saturation.
Described calcium chloride solution is with 11.76 g CaCl
22H
2O is dissolved in the 1 L water.
Described Adlerika is with 4.93 g MgSO
47H
2O is dissolved in the 1 L water.
Described sodium bicarbonate solution is with 2.59 g NaHCO
3Be dissolved in the 1 L water.
Described sodium chloride solution is that 0.23 g KCl is dissolved in the 1 L water.
Step 2) compound method of each exposure concentrations group is as follows in: get 12.5 ml E2 or EE2 storing solution in isopyknic standard dilution water, the E2 or the EE2 that are made into 1 mg/L expose liquid 25 ml; Fully become the exposure liquid of 200,40,8 and 1.6 μ g/L behind the mixing by 5 times of volume stepwise dilutions; And then the 1.6 μ g/L exposure liquid of getting 125 μ L is made into the exposure liquid of 10 mg/L in 19.875 ml standard dilution waters; 10 mg/L that get 100 μ L expose liquid in 19.9 ml dilution waters, are made into the exposure liquid of 0.05 ng/L.
In the step 3), the embryo placed in the constant incubator cultivate, under 8 ℃ of conditions, keep constant temperature.
In the step 3), adopt semi-static exposure chamber, all expose liquid and contrast liquid was changed once in per 2 days.When changing solution, draw the embryo seriatim with suction pipe and be transferred in the fresh solution.If any dead embryo or dead juvenile fish, adopt suction pipe to draw it is removed.The terminal point of test comprises that deadly (embryo is solidified, juvenile fish does not have heartbeat, juvenile fish does not have automatically), teratogenesis (yolk sac deformity, juvenile fish deformity of spine) and hatching postpone.
Exposure test is placed in the constant incubator (MGC-300H, Shanghai) to be carried out, and fish embryo uses inverted microscope, and (Zeiss Axiovert S100 USA) observes.Glass culture dish (Φ 9cm) uses for test with distilled water rinse 3 times, oven dry.
The present invention utilizes the embryo to environmental pollutants or the susceptibility that stimulates of chemical substance, under certain condition, big silverfish embryo is exposed in the standard dilute aqueous solution that contains a series of concentration of being tried thing, tests with semi-static condition.Test is exposed to from the live embryo that will be in blastula stage and contains the aqueous solution that is tried thing, and end is finished in embryo's hatchings all to control group and the exposure group, continues about 30 days.That the toxicity terminal point comprises is deadly, teratogenesis and hatching postpone.By to the observation of exposure group toxicity terminal point and with the comparison of control group, determine that this is tried the toxicity of thing to big silverfish embryonic development, comprises half lethal concentration (LC
50), terateger rate, incubation rate and hatching time delay etc.
The present invention utilizes the susceptibility of big silverfish embryo stage to chemical substance, two kinds of natural estrogen (E of research
2And EE
2) to the influence in big silverfish embryo-yolk sac stage, determining their dose-effect relationships, and estimate the potential risk that this fingerling is exposed to the existence oestrogenic hormone environmental level under to big silverfish embryonic development toxicity.
Description of drawings
Fig. 1 is blank big silverfish embryo brooding time of group and a survival rate histogram under the different temperatures;
Fig. 2 is E
2And EE
2The maximum terateger rate histogram of embryo/prelarva under the exposure condition;
The time histogram that Fig. 3 is hatched for the big silverfish prelarva of article one under the different oestrogenic hormone exposure concentrations.
Embodiment
Below in conjunction with the concrete elaboration of specific embodiments and the drawings technical characterstic of the present invention.
Biological subject is big silverfish embryo.Gather its fertilized egg in the big silverfish egg-laying period (annual mid-December is to the mid or late March in next year).Before the experiment beginning, fertilized egg is moved in the glass culture dish that the standard dilution water is housed carefully, it is scattered evenly in the culture dish bottom, place 2 ℃ of refrigerators to keep in Dark Place then.All changed a solution in per two days.
Trial test
In cosolvent methyl alcohol control group, all dead after big silverfish embryo exposes 2 days, so do not use cosolvent in the formal test, the highest exposure concentrations of oestrogenic hormone is set to 1 mg/L(and is lower than the maxima solubility of oestrogenic hormone in water).Embryo's growth and hatching situation are consistent in the basic and reservoir water of embryo's growth and hatching situation in the standard dilution water (see Table 4 and Fig. 1), therefore, in order to control experimental condition better, formal test adopts the standard dilution water as dilution and blank liquid;
Under 3,6,8 and 10 ℃, big silverfish embryo shows different growths and survival condition, as shown in Figure 1.As seen, under 8 ℃, big silverfish embryo's growth can reach brooding time short (embryo began brooding time 16 days and whole embryo's brooding time 18 days), can realize again than high viability (80%).And phenomenon (embryo began brooding time 20 ~ 28 days and whole embryo's brooding time 24 ~ 32 days) appears growing slowly in the embryo under lower temperature, can not guarantee the higher survival rate (only having 60%) of blank group under higher temperature again.Therefore, 8 ℃ of optimum temperatures that should be big silverfish embryonic development, formal experiment will adopt this temperature to carry out.
Estrogenic lethal effect
Observe the survival quantity of big silverfish embryo/prelarva every day, and record.Deadly terminal point comprises that the embryo is solidified, juvenile fish does not have automatically and juvenile fish does not have heartbeat.
Behind the experimental observation 27 days, be exposed to the E that does not plant concentration
2And EE
2Down, the survival condition of big silverfish embryo/prelarva.According to blank group embryo/prelarva survival rate, choose the relation between 5 typical period of time analysis oestrogenic hormone exposures and embryo/prelarva survival number.
Under each group exposure concentrations, E
2And EE
2Lethal effect to big silverfish embryo/prelarva just displayed after 15 days.But after 15 days, the survival rate of blank group is also reduced to 76.7% (17 ~ 23 d) and 70.0% (24 ~ 27 d), and this is because itself just there are the low characteristics of incubation rate in wild big silverfish embryo.In the time of the 23rd day, the E of 1 mg/L
2In the exposure group number of live body than blank group significantly reduce (
p＜0.05); In the time of the 27th day, the EE of 1 mg/L
2In the exposure group number of live body than the very remarkable minimizing of blank group (
p＜0.01); And be exposed to the live body number of Yangtze River Water and the live body of blank group is counted no significant difference.
Estrogenic teratogenic effect
Observe the lopsided quantity of big silverfish embryo/prelarva every day, and record, the teratogenesis terminal point comprises yolk sac deformity and prelarva deformity of spine.
Behind the experimental observation 27 days, E
2And EE
2See shown in Figure 2 to the teratogenic effect of big silverfish embryo/prelarva.Choosing big silverfish embryo in 27 days/prelarva maximum terateger rate discusses.In 27 days, blank group and Yangtze River Water control group all do not have the appearance of lopsided embryo/prelarva; And at the E of 1 mg/L
2And EE
2In the exposure group, because lethal effect is obvious, so also do not observe the individual appearance of deformity; At E
2Under the exposure condition, maximum terateger rate appears at the 16th day 8 μ g/L exposure groups; And EE
2Under the exposure condition, maximum terateger rate appears at the 18th day 0.2 mg/L exposure group.
Estrogenic hatching late effect
Write down the time that the big silverfish prelarva of article one hatches under each exposure concentrations, the result as shown in Figure 3.E at 0.2 mg/L
2And EE
2The exposure concentrations group, the time that article one prelarva hatches is compared with the blank group, tangible delay occurred; And at the E of 1 mg/L
2And EE
2The exposure concentrations group, the time that article one prelarva hatches is compared with the blank group, has occurred more significantly postponing, and is more late 7 days and 11 days than the blank group respectively; And Yangtze River Water is to the not obviously influence of hatching of big silverfish prelarva.
After formal test begins 15 days, be exposed to the E of 1 mg/L
2And EE
2In the concentration group, mortality appears in big silverfish embryo, survival rate respectively from the 15th day 90.0% and 90.0% reduce to the 23rd day 40.0% and 43.3%; And be exposed to the E of 0.2 mg/L ~ 0.05 ng/L
2And EE
2In the concentration group, survival rate was all greater than 60.0% in the 23rd day.The result shows E
2And EE
223 d-LC to big silverfish
50In the concentration range of 0.2 ~ 1 mg/L, this concentration is higher than two kinds of oestrogenic hormone detected concentration in the natural water environment far away, can judge tentatively that therefore these two kinds of oestrogenic hormone can not produce lethal effect to big silverfish embryo or prelarva in the natural water environment.This is consistent with the conclusion that people's (calendar year 2001) such as Tabata research obtains.They are exposed to E with the blue or green Medaka embryo of Japan
2In 200 ~ 230 days, found that the E of 25 μ g/L
2Exposing down, the phenomenon that survival rate significantly reduces just appears in blue or green Medaka.And people such as Westerlund (2000) are with E
2Be injected in the body of female zebra fish, found that the E of 1 μ mol/kg (0.27 mg/kg) and 1 nmol/kg (0.27 μ g/kg)
2Under the injection volume, lethality rate reaches 95% and 79% after 8 days.The difference of result of study can be owing to test fingerling to E
2The tolerance difference, perhaps since the difference of exposure chamber cause.For EE
2Lethal effect, people such as Jaser (2003) water Magna (
Ceriodaphnia reticulate) carry out acute toxic test as test organism, obtain 24h-EC
50Value is 1.814 mg/L.
No matter be to be exposed to E
2Or EE
2Under the condition, maximum terateger rate all appears between the 16th day to the 18th day, and reason is that some are most of all dead with regard to teratogenic embryo's hatching back in the shaping phase.Though terateger rate does not show tangible dose-effect relationship, even can detected E at natural environment
2And EE
2Under the concentration, the lopsided young of some all appears after the big silverfish embryo hatching.At present, many researchers begin to pay close attention to EE
2The sex reversal ability, i.e. milter feminisation.And the research by this paper, these two kinds of oestrogenic hormone teratogenesis abilities (yolk sac deformity and prelarva deformity of spine) also can not be ignored.
People such as Kishida (calendar year 2001) have reported and have been exposed to 10 μ mol/L (2.72 mg/L) E
2After, the brooding time delay has appearred in zebrafish embryo.The EE of people such as Versonnen (2004) report 100 ng/L
2After exposing 120h, the zebrafish embryo incubation rate only is 67%, and control group then has 95% incubation rate.In this test,, be not suitable for representing the influence of oestrogenic hormone, so the time that we select for use article one prelarva to hatch is estimated E to embryo's hatching with incubation rate because embryonic death quantity is more under the oestrogenic hormone high concentration
2And EE
2Influence to big silverfish embryo hatching delay.The result shows that these two kinds of oestrogenic hormone have late effect to big silverfish embryo hatching, and along with the rising of exposure concentrations, delay phenomenon is obvious more.
As can be seen, the water sample that picks up from big silverfish hatching ground, entrance of Changjiang River is to the survival of big silverfish embryo/prelarva or grow and all not have obviously to influence from result of the test.
Because environmental estrogens has the high poison of trace, widely distributed, the miscellaneous characteristics of environment, this paper chooses big silverfish embryo/prelarva and tests and have very important significance: the embryonic development stage of (1) big silverfish stimulates very responsive to external world, can be to the multiple effect terminal point of trace amount environment estrogen production; (2) big silverfish extensively is distributed in lake, Eastern China, river mouth and the Jiangkou, has important economic value and ecological significance, as model organism, can provide favourable guidance to the hydrobiological influence in this zone with it for estimating environmental estrogens.
Claims (6)
1. environmental estrogens is to big silverfish embryonic development toxotest method, and it is characterized in that: concrete steps are as follows:
1) E2 that at first takes by weighing 1 mg is dissolved in the 500 ml standard dilution waters, is mixed with the E2 storing solution of 2 mg/L, reagent bottle is sealed with film be placed on 2 ℃ of refrigerators and keep in Dark Place, and the EE2 storing solution of preparation 2 mg/L that use the same method is also preserved;
When 2) testing, take out above-mentioned storing solution, wait to return to room temperature and fully shake up after, is 1,0.2,0.04 mg/L with the standard dilution water respectively at compound concentration in the glass culture dish, 8, the E2 of 1.6 μ g/L and 10,0.05 mg/L and the exposure solution of EE2, and respectively pipette 20 ml and put into culture dish; Blank is 20 ml standard dilution waters, and other learns from else's experience Yangtze River Water 20 ml that absorbent cotton filters as the contrast of natural water environment, more than expose liquid and contrast liquid all be provided with 3 groups parallel;
3) the random choose live embryo that is in blastula stage places and carries out viewing test under the inverted microscope, in being equipped with the culture dish that exposes liquid and contrast liquid, each puts into 10 embryos, then the embryo is placed in the constant incubator and cultivate, under 2-10 ℃ of condition, keep constant temperature, unglazed photograph, test duration 30 days, use the terminal point of inverted microscope observation test every day, and record;
4) use SPSS 13.0 for Windows softwares, to big silverfish embryo under the exposure concentrations with blank under survival, g and D carry out significance analysis.
2. environmental estrogens according to claim 1 is to big silverfish embryonic development toxotest method, it is characterized in that: described standard dilution water, adopt analytically pure chemicals and full glass distilled water or deionized water, getting each 25 ml of calcium chloride solution, Adlerika, sodium bicarbonate solution and 4 kinds of solution of sodium chloride solution is mixed, be diluted to 1 L, thinned water needs through aeration to store for future use then till the oxygen saturation.
3. environmental estrogens according to claim 1 is characterized in that big silverfish embryonic development toxotest method: described calcium chloride solution is with 11.76 g CaCl
22H
2O is dissolved in the 1 L water, and described Adlerika is with 4.93 g MgSO
47H
2O is dissolved in the 1 L water, and described sodium bicarbonate solution is with 2.59 g NaHCO
3Be dissolved in the 1 L water, described sodium chloride solution is that 0.23 g KCl is dissolved in the 1 L water.
4. environmental estrogens according to claim 1 is to big silverfish embryonic development toxotest method, it is characterized in that: step 2) in the compound method of each exposure concentrations group as follows: get 12.5 ml E2 or EE2 storing solution in isopyknic standard dilution water, the E2 or the EE2 that are made into 1 mg/L expose liquid 25 ml; Fully become the exposure liquid of 200,40,8 and 1.6 μ g/L behind the mixing by 5 times of volume stepwise dilutions; And then the 1.6 μ g/L exposure liquid of getting 125 μ L is made into the exposure liquid of 10 mg/L in 19.875 ml standard dilution waters; 10 mg/L that get 100 μ L expose liquid in 19.9 ml dilution waters, are made into the exposure liquid of 0.05 ng/L.
5. environmental estrogens according to claim 1 is characterized in that big silverfish embryonic development toxotest method: in the step 3), the embryo placed in the constant incubator cultivate, keep constant temperature under 8 ℃ of conditions.
6. environmental estrogens according to claim 1 is characterized in that big silverfish embryonic development toxotest method: in the step 3), adopt semi-static exposure chamber, all expose liquid and contrast liquid was changed once in per 2 days; When changing solution, draw the embryo seriatim with suction pipe and be transferred in the fresh solution,, adopt suction pipe to draw it is removed if any dead embryo or dead juvenile fish; The terminal point of test comprises that deadly, teratogenesis and hatching postpone.
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| CN110850070A (en) * | 2019-11-22 | 2020-02-28 | 北京大学 | Method for evaluating reproductive development toxicity of chemical substances based on fluorescence labeling transgenic animal model |
| CN113804524A (en) * | 2021-08-30 | 2021-12-17 | 北京大学 | Chinese medaka-based high-toxicity-content rapid screening method for chemicals |
| CN115629172A (en) * | 2022-09-07 | 2023-01-20 | 昆明市生态环境科学研究院 | Method for quantifying harm of cyanobacterial bloom to fishes |
| CN115629172B (en) * | 2022-09-07 | 2024-01-26 | 昆明市生态环境科学研究院 | Quantification method of damage of cyanobacteria bloom to fish |
| CN115747343A (en) * | 2022-11-14 | 2023-03-07 | 吉林大学 | Water quality safety diagnosis method based on medaka embryo biomarker |
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