CN106771111A - A kind of chronotoxicity microplate analysis method - Google Patents
A kind of chronotoxicity microplate analysis method Download PDFInfo
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- CN106771111A CN106771111A CN201611114391.5A CN201611114391A CN106771111A CN 106771111 A CN106771111 A CN 106771111A CN 201611114391 A CN201611114391 A CN 201611114391A CN 106771111 A CN106771111 A CN 106771111A
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Abstract
The present invention relates to environmental contaminants toxicity detection field, a kind of chronotoxicity microplate analysis method is related in particular to.Preparation including culture medium, microplate design, microplate sample-adding, pollutant chronotoxicity are determined, it is characterised in that described culture medium prescription：0.3g NaNO3、0.08g K2HPO4·3H2O、0.08g MgSO4·7H2O、0.04g CaCl2·2H2O、0.25g KH2PO4、0.05g NaCl、0.01mL FeCl3·6H2O, 1.54mL EDTA Fe, 55mL soil extract, 1.45mL A5 solution and 942mL distilled water.The present invention more can reasonably evaluate the toxicity of pollutant and disclose environmental contaminants toxic action rule and mechanism.
Description
Technical field
The present invention relates to environmental contaminants toxicity detection field, a kind of chronotoxicity microplate analysis side is related in particular to
Method.
Background technology
With continuing to develop for modern industry, environmental pollution is on the rise, wherein, water especially prominent with the pollution of chemicals
Environment is that each pollutant finally collects place.Therefore, water environment is not only faced with the rich battalion of water body caused by traditional pollutant
Fosterization and oxygen consumption organic contamination, and also face hazardous contaminant, especially heavy metal and persistent organism pollution
Severe challenge.In order to water conservation and containment cause the continuous worsening trend of water quality, Environmental capacity and ecology by pollutant
Risk assessment management must be imperative.
Chemical analysis means can carry out quantification and qualification to pollutant in water environment, but can not directly reflect pollutant
Influence (Lee and Allen, 1998) to environment and biology.Pollutant not depends entirely on it to the toxicity of organism
Environmental content, environmental factor such as pH value, redox state etc. of some changes largely influence the biological effect of pollutant
Should.Chemical analysis data can not on the whole reflect the quality of water quality, or reflection pollutant is to organism and the ecosystem
Influence.
Parameter in bio kinetic model method can make up weak point of the chemical analysis in pollutant toxicity is evaluated.Biological assessment
Specify using the poisonous effect of sensitive biological test contaminant in standard.Primary producer and water of the green alga as ecosystem kind
The basic link of uncooked food chain, the balance to maintaining the ecosystem plays an important role.Green alga individuality is small, breeding fast, to poisonous substance
It is sensitive, be easily isolated culture and can directly in observation of cell level poisoning symptom, be commonly used for the toxicity of measure pollutant.
Algae toxotest has turned into a kind of wide variety of biological monitoring and standard method, such as International Organization for standardization ISO (8692-
2012), Organization of Economy and Cooperation Development OECD (2004), Environmental Protection Agency USEPA (1996) and China national environmental protection
Algal grown is suppressed toxicity test as standard toxotest method (2008) by general bureau.Conventional algae has pyrenoids bead
Algae, goat's horn month algae, Dunaliella salina, Phaeodactylum tricornutum and scenedesmus obliquus etc..Chlorella pyrenoidosa (Chlorella
Pyrenoidosa Chlorophyta) is belonged to, Chroococcales, Chlorella is free single cell algae, and 3~5 microns of diameter is spherical or ellipse
Circle, breeding is fast, can within a short period of time investigate influence of the pollutant to algae from generation to generation and on Population Level, be easy to culture and
Experiment；The not free settling additionally, algae solution is evenly distributed, its contact with pollutant is more abundant.Many experiments show, algae toxicity test
Result has good correlation to toxicity of compound evaluation result with other instruction biologies, even more sensitive (yellow just and Wang Jialing,
1995)。
Domestic much researchs, as the biological subject of pollutant toxotest, such as utilize pyrenoids using chlorella pyrenoidosa
Chlorella determines the surface-actives such as the toxic action (Wang waits quietly, 2011) of metal nanoparticle thing, research cetyl ammonium bromide
The growth inhibitory effect to chlorella pyrenoidosa such as agent, commodity cypermethrin agricultural chemicals, dichloromethane and dichloroethanes and medicine
(Nie Xiang equalitys, 2007；The such as Wu Shijin, 2010；Imperial court's sunshine etc., 2012；Zheng Xiangjiao and Zhou Zuoming, 2012), arsenic chromium in test water
Acute toxicity (Zhou Shiming etc., 2008) of lead, cadmium, and mercury
Using triangular glass bottle method, the shortcoming of the method more than chlorella pyrenoidosa toxotest before this：Tester is not
A large amount of parallel samples can be simultaneously determined, and test solution consumption is big, not only time-consuming, laborious but also waste reagent, and only obtain certain
The one open-assembly time terminal half effective concentration EC of such as 72 hours or 96 hours50Value.Yuan waits (2011) quietly and establishes with ELIASA
It is that detecting instrument and 96 orifice plates are the algae toxicity microplate analytic approach of carrier, open-assembly time is 96h, can multiple replicate samples poison
Property, it is allowed to statistical efficiency.Using this test system, the toxicity of multiple compounds and its mixture is have studied, wrapped
The compounds such as heavy metal, agricultural chemicals and ionic liquid are included, the complete concentration-effect toxicity information of a large amount of compounds is obtained.It is above-mentioned
Research shows that microplate oxicity analysis method of testing has simple to operate, sensitivity high, reproducible and advantages of environment protection.
However, increasing research shows that the toxicity of environmental contaminants is not only relevant with exposed dosage, the time is also
One important factor.Such as Newman (1996) proposes to utilize the eco-toxicity of real-time analysis method test contaminant first,
And point out to consider that concentration can improve the credibility of urban eco landscape forest with two factors of time simultaneously.Zhu etc. (2009) is used
(Concentration-Time-Effect Surface, CTES) CTES studies 6 kinds of triazine herbicides to photogen Q67's
Toxicity, finds the extension over time of its toxicity and gradually increases.But different poisonous substances, the increased amplitude of its toxicity is different.
Hatano etc. (2010) researchs find that heavy metal has very strong time dependence to the toxicity of bio-ligand.Grinding also
Studying carefully discovery partial contamination thing not only has Time Dependent toxicity, while the concentration effect curve of different open-assembly times also has not
Same shape.Such as Wang (2011) researchs find ionic liquid [emim] BF4To the poisonous effect of photogen Q67 over time
Extension, stimulating light emission effect is gradually converted into by suppression luminescent effect.Above-mentioned Preliminary Results show, different type and not
Isostructural pollutant may have different chronotoxicity rule and mechanism of action.Therefore, the detection of pollutant toxicity is not only
It is paid close attention in a certain special time to the biological effect of exposure, should be more conceived to it and exposure biology is changed over time
Dynamic effect.
Explore with disclose different type environmental contaminants to the dynamic effect rule of the biological time to time change of exposure with
These materials must just be carried out the chronotoxicity analysis of system by mechanism of action.Reliable chronotoxicity data are obtained, first
There must be reliable chronotoxicity analysis method, and be mostly at present traditional triangle on chlorella pyrenoidosa toxotest method
Bottle method, and these methods are substantially based on some or two open-assembly time points, still lack multiple different times of system
The toxotest method of measuring point, it is impossible to directly apply to carry out chronotoxicity test.Therefore, set up small based on green alga pyrenoids
The environmental contaminants chronotoxicity microplate analytic approach of ball algae has urgency.Chronotoxicity microplate analytic approach is not only facilitated comprehensively
Understanding pollutant poisonous effect, and can deeper into understanding pollutant toxicological effect mechanism and approach, can more embody
The exposed phenomenon of pollutant and information in actual environment.
The content of the invention
1. the invention technical problem to be solved：
For emerging pollutant concentration is low, continuous action in the deficiency and environment of traditional pollutant green alga detection method of toxicity
The features such as, the present invention emphasizes influence of the time factor to toxicity on the basis of traditional detection method of toxicity, and setup time toxicity is micro-
Plate analysis method, can effectively detect that content is low, existence time is long in the environment and has specific function site to biological subject
Pollutant toxicity.
2. technical scheme
The technical scheme of use is as follows：
A kind of chronotoxicity microplate analysis method, including culture medium preparation, microplate design, microplate sample-adding, pollutant when
Between toxicity test, it is characterised in that described culture medium prescription：0.3g NaNO3、0.08g K2HPO4 3H2O、0.08g
MgSO4 7H2O、0.04g CaCl2 2H2O、0.25g KH2PO4、0.05g NaCl、0.01mL FeCl3 6H2O、1.54mL
EDTA-Fe, 55mL soil extract, 1.45mL A5 solution and 942mL distilled water.
Described EDTA-Fe is formulated：2g Na2EDTA, 66mg FeCl3 6H2O, 50mL 0.2M HCl and 50mL
H2O。
Described A5 solution formulas are：250mg H3BO3、192mg MnCl2 4H2O、32mg ZnSO4 7H2O、
17.5mg CuSO4 5H2O, 3.5mg (NH4) 6 Mo7O24 4H2O and 100mL H2O.
Described microplate design, step is that 18 micropores on 48 hole microplate peripheries are added into 100 μ L distilled water first, surplus
Remaining choose in 30 micropores the 1st, 2,4 and 7 row totally 24 micropores used as blank, remaining 24 micropores press certain dilution gfactor
Deng than 8 concentration gradients of design, 100 μ L solution to be measured are added per hole, it is parallel per concentration 3, repeat three plates.
Described microplate sample-adding, step under the conditions of chlorella pyrenoidosa in the medium 20 ± 1 DEG C will first to cultivate to right
In number growth period, during sample-adding, will cultivate in adding blank well and treatment hole to the algae solution of logarithmic phase, it is ensured that each micropore is final
Cumulative volume be 100 μ L, and ensure mixing after algae density be about 2.0 × 108/mL, the absorbance OD690 at wavelength 690nm
Value is between 0.10~0.30.
Described pollutant chronotoxicity is determined, and step is to be added according to the design of claim 4 microplate and claim 5 microplate
Sample loading mode is completed in the microplate placement illumination box of sample-adding operation, and in 20 ± 1 DEG C of cultures, is surveyed after 0,6,12,18,24 hours
Determine the absorbance of green alga, obtain relative inhibition, calculate the growth inhibition ratio of each open-assembly time node, carry out concentration-effect
Curve matching is answered, effective concentration, confidential interval is calculated.
3. beneficial effect
Microplate oxicity analysis method determination data contrast with conventional 96 hours, finds the dense of these compounds and its mixture
Degree-effect curve (CRC) extension over time, toxicity is gradually increasing, but increased speed is different.The toxicity phase of mixture
Interaction rule changes a lot also with the component of mixture and open-assembly time, and some mixtures are with open-assembly time
Extension, collaboration or antagonism gradually strengthen, and some mixture toxicities interact and even there occurs effect class over time
The change of type is such as gradually converted into antagonism or is gradually converted into collaboration from antagonism from collaboration.Obviously, the present invention can be more reasonable
Evaluation pollutant toxicity and announcement environmental contaminants toxic action rule and mechanism.
Specific embodiment
Of the present invention is further illustrated by the following examples
A kind of chronotoxicity microplate analysis method, including culture medium preparation, microplate design, microplate sample-adding, pollutant when
Between toxicity test, it is characterised in that described culture medium prescription：0.3g NaNO3、0.08g K2HPO4(3H2O、0.08g
MgSO4(7H2O、0.04g CaCl2(2H2O、0.25g KH2PO4、0.05g NaCl、0.01mL FeCl3(6H2O、1.54mL
EDTA-Fe, 55mL soil extract, 1.45mLA5 solution and 942mL distilled water.
Described EDTA-Fe is formulated：2g Na2EDTA, 66mg FeCl3 (6H2O, 50mL 0.2M HCl and 50mL
H2O。
Described A5 solution formulas are：250mg H3BO3、192mg MnCl2(4H2O、32mg ZnSO4(7H2O、
17.5mg CuSO4 ((Mo7O24 (4H2O the and 100mL H2O of 5H2O, 3.5mg (NH4) 6.
Described microplate design, step is that 18 micropores on 48 hole microplate peripheries are added into 100 μ L distilled water first, surplus
Remaining choose in 30 micropores the 1st, 2,4 and 7 row totally 24 micropores used as blank, remaining 24 micropores press certain dilution gfactor
Deng than 8 concentration gradients of design, 100 μ L solution to be measured are added per hole, it is parallel per concentration 3, repeat three plates.
Described microplate sample-adding, step under the conditions of chlorella pyrenoidosa in the medium 20 ± 1 DEG C will first to cultivate to right
In number growth period, during sample-adding, will cultivate in adding blank well and treatment hole to the algae solution of logarithmic phase, it is ensured that each micropore is final
Cumulative volume be 100 μ L, and ensure mixing after algae density be about 2.0 × 108/mL, the absorbance OD690 at wavelength 690nm
Value is between 0.10~0.30.
Described pollutant chronotoxicity is determined, and step is to be added according to the design of claim 4 microplate and claim 5 microplate
Sample loading mode is completed in the microplate placement illumination box of sample-adding operation, and in 20 ± 1 DEG C of cultures, is surveyed after 0,6,12,18,24 hours
Determine the absorbance of green alga, obtain relative inhibition, calculate the growth inhibition ratio of each open-assembly time node, carry out concentration-effect
Curve matching is answered, effective concentration, confidential interval is calculated.
Claims (6)
1. a kind of preparation of chronotoxicity microplate analysis method, including culture medium, microplate design, microplate sample-adding, pollutant time
Toxicity test, it is characterised in that described culture medium prescription：0.3g NaNO3、0.08g K2HPO4·3H2O、0.08g
MgSO4·7H2O、0.04g CaCl2·2H2O、0.25g KH2PO4、0.05g NaCl、0.01mL FeCl3·6H2O、1.54mL
EDTA-Fe, 55mL soil extract, 1.45mL A5 solution and 942mL distilled water.
2. a kind of chronotoxicity microplate analysis method according to claim 1, it is characterised in that described EDTA-Fe matches somebody with somebody
Fang Wei：2g Na2EDTA、66mg FeCl3·6H2O, 50mL0.2M HCl and 50mL H2O。
3. a kind of chronotoxicity microplate analysis method according to claim 1, it is characterised in that described A5 solution formulas
For：250mg H3BO3、192mg MnCl2·4H2O、32mg ZnSO4·7H2O、17.5mg CuSO4·5H2O、3.5mg
(NH4)6·Mo7O24·4H2O and 100mL H2O。
4. a kind of chronotoxicity microplate analysis method according to claim 1, it is characterised in that described microplate design,
Step is that 18 micropores on 48 hole microplate peripheries are added into 100 μ L distilled water first, and the 1st, 2,4 are chosen in remaining 30 micropores
And 7 row totally 24 micropores as blank, remaining 24 micropores by certain dilution gfactor etc. than 8 concentration gradients of design, often
Hole adds 100 μ L solution to be measured, parallel per concentration 3, repeats three plates.
5. a kind of chronotoxicity microplate analysis method according to claim 1, it is characterised in that described microplate sample-adding,
Step during sample-adding, will have been cultivated will first to be cultivated to exponential phase under the conditions of chlorella pyrenoidosa in the medium 20 ± 1 DEG C
Into the algae solution addition blank well and treatment hole of logarithmic phase, it is ensured that each micropore final total volume is 100 μ L, and ensures mixing
Algae density is about 2.0 × 10 afterwards8Individual/mL, the absorbance OD at wavelength 690nm690Value is between 0.10~0.30.
6. a kind of chronotoxicity microplate analysis method according to claim 1, it is characterised in that described pollutant time
Toxicity test, step is the microplate that sample-adding operation is completed according to the design of claim 4 microplate and claim 5 microplate sample loading alternative
In placement illumination box, and in 20 ± 1 DEG C of cultures, the absorbance of green alga is determined after 0,6,12,18,24 hours, obtain phase
To inhibiting rate, calculate the growth inhibition ratio of each open-assembly time node, carry out concentration effect curve fitting, calculate effective concentration,
Confidential interval.
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| CN111796086A (en) * | 2020-07-03 | 2020-10-20 | 同济大学 | Characterization method of acute and chronic dose-effect relationship of personal care product |
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| WO2012032853A1 (en) * | 2010-09-08 | 2012-03-15 | 浜松ホトニクス株式会社 | Method for preparation of algal cells, and kit for evaluation of toxicity of chemical substance |
| US20130256561A1 (en) * | 2012-04-03 | 2013-10-03 | Ut-Battelle, Llc | Pulse amplitude modulated chlorophyll fluorometer |
| CN104390920A (en) * | 2014-11-27 | 2015-03-04 | 安徽建筑大学 | Microplate analysis method for time toxicity of environmental pollutants on basis of chlorella pyrenoidosa |
| CN104830691A (en) * | 2015-04-27 | 2015-08-12 | 长江大学 | Culture method of chlorella |
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2016
- 2016-12-05 CN CN201611114391.5A patent/CN106771111A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220389A (en) * | 2008-01-23 | 2008-07-16 | 重庆大学 | Sensitization algae determination method for venomous injurant acute toxicity response |
| WO2012032853A1 (en) * | 2010-09-08 | 2012-03-15 | 浜松ホトニクス株式会社 | Method for preparation of algal cells, and kit for evaluation of toxicity of chemical substance |
| US20130256561A1 (en) * | 2012-04-03 | 2013-10-03 | Ut-Battelle, Llc | Pulse amplitude modulated chlorophyll fluorometer |
| CN104390920A (en) * | 2014-11-27 | 2015-03-04 | 安徽建筑大学 | Microplate analysis method for time toxicity of environmental pollutants on basis of chlorella pyrenoidosa |
| CN104830691A (en) * | 2015-04-27 | 2015-08-12 | 长江大学 | Culture method of chlorella |
Non-Patent Citations (1)
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| 陈琼 等: "几种抗生素对蛋白核小球藻的时间毒性微板分析法", 《生态毒理学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111796086A (en) * | 2020-07-03 | 2020-10-20 | 同济大学 | Characterization method of acute and chronic dose-effect relationship of personal care product |
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