CN103276042A - Method for detecting genetic toxicity of organic pollutants in water - Google Patents
Method for detecting genetic toxicity of organic pollutants in water Download PDFInfo
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- CN103276042A CN103276042A CN201310200244XA CN201310200244A CN103276042A CN 103276042 A CN103276042 A CN 103276042A CN 201310200244X A CN201310200244X A CN 201310200244XA CN 201310200244 A CN201310200244 A CN 201310200244A CN 103276042 A CN103276042 A CN 103276042A
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Abstract
The invention provides a method for detecting the genetic toxicity of organic pollutants. The method comprises the following steps of: (1) treating gobiocypris rarus by a water solution of an organic-pollutant-containing sample to be detected in an exposed way; and (2) detecting the peripheral blood erythrocyte of the gobiocypris rarus which is treated in the exposed way, carrying out statistics on cell micronucleus rate and drawing a dosage-effect curve. The detection method is sensitive, simple, convenient, rapid, more visual and accurate, has good result repeatability, and has important significance for the research of genetic toxicity of water quality.
Description
Technical field
The invention belongs to the Study of Water Environment field, relate to the genotoxic method of a kind of detection organic pollutant.
Background technology
Environmental pollution increases the weight of, and brings threat for people's health.Various research methods have been carried out genetoxic have been estimated, and propose micronucleus test first so far from Heddle etc., and micronucleus test is because of advantage detections that is applied to karyomit(e)/spindle body damaging effect more and more widely such as its economy, simple and easy to do, confidence level height.
Hereditary poisonous substance in the water surrounding can bring out the fish body and produce micronucleus, can study the genetoxic of xenobiotics and estimate ambient water quality by the variation of monitoring fish micronucleus in erythrocytes rate.At present, the investigator discusses to the validity that the micronucleus in erythrocytes of multiple fish is tested, domestic scholars is used Chinese traditional fish crucian, carp, silver carp and bighead etc. and is successfully taked in situ test to estimate ambient water quality, confirms that the fish peripheral red blood cells is as the feasibility of the biomarker of the hereditary poisonous substance chromosome damage of monitoring.And along with the aggravation of Economic development and process of industrialization, make water body polluted by a large amount of organic pollutants, many carcinogenic, teratogenesis, mutagenic matters of belonging to wherein.For organic pollutant in the water body Gobiocypris rarus peripheral red blood cells genetic toxicity test method being detected research does not carry out as yet.
Summary of the invention
An object of the present invention is to provide the genotoxic detection method of organic pollutant in a kind of test sample, comprise following steps:
(1) with the aqueous solution of testing sample, exposes and handle Gobiocypris rarus;
(2) detect the peripheral red blood cells that exposes the Gobiocypris rarus after handling, statistics cell micronucleus rate.
Described detection method is to judge the genetoxic of organic pollutant in the testing sample according to the cell micronucleus rate.
In the described step (1), before described exposure was handled, the peripheral red blood cells micronuclear rates of Gobiocypris rarus was 0.1 ‰.
In the described step (1), the method that described exposure is handled is: Gobiocypris rarus is placed the described aqueous solution that contains testing sample, and every 2-3d changes and exposes treatment solution once between resting period, replaces half at every turn.
In the described step (1), lucifuge in the described exposure treating processes.
In the described step (1), the time that described exposure is handled is 21-28d; Be specially 28d.
In the described step (1), the compound method of the aqueous solution of described testing sample is: determinand is dissolved in behind the organic solvent dilute with water namely again.
In the described step (2), the method that described detection exposes the peripheral red blood cells of the Gobiocypris rarus after handling is: from step (1) expose that Gobiocypris rarus after handling, after putting to death is got blood, smear, fixes, dried, Giemsa uses liquid dyeing, clean dye liquor, dry, microscopy.
In the described method, described organic pollutant is benzo [a] pyrene or 4-nitroquinoline-1-oxide compound.
Another object of the present invention provides the application in the Gobiocypris rarus genetoxic that organic pollutant causes in detecting water.
In the described application, described organic pollutant is benzo [a] pyrene or 4-nitroquinoline-1-oxide compound.
Also purpose of the present invention provides the method for organic pollutant content in a kind of auxiliary detection water, comprises following steps:
(1) production standard curve:
The determinand of A, a series of concentration known of preparation is dissolved in the solution storing solution of organic solvent, is diluted in the determinand aqueous solution that water after the activated carbon treatment obtains various different concns;
B, expose with the determinand aqueous solution of various concentration and handle Gobiocypris rarus respectively;
C, detection expose the peripheral red blood cells of the Gobiocypris rarus after handling, statistics cell micronucleus rate;
D, being ordinate zou with the cell micronucleus rate, is X-coordinate with the concentration of determinand in the described determinand aqueous solution, the production standard curve;
(2) get water sample to be measured, according to identical method among step B and the C, Gobiocypris rarus exposed handle and statistics cell micronucleus rate; With the typical curve among the cell micronucleus rate substitution above-mentioned steps D, namely obtain the content of determinand in the water sample to be measured.
Among the described step B, before described exposure was handled, the peripheral red blood cells micronuclear rates of Gobiocypris rarus was 0.1 ‰.
Among the described step B, the method that described exposure is handled is: Gobiocypris rarus is placed the described aqueous solution that contains testing sample, places, during every 2-3d change and expose treatment solution once, replace half at every turn.
Among the described step B, lucifuge in the described exposure treating processes.
Among the described step B, the time that described exposure is handled is 21-28d; Be specially 28d.
Among the described step C, the method that described detection exposes the peripheral red blood cells of the Gobiocypris rarus after handling is: from step B expose that Gobiocypris rarus after handling, after putting to death is got blood, smear, fixes, dried, Giemsa uses liquid dyeing, clean dye liquor, dry, microscopy.
In the described method, described organic pollutant is benzo [a] pyrene or 4-nitroquinoline-1-oxide compound.
The present invention compared with prior art has following advantage:
1. Gobiocypris rarus is Chinese peculiar small-scale test fish, is easy to standardized study, reduces other experimental factor influences, and its peripheral red blood cells micronucleus test can detect organic pollutant genetoxic hazard conditions in the water well.
2. develop the micronucleus test technology at organism exposure chamber and exposure duration, and draw dose-effect curve, realize the quantitative evaluation to hereditary poisonous substance toxicity.
Therefore, the inventive method sensitivity, easy, fast, more directly perceived, accurate, favorable reproducibility as a result, significant for the research of water quality genetoxic.
Description of drawings
Fig. 1 is benzo [a] pyrene dose-effect curve.
Fig. 2 is 4-nitroquinoline-1-oxide compound dose-effect curve.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
Embodiment 1, the genotoxic detection of dimethyl sulfoxide (DMSO)
(1) detection method and step
(1) raising and train of Gobiocypris rarus: raise and train same batch, Gobiocypris rarus 14d that the body weight gain is close, during no natural death fish phenomenon, its peripheral blood micronuclear rates reaches about 0.1 ‰, namely can be used for test;
(2) trial test selects the dimethyl sulfoxide (DMSO) of suitable concn scope to handle, and is intending adopting no dead fish phenomenon under the highest exposure concentrations;
(3) expose the preparation for the treatment of solution: dimethyl sulfoxide (DMSO) is dissolved in the activated carbon treatment tap water, is mixed with volume fraction than 0.03% the aqueous solution, be the exposure treatment solution for preparing;
(4) expose processing: establish the blank that does not add dimethyl sulfoxide (DMSO); Experiment exposure group is selected 5 tail Gobiocypris rarus at random, and lucifuge exposes 28d, every 2-3d change expose treatment solution half;
(5) make the blood smear of Gobiocypris rarus erythrocyte: will expose and get blood after fish after handling is put to death, methyl alcohol is 10min fixedly, dry, through Giemsa use liquid dye 15min, immediately with the clean dye liquor of the phosphate buffered saline buffer of pH=6.8, dry namely;
(6) detection of cell micronucleus rate: blood smear places microscopically to observe, 10*100 doubly observes, counts, choose complete in the visual field, be uniformly dispersed and painted suitable cell, double-blind method is read sheet, every erythrocytic micronuclear rates of fish that smear is counted about 5000 endochylema complete displays, the result represents with permillage (‰).Dimethyl sulfoxide (DMSO) exposed volume mark is that the cell micronucleus rate of 0%, 0.03% o'clock correspondence is respectively 0.29 ± 0.04 ‰, 0.37 ± 0.06 ‰, and there was no significant difference shows that dimethyl sulfoxide (DMSO) does not show the genetoxic effect under this test condition.
(1) detection method and step
(1) raising and train of Gobiocypris rarus: raise and train same batch, Gobiocypris rarus 14d that the body weight gain is close, during no natural death fish phenomenon, its peripheral blood micronuclear rates reaches about 0.1 ‰, namely can be used for test;
(2) trial test selects the suitable concn scope to handle; Do not observe dead fish phenomenon to intend adopting the highest exposure concentrations to expose processing, show to can be used for follow-up micronucleus exposure test.
(3) preparation of exposure treatment solution: benzo [a] pyrene is dissolved in the dimethyl sulfoxide (DMSO), be mixed with concentration and be respectively the mother liquor of 0mg/L, 4/3mg/L, 20/3mg/L, 100/3mg/L, 500/3mg/L, the mother liquor of getting the same volume product then respectively is dissolved in the tap water of activated carbon filtration, be mixed with benzo [a] the pyrene concentration solution of 0 μ g/L, 0.4 μ g/L, 2.0 μ g/L, 10.0 μ g/L, 50.0 μ g/L respectively, be 5 groups of exposure treatment solutions that prepare;
(4) expose processing: establishing the dimethyl sulfoxide (DMSO) blank solution that does not add benzo [a] pyrene is control group; Every group is selected 10 tail Gobiocypris rarus; Adopt semi-static exposure chamber, namely every 2-3d changes and exposes treatment solution once, once half; Lucifuge exposes 28d;
(5) make the blood smear of Gobiocypris rarus erythrocyte: will expose and get fixedly 10min of blood, smear, methyl alcohol after fish after handling is put to death, dry, through Giemsa use liquid dye 15min, immediately with the clean dye liquor of the phosphate buffered saline buffer of pH=6.8, dry namely;
(6) detection of cell micronucleus rate: blood smear places microscopically to observe, 10*100 doubly observes, counts, choose complete in the visual field, be uniformly dispersed and painted suitable cell, double-blind method is read sheet, every erythrocytic micronuclear rates of fish that smear is counted about 5000 endochylema complete displays, the result represents with permillage (‰).The cell micronucleus rate that exposure concentrations increases successively is respectively 0.37 ± 0.06 ‰, 1.53 ± 0.11 ‰, 4.19 ± 0.08 ‰, 10.29 ± 0.93 ‰, 17.93 ± 0.43 ‰.
The result shows that when benzo [a] pyrene concentration was 0.4 μ g/L, cell micronucleus rate and blank had significant difference.The detection method of existing bibliographical information is as shown in table 1.
Table 1
Table 1 is the result show, compares with existing detection method, and detection method sensitivity provided by the invention is higher.
(2) dose-effect curve
Be X-coordinate with benzo [a] pyrene concentration, the micronucleus permillage is ordinate zou, draws dose-effect curve, as shown in Figure 2.Quantizing dose-effect relationship by dose-effect curve is following formula:
y＝2.14(1-e
-1.76x)+16.30(1-e
-0.069x)
Benzo [a] pyrene 28d exposes the matched curve formula to be changed: R
2=0.9994; Adjusted R
2=0.9974
Multiple comparisons shows and there are differences extremely significantly between each concentration group, illustrates that benzo [a] pyrene has the genetoxic effect to the Gobiocypris rarus peripheral red blood cells, and apparent in view dose-effect relationship is arranged.
Embodiment 3, the 4-nitroquinoline-genotoxic detection of 1-oxide compound (4-NQO)
(1) detection method and step
(1) raising and train of Gobiocypris rarus: raise and train same batch, Gobiocypris rarus 14d that the body weight gain is close, during no natural death fish phenomenon, its peripheral blood micronuclear rates reaches about 0.1 ‰, namely can be used for test;
(2) trial test selects the suitable concn scope to handle; Do not observe dead fish phenomenon to intend adopting the highest exposure concentrations to expose processing, show to can be used for follow-up micronucleus exposure test;
(3) preparation of exposure treatment solution: 4-nitroquinoline-1-oxide compound (4-NQO) is dissolved in the dimethyl sulfoxide (DMSO), be mixed with concentration and be respectively the mother liquor of 0mg/L, 1/3mg/L, 5/3mg/L, 25/3mg/L, 125/3mg/L, the mother liquor of getting the same volume product then respectively is dissolved in the tap water of activated carbon filtration, be mixed with the 4-NQO concentration solution of 0 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 2.5 μ g/L, 12.5 μ g/L respectively, be 5 groups of exposure treatment solutions that prepare;
(4) expose processing: establishing the dimethyl sulfoxide (DMSO) blank solution that does not add 4-nitroquinoline-1-oxide compound is control group; Every group is selected 10 tail Gobiocypris rarus; Adopt semi-static exposure chamber, namely every 2-3d changes and exposes treatment solution once, once half; Lucifuge exposes 28d;
(5) make the blood smear of Gobiocypris rarus erythrocyte: will expose and get fixedly 10min of blood, smear, methyl alcohol after fish after handling is put to death, dry, through Giemsa use liquid dye 15min, immediately with the clean dye liquor of the phosphate buffered saline buffer of pH=6.8, dry namely;
(6) detection of cell micronucleus rate: blood smear places microscopically to observe, 10*100 doubly observes, counts, choose complete in the visual field, be uniformly dispersed and painted suitable cell, double-blind method is read sheet, every erythrocytic micronuclear rates of fish that smear is counted about 5000 endochylema complete displays, the result represents with permillage (‰).The cell micronucleus rate that exposure concentrations increases successively is respectively 0.37 ± 0.06 ‰, 1.36 ± 0.08 ‰, 4.35 ± 0.40 ‰, 8.89 ± 0.26 ‰, 22.69 ± 0.56 ‰.
The result shows when 4-nitroquinoline-1-oxide concentration is 0.1 μ g/L, to have significant difference with blank.Document Valentin-Severin, I.et al.Mutation Research-Genetic Toxicology and Environmental Mutagenesis[J] .536 (1-2): the genetoxic that detects 4-nitroquinoline-1-oxide compound among the 79-90. by human liver cancer cell HepG2, experimental result shows, when the cell micronucleus rate reached two times of negative control micronuclear rates, the concentration of 4-nitroquinoline-1-oxide compound was 22.8 μ g/L.This shows with having detection method now compares, and detection method sensitivity provided by the invention is higher.
(2) dose-effect curve
Be X-coordinate with 4-nitroquinoline-1-oxide concentration, the micronucleus permillage is ordinate zou, draws dose-effect curve, as shown in Figure 2.Quantizing dose-effect relationship by dose-effect curve is following formula:
y＝4.19(1-e
-3.24x)+39.26(1-e
-0.05x)
4-nitroquinoline-1-oxide compound (4-NQO) 28d exposes the matched curve formula to be changed: R
2=0.9996; AdjustedR
2=0.9983
Multiple comparisons shows and there are differences extremely significantly between each concentration group, illustrates that 4-nitroquinoline-1-oxide compound has the genetoxic effect to the Gobiocypris rarus peripheral red blood cells, and apparent in view dose-effect relationship is arranged.
Claims (7)
1. the genotoxic detection method of organic pollutant in the test sample comprises following steps:
(1) with the aqueous solution of testing sample, exposes and handle Gobiocypris rarus;
(2) detect the peripheral red blood cells that exposes the Gobiocypris rarus after handling, statistics cell micronucleus rate.
2. method according to claim 1 is characterized in that: in the step (1), before described exposure was handled, the peripheral red blood cells micronuclear rates of Gobiocypris rarus was 0.1 ‰.
3. method according to claim 1 and 2 is characterized in that: in the step (1), the method that described exposure is handled is: Gobiocypris rarus is placed the described aqueous solution that contains testing sample, places, during every 2-3d change and expose treatment solution once, replace half at every turn.
4. the application in the Gobiocypris rarus genetoxic that organic pollutant causes in detecting water.
5. the method for organic pollutant content in the auxiliary detection water comprises following steps:
(1) production standard curve:
The determinand of A, a series of concentration known of preparation is dissolved in the solution storing solution of organic solvent, is diluted in the determinand aqueous solution that water after the activated carbon treatment obtains various different concns;
B, expose with the determinand aqueous solution of various concentration and handle Gobiocypris rarus respectively;
C, detection expose the peripheral red blood cells of the Gobiocypris rarus after handling, statistics cell micronucleus rate;
D, being ordinate zou with the cell micronucleus rate, is X-coordinate with the concentration of determinand in the described determinand aqueous solution, the production standard curve;
(2) get water sample to be measured, according to identical method among step B and the C, Gobiocypris rarus exposed handle and statistics cell micronucleus rate; With the typical curve among the cell micronucleus rate substitution above-mentioned steps D, namely obtain the content of determinand in the water sample to be measured.
6. method according to claim 5 is characterized in that: among the step B, before described exposure was handled, the peripheral red blood cells micronuclear rates of Gobiocypris rarus was 0.1 ‰.
7. according to claim 5 or 6 described methods, it is characterized in that: among the step B, the method that described exposure is handled is: Gobiocypris rarus is placed the described aqueous solution that contains testing sample, places, during every 2-3d change and expose treatment solution once, replace half at every turn.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830946A (en) * | 2015-05-13 | 2015-08-12 | 中国环境科学研究院 | Technology for detecting TK6 cell chromosome damage genetic toxicity caused by environmental water and application of technology |
| CN105301211A (en) * | 2014-07-04 | 2016-02-03 | 中国环境科学研究院 | Semi-static temperature-control type fish toxicity test apparatus |
| CN105548514A (en) * | 2015-12-25 | 2016-05-04 | 哈尔滨工业大学 | Multi-genetic terminal biological group test method for evaluating genetic toxicity of industrial wastewater reuse of water treatment plant |
-
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Non-Patent Citations (3)
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| 刘楠楠等: "稀有鮈鲫外周血红细胞微核试验方法研究", 《中国环境监测》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105301211A (en) * | 2014-07-04 | 2016-02-03 | 中国环境科学研究院 | Semi-static temperature-control type fish toxicity test apparatus |
| CN105301211B (en) * | 2014-07-04 | 2017-07-14 | 中国环境科学研究院 | Semi-static temperature controlled toxicity test device of fishes |
| CN104830946A (en) * | 2015-05-13 | 2015-08-12 | 中国环境科学研究院 | Technology for detecting TK6 cell chromosome damage genetic toxicity caused by environmental water and application of technology |
| CN104830946B (en) * | 2015-05-13 | 2018-07-17 | 中国环境科学研究院 | Environment water causes TK6 cell chromosome damages genetic toxicology assays and application |
| CN105548514A (en) * | 2015-12-25 | 2016-05-04 | 哈尔滨工业大学 | Multi-genetic terminal biological group test method for evaluating genetic toxicity of industrial wastewater reuse of water treatment plant |
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