CN101000330A - Method for investigating estrogen active constribution material in sewage - Google Patents
Method for investigating estrogen active constribution material in sewage Download PDFInfo
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- CN101000330A CN101000330A CN 200710062639 CN200710062639A CN101000330A CN 101000330 A CN101000330 A CN 101000330A CN 200710062639 CN200710062639 CN 200710062639 CN 200710062639 A CN200710062639 A CN 200710062639A CN 101000330 A CN101000330 A CN 101000330A
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Abstract
The invention relates to a method used to detect estrogen activity contribution matter in sewage. It includes the following steps: extracting estrogen activity matter form the sewage sample by solid phase column extractor; using methanol, dichloroethanes, normal hexane purging, and silica gel chromatographic column to classify into polarity, moderate and weak polarity three components; using recombination gene yeast bioassay method to measure the estrogen activity; taking out the maximum component to analyze its kind and density by gas phase color photography-mass spectrum on line. This method can fast effectively detect the main estrogen activity contribution matter in the sewage, supply foundation and support for sewerage treatment technology improvement.
Description
Technical field
The present invention relates to the detection technique field of organic contaminant in a kind of sewage, particularly relate to the detection method of the active contribution of trace estrogen in the sewage.
Background technology
Current, the incretion interferent in the water has caused extensive concern both domestic and external, and the incretion interferent that wherein has estrogen active occupies main status.This class material is hundreds of, known agricultural chemicals (Atrazine, D.D.T. (dichloro-diphenyl-trichloroethane), toxaphene etc.), natural estrogen (17-β estradiol, oestrone etc.), surfactant (APES and metabolic product thereof are as nonylphenol, octyl phenol etc.), medicine class (diethylstilbestrol, ethinyloestradiol etc.) and the industrial products (phthalate ester, bisphenol-A etc.) of mainly comprising.Along with the high speed development of industrial or agricultural and the acceleration of urbanization construction, this class material is mingled in a large amount of industrial sewage and sanitary sewage, without effective wastewater treatment, or directly enter into surface water bodies such as river, river, lake by rainwash, even be penetrated in the underground water, causing with surface water and underground water is that the drinking water resource at water source is polluted, and source quality obviously worsens.Though this class material and the exist level of secondary pollutant in sewage that produces under environmental activity thereof are μ g/L, ng/L level or lower, and they the contribution of monitoring organic compounds index is very little routinely to chemical oxygen demand (COD), biochemical oxygen demand etc., but the harmfulness of this class material is very big, and human body and Ecosystem System Influence are had disguise, potentiality and hysteresis quality.This class material overwhelming majority is long-term accumulation in human and other animal bodies easily, also can show stronger estrogen active and disturb normal endocrine metabolism of biosome and reproduction function even concentration level is the ng/L level, as: carcinogenic, metabolic disorder, inborn defect, dysplasia etc.At present, this class material has detected in the sewage Inlet and outlet water in country variant and area has higher concentration, seriously threatening hydrobiological safety, therefore, detecting the harm that whether has these incretion interferents with estrogen active, exists concentration and this class material to bring from sewage and treatment process unit thereof all is the problem that present people are concerned about.
In recent years, the instrument detecting technology of estrogen active material is enhanced, and emerges in an endless stream as gas chromatography-mass spectrometry machine (GC-MS), gas chromatography-tandem mass spectrum online (GC-MS/MS), liquid phase chromatogram-mass spectrometry combination machine (LC-MS/MS) and liquid chromatography-nuclear magnetic resonance online (LC-NMR) etc.These methods can provide higher sensitivity and degree of accuracy for analyzing the estrogen active material.Yet, instrument detecting can not be divided the influence that the estrogen active material brings to biology in the bleed, simultaneously because the estrogen active material that instrument detecting goes out is many, understand harm which material wherein brings to ecologic environment the most serious be the thing of comparison difficulty.Therefore and the biological test method that combines of instrumental analysis, developed as recombinant gene yeast method, biomarker etc., and can be used for the detection that sewage carries out estrogen active.Yet, owing to contain the activator and the antagonist of a large amount of estrogen active materials in the water, the interaction that exists between the estrogen active material of different structure (add and, collaborative or antagonism) can be covered real estrogen active effect when biological detection, thereby can cause occurring easily when the main estrogen active contribution of identification mistake.Since at present both at home and abroad the sewage quality monitoring index at the control of organic contaminant mainly with its kind and concentration as guiding, to use more be that the employing instrument carries out classification to the estrogen active material in the sewage, as high performance liquid chromatography (HPLC).Though this class methods test is more comprehensive, can carry out discrete testing to each detected estrogen active material in the sewage, yet its classification step often depends on chromatogram and detects what of material, the biological test process that the estrogen active material adopts more for a long time is comparatively loaded down with trivial details when detecting, and has increased the difficulty that finds main estrogen active contribution in the sewage.
Summary of the invention
The objective of the invention is at adopting instrument test to analyze main estrogen active contribution complex steps in the sewage for the classification technique of guiding, analyze the big deficiency that waits of difficulty, intending exploitation a kind of is that the classification technique that leads detects estrogen active material in the sewage based on biological test comparatively simply, realizes the effective mensuration to main estrogen active contribution in the sewage.
Technical scheme of the present invention is as follows:
A kind of method that detects estrogen active contribution in the sewage is characterized in that this method comprises as the lower part:
1) the sewage water sample of gathering is carried out pre-service: filter water sample with glass fiber filter earlier, the water sample after will filtering then is through solid phase extraction concentration estrogen active material wherein, afterwards with the solid phase extraction column freeze drying;
2) adopt the opposed polarity organic solvent that the estrogen active material is eluted from solid-phase extraction column, adopt silica gel chromatographic column that the estrogen active material is pressed similar polarity classification, obtain polarity, Semi-polarity and three kinds of components of low pole;
3) use the recombinant gene yeast measuring technology to analyze the estrogen active of classification component the classification component of opposed polarity;
4) utilize the gas chromatography-mass spectrometry machine to analyze the substance classes and the concentration of the maximum component of estrogen active, find main estrogen active contribution.
The organic solvent of opposed polarity of the present invention is respectively normal hexane, methylene chloride and methyl alcohol.
Silica gel chromatographic column of the present invention is the upper strata filling gel, and the two-part of aluminium oxide is filled by lower floor.
The test duration that the recombinant gene yeast method of testing that the present invention adopts is selected is 16～20h, and the wherein initial quality of saccharomycete in nutrient culture media is 5～10% than concentration, and the optical density of culture yeast under 600nm is to test in 1.0～1.5 o'clock.
The present invention with existing with the instrument test be the guiding classification technique compare, have the following advantages and the high-lighting effect: 1. this method adopts biological test simple for the classification technique flow process of guiding, can eliminate the interference effect between the opposed polarity material effectively, the legitimate reading of reflection biological test; 2. instrument test causes the estrogen active material that detects more for the classification technique of guiding, difficult judgement main contributor wherein, what this method adopted can directly analyze at the main material that produces estrogen active in the sewage for the classification technique of guiding based on biological test; 3. this method tests out main estrogen active contribution and helps sewage treatment process to improve targetedly; 4. the present recombinant gene yeast test duration is long, is generally about 48-72h, and fast culture and test are selected in the recombinant gene yeast test that this method adopts, and the shortening test duration is 20h, but does not change measurement sensitivity.
Description of drawings
Fig. 1 tests the dose-effect curve of typical estrogen active material for adopting the present invention.
Embodiment
Below in conjunction with embodiment and accompanying drawing technical scheme of the present invention is further described.
Gathered instantaneous water sample of sewage or 24h compound sample, got 1L, removed the interference of the suspended particulate substance in the water through 0.7 μ m glass fiber filter.Water sample after the filtration is with solid phase extraction column extraction estrogen active material wherein, for improving the extraction efficiency of extraction pillar, avoid the wherein interference of moisture, the extraction pillar need carry out freeze drying, with the estrogen active material in normal hexane, methylene chloride and the methanol-eluted fractions extraction pillar, merge eluent, through two-part silica gel-aluminium oxide chromatographic column, wherein silica gel 10g, aluminium oxide 10g, with normal hexane, methylene chloride and methyl alcohol difference drip washing silica gel-aluminium oxide chromatographic column, drip washing obtains low pole, Semi-polarity and three kinds of components of polarity.This stage division is to known estrogen active material, as: the recovery of estradiol, oestrone, bisphenol-A, nonylphenol etc. is between 85-95%.
The recombinant gene yeast that with initial concentration is 5～10% (weight ratios) is at 30 ℃, and rotating speed is to cultivate in the bio-incubator of 150rpm to improve rotating speed behind the 2h and cultivate 14～18h to 200rpm, measures saccharomycete OD600 and is in 1.0～1.5.Enriched sample is evenly mixed after getting 950 μ L bacteria suspensions and 50 μ L classifications.Each sample is made 3 parallel samples at least.Getting 200 μ L mixed liquors transfers in 96 orifice plates on micro oscillator 30 ℃ and exposes down and cultivate 2h, rotating speed 600rpm.Positive control (estradiol) and negative control (dimethyl sulfoxide (DMSO)) are set on every 96 orifice plate simultaneously.For avoiding the influence of enriched sample to the acute toxicity of yeast, the OD600 after needing to measure bacterium liquid and enriched sample contacts 2h, with contact before can continue to test when comparing OD600 and not having significant change, otherwise need dilute sample to expose cultivation again.The mixed liquor that pipettes behind the 50 μ L contact 2h adds 120 μ L buffer solution, and vibration 10min adds 20 μ L methenyl cholorides, 20 min that vibrate, and adds 40 μ L developer ONPG (4g/L is dissolved in buffer solution) and starts enzyme reaction.When generating, tangible yellow adds 100 μ LNa
2CO
3(1M) stop enzyme reaction, in the optical density (OD420) of measuring the supernatant of mixed liquor under the 420nm wavelength on the microplate reader, by formula U=OD420 (S)/OD600 (S)-OD420 (B)/OD600 (B), draw dose-effect curve, calculate effective concentration 50 EC50, estrogen active equivalent (EEQ) and estrogen active equivalence factor (EEF).Wherein, OD420 (S) and OD600 (S) are the optical density of water sample, the optical density of OD420 (B) and the negative contrast of OD600 (B).Table 1 is the EC50 and the EEF of typical estrogen active material.Recombinant gene yeast detects usefulness as shown in Figure 1, and detectability reaches 0.1nM EEQ/L.The estrogen active of water sample is higher than this detectability and all can tests after the enrichment.
According to the recombinant gene yeast test result of three kinds of components, the component of selecting the estrogen active maximum is accurately measured wherein estrogen active substance classes and concentration with GC-MS.According to the concentration of the estrogen active material that is measured to and the EEF of this material, by formula EEQ=∑ C
i* EEF
iCalculate to measure the EEQ of material,, find estrogen active contribution main in the sewage water sample according to the EEQ of Instrument measuring material contribution rate to biological test EEQ.Wherein, C
iBe the concentration/μ g/L of i the estrogen active material of measuring, EEF
iIt is the estrogen active equivalence factor of i the estrogen active material of measuring.
The present invention can find estrogen active contribution main in the sewage quickly and accurately, helps sewage treatment process to improve targetedly.
The EC50 of the typical estrogen active material of table 1., EEF
| Material | Estradiol | Oestrone | The 4-nonylphenol | The 4-t-octyl phenol | Bisphenol-A |
| EC50(nM) EEF | 0.380 1 | 0.605 0.63 | 620 6.2E-4 | 451 8.4E-4 | 2.93E3 1.3E-4 |
Utilize the present invention to detect the embodiment of estrogen active contribution in certain sewage:
Select the exemplary process technique unit of a sewage treatment plant, the instantaneous water sample 4L of collection in worksite is kept at and takes back the laboratory in the low temperature refrigerator and carry out Treatment Analysis.Get 1L through 0.7 μ m glass fiber filter, water sample after the filtration this (Waters) solid phase extraction column extraction of water estrogen active material wherein, extraction pillar freeze drying 12h, use normal hexane respectively, estrogen active material in methylene chloride and the methanol-eluted fractions extraction pillar, merge eluent, through two-part silica gel-aluminium oxide chromatographic column, silica gel 10g wherein, aluminium oxide 10g, packed height is 30cm, use normal hexane respectively, methylene chloride and methyl alcohol is drip washing silica gel-aluminium oxide chromatographic column respectively, drip washing obtains low pole, three kinds of components of Semi-polarity and polarity are transferred to after concentrated the drying up and are distinguished constant volume in the sample introduction bottle in normal hexane, be used for GC-MS analysis and recombinant gene yeast test in the dimethyl sulfoxide (DMSO).
The recombinant gene yeast that with initial concentration is 10% (weight ratio) is at 30 ℃, and rotating speed is to cultivate in the bio-incubator of 150rpm to improve rotating speed behind the 2h and cultivate 14h to 200rpm, and measuring saccharomycete OD600 is 1.3.Enriched sample is evenly mixed after getting 950 μ L bacteria suspensions and 50 μ L classifications.Each sample is made 3 parallel samples.Getting 200 μ L mixed liquors transfers in 96 orifice plates on micro oscillator 30 ℃ and exposes down and cultivate 2h, rotating speed 600rpm.Positive control (estradiol) and negative control (dimethyl sulfoxide (DMSO)) are set on every 96 orifice plate simultaneously.OD600 after measuring bacterium liquid and enriched sample contacting 2h, observing enriched sample does not have acute toxicity to yeast.The mixed liquor that pipettes behind the 50 μ L contact 2h adds 120 μ L buffer solution, and vibration 10min adds 20 μ L methenyl cholorides vibration 20min, adds 40 μ L developer ONPG (4g/L is dissolved in buffer solution) and starts enzyme reaction.Tangible yellow the generation arranged during reaction 2h, add 100 μ L Na
2CO
3(1M) stop enzyme reaction, on microplate reader, measure OD420,, draw dose-effect curve, calculate effective concentration 50 EC50, estrogen active equivalent (EEQ) by formula U=OD420 (S)/OD600 (S)-OD420 (B)/OD600 (B).
According to the recombinant gene yeast test result (table 2) of three kinds of components, the polarity component of selecting the estrogen active maximum is accurately measured wherein estrogen active substance classes and concentration with GC-MS.The estrogen active material that GC-MS is measured to has phthalate ester, bisphenol-A, nonylphenol, octyl phenol (other component detects D.D.T. (dichloro-diphenyl-trichloroethane) and diethylstilbestrol etc.).According to concentration 108.17 μ g/L, 1.98 μ g/L, 11.98 μ g/L and their EEF (table 1) of bisphenol-A, nonylphenol and octyl phenol material, by formula EEQ=∑ C
i* EEF
iCalculate to measure the EEQ of material, according to the EEQ of Instrument measuring material contribution rate to biological test EEQ.As shown in table 3, bisphenol-A in this sewage sample, nonylphenol and octyl phenol are the main contribution of estrogen active in the sewage sample, wherein occupy an leading position with bisphenol-A again, but show this sewage treatment process from the adsorbent Coagulation and Adsorption of removal estrogen active angle overriding concern employing, or select the degradation bacteria enhanced biological degraded of bisphenol-A the selective absorption of bisphenol-A.
The estrogen active (ng EEQ/L) of certain sewage sample opposed polarity component of table 2.
| Different components | The polarity component | The Semi-polarity component | Nonpolar component |
| Estrogen active | 59.4 | 12.2 | 12.5 |
The main contribution of estrogen active in certain sewage sample of table 3.
| Instrument test (ng EEQ/L) | Yeast test (ng EEQ/L) | Contribution rate (%) | ||
| Bisphenol-A | Octyl phenol | Nonylphenol | ||
| 61.6 | 8.1 | 7.6 | 84.1 | 92 |
Claims (4)
1. method that detects estrogen active contribution in the sewage is characterized in that this method comprises as the lower part:
1) the sewage water sample of gathering is carried out pre-service: filter water sample with glass fiber filter earlier, the water sample after will filtering then is through solid phase extraction concentration estrogen active material wherein, afterwards with the solid phase extraction column freeze drying;
2) adopt the opposed polarity organic solvent that the estrogen active material is eluted from solid-phase extraction column, adopt silica gel chromatographic column that the estrogen active material is pressed similar polarity classification, obtain polarity, Semi-polarity and three kinds of components of low pole;
3) use the recombinant gene yeast measuring technology to analyze the estrogen active of classification component the classification component of opposed polarity;
4) utilize the gas chromatography-mass spectrometry machine to analyze the substance classes and the concentration of the maximum component of estrogen active, find main estrogen active contribution.
2. according to the method for estrogen active contribution in the described detection sewage of claim 1, it is characterized in that: the organic solvent of described opposed polarity is respectively normal hexane, methylene chloride and methyl alcohol.
3. according to the method for estrogen active contribution in the described detection sewage of claim 1, it is characterized in that: described silica gel chromatographic column is the upper strata filling gel, and the two-part of aluminium oxide is filled by lower floor.
4. according to the method for estrogen active contribution in the described detection sewage of claim 1, it is characterized in that: the test duration that the recombinant gene yeast method of testing that this method adopts is selected is 16～20h, and the wherein initial mass concentration of saccharomycete in nutrient culture media is 5～10%; The optical density of culture yeast under 600nm is to test in 1.0～1.5 o'clock.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101469315B (en) * | 2007-12-29 | 2011-03-30 | 中国科学院生态环境研究中心 | Two-hybrid yeast for detecting estrogen-like compound in environment and biological test method |
| CN102175792A (en) * | 2010-12-24 | 2011-09-07 | 北京师范大学 | Method for detecting estrogen, nonyl phenol, octylphenol and bisphenol A together in water environment |
| CN102236001A (en) * | 2010-04-23 | 2011-11-09 | 北京师范大学 | Method for detecting content of trace natural and synthetic estrogen in soil environment |
| CN102253008A (en) * | 2011-06-10 | 2011-11-23 | 东华大学 | Detection method for interaction between estrogen acceptor and phthalate ester ligand |
| CN101571523B (en) * | 2009-05-07 | 2012-04-11 | 浙江省海洋水产研究所 | Method for measuring various types of unknown pollutants in sewage |
| CN104020023A (en) * | 2014-05-09 | 2014-09-03 | 中南大学 | Method for preprocessing biological sample before preservation and analysis |
| CN105891365A (en) * | 2016-04-21 | 2016-08-24 | 南京大学 | Effect-directed target/non-target androgen disruptor identification method |
| CN107764914A (en) * | 2017-12-06 | 2018-03-06 | 中国科学院山西煤炭化学研究所 | The detection method of bisphenol-A and tetrabromobisphenol A content in a kind of environmental water sample |
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2007
- 2007-01-12 CN CNB2007100626392A patent/CN100538354C/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101469315B (en) * | 2007-12-29 | 2011-03-30 | 中国科学院生态环境研究中心 | Two-hybrid yeast for detecting estrogen-like compound in environment and biological test method |
| CN101571523B (en) * | 2009-05-07 | 2012-04-11 | 浙江省海洋水产研究所 | Method for measuring various types of unknown pollutants in sewage |
| CN102236001A (en) * | 2010-04-23 | 2011-11-09 | 北京师范大学 | Method for detecting content of trace natural and synthetic estrogen in soil environment |
| CN102236001B (en) * | 2010-04-23 | 2012-05-23 | 北京师范大学 | Method for detecting content of trace natural and synthetic estrogen in soil environment |
| CN102175792A (en) * | 2010-12-24 | 2011-09-07 | 北京师范大学 | Method for detecting estrogen, nonyl phenol, octylphenol and bisphenol A together in water environment |
| CN102175792B (en) * | 2010-12-24 | 2012-04-25 | 北京师范大学 | Method for detecting estrogen, nonyl phenol, octylphenol and bisphenol A together in water environment |
| CN102253008A (en) * | 2011-06-10 | 2011-11-23 | 东华大学 | Detection method for interaction between estrogen acceptor and phthalate ester ligand |
| CN104020023A (en) * | 2014-05-09 | 2014-09-03 | 中南大学 | Method for preprocessing biological sample before preservation and analysis |
| CN105891365A (en) * | 2016-04-21 | 2016-08-24 | 南京大学 | Effect-directed target/non-target androgen disruptor identification method |
| CN105891365B (en) * | 2016-04-21 | 2018-07-03 | 南京大学 | It is a kind of using effect as be oriented to target/non-targeted androgen chaff interferent discrimination method |
| CN107764914A (en) * | 2017-12-06 | 2018-03-06 | 中国科学院山西煤炭化学研究所 | The detection method of bisphenol-A and tetrabromobisphenol A content in a kind of environmental water sample |
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| CN100538354C (en) | 2009-09-09 |
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