CN100554959C - The biomarker monitoring method of environment As pollution - Google Patents
The biomarker monitoring method of environment As pollution Download PDFInfo
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- CN100554959C CN100554959C CNB2006100258748A CN200610025874A CN100554959C CN 100554959 C CN100554959 C CN 100554959C CN B2006100258748 A CNB2006100258748 A CN B2006100258748A CN 200610025874 A CN200610025874 A CN 200610025874A CN 100554959 C CN100554959 C CN 100554959C
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Abstract
A kind of biomarker monitoring method of environment As pollution of environmental technology field.The present invention designs the soil sampling scheme at first according to the place that will monitor; Gather earthworm at each sampled point then, it is packed into contain in the container of sampling site soil; Get adopt earthworm, carry out gut purge earlier, homogenate then, centrifugal, supernatant be the required extract that contains P450; In fluorospectrophotometer, measure the activity of 7-methoxyl-3-Yi Fen azolactone-O-debenzylation enzyme; According to its active soil total arsenic content that calculates, thus the arsenic contamination degree of monitoring soil.The present invention both can understand the degree that arsenic in soil pollutes, and can understand the biochemical effect of arsenic contamination again, and biosome was to the reaction of arsenic contamination.Have more practical value than directly measuring arsenic in soil content.And highly sensitive, be suitable for long term monitoring.
Description
Technical field
What the present invention relates to is a kind of method of green technology, specifically be a kind of biomarker monitoring method of environment As pollution, promptly utilize Cytochrome P450 to monitor in the surrounding environment of pig farm and used the method for arsenic contamination in the soil environment of pig manure as biomarker.
Background technology
Arsenic is the essential a kind of trace element of animal body, has multiple vital role.Arsenic preparation has become the widely used feed in countries in the world and has added medicine in recent years, has promoted Developing of Animal Industry, has obtained the good social economic benefit.But the farm animal excrement that contains arsenic simultaneously in a large number enters environment, and environment has been caused pollution.Its contaminated object mainly is plant's surrounding environment and the soil environment of using pig manure.For the monitoring of environment As pollution, main at present still chemical assay is promptly understood the arsenic contamination situation by the content that detects arsenic in the soil.But chemical assay has its defective, only can't react the biological effect of these pollutants with content.Biomarker monitoring rule can compensate its defect, and not only can react the soil pollution situation, can also react biological adaptability, and the poisonous effect of pollutant on molecular level; Or the like.
Biomarker (also claiming biomarker) is a kind of newer method of environmental monitoring of rising in recent years, and Cytochrome P450 (CYP450) is wherein most widely used a kind of.Cytochrome P 450 enzymes system is the class metabolic enzyme system that extensively is present in the different biosomes such as various animals, plant and microorganism, has extremely important physiological function.With inducing or inhibiting effect of P450 enzyme system, can be used as biomarker responsive on the molecular level, monitor the influence of pollutant to soil ecosystem.Have at present in a large number to be used as industrial pollutants such as polychlorinated biphenyl about P450, the research report of the biomarker of environmental pollutions such as palycyclic aromatic and heavy metal, but do not see that as yet it is used for the research that arsenic in soil pollutes.7-methoxyl-3-Yi Fen azolactone-O-debenzylation enzyme (MROD) is a kind of in the CYP450 enzyme system, i.e. CYP4501A2.It is responsive to find after deliberation in the earthworm body that this enzyme pollutes arsenic in soil, and the repressed degree of this enzyme and arsenic in soil content have correlativity within the specific limits.Can monitor arsenic in soil with the activity change degree of this enzyme pollutes.
Through new, do not find and the identical or similar bibliographical information of the biomarker monitoring method of theme arsenic contamination of the present invention as yet looking into of prior art.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of biomarker monitoring method of environment As pollution is provided, make it can monitor the pig excrements that contains arsenic and enter the pig farm surrounding environment, perhaps use
In agricultural land soil and behind the as fertilizer sources, the arsenic in soil that causes pollutes.
The present invention is achieved by the following technical solutions, and the present invention is at first according to the place that will monitor, earth sampling plan in the design; Gather earthworm at each sampled point then, it is packed into contain in the container of sampling site soil; Get adopt earthworm, carry out gut purge earlier, homogenate then, centrifugal, supernatant be the required extract that contains P450; In fluorospectrophotometer, measure the activity of MROD; According to the active soil total arsenic content that calculates of MROD, thus the arsenic contamination degree of monitoring soil.
Described at first according to the place that will monitor, design soil sampling scheme is specially: at first will understand the background information in monitoring place, determine the fluid discharge or the ight soil fertilising situation of pollution source and plant.The former can understand the distance of the residual pollution source of discharge arsenical apart from the monitoring point earlier, and the latter can understand the scheme of monitoring point fertilising earlier.Determine the concrete grammar of sampling then: when polluting, can arrange sampled point along the different distance of polluting direction for point-source pollution; When polluting, can evenly arrange sampled point for pollution of area source.
Described collection earthworm is specially: gather the soil of sampled point earlier, in the container of packing into; Then the earthworm of extraction is put into this container, take back the laboratory, do not change with the living environment that guarantees earthworm, experimental result has more reliability.
The described extract that contains P450, it specifically is prepared as: the earthworm in the container is taken out, be placed on the wet filter paper, at room temperature placed 1 to 2 day, to discharge intestinal contents; Get the earthworm after the gut purge, use the filter paper suck dry moisture, place glass homogenizer, pour the earthworm homogenate buffer of precooling in 4 ℃ of refrigerators into, homogenate; Homogenate is transferred in the 1.5ml centrifuge tube centrifugal 30min.Discard precipitation, supernatant is the required extract that contains P450.
The activity of described mensuration MROD, be specially: in the fluorescence cuvette, add each reacted constituent, concentration is respectively trishydroxymethylaminomethane (Tris) 100mmol/L, niacinamide dinucleotide phosphoric acid (NADPH) 0.5mmol/L, the about 10 μ mol/L of 7-methoxyl Yi Fen azolactone, the sample extracting solution that contains P450 of about 0.5mg albumen.NADPH adds at last during reaction, adds afterreaction and begins immediately.After accurately carrying out 10min, adding 0.5ml methyl alcohol stopped reaction, cuvette is put into the fluorescence colorimetric pool, is 58lnm at excitation wavelength Ex, and emission wavelength Em measures relative intensity of fluorescence under the 593nm condition.Quantitative with typical curve.
Described according to the active soil total arsenic content that calculates of MROD, be specially: at first set up the relational expression of arsenic in soil content and MROD activity, after this can calculate arsenic in soil content according to enzymatic activity, thus the arsenic contamination degree of monitoring soil.
The present invention monitors the arsenic in soil pollution situation by the mensuration of MROD activity in the earthworm body, both can understand the arsenic in soil pollution level, can understand the biochemical effect of arsenic contamination again, and biosome is to the reaction of arsenic contamination.Have more practical value than directly measuring arsenic in soil content, and highly sensitive, be suitable for long term monitoring.
Description of drawings
Fig. 1 is the inventive method process flow diagram
Embodiment
As shown in Figure 1, embodiment of the invention implementation step is as follows:
1. at first understand the background information in monitoring place, determine the fluid discharge or the ight soil fertilising situation of pollution source and plant.Determine the concrete grammar of sampling then.
2. gather the soil of sampled point earlier, in the container of packing into; Then the earthworm of extraction is put into this container, take back the laboratory.
3. the earthworm in the container is taken out, be placed on the wet filter paper, at room temperature placed 1 to 2 day, to discharge intestinal contents; Get the earthworm after the gut purge, use the filter paper suck dry moisture, place glass homogenizer, pour the earthworm homogenate buffer of precooling in 4 ℃ of refrigerators into, homogenate; Homogenate is transferred in the 1.5ml centrifuge tube centrifugal 30min.Discard precipitation, supernatant is the required extract that contains P450.
4. in the fluorescence cuvette, add each reacted constituent, make various concentration of reactants reach Tris100mmol/L respectively, NADPH 0.5mmol/L, the about 10 μ mol/L of 7-methoxyl Yi Fen azolactone, the sample extracting solution that contains P450 of about 0.5mg albumen.NADPH adds at last during reaction, adds afterreaction and begins immediately.After accurately carrying out 10min, adding 0.5ml methyl alcohol stopped reaction, cuvette is put into the fluorescence colorimetric pool, is 58lnm at excitation wavelength Ex, and emission wavelength Em measures relative intensity of fluorescence under the 593nm condition.Quantitative with typical curve.
5. at first set up the relational expression of arsenic in soil content and MROD activity, after this can calculate arsenic in soil content according to enzymatic activity, thus the arsenic contamination degree of monitoring soil.
Providing embodiment below in conjunction with content of the present invention, is example with the vegetable plot of the residual pig manure of chronic administration arsenical around certain pig farm, monitoring arsenic contamination situation wherein.
The long-term planting vegetable in vegetable plot, edible for the pig farm employee.Contain the arsenic adjuvant in the feed on this pig farm, fertilizer is made with pig manure in the vegetable plot.According to the foursquare shape of being approximately of vegetable plot, designed 1 point in center, on every side 4 points totally 5 sampled points.Gather earthworm (being white neck Pheretimatschiliensis) wherein.Take back the activity of MROD in its body of experimental determination.Before this in the laboratory with identical quality uncontaminated soil, carry out artificial contamination experiment, the relational expression of the interior MROD activity of earth total arsenic content and earthworm body: A in foundations
Earthworm=-0.038C
Soil+ 10.37 C wherein
SoilBe soil total arsenic content, A
EarthwormBe the activity of MROD in the earthworm body, this formula is applicable to C
SoilIn the scope of 20mg/kg～200mg/kg.The mean value of this enzymatic activity is 9.24 ± 0.85p mol/mgprotein.min (activity of interior this enzyme of earthworm body is 48.63 ± 1.11p mol/mgprotein.min in the not comtaminated soil) in the sample earthworm body of vegetable plot, then the soil total arsenic content be 28.74mg/kg. and away from the pig farm be not subjected to that total arsenic content is 8.75mg/kg in the Luo Kesha arsine contaminated soil, illustrate that vegetable field soil has been subjected to being higher than the arsenic contamination of 3 to 4 times of original self content.This result shows that simultaneously under the coercing of arsenic contamination, the P450 enzyme in the earthworm body has been subjected to inhibition, and inhibiting rate reaches 81%.This might cause the change of earthworm to other metabolisms.Carry out relatively sensitivity of long term monitoring with this method.
Claims (4)
1. the biomarker monitoring method of an environment As pollution is characterized in that: at first according to the place that will monitor, design the soil sampling scheme; Gather earthworm at each sampled point then, it is packed into contain in the container of sampling site soil; Get adopt earthworm, carry out gut purge earlier, homogenate then, centrifugal, supernatant be the required extract that contains P450; In fluorospectrophotometer, measure the activity of 7-methoxyl-3-Yi Fen azolactone-O-debenzylation enzyme; According to its active soil total arsenic content that calculates, thus the arsenic contamination degree of monitoring soil.
2. the biomarker monitoring method of environment As pollution according to claim 1, it is characterized in that, the place that described basis will be monitored, design soil sampling scheme, be specially: the background information of at first understanding the monitoring place, determine the fluid discharge or the ight soil fertilising situation of pollution source and plant, determine the concrete grammar of sampling then: when polluting, arranging sampled point along the different distance of polluting direction for point-source pollution; When polluting, evenly arrange sampled point for pollution of area source.
3. the biomarker monitoring method of environment As pollution according to claim 1, it is characterized in that, the activity of described mensuration 7-methoxyl-3-Yi Fen azolactone-O-debenzylation enzyme, be specially: in the fluorescence cuvette, add each reacted constituent, concentration is respectively trishydroxymethylaminomethane 100mmol/L, niacinamide dinucleotide phosphoric acid 0.5mmol/L, 7-methoxyl Yi Fen azolactone 10 μ mol/L, 0.5mg the sample extracting solution that contains P450 of albumen, niacinamide dinucleotide phosphoric acid adds at last during reaction, adds afterreaction and begins immediately; After accurately carrying out 10min, adding 0.5ml methyl alcohol stopped reaction, cuvette is put into the fluorescence colorimetric pool, is 581nm at excitation wavelength Ex, and emission wavelength Em measures relative intensity of fluorescence under the 593nm condition, and is quantitative with typical curve.
4. the biomarker monitoring method of environment As pollution according to claim 1, it is characterized in that, described according to 7-methoxyl-3-Yi Fen azolactone-O-debenzylation enzymatic activity calculating soil total arsenic content, be specially: at first set up arsenic in soil content and 7-methoxyl-3-Yi Fen azolactone-O-debenzylation activity relationship formula, calculate arsenic in soil content according to enzymatic activity again, thus the arsenic contamination degree of monitoring soil.
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| CN105866231B (en) * | 2015-01-19 | 2019-07-30 | 上海市食品药品检验所 | The method for measuring total amount arsenic and valence state arsenic content in living organism tissue and internal organs |
| CN106248897A (en) * | 2016-07-12 | 2016-12-21 | 广西大学 | The method utilizing the CAT Activity determination degree of water pollution of fish |
| CN109609469A (en) * | 2018-11-26 | 2019-04-12 | 浙江海洋大学 | A kind of novel sea biological pollution detection marker |
| CN110221041B (en) * | 2019-06-06 | 2022-01-21 | 生态环境部南京环境科学研究所 | Method for detecting heavy metal contaminated soil ecotoxicity by using luminous earthworms |
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Non-Patent Citations (4)
| Title |
|---|
| 2种含砷兽药对蚯蚓的急性毒性试验. 李银生等.中国兽医学报，第25卷第4期. 2005 |
| 2种含砷兽药对蚯蚓的急性毒性试验. 李银生等.中国兽医学报，第25卷第4期. 2005 * |
| 火焰原子吸收法测定蚯蚓中的微量元素. 叶兴福等.分析测试技术与仪器，第3卷第2期. 1997 |
| 火焰原子吸收法测定蚯蚓中的微量元素. 叶兴福等.分析测试技术与仪器，第3卷第2期. 1997 * |
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