CN109580913A - Organic phosphorus, pyrethroid insecticides residual contamination soil ecology toxicity measuring method - Google Patents
Organic phosphorus, pyrethroid insecticides residual contamination soil ecology toxicity measuring method Download PDFInfo
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- CN109580913A CN109580913A CN201910015195.XA CN201910015195A CN109580913A CN 109580913 A CN109580913 A CN 109580913A CN 201910015195 A CN201910015195 A CN 201910015195A CN 109580913 A CN109580913 A CN 109580913A
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Abstract
The invention belongs to field of environment protection fields, it is specially a kind of organic phosphorus, the measuring method of pyrethroid insecticides residual contamination soil ecology toxicity, it is characterized in that this method chooses Eisenia Foetida, it will carry out exposing experiment in Eisenia Foetida soil to be measured after raising and train, through protein extraction, SDS-PAGE protein isolate and Western Blotting test and analyze purity step, if test group is with control group, there are significant difference p < 0.05, show that pesticide residue has toxicity to geobiont growth in testing soil, significant difference is such as not present, then testing soil does not have embryotoxicity.This method is suitble to the early diagnosis and early warning of contaminated soil, and greatly shortens the minute of eco-toxicity, effectively improves the diagnosis efficiency of soil ecology toxicity.
Description
Technical field
The invention belongs to field of environment protection field, specially a kind of organic phosphorus, pyrethroid insecticides residual dirt
Contaminate the measuring method of soil ecology toxicity.
Background technique
Organic phosphorus, pyrethroid insecticides are the most commonly used insecticide type pesticide in China, these two types of agricultures
Medicine is mostly nonpolar insecticide, has very strong bio-toxicity, easily by adsorption by soil and fixation.Organic phosphorus, pyrethroid
A large amount of uses of insecticide, are easy to cause Pesticide-Polluted Soil to pollute, and directly threaten human food healthy and safe and soil ecology
The sustainable development of function.
Molecular labeling is one kind of biomarker, can be as the early signal of hazard of contaminant effect, to pollutional condition
Effect more than lower individual level and individual level is predicted and is forecast.Heat shock protein molecular labeling is as biomarker
Main advantage is quickly and effectively integrate all negative effects and be embodied in the integrality of albumen, i.e. " albumen poison
Property ", to hazard of contaminant effect sensibility with higher.Heat shock protein is that organism answers in physics, chemistry and microorganism etc.
Swash under effect, starts special gene-heat shock gene and specific proteins very conservative in one group of structure generating, extensively
It is present in the biology from protokaryon to eukaryon and mitochondria, chloroplaset and hydrogenosome.
Currently, focusing mostly in relation to the research using stress protein as biomarker in vertebrate, for no vertebra
The stress protein of animal is only just paid attention in the past few years as the research of biomarker, and focuses mostly on to aquatic ring
The research in border, terrestrial invertebrate are few by the stress protein expression report that poisonous substance mediates.
Publication No. 103215343A, entitled " springtail heat shock protein gene hsp70 is in soil environment quality monitoring
Using " patent application, the invention provides a kind of methods for soil environment quality monitoring, and this method is by springtail
F.candida is exposed in soil to be measured, the expression of hsp70 gene in the springtail F.candida after then detection exposes,
But hsp70 gene expression amount of this method in 48 hours most fast using springtail be the index of monitoring soil quality, time-consuming longer；
In addition this method is only applicable to the organophosphorus insecticide in detection soil, i.e. chlopyrifos ingredient, has no any document surface, springtail
Heat shock protein gene hsp70 organic phosphorous insecticide suitable for soil detects.
Summary of the invention
For existing pesticide residual contamination soil ecology toxicity detection method it is time-consuming it is longer, detected value is single
The problem of, the present invention proposes the measuring method of organic phosphorus pyrethroid insecticides residual contamination soil ecology toxicity.
Organic phosphorus, pyrethroid insecticides residual contamination soil ecology toxicity measuring method of the invention, passes through
Following manner is realized:
1) experiment earthworm is Eisenia Foetida (Eisenia foetida), and experiment earthworm is placed in uncontaminated soil and is carried out
It raises and train 7-10 days, selects three more than the monthly age, the long 5~6cm of body, the earthworm of 350~400mg of weight is experiment earthworm；
2) experiment earthworm is placed on and carries out raising and train 7-10 days in uncontaminated soil, selected three more than the monthly age, body grows 5~6cm, body
The earthworm of 350~400mg is weighed as experiment earthworm；
3) exposure test: placing a certain number of experiment earthworms in the incubator equipped with soil to be measured and carry out exposing experiment, with
The experiment earthworm of identical quantity is control group in incubator equipped with uncontaminated soil, and exposure duration is 4 hours；
4) protein extraction: after the earthworm of exposure test processing washes away remains of pesticide with high purity water, being added 5ml protein extract, into
Row ice bath milling and extracting, homogenate centrifugation 12000rpm, 4 DEG C of centrifugation 5min take supernatant；
5) SDS-PAGE protein isolate: it is as follows that the concentration of separation gel is selected as 14%, 10ml formula: A liquid: 4.7ml；B liquid:
2.5ml；High purity water: 2.8ml；It is as follows that the concentration of compression glue is selected as 5%, 4ml formula: high purity water: 2.3ml；A liquid:
0.67ml；C liquid: 1.0ml；A liquid, B liquid, the composition of C liquid are as follows:
A liquid: weighing 29.2g acrylamide and 0.8g methylene diacrylamide, after being dissolved in water, is settled to 100ml with water, is placed in ice
It is saved backup at 4 DEG C in case；
B liquid: 10% dodecyl sodium sulfonate of three (methylol) aminomethane hydrochlorides and 4ml of 75ml, 2mol/L, pH8.0 are measured
Sodium is settled to 100ml with water, is placed in refrigerator and saves backup at 4 DEG C；
C liquid: 10% dodecyl sodium sulfonate of three (methylol) aminomethane hydrochlorides and 4ml of 50ml, 1mol/L, pH6.8 are measured
Sodium is settled to 100ml with water, is placed in refrigerator and saves backup at 4 DEG C；
The protein extract of step 3) is heated into 5min in 100 DEG C of boiling water baths, is centrifuged loading after 1min under 13000rpm, electric current is set
It sets in 180V, bromophenol blue forward position is improved into voltage after separation gel to 200V, is separated by electrophoresis to Bromophenol Blue dye and reaches separation
Power supply is closed in glue bottom.Gel is taken out, is dyed using coomassie brilliant blue R_250, stained over night is vibrated on shaking table, pour out dye
Gel is immersed in destainer and is decolourized by color liquid；
6) Western Blotting is detected: Whatman filter paper and NC film is cut by the gel size of step 4), after cutting
Whatman filter paper and NC film are respectively put into transfer buffer and balance 10~15min and 30min immersion balance.By gel size
Whatman filter paper is cut, is slowly put at 45 degree of angles in transfer buffer and balances 10~15min.By gel size but each side ratio
The big 1mm of gel cuts NC film, and cuts Yi little Jiao in the lower right corner of NC film, and to make sequence notation, the NC film after label is at 45 degree of angles
It is slowly put into transfer buffer and impregnates balance 30min；
From top to bottom successively by the Whatman filter paper of the big 2mm of length-width ratio gel, NC film, gel, length-width ratio gel small 1mm
Whatman filter paper sequence neatly stacks, and prepares transfer membrane；The transfer membrane made is transferred in half-dried transfer groove, voltage is set
For pressure stabilizing 20V, transfer time is that 50min carries out transferring film；NC film after transfer is shaken into cleaning with TBS-T liquid on constant-temperature table
3 times, each 15min, later with 10% Bovine serum albumin-TBS-T at 4 DEG C soaked overnight, cleaned and blocked,
Liquid for plugging out, with TBS-T liquid shaking table cleaning 3 times after, NC film is put into primary antibody working solution, in 37 DEG C of incubation 2h of constant-temperature table into
Row primary antibody is incubated for, then is cleaned NC film with TBS-T liquid and be put into the secondary antibody working solution of 1:1000 after cleaning removes unbonded primary antibody
In, in 37 DEG C of incubation 1.5h of constant-temperature table, colour developing working solution is put into after washing film 3 times, develop the color 10~30min, if no signal occurs
It can then continue to develop the color；It is terminated and is reacted with high-purity water washing NC film after colour developing, NC film uses scanner scanning after drying；Trace
As a result after scanning, using Total Version software purity assay.Data processing uses the One way of SPSS software
ANOVA is for statistical analysis, and test group and control group show pesticide residue pair in testing soil there are significant difference p < 0.05
Geobiont growth has toxicity, and significant difference is such as not present, then testing soil does not have embryotoxicity.
Earthworm is the representative monoid of soil fauna, is played an important role in soil ecosystem.From ecology
On from the point of view of, earthworm is in the front end of land ecologic food chain, using earthworm as soil environment instruction biology, one can be provided
The safe thresholding of a entire soil fauna of protection.Therefore earthworm is often used as a kind of important instruction biology in soil
The eco-toxicity of earth pollutant is tested.Since its growth, development and breeding environmental pollution are more sensitive, these indexs can be with
It is used for soil pollution diagnosis and ecological risk assessment as diagnosis terminal, but the measurement of these indexs generally to need longer test
Period, it is difficult to meet the requirement of the timely diagnosis and ecological risk assessment of soil pollution.
HSP27 is a kind of HSP that molecular weight is 27kD, and gene has heat shock regulating element (Heat in promoter region
Shock Regulatory Element, HSE) and stress associated regulatory elements (Stress-related Regulatory
Element, STRE), amino acid sequence has the N-terminal sequence similar with alpha-crystal albumen.HSP27 and other HSP mono-
Sample, composition can be divided into induction type and composition type.Wherein, the molding HSP of structure is generally expressed in the physiological state, point with cell
Change, develop it is closely related.Induction type HSP is mainly expressed under the stimulation of external environment, and important biological function is protection
Damage of the cell from various stress factors.Our experiment discovery, organic phosphorus, pyrethroid insecticides pesticide can induce
The expression of earthworm HSP27 has centainly indicative to the poisonous effect of the pesticide residue, but reaction mechanism is still not clear.
Using earthworm HSP27 as molecular labeling, to Organic phosphate, pyrethroid insecticides pesticide residual contaminations
Eco-toxicity measurement has not been reported.As can monitoring Pesticide Residue in Soil residual to earthworm using earthworm HSP27 as biomarker
The inducing expression amount of earthworm HSP27, can in time in qualitative evaluation soil organic phosphorus, pyrethroid insecticides pesticide residue to soil
The toxic effect of earth biology, will be latent with important application in terms of the monitoring of the ecological risk of Soil Contamination by Chemical Pesticides and safety evaluation
Power.
Compared with prior art, the present invention have following features and the utility model has the advantages that
1) HSP27 is a kind of molecular labeling of sensitivity, and low-dose polluted object present in environment can induce its synthesis, than individual
Horizontal more sensitive, the present invention is suitble to the early diagnosis and early warning of contaminated soil；
2) present invention detection is time-consuming short, with the common LC of soil pollutant ecotoxicity assay50, the indexs such as increment compare, earthworm
Earthworm HSP27 is issuable to earthworm acute (lethal), chronic (raw with regard to organic phosphorus, pyrethroid insecticides pesticide residue
It is long) toxicity instruction has the characteristics that sensitive, quick, it tests after pesticide stress handle 4 hours, so that it may detect earthworm HSP27's
Unconventionality expression, and LCso, the common Testing index such as increment, detection time is up to 14d and 56d or even longer.Using this hair
Bright measuring method can greatly shorten the minute of eco-toxicity, effectively improve the diagnosis efficiency of soil ecology toxicity.
3) the invention belongs to long term toxicity tests, time-consuming short.
Specific embodiment
Example given below is quasi- with the invention will be further described, but is not to be construed as protecting model to the present invention
It encloses.
Embodiment 1: application of the earthworm HSP27 in the monitoring of organic phosphorus chlopyrifos soil pollution eco-toxicity is broadly divided into
Three steps, it may be assumed that the exposure of earthworm；The extraction of earthworm HSP27 separates；The detection of HSP27 expression quantity.Resulting result such as table 1
Shown, HSP27 abnormal expression value is 20mg/kg.
The expression of table 1 chlopyrifos pesticides various dose processing group and control group earthworm HSP27
There are significant differences with control group for a: α=0.05 horizontal lower processing group
Using existing earthworm embryotoxicity measuring method, chlopyrifos soil pollution is measured to the embryotoxicity of earthworm, side
For method referring to Khalil et al. (1996), test result is shown in Table 2.Table 2 the result shows that, the dead tick pesticide concentration of soil poisoning reaches
To 20.0mg/kg or more, there are significant difference, it is right in soil to show for the growth of earthworm and control group earthworm in processing group of contaminating
The minimum effective concentration that there is earthworm embryotoxicity to influence is 20.0mg/kg.
The dead tick pesticide of 2 soil poisoning of table tests the embryotoxicity of earthworm
: P < 0.05, there are significant differences with control group for processing group
In conjunction with table 1, table 2 result from the point of view of, chlopyrifos pesticides are to the inducing expression amount of earthworm HSP27 and earthworm embryotoxicity
It is consistent to analyze result, shows that the inducing expression of earthworm HSP27 and chlopyrifos have the issuable embryotoxicity effect of earthworm
Preferable correlation qualitative can reflect the dead tick pesticide residue of soil poisoning to the embryotoxicity of earthworm.
Embodiment 2: earthworm HSP27 is in the monitoring of pyrethroid insecticides cypermethrin soil pollution eco-toxicity
Using being broadly divided into three steps, it may be assumed that the exposure of earthworm；The extraction of earthworm HSP27 separates；The detection of HSP27 expression quantity.Institute
The results are shown in Table 1, HSP27 abnormal expression value be 40mg/kg.
The expression of table 3 cypermethrin pesticide various dose processing group and control group earthworm HSP27
There are significant differences with control group for a: α -0.05 horizontal lower processing group
Using existing earthworm embryotoxicity measuring method, cypermethrin soil pollution is measured to the embryotoxicity of earthworm,
For method referring to Khalil et al. (1996), test result is shown in Table 4.Table 4 the result shows that, cypermethrin pesticide is dense in soil
Degree reaches 40.0mg/kg or more, and there are significant differences for the growth of earthworm and control group earthworm in processing group of contaminating, and shows in soil
It is 40.0mg/kg on the minimum effective concentration that there is earthworm embryotoxicity to influence.
Embryotoxicity real-turn of the dead tick pesticide of 4 soil poisoning of table to earthworm
In conjunction with table 3, table 4 result from the point of view of, chlopyrifos pesticides are to the inducing expression amount of earthworm HSP27 and earthworm embryotoxicity
Analysis result is consistent, shows that the inducing expression of earthworm HSP27 and cypermethrin have the issuable embryotoxicity effect of earthworm
Have preferable correlation, can in qualitative reflection soil cypermethrin pesticide residue to the embryotoxicity of earthworm, with the application of the invention,
The diagnosis efficiency of geobiont toxicity can effectively be promoted.
Claims (1)
1. the measuring method of organic phosphorus pyrethroid insecticides residual contamination soil ecology toxicity, it is characterised in that the party
Method is accomplished by the following way:
1) experiment earthworm is Eisenia Foetida, and experiment earthworm is placed on and carries out raising and train 7-10 days in uncontaminated soil, selects three
It is more than the monthly age, the earthworm of body long 5~6cm, 350~400mg of weight be experiment earthworm；
2) experiment earthworm is placed on and carries out raising and train 7-10 days in uncontaminated soil, selected three more than the monthly age, body grows 5~6cm, body
The earthworm of 350~400mg is weighed as experiment earthworm；
3) exposure test: placing a certain number of experiment earthworms in the incubator equipped with soil to be measured and carry out exposing experiment, with
The experiment earthworm of identical quantity is control group in incubator equipped with uncontaminated soil, and exposure duration is 4 hours；
4) protein extraction: after the earthworm of exposure test processing washes away remains of pesticide with high purity water, being added 5ml protein extract, into
Row ice bath milling and extracting, homogenate centrifugation 12000rpm, 4 DEG C of centrifugation 5min take supernatant；
5) SDS-PAGE protein isolate: it is as follows that the concentration of separation gel is selected as 14%, 10ml formula: A liquid: 4.7ml；B liquid:
2.5ml；High purity water: 2.8ml；It is as follows that the concentration of compression glue is selected as 5%, 4ml formula: high purity water: 2.3ml；A liquid:
0.67ml；C liquid: 1.0ml；A liquid, B liquid, the composition of C liquid are as follows:
A liquid: weighing 29.2g acrylamide and 0.8g methylene diacrylamide, after being dissolved in water, is settled to 100ml with water, is placed in ice
It is saved backup at 4 DEG C in case；
B liquid: 10% dodecyl sodium sulfonate of three (methylol) aminomethane hydrochlorides and 4ml of 75ml, 2mol/L, pH8.0 are measured
Sodium is settled to 100ml with water, is placed in refrigerator and saves backup at 4 DEG C；
C liquid: 10% dodecyl sodium sulfonate of three (methylol) aminomethane hydrochlorides and 4ml of 50ml, 1mol/L, pH6.8 are measured
Sodium is settled to 100ml with water, is placed in refrigerator and saves backup at 4 DEG C；
The protein extract of step 3) is heated into 5min in 100 DEG C of boiling water baths, is centrifuged loading after 1min under 13000rpm, electric current is set
It sets in 180V, bromophenol blue forward position is improved into voltage after separation gel to 200V, is separated by electrophoresis to Bromophenol Blue dye and reaches separation
Power supply is closed in glue bottom；Gel is taken out, is dyed using coomassie brilliant blue R_250, stained over night is vibrated on shaking table, pour out dye
Gel is immersed in destainer and is decolourized by color liquid；
6) Western Blotting is detected: Whatman filter paper and NC film is cut by the gel size of step 4), after cutting
Whatman filter paper and NC film are respectively put into transfer buffer and balance 10 ~ 15min and 30min immersion balance；By gel size
Whatman filter paper is cut, is slowly put at 45 degree of angles in transfer buffer and balances 10 ~ 15min；NC film, NC are cut by gel size
Film, which is slowly put into transfer buffer at 45 degree of angles, impregnates balance 30min；From top to bottom successively by Whatman filter paper, NC film, solidifying
Glue, Whatman filter paper sequence neatly stack, and prepare transfer membrane；The transfer membrane made is transferred in half-dried transfer groove, setting electricity
Pressure is pressure stabilizing 20V, and transfer time is that 50min carries out transferring film；NC film after transfer is shaken clearly with TBS-T liquid on constant-temperature table
Wash 3 times, each 15min, later with 10% Bovine serum albumin-TBS-T at 4 DEG C soaked overnight, cleaned and blocked,
Liquid for plugging is poured out, after TBS-T liquid shaking table cleaning 3 times, NC film is put into primary antibody working solution, in 37 DEG C of incubation 2h of constant-temperature table
Primary antibody incubation is carried out, then NC film is cleaned with TBS-T liquid, after cleaning removes unbonded primary antibody, is put into the secondary antibody work of 1:1000
In liquid, in 37 DEG C of incubation 1.5h of constant-temperature table, colour developing working solution is put into after washing film 3 times, develop the color 10 ~ 30min, if no signal occurs
It can then continue to develop the color；It is terminated and is reacted with high-purity water washing NC film after colour developing, NC film uses scanner scanning after drying；Trace knot
Fruit scans post analysis purity；If test group and control group show pesticide residue pair in testing soil there are significant difference p < 0.05
Geobiont growth has toxicity, and significant difference is such as not present, then testing soil does not have embryotoxicity.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111596048A (en) * | 2020-04-07 | 2020-08-28 | 浙江省农业科学院 | Application method of two insecticides |
| CN112326616A (en) * | 2020-11-02 | 2021-02-05 | 云南大学 | Method for detecting heavy metal contaminated soil ecotoxicity by using luminous earthworms |
| CN112946242A (en) * | 2021-02-03 | 2021-06-11 | 生态环境部南京环境科学研究所 | Method and device for diagnosing ecological toxicity of solid waste soil in pesticide production field |
-
2019
- 2019-01-08 CN CN201910015195.XA patent/CN109580913A/en active Pending
Non-Patent Citations (12)
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111596048A (en) * | 2020-04-07 | 2020-08-28 | 浙江省农业科学院 | Application method of two insecticides |
| CN112326616A (en) * | 2020-11-02 | 2021-02-05 | 云南大学 | Method for detecting heavy metal contaminated soil ecotoxicity by using luminous earthworms |
| CN112946242A (en) * | 2021-02-03 | 2021-06-11 | 生态环境部南京环境科学研究所 | Method and device for diagnosing ecological toxicity of solid waste soil in pesticide production field |
| US11383233B1 (en) | 2021-02-03 | 2022-07-12 | Nanjing Institute Of Environmental Sciences, Mee | Diagnosis method and diagnosis device of ecotoxicity of solid waste soil in pesticide production site |
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Application publication date: 20190405 |