CN112268966A - Isotope analysis method for pyrethroid microbial degradation in soil - Google Patents
Isotope analysis method for pyrethroid microbial degradation in soil Download PDFInfo
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- 239000002689 soil Substances 0.000 title claims abstract description 51
- 230000000813 microbial effect Effects 0.000 title claims abstract description 27
- 238000004458 analytical method Methods 0.000 title claims abstract description 26
- 230000015556 catabolic process Effects 0.000 title claims abstract description 25
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 25
- 239000002728 pyrethroid Substances 0.000 title claims abstract description 16
- 239000000203 mixture Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000002156 mixing Methods 0.000 claims abstract description 11
- 238000006065 biodegradation reaction Methods 0.000 claims abstract description 10
- 239000004698 Polyethylene Substances 0.000 claims abstract description 8
- 239000002985 plastic film Substances 0.000 claims abstract description 8
- 229920006255 plastic film Polymers 0.000 claims abstract description 8
- -1 polyethylene Polymers 0.000 claims abstract description 8
- 229920000573 polyethylene Polymers 0.000 claims abstract description 8
- 238000007605 air drying Methods 0.000 claims abstract description 4
- 239000012535 impurity Substances 0.000 claims abstract description 4
- 238000007873 sieving Methods 0.000 claims abstract description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 63
- KAATUXNTWXVJKI-UHFFFAOYSA-N cypermethrin Chemical compound CC1(C)C(C=C(Cl)Cl)C1C(=O)OC(C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 KAATUXNTWXVJKI-UHFFFAOYSA-N 0.000 claims description 45
- 229910052799 carbon Inorganic materials 0.000 claims description 24
- 238000002347 injection Methods 0.000 claims description 24
- 239000007924 injection Substances 0.000 claims description 24
- 239000012086 standard solution Substances 0.000 claims description 24
- 230000000155 isotopic effect Effects 0.000 claims description 22
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 21
- 239000003480 eluent Substances 0.000 claims description 18
- 238000000944 Soxhlet extraction Methods 0.000 claims description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- 238000000605 extraction Methods 0.000 claims description 12
- 238000005303 weighing Methods 0.000 claims description 10
- 241001411320 Eriogonum inflatum Species 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 9
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 claims description 9
- 150000002500 ions Chemical class 0.000 claims description 9
- 239000008399 tap water Substances 0.000 claims description 9
- 235000020679 tap water Nutrition 0.000 claims description 9
- 238000005507 spraying Methods 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 229920000742 Cotton Polymers 0.000 claims description 6
- 239000005946 Cypermethrin Substances 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 6
- 239000012159 carrier gas Substances 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 6
- 239000003365 glass fiber Substances 0.000 claims description 6
- 239000001307 helium Substances 0.000 claims description 6
- 229910052734 helium Inorganic materials 0.000 claims description 6
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 6
- 238000002307 isotope ratio mass spectrometry Methods 0.000 claims description 6
- 238000001819 mass spectrum Methods 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 6
- BKIMMITUMNQMOS-UHFFFAOYSA-N nonane Chemical compound CCCCCCCCC BKIMMITUMNQMOS-UHFFFAOYSA-N 0.000 claims description 6
- 239000010453 quartz Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 4
- KAATUXNTWXVJKI-NSHGMRRFSA-N (1R)-cis-(alphaS)-cypermethrin Chemical compound CC1(C)[C@@H](C=C(Cl)Cl)[C@H]1C(=O)O[C@H](C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 KAATUXNTWXVJKI-NSHGMRRFSA-N 0.000 claims description 3
- 239000005877 Alpha-Cypermethrin Substances 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 claims description 3
- 238000007664 blowing Methods 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000002485 combustion reaction Methods 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 claims description 3
- 238000005194 fractionation Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- 238000010926 purge Methods 0.000 claims description 3
- 230000000630 rising effect Effects 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- FKHIFSZMMVMEQY-UHFFFAOYSA-N talc Chemical compound [Mg+2].[O-][Si]([O-])=O FKHIFSZMMVMEQY-UHFFFAOYSA-N 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 2
- 239000000575 pesticide Substances 0.000 abstract description 14
- 238000011160 research Methods 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000004451 qualitative analysis Methods 0.000 abstract 1
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- 239000007857 degradation product Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 238000011158 quantitative evaluation Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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Abstract
An isotope analysis method for pyrethroid biodegradation in soil comprises the following steps: firstly, a layer of polyethylene plastic film is padded on a laboratory table which is dry in the shade and well ventilated, a collected soil sample is placed on the polyethylene plastic film to be flattened, soil blocks are crushed, impurities such as stones, animal and plant residues and the like are removed, then the soil sample is paved into a thin layer, and the soil sample is frequently turned over to be naturally dried; and step two, after air drying, sieving by a sieve of 2mm, uniformly mixing, taking two parts of opposite angles by a quartering method, uniformly mixing, and taking two parts of opposite angles by the quartering method. The isotope analysis method for pyrethroid microbial degradation in soil can utilize stable isotope analysis technology to research the relation between isotope composition and biodegradation rate, and carry out qualitative and quantitative analysis on the microbial degradation of pesticide, thereby obtaining real and accurate microbial degradation data of pesticide.
Description
Technical Field
The invention relates to an isotope analysis method for pyrethroid microbial degradation in soil.
Background
In environmental research, quantitative evaluation of the microbial degradation of pesticides has been a research hotspot of scholars at home and abroad. The traditional method is to determine qualitative and quantitative indexes of microbial activity, such as reduction of substrate and electron acceptor concentration, increase of microbial biomass and degradation products, and the like, when evaluating the microbial degradation of pesticides in environment. However, these concentration-based indicators do not truly reflect the microbial degradation of pesticides, and natural processes such as volatilization, dilution, adsorption and the like also cause the reduction of pesticide concentration, while the pesticide itself is not degraded. In addition, the material balance of the substrate, electron acceptor and degradation product is difficult to obtain accurately in a complex natural environment system.
Disclosure of Invention
The invention aims to solve the technical problem of providing an isotope analysis method for the microbial degradation of pyrethroid in soil, which can research the relation between isotope composition and biodegradation rate by using a stable isotope analysis technology and qualitatively and quantitatively analyze the microbial degradation of pesticide so as to obtain real and accurate microbial degradation data of pesticide.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the invention discloses an isotope analysis method for pyrethroid microbial degradation in soil, which is characterized by comprising the following steps: firstly, a layer of polyethylene plastic film is padded on a laboratory table which is dry in the shade and well ventilated, a collected soil sample is placed on the polyethylene plastic film to be flattened, soil blocks are crushed, impurities such as stones, animal and plant residues and the like are removed, then the soil sample is paved into a thin layer, and the soil sample is frequently turned over to be naturally dried;
step two, after air drying, sieving the mixture by a sieve of 2mm, uniformly mixing the mixture, taking two opposite angles by a quartering method, uniformly mixing the two opposite angles, taking two opposite angles by the quartering method, wherein one part is used for an experiment, and the other part is packed by a sample bag and then stored in a dark and dry place;
thirdly, weighing 0.01000 +/-0.00005 g of a cis-cypermethrin solid standard sample by using a trace electronic balance, dissolving the cis-cypermethrin solid standard sample in n-hexane, carrying out constant volume in a 100mL volumetric flask to prepare a 100mg/L cis-cypermethrin standard solution, shaking the solution uniformly for later use, respectively weighing 10mL and 50mL cis-cypermethrin standard solutions, diluting the solution by using n-hexane in a 100mL volumetric flask to realize constant volume, and respectively preparing 10mg/L and 50mg/L cis-cypermethrin standard solutions;
fourthly, diluting the cis-cypermethrin standard solution with the concentration of 100mg/L by normal hexane according to a certain gradient, respectively preparing the cis-cypermethrin standard solutions with the concentrations of 500 mug/L, 250 mug/L, 100 mug/L, 50 mug/L, 25 mug/L and 10 mug/L, analyzing and determining by GC-MS, and drawing a standard curve;
fifthly, taking the cis-cypermethrin standard solution with the concentration of 50mg/L, analyzing by GC-C-IRMS, and determining the carbon isotope composition (delta)0 13C)；
Sixth, measure (δ)0 13C) Then, calculating the isotopic enrichment factor epsilon;
seventhly, combining the isotope enrichment factor epsilon, the biodegradation rate (B) of the alpha-cypermethrin in the environment can be obtained through a formula
And (6) performing calculation.
In quantitative isotope fractionation, Rayleigh model formula is generally adopted
In the formula Rt，R0And Ct，C0The isotopic ratio and the concentration of the compound at time t and at the beginning of the reaction, respectively, epsilon is an isotopic enrichment factor which relates the isotopic ratio variation to the concentration variation, the stable carbon isotopic ratio for natural abundance (R ═ c)13C/12C) It can be assumed that R +1 ≈ 1, and thus the above formula can be simplified to
The simplified Rayleigh equation
Taking logarithm to obtain a carbon isotope enrichment factor epsilon;finally, ln (C)t/C0) For ln [ (delta)t13C+1)/(δ013C+1)]And (3) plotting, as shown in the attached figure 1 of the specification, wherein the slope of the straight line after linear fitting is the carbon isotope enrichment factor epsilon.
Carbon isotope composition (delta)0 13C) Can be obtained by the activation of soil microorganisms and the analysis of a control setting mode; when the soil microorganism activation and contrast setting mode is used, firstly, 96 50mL conical flasks (with rubber stoppers) are taken, 5.0000 +/-0.0250 g of soil sample is weighed by an analytical electronic balance and respectively added into the conical flasks and flattened at the bottom of the flasks, 3mL of tap water is uniformly sprayed into each conical flask by a 1mL liquid-transferring gun, and the total mass of each conical flask is weighed and recorded after the rubber stoppers are plugged;
then, placing the conical flask into a constant temperature shaking table, oscillating for 30min at 30 ℃ and 120r/min, then placing the conical flask into a constant temperature incubator, and culturing for 2 weeks at 30 ℃ in a dark place;
then, taking out the conical flasks from the constant-temperature incubator, dividing the conical flasks into A, B groups, placing the group A in an autoclave, sterilizing the conical flasks at 121 ℃ for 3 times for 20min each time, dividing the A, B group into A1 group, A2 group, B1 group and B2 group, respectively, uniformly spraying 1mL of a cis-cypermethrin standard solution with the concentration of 10mg/L into each of the A1 bottle and the B1 bottle by using a 1mL liquid transfer gun, and uniformly spraying 1mL of a cis-cypermethrin standard solution with the concentration of 50mg/L into the A2 bottle and the B2 bottle;
and then, placing the conical flask in a constant-temperature shaking table, oscillating for 10min at the temperature of 30 ℃ and at the speed of 120r/min to ensure that the cis-cypermethrin is uniformly distributed in the soil sample, loosening the bottle stopper to ensure that n-hexane is volatilized completely, then plugging the bottle stopper, weighing A, B groups, respectively adding sterilized tap water and non-sterilized tap water, and then plugging the bottle stopper. Then placing into a constant temperature incubator, and culturing at 30 ℃ in a dark place;
finally, three samples each of A1, A2, B1 and B2 were taken from the incubator every 10 days for analysis of the concentration of cis-cypermethrin therein (C)t) And carbon isotope composition (delta)0 13C)。
Firstly, selecting a Soxhlet extraction method to extract cis-cypermethrin in soil, connecting a Soxhlet extraction device, using n-hexane to soxhlet and wash a glass fiber filter cartridge and cotton for 8 hours, then changing the n-hexane into 160mL of Soxhlet extraction solution (n-hexane: acetone/v: 7:1), adding a proper amount of anhydrous sodium sulfate into a conical flask, uniformly stirring the mixture with a soil sample, then completely transferring the mixture into the glass fiber filter cartridge, plugging the cotton, connecting the Soxhlet extraction device, transferring the extraction solution in a soxhlet tube and the filter cartridge into a flat-bottomed flask after Soxhlet extraction for 24 hours, concentrating the extraction solution by using a rotary evaporator at the water bath temperature of 45 ℃, the rotation speed of 75r/min and the initial pressure of 450hPa, gradually reducing the pressure along with the reduction of the extraction solution, and finally concentrating the extraction solution to 1-2 mL under the pressure of 360 hPa;
then, the column was packed with n-hexane by a wet method, and a glass chromatography column was packed with a packing material comprising, from bottom to top, 5g of anhydrous sodium sulfate, 5g of Florisil, 3.5g of silica gel, and 5g of anhydrous sodium sulfate. The concentrated extract was then transferred to a chromatography column, eluted with 100mL of an eluent (n-hexane: acetone/v: v ═ 9:1), and collected in a flat-bottomed flask. Concentrating the eluent after the column by using a rotary evaporator at the water bath temperature of 45 ℃, the rotating speed of 75r/min and the initial pressure of 400hPa, gradually reducing the pressure along with the continuous reduction of the eluent, and finally concentrating to 1-2 mL under the pressure of 360 hPa.
Then transferring the concentrated post-column eluent to a 10mL colorimetric tube with scales, adding 1 dropper of n-hexane into the flat-bottomed flask, fully purging the inner wall of the flat-bottomed flask by using the dropper, transferring to the colorimetric tube, repeating for 2 times, finally fixing the volume to 5mL, and uniformly mixing by using a vortex oscillator;
then, 200. mu.L (group A1, group B1) or 50. mu.L (group A2, group B2) of 200. mu.L pipette is respectively taken from the colorimetric cylinder and added into a sample injection bottle, 800. mu.L or 950. mu.L of n-hexane is respectively added into 1mL pipette to be constant volume to 1mL, and the concentration of the cis-cypermethrin (C) is analyzed by GC-MSt)；
Finally, the eluent after the residual column in the colorimetric tube is blown to be nearly dry in a nitrogen blowing device, 100 mu L of n-nonane is added, then the eluent is completely transferred to a 150 mu L glass lining tube embedded in a sample injection bottle, and GC-C-IRMS is carried out to analyze the isotope composition (delta) of the cis-cypermethrin0 13C)。
Concentration of cis-cypermethrin (C)t) Using Agilent 7890A gas chromatographThe detection is carried out by combining Agilent 5975C mass spectrum, a chromatographic column is an HP-5ms quartz capillary column (30m multiplied by 0.25mm i.d. multiplied by 0.25 mu m), the injection inlet temperature is 260 ℃, the injection amount is 1 mu L, the injection mode is non-shunting, high-purity helium (99.999%) is used as carrier gas, and the flow rate is 1.2 mL/min; the temperature rising procedure of the chromatographic column is as follows: the initial temperature is 100 ℃, the temperature is kept for 4min, the temperature is increased to 200 ℃ at the speed of 25 ℃/min, the temperature is kept for 10min, the temperature is increased to 270 ℃ at the speed of 10 ℃/min, the temperature is kept for 5min, and finally the temperature is increased to 280 ℃ at the speed of 20 ℃/min, and the temperature is kept for 5 min; the ionization mode was EI mode (70eV) and the scan mode was selective ion scan (qualitative ion m/z 163,181,209, where 163 is a quantitative ion).
Carbon isotope composition (delta) of cis-cypermethrin0 13C) The measurement is carried out by an Agilent 7890A gas chromatograph through a GC5 combustion interface by using a GV Isoprotime isotope mass spectrum, the chromatographic column is an HP-5ms quartz capillary column (30m multiplied by 0.25mm i.d. multiplied by 0.25 mu m), the injection port temperature is 260 ℃, the injection amount is 2 mu L, the injection mode is non-split flow, high-purity helium (99.999%) is used as carrier gas, and the flow rate is 1.2 mL/min. The temperature procedure of the chromatographic column is the same as that of GC-MS; all samples were assayed in triplicate and the uncertainty of the assay was made less than 0.5% o.
The invention has the beneficial effects that:
compared with the prior art, the isotope analysis method for the microbial degradation of the pyrethroid in the soil can research the relation between the isotope composition and the biodegradation rate by using a stable isotope analysis technology, and qualitatively and quantitatively analyze the microbial degradation of the pesticide, thereby obtaining real and accurate microbial degradation data of the pesticide.
Compared with the prior art, the isotope analysis method for the microbial degradation of the pyrethroid in the soil can provide certain guidance for the follow-up monitoring and control of the pesticide residue in the soil.
Drawings
FIG. 1 is a Rayleigh logarithmic model diagram.
Detailed Description
The invention is described in further detail below with reference to the following figures and detailed description:
referring to fig. 1, the present invention provides an isotope analysis method for pyrethroid biodegradation in soil, which is characterized by comprising the following steps: firstly, a layer of polyethylene plastic film is padded on a laboratory table which is dry in the shade and well ventilated, a collected soil sample is placed on the polyethylene plastic film to be flattened, soil blocks are crushed, impurities such as stones, animal and plant residues and the like are removed, then the soil sample is paved into a thin layer, and the soil sample is frequently turned over to be naturally dried;
step two, after air drying, sieving the mixture by a sieve of 2mm, uniformly mixing the mixture, taking two opposite angles by a quartering method, uniformly mixing the two opposite angles, taking two opposite angles by the quartering method, wherein one part is used for an experiment, and the other part is packed by a sample bag and then stored in a dark and dry place;
thirdly, weighing 0.01000 +/-0.00005 g of a cis-cypermethrin solid standard sample by using a trace electronic balance, dissolving the cis-cypermethrin solid standard sample in n-hexane, carrying out constant volume in a 100mL volumetric flask to prepare a 100mg/L cis-cypermethrin standard solution, shaking the solution uniformly for later use, respectively weighing 10mL and 50mL cis-cypermethrin standard solutions, diluting the solution by using n-hexane in a 100mL volumetric flask to realize constant volume, and respectively preparing 10mg/L and 50mg/L cis-cypermethrin standard solutions;
fourthly, diluting the cis-cypermethrin standard solution with the concentration of 100mg/L by normal hexane according to a certain gradient, respectively preparing the cis-cypermethrin standard solutions with the concentrations of 500 mug/L, 250 mug/L, 100 mug/L, 50 mug/L, 25 mug/L and 10 mug/L, analyzing and determining by GC-MS, and drawing a standard curve;
fifthly, taking the cis-cypermethrin standard solution with the concentration of 50mg/L, analyzing by GC-C-IRMS, and determining the carbon isotope composition (delta)0 13C)；
Sixth, measure (δ)0 13C) Then, calculating the isotopic enrichment factor epsilon;
seventhly, combining the isotope enrichment factor epsilon, the biodegradation rate (B) of the alpha-cypermethrin in the environment can be obtained through a formula
Calculation was carried out by isotopic composition(δ0 13C) After the accurate biodegradation rate is obtained, the microbial degradation of the pesticide can be accurately qualitatively and quantitatively analyzed, so that real and accurate microbial degradation data of the pesticide can be obtained.
In quantitative isotope fractionation, Rayleigh model formula is generally adopted
In the formula Rt，R0And Ct，C0The isotopic ratio and the concentration of the compound at time t and at the beginning of the reaction, respectively, epsilon is an isotopic enrichment factor which relates the isotopic ratio variation to the concentration variation, the stable carbon isotopic ratio for natural abundance (R ═ c)13C/12C) It can be assumed that R +1 ≈ 1, and thus the above formula can be simplified to
The simplified Rayleigh equation
Taking logarithm to obtain a carbon isotope enrichment factor epsilon; finally, ln (C)t/C0) For ln [ (delta)t13C+1)/(δ013C+1)]And (3) plotting, as shown in the attached figure 1 of the specification, wherein the slope of the straight line after linear fitting is the carbon isotope enrichment factor epsilon.
Carbon isotope composition (delta)0 13C) Can be obtained by the activation of soil microorganisms and the analysis of a control setting mode; when the soil microorganism activation and contrast setting mode is used, firstly, 96 50mL conical flasks (with rubber stoppers) are taken, 5.0000 +/-0.0250 g of soil sample is weighed by an analytical electronic balance and respectively added into the conical flasks and flattened at the bottom of the flasks, 3mL of tap water is uniformly sprayed into each conical flask by a 1mL liquid-transferring gun, and the total mass of each conical flask is weighed and recorded after the rubber stoppers are plugged;
then, placing the conical flask into a constant temperature shaking table, oscillating for 30min at 30 ℃ and 120r/min, then placing the conical flask into a constant temperature incubator, and culturing for 2 weeks at 30 ℃ in a dark place;
then, taking out the conical flasks from the constant-temperature incubator, dividing the conical flasks into A, B groups, placing the group A in an autoclave, sterilizing the conical flasks at 121 ℃ for 3 times for 20min each time, dividing the A, B group into A1 group, A2 group, B1 group and B2 group, respectively, uniformly spraying 1mL of a cis-cypermethrin standard solution with the concentration of 10mg/L into each of the A1 bottle and the B1 bottle by using a 1mL liquid transfer gun, and uniformly spraying 1mL of a cis-cypermethrin standard solution with the concentration of 50mg/L into the A2 bottle and the B2 bottle;
and then, placing the conical flask in a constant-temperature shaking table, oscillating for 10min at the temperature of 30 ℃ and at the speed of 120r/min to ensure that the cis-cypermethrin is uniformly distributed in the soil sample, loosening the bottle stopper to ensure that n-hexane is volatilized completely, then plugging the bottle stopper, weighing A, B groups, respectively adding sterilized tap water and non-sterilized tap water, and then plugging the bottle stopper. Then placing into a constant temperature incubator, and culturing at 30 ℃ in a dark place;
finally, three samples each of A1, A2, B1 and B2 were taken from the incubator every 10 days for analysis of the concentration of cis-cypermethrin therein (C)t) And carbon isotope composition (delta)0 13C)。
Firstly, selecting a Soxhlet extraction method to extract cis-cypermethrin in soil, connecting a Soxhlet extraction device, using n-hexane to soxhlet and wash a glass fiber filter cartridge and cotton for 8 hours, then changing the n-hexane into 160mL of Soxhlet extraction solution (n-hexane: acetone/v: 7:1), adding a proper amount of anhydrous sodium sulfate into a conical flask, uniformly stirring the mixture with a soil sample, then completely transferring the mixture into the glass fiber filter cartridge, plugging the cotton, connecting the Soxhlet extraction device, transferring the extraction solution in a soxhlet tube and the filter cartridge into a flat-bottomed flask after Soxhlet extraction for 24 hours, concentrating the extraction solution by using a rotary evaporator at the water bath temperature of 45 ℃, the rotation speed of 75r/min and the initial pressure of 450hPa, gradually reducing the pressure along with the reduction of the extraction solution, and finally concentrating the extraction solution to 1-2 mL under the pressure of 360 hPa;
then, the column was packed with n-hexane by a wet method, and a glass chromatography column was packed with a packing material comprising, from bottom to top, 5g of anhydrous sodium sulfate, 5g of Florisil, 3.5g of silica gel, and 5g of anhydrous sodium sulfate. The concentrated extract was then transferred to a chromatography column, eluted with 100mL of an eluent (n-hexane: acetone/v: v ═ 9:1), and collected in a flat-bottomed flask. Concentrating the eluent after the column by using a rotary evaporator at the water bath temperature of 45 ℃, the rotating speed of 75r/min and the initial pressure of 400hPa, gradually reducing the pressure along with the continuous reduction of the eluent, and finally concentrating to 1-2 mL under the pressure of 360 hPa.
Then transferring the concentrated post-column eluent to a 10mL colorimetric tube with scales, adding 1 dropper of n-hexane into the flat-bottomed flask, fully purging the inner wall of the flat-bottomed flask by using the dropper, transferring to the colorimetric tube, repeating for 2 times, finally fixing the volume to 5mL, and uniformly mixing by using a vortex oscillator;
then, 200. mu.L (group A1, group B1) or 50. mu.L (group A2, group B2) of 200. mu.L pipette is respectively taken from the colorimetric cylinder and added into a sample injection bottle, 800. mu.L or 950. mu.L of n-hexane is respectively added into 1mL pipette to be constant volume to 1mL, and the concentration of the cis-cypermethrin (C) is analyzed by GC-MSt)；
Finally, the eluent after the residual column in the colorimetric tube is blown to be nearly dry in a nitrogen blowing device, 100 mu L of n-nonane is added, then the eluent is completely transferred to a 150 mu L glass lining tube embedded in a sample injection bottle, and GC-C-IRMS is carried out to analyze the isotope composition (delta) of the cis-cypermethrin0 13C)。
Concentration of cis-cypermethrin (C)t) The method is characterized by using an Agilent 7890A gas chromatograph combined with an Agilent 5975C mass spectrum for determination, wherein a chromatographic column is an HP-5ms quartz capillary column (30m multiplied by 0.25mm i.d. multiplied by 0.25 mu m), the injection inlet temperature is 260 ℃, the injection amount is 1 mu L, the injection mode is non-split flow, and the carrier gas is high-purity helium (99.999%) and the flow rate is 1.2 mL/min; the temperature rising procedure of the chromatographic column is as follows: the initial temperature is 100 ℃, the temperature is kept for 4min, the temperature is increased to 200 ℃ at the speed of 25 ℃/min, the temperature is kept for 10min, the temperature is increased to 270 ℃ at the speed of 10 ℃/min, the temperature is kept for 5min, and finally the temperature is increased to 280 ℃ at the speed of 20 ℃/min, and the temperature is kept for 5 min; ionization mode is EI mode (70eV), scan mode is selective ion scan (qualitative ion m/z 163,181,209, where 163 is quantitative ion); carbon isotope composition (delta) of cis-cypermethrin0 13C) The detection is carried out by Agilent 7890A gas chromatograph through a GC5 combustion interface by using GV Isoprime isotope mass spectrum, the chromatographic column is an HP-5ms quartz capillary column (30m multiplied by 0.25mm i.d. multiplied by 0.25 mu m), the injection port temperature is 260 ℃, the injection amount is 2 mu L, the injection mode is non-split flow, the carrier gas is high-purity helium (99.999 percent) and the flow is high-purity heliumThe speed was 1.2 mL/min. The temperature procedure of the chromatographic column is the same as that of GC-MS; all samples are measured in parallel for three times, and the uncertainty of analysis is less than 0.5 per thousand, so that real and accurate microbial degradation data of the pesticide can be effectively obtained.
Claims (6)
1. An isotope analysis method for pyrethroid biodegradation in soil is characterized by comprising the following steps: firstly, a layer of polyethylene plastic film is padded on a laboratory table which is dry in the shade and well ventilated, a collected soil sample is placed on the polyethylene plastic film to be flattened, soil blocks are crushed, impurities such as stones, animal and plant residues and the like are removed, then the soil sample is paved into a thin layer, and the soil sample is frequently turned over to be naturally dried;
step two, after air drying, sieving the mixture by a sieve of 2mm, uniformly mixing the mixture, taking two opposite angles by a quartering method, uniformly mixing the two opposite angles, taking two opposite angles by the quartering method, wherein one part is used for an experiment, and the other part is packed by a sample bag and then stored in a dark and dry place;
thirdly, weighing 0.01000 +/-0.00005 g of a cis-cypermethrin solid standard sample by using a trace electronic balance, dissolving the cis-cypermethrin solid standard sample in n-hexane, carrying out constant volume in a 100mL volumetric flask to prepare a 100mg/L cis-cypermethrin standard solution, shaking the solution uniformly for later use, respectively weighing 10mL and 50mL cis-cypermethrin standard solutions, diluting the solution by using n-hexane in a 100mL volumetric flask to realize constant volume, and respectively preparing 10mg/L and 50mg/L cis-cypermethrin standard solutions;
fourthly, diluting the cis-cypermethrin standard solution with the concentration of 100mg/L by normal hexane according to a certain gradient, respectively preparing the cis-cypermethrin standard solutions with the concentrations of 500 mug/L, 250 mug/L, 100 mug/L, 50 mug/L, 25 mug/L and 10 mug/L, analyzing and determining by GC-MS, and drawing a standard curve;
fifthly, taking the cis-cypermethrin standard solution with the concentration of 50mg/L, analyzing by GC-C-IRMS, and determining the carbon isotope composition (delta)0 13C)；
Sixth, measure (δ)0 13C) Then, calculating the isotopic enrichment factor epsilon;
seventh step, combining isotopic enrichment factor epsilonThe biodegradation rate (B) of the alpha-cypermethrin in the environment can be obtained by the formula
And (6) performing calculation.
2. An isotopic analysis method for pyrethroid microbial degradation in soil according to claim 1, characterized by: in quantitative isotope fractionation, Rayleigh model formula is generally adopted
In the formula Rt，R0And Ct，C0The isotopic ratio and the concentration of the compound at time t and at the beginning of the reaction, respectively, epsilon is an isotopic enrichment factor which relates the isotopic ratio variation to the concentration variation, the stable carbon isotopic ratio for natural abundance (R ═ c)13C/12C) It can be assumed that R +1 ≈ 1, and thus the above formula can be simplified to
The simplified Rayleigh equation
Taking logarithm to obtain a carbon isotope enrichment factor epsilon; finally, ln (C)t/C0) For ln [ (delta)t13C+1)/(δ013C+1)]And (3) plotting, as shown in the attached figure 1 of the specification, wherein the slope of the straight line after linear fitting is the carbon isotope enrichment factor epsilon.
3. An isotopic analysis method for pyrethroid microbial degradation in soil according to claim 1, characterized by: carbon isotope composition (delta)0 13C) Can be obtained by the activation of soil microorganisms and the analysis of a control setting mode; when the soil microorganism activation and control setting mode is used, 96 50mL conical flasks (with rubber stoppers) are taken, 5.0000 +/-0.0250 g of soil sample is weighed by an analytical electronic balance and is added into the cones respectivelyFlattening the flask in the bottom of the flask, uniformly spraying 3mL of tap water into each conical flask by using a 1mL liquid-transferring gun, plugging a rubber plug, and weighing and recording the total mass of each conical flask;
then, placing the conical flask into a constant temperature shaking table, oscillating for 30min at 30 ℃ and 120r/min, then placing the conical flask into a constant temperature incubator, and culturing for 2 weeks at 30 ℃ in a dark place;
then, taking out the conical flasks from the constant-temperature incubator, dividing the conical flasks into A, B groups, placing the group A in an autoclave, sterilizing the conical flasks at 121 ℃ for 3 times for 20min each time, dividing the A, B group into A1 group, A2 group, B1 group and B2 group, respectively, uniformly spraying 1mL of a cis-cypermethrin standard solution with the concentration of 10mg/L into each of the A1 bottle and the B1 bottle by using a 1mL liquid transfer gun, and uniformly spraying 1mL of a cis-cypermethrin standard solution with the concentration of 50mg/L into the A2 bottle and the B2 bottle;
and then, placing the conical flask in a constant-temperature shaking table, oscillating for 10min at the temperature of 30 ℃ and at the speed of 120r/min to ensure that the cis-cypermethrin is uniformly distributed in the soil sample, loosening the bottle stopper to ensure that n-hexane is volatilized completely, then plugging the bottle stopper, weighing A, B groups, respectively adding sterilized tap water and non-sterilized tap water, and then plugging the bottle stopper. Then placing into a constant temperature incubator, and culturing at 30 ℃ in a dark place;
finally, three samples each of A1, A2, B1 and B2 were taken from the incubator every 10 days for analysis of the concentration of cis-cypermethrin therein (C)t) And carbon isotope composition (delta)0 13C)。
4. An isotopic analysis method for pyrethroid microbial degradation in soil according to claim 3, characterized by: firstly, selecting a Soxhlet extraction method to extract cis-cypermethrin in soil, connecting a Soxhlet extraction device, using n-hexane to soxhlet and wash a glass fiber filter cartridge and cotton for 8 hours, then changing the n-hexane into 160mL of Soxhlet extraction solution (n-hexane: acetone/v: 7:1), adding a proper amount of anhydrous sodium sulfate into a conical flask, uniformly stirring the mixture with a soil sample, then completely transferring the mixture into the glass fiber filter cartridge, plugging the cotton, connecting the Soxhlet extraction device, transferring the extraction solution in a soxhlet tube and the filter cartridge into a flat-bottomed flask after Soxhlet extraction for 24 hours, concentrating the extraction solution by using a rotary evaporator at the water bath temperature of 45 ℃, the rotation speed of 75r/min and the initial pressure of 450hPa, gradually reducing the pressure along with the reduction of the extraction solution, and finally concentrating the extraction solution to 1-2 mL under the pressure of 360 hPa;
then, the column was packed with n-hexane by a wet method, and a glass chromatography column was packed with a packing material comprising, from bottom to top, 5g of anhydrous sodium sulfate, 5g of Florisil, 3.5g of silica gel, and 5g of anhydrous sodium sulfate. The concentrated extract was then transferred to a chromatography column, eluted with 100mL of an eluent (n-hexane: acetone/v: v ═ 9:1), and collected in a flat-bottomed flask. Concentrating the eluent after the column by using a rotary evaporator at the water bath temperature of 45 ℃, the rotating speed of 75r/min and the initial pressure of 400hPa, gradually reducing the pressure along with the continuous reduction of the eluent, and finally concentrating to 1-2 mL under the pressure of 360 hPa.
Then transferring the concentrated post-column eluent to a 10mL colorimetric tube with scales, adding 1 dropper of n-hexane into the flat-bottomed flask, fully purging the inner wall of the flat-bottomed flask by using the dropper, transferring to the colorimetric tube, repeating for 2 times, finally fixing the volume to 5mL, and uniformly mixing by using a vortex oscillator;
then, 200. mu.L (group A1, group B1) or 50. mu.L (group A2, group B2) of 200. mu.L pipette is respectively taken from the colorimetric cylinder and added into a sample injection bottle, 800. mu.L or 950. mu.L of n-hexane is respectively added into 1mL pipette to be constant volume to 1mL, and the concentration of the cis-cypermethrin (C) is analyzed by GC-MSt)；
Finally, the eluent after the residual column in the colorimetric tube is blown to be nearly dry in a nitrogen blowing device, 100 mu L of n-nonane is added, then the eluent is completely transferred to a 150 mu L glass lining tube embedded in a sample injection bottle, and GC-C-IRMS is carried out to analyze the isotope composition (delta) of the cis-cypermethrin0 13C)。
5. An isotopic analysis method for pyrethroid microbial degradation in soil according to claim 1, characterized by: concentration of cis-cypermethrin (C)t) The measurement is carried out by Agilent 7890A gas chromatograph combined with Agilent 5975C mass spectrum, the chromatographic column is HP-5ms quartz capillary column (30m × 0.25mm i.d. × 0.25 μm), the injection inlet temperature is 260 ℃, the injection amount is 1 μ L, the injection mode is non-split flow, the carrier gas is highPure helium (99.999%) at a flow rate of 1.2 mL/min; the temperature rising procedure of the chromatographic column is as follows: the initial temperature is 100 ℃, the temperature is kept for 4min, the temperature is increased to 200 ℃ at the speed of 25 ℃/min, the temperature is kept for 10min, the temperature is increased to 270 ℃ at the speed of 10 ℃/min, the temperature is kept for 5min, and finally the temperature is increased to 280 ℃ at the speed of 20 ℃/min, and the temperature is kept for 5 min; the ionization mode was EI mode (70eV) and the scan mode was selective ion scan (qualitative ion m/z 163,181,209, where 163 is a quantitative ion).
6. An isotopic analysis method for pyrethroid microbial degradation in soil according to claim 1, characterized by: carbon isotope composition (delta) of cis-cypermethrin0 13C) The measurement is carried out by an Agilent 7890A gas chromatograph through a GC5 combustion interface by using a GV Isoprotime isotope mass spectrum, the chromatographic column is an HP-5ms quartz capillary column (30m multiplied by 0.25mm i.d. multiplied by 0.25 mu m), the injection port temperature is 260 ℃, the injection amount is 2 mu L, the injection mode is non-split flow, high-purity helium (99.999%) is used as carrier gas, and the flow rate is 1.2 mL/min. The temperature procedure of the chromatographic column is the same as that of GC-MS; all samples were assayed in triplicate and the uncertainty of the assay was made less than 0.5% o.
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| CN103472150A (en) * | 2013-09-22 | 2013-12-25 | 邬金飞 | Gas chromatography method for detecting pesticide residue amounts of seven pyrethroids in soil |
| US20140171309A1 (en) * | 2012-12-19 | 2014-06-19 | Dow Agrosciences Llc | Pesticidal compositions and processes related thereto |
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| US20140171309A1 (en) * | 2012-12-19 | 2014-06-19 | Dow Agrosciences Llc | Pesticidal compositions and processes related thereto |
| CN103472150A (en) * | 2013-09-22 | 2013-12-25 | 邬金飞 | Gas chromatography method for detecting pesticide residue amounts of seven pyrethroids in soil |
| CN110256285A (en) * | 2019-07-09 | 2019-09-20 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | A kind of synthetic method of stable isotope labeling pyrethroid |
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