CN109916938A - A method of the measurement soil organism adsorbs organic pollutant spatial distribution - Google Patents
A method of the measurement soil organism adsorbs organic pollutant spatial distribution Download PDFInfo
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Abstract
The invention discloses a kind of methods of measurement soil organism absorption organic pollutant spatial distribution.This method uses13The organic pollutants such as the antibiotic of C flag are adsorbed on the soil organism, then with the spatial distribution characteristic of NanoSIMS technology analysis soil organism absorption organic pollutant.This method can not only effectively overcome the carbon distribution problem distinguished in the soil organism and pollutant, but also the intuitive displaying of organic matter two-dimensional spatial distribution feature may be implemented, and can also be measured to three-dimensional spatial distribution.
Description
Technical field
After adsorbing organic pollutant the present invention relates to a kind of measurement soil organism, organic pollutant is on the soil organism
Spatial distribution method.
Background technique
Soil is the carrier of Agro-ecological System exchanges of mass and energy, is the material base and land life of plant growth
Basis.Important component of the soil as environment, can not only accept the pollution from other surrounding mediums, be also simultaneously
The pollution sources of other surrounding mediums.Soil is once contaminated, it will seriously threatens human health and Environmental security.Therefore, to soil
Earth pollution and the research of mechanism cause the concern of researcher more and more.Organic pollutant is generally existing in current environment
One of major pollutants, mainly include antibiotic, organic agricultural chemicals, phenols, synthetic detergent and by municipal sewage, sludge and
Apply fertilizer bring harmful microorganism etc..Although number of types of organic chemical reagent has disabled for many years now, due to its change
Learn that property is stable, the secondary species after difficult to degrade or degradation remains unchanged toxic effect to environment, the residual of these pollutants with
It pollutes still extremely serious.The soil organism is the important component of soil, is played a significant role to the absorption of organic pollutant.
The soil organism can be divided into active organic matter, inertia organic matter and stability organic matter according to its stability difference, different steady
The organic pollutant of qualitative soil organism absorption is different to the toxic action of environment, such as adding with degree of humification
Deep, activity is higher, the organic pollutant of the lower particulate organic matter absorption of stability may discharge to form secondary dirt again
Dye etc..The soil organism has a complicated space structure, these structures are to the absorption mechanism of ammonium fixation of soil organic pollutant not phase
Together, the movable effect of animals and plants resisted in soil environment is also different, for example enters organic matter and close the pollution stored in state hole
Object is more difficult to be released.Therefore, the spatial structure characteristic for specifying soil organism absorption organic pollutant can be soil
The polluting effect of organic pollutant and environmental risk assessment provide data basis and scientific basis.
Currently there is researcher to combine synchrotron radiation spectroscopy, using energy storehouse technique study pollutant in the soil
Two-dimensional spatial distribution.The control of this method experiment condition requires harshness, hardly results in ideal distribution of organic data.And
Seemed beam using the research that this method is distributed (pollutant and organic matter contain carbon simultaneously) for organic pollutant on organic matter
Hand is without plan.
Summary of the invention
In order to solve the predicament of current research, the present invention provides a kind of measurement soil organism absorption organic pollutant is empty
Between the new method that is distributed.This method uses13The organic pollutants such as the antibiotic of C flag are adsorbed on the soil organism, so
Afterwards with the spatial distribution characteristic of NanoSIMS technology analysis soil organism absorption organic pollutant.This method not only can be effective
Ground overcomes the carbon distribution problem distinguished in the soil organism and pollutant, and it is special that organic matter two-dimensional spatial distribution may be implemented
The intuitive displaying of sign, can also be measured three-dimensional spatial distribution.
The technical scheme is that a kind of method of measurement soil organism absorption organic pollutant spatial distribution,
It is characterized in,
1) it uses13C flag organic pollutant, is adsorbed with the soil organism13The organic pollutant of C flag, concussion absorption reach
Equilibrium state is adsorbed13The soil organism of the organic pollutant of C flag；
2) production is not added13The soil organism of C flag organic pollutant is as blank control；
3) by the soil organism in step 1) and step 2), freezing forming；
4) soil organism by freezing forming in step 3) makes slice, then carries out tem observation；
5) position being distributed according to tem observation result selection target organic matter records the coordinate points of object organic matter；
6) utilize nanoparticle probe (NanoSIMS) scanning step 5) in target organic matter, according to object organic matter
Coordinate point location scanning area, respectively obtain the soil organism NanoSIMS picture and absorption label13C organic pollutant
Soil organism picture, absorption label13The soil organism picture of C organic pollutant and unadsorbed soil organism picture pair
Than having apparent13C enrichment, then obtain organic pollutant in the distribution map of soil organism particle upper surface；
7) using the target organic matter in nanoparticle probe (NanoSIMS) multiple scanning step 5), every run-down,
Being etched with a thickness of 15 ± 5nm for organic matter sample surface, repeats at least 8 times, prepares organic dirt in the thickness of 100-500nm
The three-dimensional spatial distribution figure of object is contaminated, this thickness can need voluntarily to adjust according to experiment.
Wherein, organic pollutant is to be easy the substance that is adsorbed by the soil organism, such as antibiotic (such as Ciprofloxacin), have
Machine pesticide, phenols, synthetic detergent etc. can also also wrap for by municipal sewage, sludge and fertilising bring harmful microorganism etc.
Include persistence organic pollutant.
Preferably, the step 1) is adsorbed with the soil organism13The specific method is as follows for the organic pollutant of C flag: matching
Background solution of the NaCl solution processed as adsorption experiment, is prepared with background solution13The adsorption liquid of the organic pollutant of C flag；So
Afterwards in soil organism absorption adsorption liquid13The organic pollutant of C flag, concussion absorption reach equilibrium state.
Preferably, the step 2) are as follows: repeat the process of step 1) and adsorption liquid is not add in step 1)13C flag has
The background solution of machine pollutant.
Wherein, step 1) can be spaced different period a certain amount of absorption of taking-up in adsorption process13C flag has
The soil organism of machine pollutant, freezen protective, adsorption process of the dynamic monitoring Ciprofloxacin on soil particle state organic matter.
Preferably, the freezing forming of the step 3) are as follows: by the soil organism in -20 DEG C of freezing wedgewises, it is furthermore preferred that
Before freezing the soil organism is fixed into shape in aluminium-foil paper, be put into -20 DEG C, then aluminium-foil paper is peelled off in freezing forming.
Preferably, the step 4) production slice are as follows: in liquid nitrogen environment with come card ultracryotomy machine will freezing
The slice that the soil organism of forming is cut into a thickness of 500nm.
Preferably, the step 4) is to prevent piece cutting structure from damaging, and often cuts a piece of, slice need to be dipped with eyelash pencil, cutting
Piece is transferred on hydrophily silicon wafer and tiles, and avoids organic matter that " curling " occurs, silicon wafer is micro slide, then carries out tem observation.
Preferably, step 7) the organic matter sample surface is etched with a thickness of 15nm, is repeated 10 times, and prepares 150nm
Thickness in organic pollutant three-dimensional spatial distribution figure.
NanoSIMS (Nano-scale secondary ion mass spectrometry) technology uses newest
SIMS instrument, which (flown in SIMS (Ion mass spectrometry, ion microprobe mass spectrometer) and TOF-SIMS
Row time mass spectrum instrument) on the basis of grow up, can be used for simulating microbial activities in soil and original position soil solution visual
The technological means of change.7 kinds of ionic types, sensibility with higher can be analyzed simultaneously, and resolution ratio can reach 50nm, therefore can
The spatial distribution of carbon and nitrogen stable isotope is accurately positioned.Area is also effectively overcomed using the technology of isotope labelling pollutant
The carbon divided in organic matter and pollutant is distributed problem.
Isotope fractionation feature can be used to tracer soil microbial activity and organic matter migration, conversion etc. and feed back
Journey, more application is in soil organism humification process, Forming Mechanism of aggregate etc. are studied at present.Shown at present with isotope
Track studies pollutant distribution, mostly uses radioactive isotope, and radioactive isotope requires harshness to experiment condition, needs to be equipped with specially
The hot laboratory of door could be completed by the laboratory technician of special training, therefore the tracer for being not suitable for needing in routine experimentation is ground
Study carefully.Therefore, the present invention marks organic pollutant using stable isotope, then with the pollutant of label in the soil organism
Adsorption experiment is carried out, it can be with difficulty of the current organic pollutant of effective solution in organic matter in distribution characteristics research.
Be sliced both at home and abroad at present mostly using the embedding mediums such as paraffin, resin, with embedding medium it is fixed-type after can just be cut into
Piece, but in these embedding mediums contain organic matter (carbon), with the carbon in the soil organism and organic pollutant in the present invention without
Method is distinguished, therefore not applicable.In order to study organic matter slice, foreign countries have researcher to propose to do embedding medium with sulphur and indium foil, need height
Temperature is heated to sulphur powder fusing, then organic matter is put into high-temperature molten-state sulphur, since part soil organic pollutant high temperature is easy
It degrades (for example antibiotic is more than 40 DEG C i.e. degradable).In addition, indium foil needs for organic matter to be pressed into indium foil when fixing organic matter,
The space structure of the process meeting destruction of organic material primitive nature of pressure, in conclusion the method that production is sliced both at home and abroad at present is not
Suitable for the present invention.
In consideration of it, being improved present inventor has performed following:
1, proposition of the present invention does embedding medium with water, and freezing forms organic matter, both will not destruction of organic material space structure,
The interference of other organic matters will not be introduced.
2, the slice that conventional embedding medium is done picks up the analysis that is available on the machine with copper mesh in water, and organic matter slice is met water and melted,
And the organic pollutant of absorption may be diluted desorption, influence subsequent measurements result.Therefore inventor devises eyelash pencil and (uses
The eyelash pencil that a piece eyelashes are fabricated to) it is sliced to shift the soil organism, with eyelash pencil transfer slice departing from the influence of water, and
And slice will not be caused to damage.
3, since slice thickness only has 500nm, it is easy to drying at room temperature, causes organic matter slice that " curling " occurs existing
As that smooth slice can not be transferred to silicon wafer, therefore the present invention is (low in liquid nitrogen environment using entire slice and transfer process
Temperature guarantee organic matter slice molding), while to silicon wafer carry out hydrophilic treated (because conventional silicon wafers on place organic matter slice, by
It before NanoSIMS experimental analysis needs that sample is dried, and experimentation sample room is vacuum state, organic matter is cut
It is easy to fall off from silicon wafer when vacuumizing after piece is dry, after carrying out hydrophilic treated to silicon wafer, organic matter slice is coated with by ice, room temperature
The process of lower ice dissolution can make the good docile of organic matter on silicon wafer, will not fall off vacuumizing.
The beneficial effects of the present invention are: method of the invention, which can not only effectively overcome, distinguishes the soil organism and dirt
The carbon contaminated in object is distributed problem, and the intuitive displaying of organic matter two-dimensional spatial distribution feature may be implemented, can also be to three-dimensional
Spatial distribution is measured, so that the spatial distribution characteristic for adsorbing organic pollutant to probe into the soil organism provides directly fastly
Prompt method has established method basis to inquire into the harm of organic pollutant in the soil in environment.
Detailed description of the invention
Fig. 1 is slice distribution map of the soil particle state organic matter under optical electron microscope, black and dark brown in picture
The particle of color is soil particle state organic matter；
Fig. 2 is unadsorbed soil particle state organic matter, is scanned using nanoparticle probe technique13The NanoSIMS of C schemes；
Fig. 3 is absorption label13The soil particle state organic matter of C Ciprofloxacin is scanned using nanoparticle probe technique13C
NanoSIMS figure, darker regions are in figure13C flag rich region, orange red enrichment are most；
Fig. 4 is absorption label13The soil particle state organic matter of C Ciprofloxacin is scanned using nanoparticle probe technique13C
Three-dimensional spatial distribution figure.
Fig. 5 is wedge shape Noah's ark structural schematic diagram made of aluminium-foil paper, i.e., rectangular one end is 45 degree of inclined-planes.
Specific embodiment
Embodiment 1:
1) background solution of the NaCl solution of 0.01mol/L as adsorption experiment is accurately prepared, the background of certain volume is taken
Solution is prepared13The concentration of C flag Ciprofloxacin is the adsorption liquid of 40mg/kg.Accurately weigh 0.100g soil particle state organic matter
In polytetrafluoroethylene (PTFE) centrifuge tube, solid-to-liquid ratio 1:10, with soil particle state organic matter (activity is higher) absorption13The ring of C flag
Third husky star, under the conditions of 25 DEG C, for 24 hours, absorption can reach equilibrium state for concussion, within for 24 hours, settable 12 different samplings
Time, adsorption process of the dynamic monitoring Ciprofloxacin on soil particle state organic matter, setting sample time are respectively when adsorbing
Between for 2min, 5min, 10min, 15min, 20min, 30min, 1h, 2h, 4h, 6h, 10h, for 24 hours when, freezen protective sampling
Soil particle state organic matter sample；
13The Ciprofloxacin of C flag purchased from Cambridge Isotope Laboratories (Cambridge Isotope Laboratories,
Inc)；The flag parameters of Ciprofloxacin: min.99%atom%13C；
2) soil particle state organic matter blank control is done, i.e., repeatedly the process of step 1) and adsorbent are not add step 1)
In13The background solution of C flag Ciprofloxacin；
3) the soil particle state organic matter sample that will be taken out in step 1) and step 2), is respectively put into wedge made of aluminium-foil paper
In shape Noah's ark (such as Fig. 5), fixed shape is put into -20 DEG C, and then aluminium-foil paper is peelled off in freezing forming, this process is without adding other
Embedding medium does embedding medium with water, and cryogenic freezing Cheng Bing operates microsection manufacture process in low temperature environment；
4) wedge-shaped soil particle state organic matter in step 3) is made into slice, with coming the ultra-thin freezing of card in liquid nitrogen environment
The slice that the soil organism is cut into a thickness of 500nm by slicer, since soil particle state organic matter particle is very small and quality
It is soft, it to prevent piece cutting structure from damaging, often cuts a piece of, slice need to be dipped with eyelash pencil, slice, which is transferred to catercorner length, is
It tiles on square (or diameter is the circle of 10mm) hydrophily silicon wafer of 10mm, avoids organic matter that " curling " occurs, silicon wafer is
Micro slide, the process carry out under the microscope, should avoid organic matter sample particle cluster during microsection manufacture or overlap
Together, tem observation is then carried out, as a result as shown in Figure 1；
5) position for being distributed tem observation result selection target organic matter, records the coordinate points of object；
6) sample is transferred to nanoparticle probe instrument Sample Room, sample room vacuumizes, and sample is dried, and utilizes
Nanoparticle probe (NanoSIMS) analyzer scanning step 5) in target organic matter, according to coordinate point location scanning area,
The NanoSIMS picture (Fig. 2) and absorption label of the soil organism are obtained respectively13Soil organism picture (the figure of C Ciprofloxacin
3), absorption label13The soil particle state organic matter picture of C Ciprofloxacin and unadsorbed soil particle state organic matter compare, and have
Significantly13C enrichment (darkens, is enriched with13C is more, more partially orange red), then obtain Ciprofloxacin has in soil particle state
The distribution map of machine matter particle upper surface；
The experiment of nanoparticle probe analysis is complete in Chinese Academy of Sciences's geology and Geophysics Institute's nanoparticle probe laboratory
At.Experiment initial ion source is Cs+Ion, initial ion excitation can be 6kev, initial electron beam be 1.2~3.8pA, it is secondary from
It is 8kev that son, which draws pressure, and lateral resolution is 100~200nm, corroding thickness 15nm, and scanning sample coverage area is 20 × 20
μm2.Initial ion beam constant current hold in experimentation.
The electron gun of NanoSIMS can continue to generate electron beam compensation electric current.All testing results are with image model
It provides, the retention time of ion image is 15ms/ pixel, and secondary ion image is 256 × 256 pixels.
7) using the target organic matter in nanoparticle probe (NanoSIMS) multiple scanning step 5), every run-down,
Being etched with a thickness of 15nm for organic matter sample surface, is repeated 10 times, and the organic pollutant in the thickness of 150nm can be obtained
Three-dimensional spatial distribution figure (such as Fig. 4).
Embodiment 2:
Step 7) utilizes the target organic matter in nanoparticle probe (NanoSIMS) multiple scanning step 5), every scanning one
Secondary, being etched with a thickness of 15nm for organic matter sample surface is repeated 20 times, the organic contamination in the thickness of 300nm can be obtained
The three-dimensional spatial distribution figure of object.
Claims (10)
1. a kind of method of measurement soil organism absorption organic pollutant spatial distribution, characterized in that the following steps are included:
1) it uses13C flag organic pollutant, is adsorbed with the soil organism13The organic pollutant of C flag, concussion absorption reach balance
State is adsorbed13The soil organism of the organic pollutant of C flag；
2) production is not added13The soil organism of C flag organic pollutant is as blank control；
3) by the soil organism in step 1) and step 2), freezing forming；
4) soil organism by freezing forming in step 3) makes slice, then carries out tem observation；
5) position being distributed according to tem observation result selection target organic matter records the coordinate points of object organic matter；
6) utilize nanoparticle probe NanoSIMS scanning step 5) in target organic matter, according to the coordinate of object organic matter
Point location scanning area obtains the NanoSIMS picture and absorption label of the soil organism respectively13The soil of C organic pollutant has
Machine matter picture, absorption label13The soil organism picture of C organic pollutant and unadsorbed soil organism picture compare, and have
Significantly13C enrichment, then obtain organic pollutant in the distribution map of soil organism particle upper surface.
2. a kind of method of measurement soil organism absorption organic pollutant spatial distribution as described in claim 1, feature
It is to utilize the target organic matter in nanoparticle probe NanoSIMS multiple scanning step 5), every run-down, organic matter print
Being etched with a thickness of 15 ± 5nm for surface, repeats at least 8 times, prepares the three-dimensional of the organic pollutant in the thickness of 100-500nm
Spatial distribution map.
3. a kind of method of measurement soil organism absorption organic pollutant spatial distribution as claimed in claim 2, feature
It is that being etched with a thickness of 15nm for the organic matter sample surface is repeated 10 times, prepares the organic contamination in the thickness of 150nm
The three-dimensional spatial distribution figure of object.
4. a kind of measurement soil organism absorption organic pollutant spatial distribution as described in any one of claim 1-3
Method, characterized in that the organic pollutant is antibiotic, organic agricultural chemicals, phenols, synthetic detergent and by municipal sewage, dirt
Mud and fertilising bring harmful microorganism in any one.
5. a kind of measurement soil organism absorption organic pollutant spatial distribution as described in any one of claim 1-3
Method, characterized in that the step 1) is adsorbed with the soil organism13The specific method is as follows for the organic pollutant of C flag: preparing
Background solution of the NaCl solution as adsorption experiment, is prepared with background solution13The adsorption liquid of the organic pollutant of C flag；Then
With in soil organism absorption adsorption liquid13The organic pollutant of C flag, concussion absorption reach equilibrium state.
6. a kind of method of measurement soil organism absorption organic pollutant spatial distribution as claimed in claim 5, feature
It is the step 2) are as follows: repeat the process of step 1) and adsorption liquid is not add in step 1)13The back of C flag organic pollutant
Scape solution.
7. a kind of measurement soil organism absorption organic pollutant spatial distribution as described in any one of claim 1-3
Method, characterized in that the freezing of the step 3) shapes are as follows: by the soil organism in -20 DEG C of freezing wedgewises.
8. a kind of method of measurement soil organism absorption organic pollutant spatial distribution as claimed in claim 7, feature
It is that the soil organism is fixed into shape in aluminium-foil paper before freezing, is put into -20 DEG C, peels off aluminium-foil paper after freezing wedgewise.
9. a kind of measurement soil organism absorption organic pollutant spatial distribution as described in any one of claim 1-3
Method, characterized in that the production of the step 4) is sliced are as follows: the soil organism in liquid nitrogen environment by freezing forming is cut into thickness
Degree is the slice of 500nm.
10. a kind of method of measurement soil organism absorption organic pollutant spatial distribution as described in claim 1 or 9,
It is characterized in, the step 4) is often cut a piece of, and slice need to be dipped with eyelash pencil, slice is transferred on hydrophily silicon wafer and is tiled, so
After carry out tem observation.
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| PCT/CN2019/079437 WO2020181578A1 (en) | 2019-03-08 | 2019-03-25 | Method for determining spatial distribution of organic pollutants adsorbed by soil organic matter |
| AU2019411708A AU2019411708B2 (en) | 2019-03-08 | 2019-03-25 | Method for determining spatial distribution of organic pollutants adsorbed by soil organic matter |
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| 周广荣: "样品切片制备及电镜观察技巧", 《分析仪器》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2020181578A1 (en) | 2020-09-17 |
| AU2019411708A1 (en) | 2020-09-24 |
| CN109916938B (en) | 2021-09-21 |
| AU2019411708B2 (en) | 2022-01-27 |
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