CN110186886A - The inversion method of Microcystins in Water MC-LR concentration - Google Patents
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Abstract
The invention discloses the inversion methods of Microcystins in Water MC-LR concentration, it include: using the normalized characteristic fluorescence peak intensity of the extracellular organic matter of the green Microcystis aeruginosa of Copper in Water Samples as independent variable, normalized microcapsule phycotoxin MC-LR concentration is that dependent variable constructs unitary linear inversion equation in water sample, wherein, characteristic fluorescence peak intensity and microcapsule phycotoxin MC-LR concentration are normalized with water sample Determination of Chlorophyll a concentration;Take water sample to be detected, measure the three-dimensional excitation-emission matrix fluorescence spectrum of the extracellular organic matter of the green Microcystis aeruginosa of Copper in Water Samples, the characteristic fluorescence peak intensity of the extracellular organic matter of microcystic aeruginosa is obtained, then substitutes into unitary linear inversion equation, obtains the concentration of microcapsule phycotoxin MC-LR.This method is not necessarily to sample pre-treatments, it can be achieved that the quick on-line monitoring of microcapsule phycotoxin MC-LR concentration provides new thinking for inversion method using the extracellular organic matter characteristic fluorescence peak intensity of microcystic aeruginosa as the variable of building equation.
Description
Technical field
The present invention relates to water body environment detection more particularly to a kind of inverting sides of Microcystins in Water MC-LR concentration
Method.
Background technique
A large amount of microcapsule phycotoxin MC-LRs enter water body, seriously threaten drinking water safety.Microcapsule phycotoxin MC-LR mainly by
Microcystic aeruginosa in water body generates.Currently, conventional concentration detection method be divided into bioassay method, physical chemistry detection method,
Biochemistry detection method, molecular biological assay, each detection method water sample pre-treatment is cumbersome, time-consuming, at high cost, can not
Realize quickly on-line monitoring.And inversion method can use and construct inverse model with the highly relevant impact factor of MC-LR concentration,
The variation of indirect reaction Microcystins in Water MC-LR concentration.Numerous researchers using nutrient concentrations in water body (such as
Nitrogen, phosphorus etc.), temperature, the impact factors such as dissolved oxygen establish multiple linear regression equations, microcapsule phycotoxin MC-LR concentration is carried out
Inverting, but its concentration has different correlations in different time, different location from each impact factor.Therefore, inverse model
It can not be general.
Summary of the invention
Goal of the invention: the problems of the prior art are directed to, the present invention provides a kind of Microcystins in Water MC-LR is dense
The inversion method of degree, is not required to complicated pre-treatment, and variable uses the extracellular organic matter of source microcystic aeruginosa physiological metabolism.
Technical solution: the inversion method of Microcystins in Water MC-LR concentration of the present invention, comprising:
(1) using the normalized characteristic fluorescence peak intensity of the extracellular organic matter of the green Microcystis aeruginosa of Copper in Water Samples as independent variable, water sample
The middle normalized MC-LR concentration of Microcystin is dependent variable, constructs unitary linear inversion equation, wherein characteristic fluorescence peak intensity
Degree and microcapsule phycotoxin MC-LR concentration are normalized with water sample Determination of Chlorophyll a concentration;
(2) water sample to be detected is taken, the three-dimensional excitation-emission matrix for measuring the extracellular organic matter of the green Microcystis aeruginosa of Copper in Water Samples is glimmering
Light spectrum obtains the characteristic fluorescence peak intensity of the extracellular organic matter of microcystic aeruginosa;
(3) the characteristic fluorescence peak intensity that step (2) obtain is substituted into the unitary linear inversion equation built, is obtained
Obtain the concentration of microcapsule phycotoxin MC-LR.
Specifically, in step (1), the construction method of unitary linear inversion equation includes:
(a) water sampling;
(b) reference standard method is measured the content of the microcapsule phycotoxin MC-LR in water sample;
(c) the extracellular organic matter of microcystic aeruginosa in water sample is extracted, the measurement extracellular organic matter of the green Microcystis aeruginosa of Copper in Water Samples
Three-dimensional excitation-emission matrix fluorescence spectrum obtains the characteristic fluorescence peak intensity of the extracellular organic matter of microcystic aeruginosa;
(d) the algae chlorophyll-a concentration in water sample is measured;
(e) green with leaf to the characteristic fluorescence peak intensity and microcapsule phycotoxin MC-LR concentration of the extracellular organic matter of microcystic aeruginosa
Plain a concentration is normalized;
(f) using normalized characteristic fluorescence peak intensity as independent variable, normalized microcapsule phycotoxin MC-LR concentration be because
Variable establishes unitary linear inversion equation.
When constructing unitary linear inversion equation, standard method described in step (b) refers to that other are different more reliable
Detection method, such as national standard GB/T20466-2006 method can be used.
According to the tightness degree in conjunction with frustule, the extracellular organic constituents such as albuminoid, humic-like substance can divide in water body
For reference state (bEOM) and solubilised state (dEOM) extracellular organic matter, verdigris micro-capsule is mainly derived from microcapsule phycotoxin MC-LR
The physiological metabolism process of algae, and release rule is similar.
In step (c), the extracting method of extracellular organic matter includes: that water sampling is centrifuged in 0~6 DEG C, obtains precipitating and supernatant,
The supernatant 0.22~0.45 μm of aperture membrane filtration, filtrate are the extracellular organic solution of solubilised state;It is rinsed with water filter membrane
It is mixed again with the precipitating afterwards, 60~70 DEG C of 20~30min of heating, after centrifugation, supernatant 0.22~0.45 μm of aperture filter membrane
Filtering, filtrate are the extracellular organic solution of reference state.When above-mentioned centrifugation, 5~10min can be centrifuged in 8000~9000g.In step
(2) it in, for water sample to be detected, is also extracted using this method, three-dimensional fluorescence spectrum detection is carried out after extraction.
In step (c), when three-dimensional fluorescence spectrum measures, parameter setting is as follows: excitation wavelength scanning range: 220~
660nm, launch wavelength scanning range: 240~700nm.In step (3), for water sample to be detected, also carried out using the parameter
Three-dimensional fluorescence spectrum detection.
In step (c) or in step (2), data prediction need to be carried out by obtaining fluorescence spectrum, need to generally be eliminated Rayleigh and be dissipated
It penetrates with after Raman scattering, obtains characteristic fluorescence peak intensity.
In step (c), organic matter extracellular for solubilised state, characteristic fluorescence peak is selected from fluorescence peak S, T, A or C, for combining
The extracellular organic matter of state, characteristic fluorescence peak are selected from fluorescence peak D, T or C, and wherein fluorescence peak A represents ultraviolet humic-like substance, fluorescence peak C generation
Table visible light humus, excitation wavelength are 250~260nm, and launch wavelength is 380~460nm;Fluorescence peak S represents low excitation wave
Long class tryptophan, excitation wavelength are 220~230nm, and launch wavelength is 320~350nm;Fluorescence peak T represents high excitation wavelength class
Tryptophan, excitation wavelength are 270~280nm, and launch wavelength is 320~350nm;Fluorescence peak D represents low excitation wavelength class color ammonia
Acid, excitation wavelength are 220~230nm, and launch wavelength is 300~310nm.Features described above peak can construct unitary linear inversion
Equation, wherein best with the equation efficiency of inverse process that the fluorescence peak T of the extracellular organic matter of reference state is constructed.It is obtained needed for water sample to be detected
The characteristic fluorescence peak intensity taken is corresponding with equation.
When being measured to the algae chlorophyll-a concentration in water sample, " lake eutrophication investigation specifications " method can be used.
The normalized each characteristic fluorescence peak intensity of chlorophyll a is to microcapsule phycotoxin MC-LR concentration in extremely significant related, utilization
Each characteristic fluorescence peak intensity establishes the unitary linear inversion equation such as following table of MC-LR concentration, and wherein any one use may be selected,
In unitary linear inversion equation, CbEOMDRepresent the intensity of fluorescence peak D in bEOM, CbEOMTThe intensity of fluorescence peak T in bEOM is represented,
CbEOMCRepresent the intensity of fluorescence peak C in bEOM, CdEOMSRepresent the intensity of fluorescence peak S in dEOM, CdEOMTRepresent fluorescence peak in dEOM
The intensity of T, CdEOMARepresent the intensity of fluorescence peak A in dEOM, CdEOMCRepresent the intensity of fluorescence peak C in dEOM, CchlaRepresent water sample
The concentration of Determination of Chlorophyll a, unit are μ gL-1, CMC-LRRepresent the concentration of microcapsule phycotoxin MC-LR in water sample, μ gL-1:
The utility model has the advantages that
The inversion method of Microcystins in Water MC-LR concentration of the present invention utilizes three-dimensional excitation-emission matrix fluorescence light
Spectral technology establishes the unitary linear inversion equation of microcapsule phycotoxin MC-LR, which does not need to carry out complexity to water body example
Preprocessing process, detection are quick.
The present invention is inverting side using the extracellular organic matter characteristic fluorescence peak intensity of microcystic aeruginosa as the variable of building equation
Method provides a kind of new thinking, and the extracellular organic matter of microcystic aeruginosa is related with frond growth metabolism itself, can avoid using
The factors, accuracy and the stability such as nitrogen, phosphorus, temperature, dissolved oxygen vulnerable to external environment variation are more preferable.
After chlorophyll a normalized, the characteristic fluorescence peak and Microcystin MC- of the extracellular organic matter of microcystic aeruginosa
The concentration of LR is highly relevant, passes through 3 coefficient of determination, root-mean-square error and root mean square relative error metrics evaluation the method for the present invention
The regression equation of building, efficiency of inverse process is good, and wherein the efficiency of inverse process of reference state high excitation wavelength tryptophan-like fluorescence peak T is best,
Its coefficient of determination, root-mean-square error, root mean square relative error are respectively 0.841,0.579 μ gL-1, 37.10%, inversion equation
Regression coefficient Relatively centralized, convenient for being constantly regulate in practical applications, to obtain best efficiency of inverse process.
Detailed description of the invention
Fig. 1 is the three-dimensional fluorescence spectrum figure of the extracellular organic matter of Taihu Lake microcystic aeruginosa solubilised state;
Fig. 2 is the three-dimensional fluorescence spectrum figure of the extracellular organic matter of Taihu Lake microcystic aeruginosa reference state;
Fig. 3 is the MC-LR concentration of each fluorescence peak inverting of dEOM compared with measured concentration variation tendency;
Fig. 4 is the MC-LR concentration of each fluorescence peak inverting of bEOM compared with measured concentration variation tendency.
Specific embodiment
Combined with specific embodiments below, the present invention is furture elucidated.
The detection method of Microcystins in Water MC-LR, comprising:
(1) sample: the microcystic aeruginosa outbreak period, in the monitoring point in Wuxi Taihu Lake, with sampler, 20~30cm takes 1L under water
Water sample, quantity of sampling quantity are 31.It randomly selects 9 samples to be used as to inverting sample, other samples are for constructing inversion equation.
(2) referring to national standard GB/T20466-2006 method, using 1200 liquid chromatograph of Agilent to micro- in water sample sample
The content of capsule algae toxin MC-LR is measured, and the foundation and inverting for inversion equation detect;
(3) it extracts the extracellular organic matter of microcystic aeruginosa in water sample: the extracellular organic matter of microcystic aeruginosa is divided into dEOM
(the extracellular organic matter of solubilised state) and bEOM (the extracellular organic matter of reference state) two parts, extracting method are as follows: take 5mL water sample in 4 DEG C of items
9000g is centrifuged 10min under part, takes the cellulose acetate film filtering of 0.45 μm of supernatant aperture to save, filtrate is for measuring dEOM;It takes
5mL ultrapure water cellulose acetate film is simultaneously mixed with the centrifugal deposition object of upper step centrifugation, 20min is heated in 70 DEG C of water-baths, through 4
9000g is centrifuged 10min under the conditions of DEG C, takes supernatant to be saved with 0.45 μm of cellulose acetate film filtering, filtrate is for measuring bEOM.
(4) three-dimensional fluorescence spectrum (the three-dimensional excitation-emission matrix fluorescence of the extracellular organic matter of the green Microcystis aeruginosa of Copper in Water Samples is obtained
Spectrum) data: using Hitachi's F-7000 sepectrophotofluorometer measure respectively each water sample the extracellular organic matter of solubilised state or
The three-dimensional fluorescence spectrum of the extracellular organic matter of reference state, parameter setting are as follows: excitation wavelength scanning range: 220~660nm, transmitting
Wavelength scanning range: 240~700nm, excitation wavelength step-length and scanning wavelength step-length are respectively 10nm and 2nm, exciting slit and
Transmite slit is 5nm, scanning speed 12000nm/min, Photomultiplier tube voltage 700V, and spectrum does not correct.To three-dimensional glimmering
Light spectrum data matrix eliminates the influence of Rayleigh scattering by numerical value zero setting, while with Milli-Q ultrapure water spectroscopic data deduction
Blank, to eliminate the influence of Raman scattering.
(4) bright referring to metallographic, " the lake eutrophication investigation specifications " of Tu Qingying chief editor are green to the algae leaf of each water sample
Plain a concentration is measured and is segmented according to chlorophyll-a concentration to sample.
(5) each fluorescence peak intensity and microcapsule phycotoxin MC-LR concentration are normalized with chlorophyll-a concentration.MC-
LR concentration and the unit of chlorophyll-a concentration are μ gL-1。
(6) utilize SPSS software, using normalized fluorescence peak intensity as independent variable, normalized MC-LR concentration for because
Variable establishes unary linear regression equation;
(7) according to 3 coefficient of determination, root-mean-square error and root mean square relative error metrics evaluation regression equations.
Fig. 1, Fig. 2 are respectively the extracellular organic matter of Taihu Lake microcystic aeruginosa solubilised state (dEOM), the extracellular organic matter of reference state
(bEOM) three-dimensional fluorescence spectrum figure.In dEOM three-dimensional spectrogram 4 stronger fluorescence peaks be respectively fluorescence peak S (Ex/Em:
230/320)、T(Ex/Em:280/330)、A(Ex/Em:270/440)、C
(Ex/Em:350/440), what wherein fluorescence peak S, T respectively represented is low excitation wavelength tryptophan-like fluorescence peak, height
Excitation wavelength tryptophan-like fluorescence peak, it is related with the substances such as albumen, amino acid, polypeptide, aromatic ring amino acid is contained in structure, mainly
From the secretion and subsequent biodegrade of frustule;Fluorescence peak A, C represent be ultra-violet (UV) band humic-like fluorescence peak, can
See area humic-like fluorescence peak, it is related to the components such as class fulvic acid and class humic acid, carboxyl, hydroxyl and phenolic hydroxyl group are contained in structure
Deng the extracellular organic matter for being generally considered to be frustule release is formed through humification.In addition to fluorescence peak T, A, C in bEOM
Except, have also appeared a center excitation wavelength 220nm, the class tyrosine fluorescence peak D of launch wavelength 300nm, source and class color
Propylhomoserin is similar, and fluorescence peak position is smaller by microenvironment.Major fluorescent peak position is shown in Table 1 in water body.
Major fluorescent peak position in 1. water body of table
Correlation analysis is carried out with MC-LR concentration respectively to each major fluorescent peak intensity, the results are shown in Table 2, each fluorescence peak intensity
For degree with MC-LR concentration without apparent correlation, each fluorescence peak cannot be directly as the independent variable of regression analysis.Therefore, with leaf
Each fluorescence peak intensity and MC-LR concentration is normalized in green element a concentration, and in addition to fluorescence peak A in bEOM, other are each glimmering
The normalized fluorescence peak intensity of photopeak is in extremely significant related (table 3) to MC-LR concentration.
The correlation of 2 Taihu Lake microcystic aeruginosa of table each fluorescence peak intensity and MC-LR concentration
The correlation of 3 Taihu Lake microcystic aeruginosa of table normalized each fluorescence peak intensity and MC-LR concentration
Unitary linear inversion equation is established using the bEOM and dEOM each fluorescence peak highly relevant with MC-LR concentration, as a result
It is shown in Table 4, in unitary linear inversion equation, CbEOMDRepresent the intensity of fluorescence peak D in bEOM, CbEOMTRepresent fluorescence peak T in bEOM
Intensity, CbEOMCRepresent the intensity of fluorescence peak C in bEOM, CdEOMSRepresent the intensity of fluorescence peak S in dEOM, CdEOMTIt represents in dEOM
The intensity of fluorescence peak T, CdEOMARepresent the intensity of fluorescence peak A in dEOM, CdEOMCRepresent the intensity of fluorescence peak C in dEOM, CchlaGeneration
The concentration of table water sample Determination of Chlorophyll a, unit are μ gL-1, CMC-LRRepresent the concentration of microcapsule phycotoxin MC-LR in water sample, μ gL-1。
The unitary linear inversion equation of 4 Taihu Lake microcapsule phycotoxin MC-LR concentration of table
According to the coefficient of determination, 3 metrics evaluation tables 4 of root-mean-square error and root mean square relative error construct regression equation,
As a result such as table 5, table 6, table 7.Fig. 3, Fig. 4 are respectively the MC-LR concentration of each fluorescence peak inverting compared with measured concentration variation tendency,
The MC-LR concentration and measured concentration variation tendency of each fluorescence peak inverting are almost the same.Wherein dEOM fluorescence peak S, T and bEOM fluorescence
The MC-LR concentration of peak D, T inverting and the coefficient of determination R of measured concentration2Respectively 0.722,0.663,0.744,0.841, just
Root error is 0.761 μ gL-1、0.806μg·L-1、0.715μg·L-1、0.579μg·L-1, root mean square relative error is respectively
50.35%, 52.19%, 47.11%, 37.10% (table 5, table 6, table 7).Wherein, the efficiency of inverse process of bEOM fluorescence peak T is best,
Sampling period the 14th, 20,24,29,30 day measured value and inverting concentration value is closer to, and measured value is respectively as follows: 0.58 μ gL-1、
2.07μg·L-1、3.87μg·L-1、0.72μg·L-1、0.69μg·L-1, corresponding inverting concentration value are as follows: 0.62 μ gL-1、
2.18μg·L-1、2.92μg·L-1、0.79μg·L-1、0.67μg·L-1.And dEOM fluorescence peak A, C and bEOM fluorescence peak C inverting
The 2nd concentration of specimens value deviation it is larger, lead to the reduction of its coefficient of determination, root-mean-square error and root mean square relative error increase.
Exclude the 2nd sample, the coefficient of determination of inverting concentration and measured concentration rises to 0.697,0.739,0.505 respectively, just
Root error is reduced to 1 μ gL-1Hereinafter, and root mean square relative error reduces 39.96%, 48.87%, 14.97% respectively.
The coefficient of determination of table 5 MC-LR inverting concentration and measured concentration
The root-mean-square error of table 6 MC-LR inverting concentration and measured concentration
The root mean square relative error of table 7 MC-LR inverting concentration and measured concentration
Claims (7)
1. a kind of inversion method of Microcystins in Water MC-LR concentration characterized by comprising
(1) micro- in water sample using the normalized characteristic fluorescence peak intensity of the extracellular organic matter of the green Microcystis aeruginosa of Copper in Water Samples as independent variable
The normalized MC-LR concentration of capsule algae toxin be dependent variable, construct unitary linear inversion equation, wherein characteristic fluorescence peak intensity and
Microcapsule phycotoxin MC-LR concentration is normalized with water sample Determination of Chlorophyll a concentration;
(2) water sample to be detected is taken, the three-dimensional excitation-emission matrix fluorescence light of the extracellular organic matter of the green Microcystis aeruginosa of Copper in Water Samples is measured
Spectrum obtains the characteristic fluorescence peak intensity of the extracellular organic matter of microcystic aeruginosa;
(3) the characteristic fluorescence peak intensity that step (2) obtain is substituted into the unitary linear inversion equation built, is obtained micro-
The concentration of capsule algae toxin MC-LR.
2. the inversion method of Microcystins in Water MC-LR concentration according to claim 1, which is characterized in that step
(1) in, the construction method of unitary linear inversion equation includes:
(a) water sampling;
(b) reference standard method is measured the content of the microcapsule phycotoxin MC-LR in water sample;
(c) the extracellular organic matter of microcystic aeruginosa in water sample is extracted, the three-dimensional of the extracellular organic matter of the green Microcystis aeruginosa of Copper in Water Samples is measured
Excitation-emission matrix fluorescence spectrum obtains the characteristic fluorescence peak intensity of the extracellular organic matter of microcystic aeruginosa;
(d) the algae chlorophyll-a concentration in water sample is measured;
(e) dense with chlorophyll a to the characteristic fluorescence peak intensity and microcapsule phycotoxin MC-LR concentration of the extracellular organic matter of microcystic aeruginosa
Degree is normalized;
(f) using normalized characteristic fluorescence peak intensity as independent variable, normalized microcapsule phycotoxin MC-LR concentration is because becoming
Amount, establishes unitary linear inversion equation.
3. the inversion method of Microcystins in Water MC-LR concentration according to claim 1, which is characterized in that step
(1) or in step (2), the extracellular organic matter of microcystic aeruginosa is the extracellular organic matter of solubilised state or the extracellular organic matter of reference state;For
The extracellular organic matter of solubilised state, characteristic fluorescence peak are selected from fluorescence peak S, T, A or C, organic matter extracellular for reference state, characteristic fluorescence peak
Selected from fluorescence peak D, T or C, wherein fluorescence peak A represents ultraviolet humic-like substance, and fluorescence peak C represents visible light humus, fluorescence peak S
Low excitation wavelength class tryptophan is represented, fluorescence peak T represents high excitation wavelength class tryptophan, and fluorescence peak D represents low excitation wavelength class
Tryptophan.
4. the inversion method of Microcystins in Water MC-LR concentration according to claim 1, which is characterized in that extracellular
The extracting method of organic matter includes: water sampling to be centrifuged in 0~6 DEG C, obtains precipitating and supernatant, the supernatant with aperture 0.22~
0.45 μm of membrane filtration, filtrate are the extracellular organic solution of solubilised state;It is rinsed with water after filter membrane and is mixed again with the precipitating, 60~
70 DEG C of 20~30min of heating, after centrifugation, supernatant 0.22~0.45 μm of aperture membrane filtration, filtrate, which is that reference state is extracellular, to be had
Machine object solution.
5. the inversion method of Microcystins in Water MC-LR concentration according to claim 1 or 2, which is characterized in that step
Suddenly in (2) or step (c), when three-dimensional excitation-emission matrix fluorescence spectral measuring, parameter setting is as follows: excitation wavelength scans model
It encloses: 220~660nm, launch wavelength scanning range: 240~700nm.
6. the inversion method of Microcystins in Water MC-LR concentration according to claim 1 or 2, which is characterized in that three
After tieing up excitation-emission matrix fluorescence data elimination Rayleigh scattering and Raman scattering, characteristic fluorescence peak intensity is obtained.
7. the inversion method of Microcystins in Water MC-LR concentration according to claim 1, which is characterized in that unitary
Linear inversion equation in following table any one, in unitary linear inversion equation, CbEOMDRepresent the strong of fluorescence peak D in bEOM
Degree, CbEOMTRepresent the intensity of fluorescence peak T in bEOM, CbEOMCRepresent the intensity of fluorescence peak C in bEOM, CdEOMSIt represents glimmering in dEOM
The intensity of photopeak S, CdEOMTRepresent the intensity of fluorescence peak T in dEOM, CdEOMARepresent the intensity of fluorescence peak A in dEOM, CdEOMCIt represents
The intensity of fluorescence peak C, C in dEOMchlaThe concentration of water sample Determination of Chlorophyll a is represented, unit is μ gL-1, CMC-LRIt represents micro- in water sample
The concentration of capsule algae toxin MC-LR, μ gL-1:
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CN113655042A (en) * | 2021-09-07 | 2021-11-16 | 中冶华天工程技术有限公司 | Method for quickly identifying organic matters in source water algae and application |
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