CN110186886B - Inversion method of microcystin MC-LR concentration in water body - Google Patents
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Abstract
The invention discloses an inversion method of microcystin MC-LR concentration in water, which comprises the following steps: the method comprises the following steps of (1) constructing a unitary linear inversion equation by taking the normalized characteristic fluorescence peak intensity of extracellular organic matters of Microcystis aeruginosa in a water sample as an independent variable and the normalized microcystin MC-LR concentration in the water sample as a dependent variable, wherein the characteristic fluorescence peak intensity and the microcystin MC-LR concentration are normalized by the chlorophyll a concentration in the water sample; taking a water sample to be detected, measuring a three-dimensional excitation emission matrix fluorescence spectrum of the extracellular organic matters of the microcystis aeruginosa in the water sample to obtain the characteristic fluorescence peak intensity of the extracellular organic matters of the microcystis aeruginosa, and then substituting the characteristic fluorescence peak intensity into a unitary linear inversion equation to obtain the concentration of microcystin MC-LR. The method does not need sample pretreatment, can realize the rapid on-line monitoring of the concentration of microcystin MC-LR, takes the characteristic fluorescence peak intensity of extracellular organic matters of the microcystin aeruginosa as a variable for constructing an equation, and provides a new idea for an inversion method.
Description
Technical Field
The invention relates to water environment detection, in particular to an inversion method of microcystin MC-LR concentration in water.
Background
A large amount of microcystin MC-LR enters the water body, and seriously threatens the safety of drinking water. The microcystin MC-LR is mainly generated by microcystis aeruginosa in water. At present, conventional concentration detection methods are classified into a biological detection method, a physicochemical detection method, a biochemical detection method and a molecular biological detection method, and the detection methods are complex in water sample pretreatment, long in time consumption and high in cost, and cannot realize rapid online monitoring. The inversion method can utilize influence factors highly related to the concentration of MC-LR to construct an inversion model to indirectly reflect the change of the concentration of MC-LR of microcystin in the water body. A plurality of researchers establish a multiple linear regression equation by utilizing influence factors such as the concentration of nutrient substances (such as nitrogen, phosphorus and the like), temperature, dissolved oxygen and the like in a water body, and invert the concentration of the microcystin MC-LR, but the concentration of the microcystin MC-LR and each influence factor have different interrelations at different time and different places. Therefore, the inversion model cannot be used universally.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides an inversion method of microcystin MC-LR concentration in water, complex pretreatment is not needed, and extracellular organic matters from physiological metabolism of microcystin aeruginosa are adopted as variables.
The technical scheme is as follows: the inversion method of microcystin MC-LR concentration in water comprises the following steps:
(1) taking the normalized characteristic fluorescence peak intensity of extracellular organic matters of the microcystis aeruginosa in the water sample as an independent variable and the normalized MC-LR concentration of the microcystis toxins in the water sample as a dependent variable, and constructing a unitary linear inversion equation, wherein the normalized characteristic fluorescence peak intensity and the normalized MC-LR concentration of the microcystis toxins are normalized according to the chlorophyll a concentration in the water sample;
(2) taking a water sample to be detected, and measuring a three-dimensional excitation emission matrix fluorescence spectrum of the extracellular organic matters of the Microcystis aeruginosa in the water sample to obtain the characteristic fluorescence peak intensity of the extracellular organic matters of the Microcystis aeruginosa;
(3) and (3) substituting the characteristic fluorescence peak intensity obtained in the step (2) into the constructed unitary linear inversion equation to obtain the concentration of the microcystin MC-LR.
Specifically, in the step (1), the construction method of the unary linear inversion equation includes:
(a) taking a water sample;
(b) determining the content of microcystin MC-LR in a water sample by reference to a standard method;
(c) extracting the extracellular organic matters of the Microcystis aeruginosa in the water sample, and measuring the three-dimensional excitation emission matrix fluorescence spectrum of the extracellular organic matters of the Microcystis aeruginosa in the water sample to obtain the characteristic fluorescence peak intensity of the extracellular organic matters of the Microcystis aeruginosa;
(d) measuring the concentration of algae chlorophyll a in a water sample;
(e) carrying out normalization treatment on the characteristic fluorescence peak intensity of the extracellular organic matters of the microcystins aeruginosa and the concentration of MC-LR of the microcystins by the concentration of chlorophyll a;
(f) and establishing a unitary linear inversion equation by taking the normalized characteristic fluorescence peak intensity as an independent variable and the normalized microcystin MC-LR concentration as a dependent variable.
When a unitary linear inversion equation is constructed, the standard method in the step (b) refers to other different reliable detection methods, for example, a national standard GB/T20466-2006 method can be adopted.
According to the tight degree of combination with algae cells, extracellular organic matter components such as albuminoid, humoid and the like in the water body can be divided into combined state (bEOM) and dissolved state (dEOM) extracellular organic matter, and the extracellular organic matter components and microcystin MC-LR mainly come from the physiological metabolic process of microcystis aeruginosa, and the release rule is similar.
In the step (c), the method for extracting extracellular organic substances comprises the following steps: centrifuging a water sample at 0-6 ℃ to obtain a precipitate and a supernatant, filtering the supernatant by using a filter membrane with the pore diameter of 0.22-0.45 mu m, wherein the filtrate is a dissolved extracellular organic matter solution; washing a filter membrane with water, mixing with the precipitate, heating at 60-70 ℃ for 20-30 min, centrifuging, filtering the supernatant with a filter membrane with the pore diameter of 0.22-0.45 mu m, and obtaining a filtrate which is a combined extracellular organic matter solution. The centrifugation can be carried out at 8000-9000 g for 5-10 min. In the step (2), the water sample to be detected is extracted by the method, and three-dimensional fluorescence spectrum detection is carried out after extraction.
In the step (c), when the three-dimensional fluorescence spectrum is measured, the parameters are set as follows: excitation wavelength scan range: 220-660 nm, emission wavelength scanning range: 240-700 nm. In the step (3), the parameters are also adopted to carry out three-dimensional fluorescence spectrum detection on the water sample to be detected.
In the step (c) or the step (2), data preprocessing is required for obtaining the fluorescence spectrum, and the characteristic fluorescence peak intensity is generally obtained after Rayleigh scattering and Raman scattering are eliminated.
In the step (C), for dissolved extracellular organic matters, the characteristic fluorescence peak is selected from S, T, A or C, and for combined extracellular organic matters, the characteristic fluorescence peak is selected from D, T or C, wherein the fluorescence peak A represents ultraviolet humus, the fluorescence peak C represents visible light humus, the excitation wavelength is 250-260 nm, and the emission wavelength is 380-460 nm; the fluorescence peak S represents low excitation wavelength tryptophan, the excitation wavelength is 220-230 nm, and the emission wavelength is 320-350 nm; the fluorescence peak T represents tryptophan with high excitation wavelength, the excitation wavelength is 270-280 nm, and the emission wavelength is 320-350 nm; the fluorescence peak D represents low excitation wavelength tryptophan, the excitation wavelength is 220-230 nm, and the emission wavelength is 300-310 nm. The characteristic peaks can be used for constructing a unitary linear inversion equation, wherein the equation constructed by the fluorescence peak T of the combined extracellular organic matter has the best inversion effect. The characteristic fluorescence peak intensity required to be obtained of the water sample to be detected corresponds to the equation.
When the concentration of the algae chlorophyll a in the water sample is measured, a 'lake eutrophication survey standard' method can be adopted.
The intensities of all characteristic fluorescence peaks of chlorophyll a normalization are extremely obviously related to the concentration of MC-LR of microcystin, a unitary linear inversion equation of the concentration of MC-LR is established by utilizing the intensities of all characteristic fluorescence peaks and can be selected for use as shown in the following table, and in the unitary linear inversion equation, C isbEOMDRepresents the intensity of the fluorescence peak D in bEOM, CbEOMTRepresenting the intensity of the fluorescence peak T in bEOM, CbEOMCRepresents the intensity of fluorescence peak C in bEOM, CdEOMSRepresents the intensity of the fluorescence peak S in dEOM, CdEOMTRepresents the intensity of the fluorescence peak T in dEOM, CdEOMARepresents the intensity of fluorescence peak A in dEOM, CdEOMCRepresents the intensity of the fluorescence peak C in dEOM, CchlaRepresents the concentration of chlorophyll a in a water sample with the unit of mu g.L-1，CMC-LRThe concentration of microcystin MC-LR in water sample is represented as mu g.L-1：
Has the advantages that:
the inversion method of the microcystin MC-LR concentration in the water body establishes a unitary linear inversion equation of the microcystin MC-LR by utilizing a three-dimensional excitation emission matrix fluorescence spectrum technology, does not need to carry out a complex pretreatment process on a water body sample, and is quick in detection.
The invention provides a new idea for an inversion method by taking the characteristic fluorescence peak intensity of the extracellular organic matter of the Microcystis aeruginosa as a variable of a construction equation, and the extracellular organic matter of the Microcystis aeruginosa is related to the growth and metabolism of the alga body, so that the adoption of factors such as nitrogen, phosphorus, temperature, dissolved oxygen and the like which are easy to change by external environment can be avoided, and the accuracy and the stability are better.
After chlorophyll a normalization treatment, the characteristic fluorescence peak of the extracellular organic matter of the Microcystis aeruginosa is highly related to the concentration of microcystin MC-LR, the regression equation constructed by the method is evaluated by 3 indexes of the determinant coefficient, the root mean square error and the root mean square relative error, the inversion effect is good, wherein the inversion effect of the combined high excitation wavelength tryptophan fluorescence peak T is best, and the determinant coefficient, the root mean square error and the root mean square relative error are respectively 0.841 mu g.L and 0.579 mu g.L-137.10%, and the regression coefficients of the inversion equation are relatively concentrated, so that continuous adjustment can be conveniently carried out in practical application to obtain the best inversion effect.
Drawings
FIG. 1 is a three-dimensional fluorescence spectrum of dissolved extracellular organic substances of Microcystis aeruginosa in Taihu lake;
FIG. 2 is a three-dimensional fluorescence spectrum of the extracellular organic substance of Microcystis aeruginosa in Taihu lake;
FIG. 3 is a comparison of the variation trend of the MC-LR concentration inverted by each fluorescence peak of dEOM with the actually measured concentration;
FIG. 4 is a comparison of the inverted MC-LR concentration of each fluorescence peak of bEOM with the observed concentration variation trend.
Detailed Description
The invention will be further elucidated with reference to the following specific examples.
The method for detecting microcystin MC-LR in water body comprises the following steps:
(1) sampling: in the outbreak period of the microcystis aeruginosa, 1L of water sample is taken at a monitoring point of the Wuxi Taihu lake by a sampler at 20-30 cm under water, and the number of the samples is 31. And randomly extracting 9 samples as samples to be inverted, and using other samples to construct an inversion equation.
(2) According to the national standard GB/T20466-2006 method, the Agilent 1200 liquid chromatograph is used for measuring the content of microcystin MC-LR in a water sample, and is used for establishing an inversion equation and performing inversion detection;
(3) extracting extracellular organic matters of the Microcystis aeruginosa in a water sample: the extracellular organic matter of the microcystis aeruginosa is divided into two parts of dEOM (dissolved extracellular organic matter) and bEOM (combined extracellular organic matter), and the extraction method comprises the following steps: centrifuging 5mL of water sample at 4 deg.C for 10min at 9000g, collecting supernatant, filtering with cellulose acetate membrane with pore diameter of 0.45 μm, and collecting filtrate for determining dEOM; rinsing cellulose acetate membrane with 5mL of ultrapure water, mixing with the centrifuged sediment obtained in the previous step, heating in 70 deg.C water bath for 20min, centrifuging at 4 deg.C for 10min at 9000g, collecting supernatant, filtering with 0.45 μm cellulose acetate membrane, and storing, wherein the filtrate is used for measuring bEOM.
(4) Acquiring three-dimensional fluorescence spectrum (three-dimensional excitation emission matrix fluorescence spectrum) data of extracellular organic matters of the Microcystis aeruginosa in a water sample: the three-dimensional fluorescence spectrum of the dissolved extracellular organic matter or the combined extracellular organic matter of each water sample is respectively measured by using a Hitachi F-7000 fluorescence spectrophotometer, and the parameters are set as follows: excitation wavelength scan range: 220-660 nm, emission wavelength scanning range: 240-700 nm, the excitation wavelength step length and the scanning wavelength step length are respectively 10nm and 2nm, the excitation slit and the emission slit are both 5nm, the scanning speed is 12000nm/min, the voltage of the photomultiplier is 700V, and the spectrum is not corrected. And (3) for the three-dimensional fluorescence spectrum data matrix, eliminating the influence of Rayleigh scattering by setting the numerical value to zero, and simultaneously deducting blank by the Milli-Q ultrapure water spectrum data to eliminate the influence of Raman scattering.
(4) And (3) determining the concentration of the chlorophyll a of algae in each water sample according to lake eutrophication survey standard compiled by the Ministry of metallographic British and Ministry of Tuqing, and segmenting the sample according to the concentration of the chlorophyll a.
(5) And normalizing the intensity of each fluorescence peak and the concentration of microcystin MC-LR by the concentration of chlorophyll a. The unit of MC-LR concentration and chlorophyll a concentration is μ g.L-1。
(6) Establishing a unitary linear regression equation by using SPSS software and taking the normalized fluorescence peak intensity as an independent variable and the normalized MC-LR concentration as a dependent variable;
(7) and evaluating the regression equation according to 3 indexes of the decision coefficient, the root mean square error and the root mean square relative error.
FIG. 1 and FIG. 2 are three-dimensional fluorescence spectra of dissolved extracellular organic substance (dEOM) and bound extracellular organic substance (bEOM) of Microcystis aeruginosa in Taihu lake, respectively. 4 strong fluorescence peaks in the dEOM three-dimensional spectrogram are respectively fluorescence peak S (Ex/Em:230/320), T (Ex/Em:280/330), A (Ex/Em:270/440) and C
(Ex/Em:350/440), wherein the fluorescence peak S, T represents a low excitation wavelength tryptophan fluorescence peak and a high excitation wavelength tryptophan fluorescence peak respectively, is related to proteins, amino acids, polypeptides and other substances, contains aromatic ring amino acids in the structure, and is mainly derived from the secretion and subsequent biodegradation of algae cells; the fluorescence peak A, C represents the ultraviolet region humoid fluorescence peak and the visible region humoid fluorescence peak, is related to components such as fulvic acid and humic acid, contains carboxyl, hydroxyl, phenolic hydroxyl and the like in the structure, and is generally considered to be formed by the humification of extracellular organic substances released by algae cells. besides the fluorescence peak T, A, C, the bEOM also has a tyrosine-like fluorescence peak D with the central excitation wavelength of 220nm and the emission wavelength of 300nm, the source of the tyrosine-like fluorescence peak D is similar to that of tryptophan-like, and the fluorescence peak position of the tyrosine-like fluorescence peak D is slightly influenced by the microenvironment. The main fluorescence peak positions in the water body are shown in table 1.
TABLE 1 main fluorescence peak position in water
The main fluorescence peak intensities are respectively subjected to correlation analysis with the MC-LR concentration, the results are shown in Table 2, the fluorescence peak intensities have no obvious correlation with the MC-LR concentration, and the fluorescence peaks cannot be directly used as independent variables of regression analysis. Therefore, the fluorescence peak intensities normalized by the chlorophyll a concentration and the MC-LR concentration were significantly correlated with the MC-LR concentration, except for the fluorescence peak a in bbeom (table 3).
TABLE 2 correlation of the intensity of each fluorescence peak of Microcystis aeruginosa in Taihu lake with the MC-LR concentration
TABLE 3 correlation of normalized intensity of each fluorescence peak of Microcystis aeruginosa in Taihu lake with MC-LR concentration
A unary linear inversion equation is established by utilizing the bEOM and dEOM fluorescence peaks highly correlated with the MC-LR concentration, and the result is shown in Table 4, wherein in the unary linear inversion equation, CbEOMDRepresents the intensity of the fluorescence peak D in bEOM, CbEOMTRepresenting the intensity of the fluorescence peak T in bEOM, CbEOMCRepresents the intensity of fluorescence peak C in bEOM, CdEOMSRepresents the intensity of the fluorescence peak S in dEOM, CdEOMTRepresents the intensity of the fluorescence peak T in dEOM, CdEOMARepresents the intensity of fluorescence peak A in dEOM, CdEOMCRepresents the intensity of the fluorescence peak C in dEOM, CchlaRepresents the concentration of chlorophyll a in a water sample with the unit of mu g.L-1，CMC-LRThe concentration of microcystin MC-LR in water sample is represented as mu g.L-1。
TABLE 4 one-dimensional linear inversion equation for concentration of microcystin MC-LR in Taihu lake
The regression equation constructed in table 4 was evaluated based on 3 indices of the coefficient of determination, the root mean square error and the root mean square relative error, and the results are shown in tables 5, 6 and 7. FIG. 3 and FIG. 4 are respectively a comparison of the MC-LR concentration inverted by each fluorescence peak and the actually measured concentration variation trend, and the MC-LR concentration inverted by each fluorescence peak and the actually measured concentration variation trend are basically consistent. MC with inversion of dEOM fluorescence peak S, T and bEOM fluorescence peak D, T-coefficient of determination of LR concentration versus measured concentration R20.722, 0.663, 0.744 and 0.841 respectively, and the root mean square error is 0.761 mug. L-1、0.806μg·L-1、0.715μg·L-1、0.579μg·L-1The relative root mean square errors were 50.35%, 52.19%, 47.11%, and 37.10%, respectively (tables 5, 6, and 7). Wherein, the inversion effect of the bEOM fluorescence peak T is the best, and the measured values of the 14 th, 20 th, 24 th, 29 th and 30 th days in the sampling period are closer to the inversion concentration values, and the measured values are respectively: 0.58. mu.g.L-1、2.07μg·L-1、3.87μg·L-1、0.72μg·L-1、0.69μg·L-1The corresponding inversion concentration values are: 0.62. mu.g.L-1、2.18μg·L-1、2.92μg·L-1、0.79μg·L-1、0.67μg·L-1. While the 2 nd sample concentration values inverted for dEOM fluorescence peak A, C and bEOM fluorescence peak C are more biased, resulting in a lower determinant and an increased root mean square error and relative root mean square error. The decision coefficients of the inversion concentration and the measured concentration of the 2 nd sample are respectively increased to 0.697, 0.739 and 0.505, and the root mean square error is reduced to 1 mug. L-1The relative root mean square errors were reduced by 39.96%, 48.87%, and 14.97%, respectively.
TABLE 5 determination of MC-LR inversion concentration and measured concentration
TABLE 6 root mean square error of MC-LR inversion concentration and measured concentration
TABLE 7 root mean square relative error of MC-LR inversion concentration and measured concentration
Claims (6)
1. An inversion method of microcystin MC-LR concentration in water is characterized by comprising the following steps:
(1) taking the normalized characteristic fluorescence peak intensity of extracellular organic matters of the microcystis aeruginosa in the water sample as an independent variable and the normalized MC-LR concentration of the microcystis toxins in the water sample as a dependent variable, and constructing a unitary linear inversion equation, wherein the normalized characteristic fluorescence peak intensity and the normalized MC-LR concentration of the microcystis toxins are normalized according to the chlorophyll a concentration in the water sample;
(2) taking a water sample to be detected, and measuring a three-dimensional excitation emission matrix fluorescence spectrum of the extracellular organic matters of the Microcystis aeruginosa in the water sample to obtain the characteristic fluorescence peak intensity of the extracellular organic matters of the Microcystis aeruginosa;
(3) substituting the characteristic fluorescence peak intensity obtained in the step (2) into a constructed unitary linear inversion equation to obtain the concentration of microcystin MC-LR;
the unary linear inversion equation is selected from any one of the following tables, and in the unary linear inversion equation, CbEOMDRepresents the intensity of the fluorescence peak D in bEOM, CbEOMTRepresenting the intensity of the fluorescence peak T in bEOM, CbEOMCRepresents the intensity of fluorescence peak C in bEOM, CdEOMSRepresents the intensity of the fluorescence peak S in dEOM, CdEOMTRepresents the intensity of the fluorescence peak T in dEOM, CdEOMARepresents the intensity of fluorescence peak A in dEOM, CdEOMCRepresents the intensity of the fluorescence peak C in dEOM, CchlaRepresents the concentration of chlorophyll a in a water sample with the unit of mu g.L-1，CMC-LRThe concentration of microcystin MC-LR in water sample is represented as mu g.L-1：
2. The inversion method of microcystin MC-LR concentration in water body according to claim 1, wherein in step (1), the construction method of the unitary linear inversion equation comprises:
(a) taking a water sample;
(b) determining the content of microcystin MC-LR in a water sample by reference to a standard method;
(c) extracting the extracellular organic matters of the Microcystis aeruginosa in the water sample, and measuring the three-dimensional excitation emission matrix fluorescence spectrum of the extracellular organic matters of the Microcystis aeruginosa in the water sample to obtain the characteristic fluorescence peak intensity of the extracellular organic matters of the Microcystis aeruginosa;
(d) measuring the concentration of algae chlorophyll a in a water sample;
(e) carrying out normalization treatment on the characteristic fluorescence peak intensity of the extracellular organic matters of the microcystins aeruginosa and the concentration of MC-LR of the microcystins by the concentration of chlorophyll a;
(f) and establishing a unitary linear inversion equation by taking the normalized characteristic fluorescence peak intensity as an independent variable and the normalized microcystin MC-LR concentration as a dependent variable.
3. The inversion method of microcystin MC-LR concentration in water body according to claim 1, wherein in step (1) or step (2), the extracellular organic matter of Microcystis aeruginosa is dissolved extracellular organic matter or combined extracellular organic matter; for dissolved extracellular organic matter, the characteristic fluorescence peak is selected from S, T, A or C, and for bound extracellular organic matter, the characteristic fluorescence peak is selected from D, T or C, wherein fluorescence peak A represents ultraviolet humus, fluorescence peak C represents visible light humus, fluorescence peak S represents low excitation wavelength tryptophan, fluorescence peak T represents high excitation wavelength tryptophan, and fluorescence peak D represents low excitation wavelength tryptophan.
4. The inversion method of microcystin MC-LR concentration in water body according to claim 1, wherein the extraction method of extracellular organic substances comprises: centrifuging a water sample at 0-6 ℃ to obtain a precipitate and a supernatant, filtering the supernatant by using a filter membrane with the pore diameter of 0.22-0.45 mu m, wherein the filtrate is a dissolved extracellular organic matter solution; washing a filter membrane with water, mixing with the precipitate, heating at 60-70 ℃ for 20-30 min, centrifuging, filtering the supernatant with a filter membrane with the pore diameter of 0.22-0.45 mu m, and obtaining a filtrate which is a combined extracellular organic matter solution.
5. The inversion method of microcystin MC-LR concentration in water body according to claim 1 or 2, wherein in step (2) or step (c), the parameters for the fluorescence spectrum measurement of the three-dimensional excitation emission matrix are set as follows: excitation wavelength scan range: 220-660 nm, emission wavelength scanning range: 240-700 nm.
6. The inversion method of microcystin MC-LR concentration in water according to claim 1 or 2, characterized in that the characteristic fluorescence peak intensity is obtained after Rayleigh scattering and Raman scattering are eliminated from the three-dimensional excitation emission matrix fluorescence spectrum data.
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