CN110441223A - Method for evaluating toxicity of solid particles by using luminous bacteria - Google Patents

Method for evaluating toxicity of solid particles by using luminous bacteria Download PDF

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CN110441223A
CN110441223A CN201910623602.5A CN201910623602A CN110441223A CN 110441223 A CN110441223 A CN 110441223A CN 201910623602 A CN201910623602 A CN 201910623602A CN 110441223 A CN110441223 A CN 110441223A
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photobacteria
correction
bacterium
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袁楠楠
鲍安平
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Nanjing Vocational College Of Information Technology
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34Purifying; Cleaning
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38Diluting, dispersing or mixing samples
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Optical investigation techniques, e.g. flow cytometry
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76Chemiluminescence; Bioluminescence
    • G01N21/763Bioluminescence

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Abstract

The invention discloses a method for evaluating toxicity of solid particles by utilizing luminous bacteria, which comprises the following steps of collecting solid sediments to be evaluated, diluting the solid sediments with a solvent after pretreatment to obtain experimental samples with different concentrations, taking the solvent used for dilution as a blank sample, adding luminous bacteria liquid into each experimental sample and the blank sample, filtering the samples with a filter membrane after exposing for 15 ~ 20min to obtain filtrate, taking the filtrate with the same volume and placing the filtrate in a water quality rapid toxicity detector, measuring the luminous value of the luminous bacteria after 5min, calculating the bacterial loss rate of each experimental sample and the blank sample based on a flow cytometry method, calculating the luminous value of the luminous bacteria after correction of each experimental sample and the blank sample according to the bacterial loss rate, calculating the luminous value inhibition rate after correction according to the luminous value of the luminous bacteria after correction, and evaluating toxicity of the solid particles.

Description

A method of solid particle toxicity evaluation is carried out using photobacteria
Technical field
The invention belongs to technical field of environmental detection, and in particular to a kind of to be commented using photobacteria progress solid particle toxicity The method estimated.
Background technique
Photobacteria is used for environment pollution detection, and photobacteria is exposed to be measured by traditional photobacteria toxicity test After substance, the variation of photobacteria luminous intensity is detected, the toxicity of tested substance is characterized.
The photobacteria detection method of toxicity of solid matter at present usually first prepares leaching liquor, i.e. solid matter and extraction The mixed filtered fluid of agent, then carries out photobacteria toxicity test with leaching liquor.As application for a patent for invention one kind is based on shining The soil comprehensive toxicity detection method (application No. is 201710407732.6, Publication No. CN107238599A) of bacterium method, packet It includes following steps: pedotheque is pre-processed;Soil sample is weighed, uses methanol Soxhlet extraction after crossing nylon mesh, decompression rotation is steamed Hair is concentrated, and continues to be concentrated by the mixture after addition ethylene glycol spare;Sample leaching is added in photobacteria culture solution after taking culture Extract, NaCl solution and pure water are stood after mixing, measure luminous intensity;The expression of sample toxicity: mixed liquor after measurement is stood Luminous inhibiting rate, mercury chloride equivalent concentration.The application for a patent for invention constructs a kind of convenient side of soil comprehensive toxicity detection Method, still, the toxicity of photobacteria are based on leaching liquor, and this method is practical, and reflect is that digestion agent can be turned in solid matter Fractions toxicity, and do not detect the toxicity of ingredient that is in conjunction with solid particle and not turning to digestion agent, can not assess The authentic virulence of solid matter keeps experimental result inaccurate.
Summary of the invention
It is an object of the invention to overcome deficiency in the prior art, provide it is a kind of utilize photobacteria carry out solid particle The method of toxicity evaluation can really assess the toxicity of solid particle, as a result accurately and reliably.
The present invention provides the following technical solutions:
A method of solid particle toxicity evaluation is carried out using photobacteria, comprising the following steps:
Solid deposits to be measured are acquired, the laboratory sample for obtaining various concentration are diluted after pretreatment, with solvent to dilute Solvent is blank sample;
Photobacteria bacterium solution is added in each laboratory sample and blank sample, exposure 15~20min, preferably 15min, with Membrane filtration obtains filtrate, takes the filtrate of same volume to be placed in water quality immediate toxicities detector, photobacteria is measured after 5min Luminous value;
The bacterium loss late of each laboratory sample and blank sample is calculated based on flow cytometry assay;
Photobacteria luminous value after calculating each laboratory sample and blank sample correction according to the bacterium loss late;
The luminous value inhibiting rate after correction is calculated according to the photobacteria luminous value after correction, toxicity is carried out to solid particle Assessment.
Preferably, the preprocess method of the solid deposits to be measured includes: to remove impurity with mesh screen, air-dries, then breaks Diameter is broken to less than 2mm, obtains solid particle to be measured.
Preferably, the preparation method of the laboratory sample include: by the solid particle to be measured and 3% sodium chloride solution by The ratio of 1:10 (w/v) is uniformly mixed, and then dilutes the laboratory sample for obtaining various concentration as solvent using 3% sodium chloride solution.
Preferably, the flow cytometry assay the following steps are included:
It filters to get filtrate after photobacteria is added into each laboratory sample and blank sample;
Another group of laboratory sample and blank sample are directly filtered to get filtrate;
Dyestuff Syto13 is added into gained filtrate, until its concentration is 5 μm of ol/L, cultivates 15min at dark at room temperature, so It distinguishes and counts using bacterial cell of the flow cytometer to dyeing afterwards.
Preferably, the bacterium loss late is calculated by formula (1):
RLoss=[1- (CExperiment 1-CExperiment 0)/(CBlank 1-CBlank 0)] × 100% (1)
R in formula (1)LossIndicate bacterium loss late, CExperiment 1And CBlank 1It respectively indicates laboratory sample and hair is added in blank sample The quantity of bacterium, C in photobacteria filtering gained filtrateExperiment 0And CBlank 0It respectively indicates laboratory sample and blank sample and directly filters institute Obtain the quantity of bacterium in filtrate.
Preferably, the photobacteria luminous value after the correction is calculated by formula (2):
LCorrection=LMeasurement×(1+RLoss) (2)
L in formula (2)CorrectionPhotobacteria luminous value after indicating correction, LMeasurementIndicate the luminous value of the photobacteria of measurement, RLossIndicate bacterium loss late.
Preferably, the luminous value inhibiting rate after the correction is calculated by formula (3):
RCorrection=(La-Lb)/La× 100% (3)
R in formula (3)CorrectionLuminous value inhibiting rate after indicating correction, LaPhotobacteria hair after indicating blank sample correction Light value, LbPhotobacteria luminous value after indicating laboratory sample correction.
Preferably, the photobacteria includes Fei Shi orphan bacterium.
Compared with prior art, the beneficial effects of the present invention are:
(1) photobacteria bacterium solution is added in the laboratory sample with solid particle and solvent the present invention, then measurement hair The luminous value of photobacteria while capable of turning to digestion agent some toxic in detecting solid particle, detects in solid particle not The toxicity for turning to the part of digestion agent, can really assess the toxicity of solid particle, evade the deficiency of conventional method;
(2) the present invention is based on the bacterium loss lates that flow cytometry assay calculates each laboratory sample and blank sample, in turn Photobacteria luminous value and luminous value inhibiting rate after calculating correction, as a result accurately and reliably;
(3) process of the present invention is simple, at low cost, easy to operate.
Detailed description of the invention
Fig. 1 is the flow cytometry scattergram that the laboratory sample that dilution is 25% in embodiment is shown;
Fig. 2 is the bacterium loss late schematic diagram of laboratory sample in embodiment;
Fig. 3 is the relation schematic diagram of the loss late of Fei Shi orphan bacterium and solid particle ratio in embodiment laboratory sample;
Fig. 4 is the luminous value inhibiting rate schematic diagram after correcting in embodiment;
Fig. 5 is the solid phase toxicity test of deposit 1 in comparative example and the luminous intensity inhibition ratio pair of leaching liquor toxicity test Than figure;
Fig. 6 is the solid phase toxicity test of deposit 2 in comparative example and the luminous intensity inhibition ratio pair of leaching liquor toxicity test Than figure;
Fig. 7 is the solid phase toxicity test of deposit 3 in comparative example and the luminous intensity inhibition ratio pair of leaching liquor toxicity test Than figure;
Fig. 8 is the solid phase toxicity test of deposit 4 in comparative example and the luminous intensity inhibition ratio pair of leaching liquor toxicity test Than figure.
Specific embodiment
The invention will be further described below in conjunction with the accompanying drawings.Following embodiment is only used for clearly illustrating the present invention Technical solution, and not intended to limit the protection scope of the present invention.
Embodiment-solid phase toxicity test
A method of solid particle toxicity evaluation is carried out using photobacteria, comprising the following steps:
Step 1: selection 4 different lakes, acquire 0~10cm of surface layer deposit with grab sampler, cross 1.8mm mesh screen Impurity is removed, is air-dried, partial size is crushed to less than 2mm, obtains solid particle to be measured, is denoted as sediment 1, sediment 2, precipitating respectively Object 3 and sediment 4;3g solid particle to be measured is mixed with 30mL solvent (3% (w/v) sodium chloride solution), in magnetic stirring apparatus Upper stirring 10min, mixes well it, as mother liquor (100% solution), then using 3% sodium chloride solution as solvent, passes through 1:2 continuous gradient dilutions method prepares the laboratory sample of 50%, 25% and 12.5% concentration, using 3% sodium chloride solution as blank sample Product.
Step 2: Fei Shi orphan's bacterium bacterium solution is added in the laboratory sample and blank sample of each concentration, after exposure 15min with 15 μm of membrane filtrations obtain filtrate, and the filtrate of same volume is taken to be placed in water quality immediate toxicities detector, measure and shine after 5min The luminous value of bacterium.
Step 3: carrying out flow cytometry, filtered after Fei Shi orphan bacterium is added into each laboratory sample and blank sample Filtrate;Another group of laboratory sample and blank sample are directly filtered to get filtrate;It is added after Fei Shi orphan bacterium only into blank sample Filter to obtain blank bacterium solution;Dyestuff Syto13 is added into gained filtrate and blank bacterium solution, until its concentration is 5 μm of ol/L, it is black at room temperature 15min is cultivated in dark place, is then distinguished and is counted using bacterial cell of the flow cytometer to dyeing.
Step 4: calculating bacterium loss late by formula (1):
RLoss=[1- (CExperiment 1-CExperiment 0)/(CBlank 1-CBlank 0)] × 100% (1)
R in formula (1)LossIndicate bacterium loss late, CExperiment 1And CBlank 1It respectively indicates laboratory sample and hair is added in blank sample The quantity of bacterium, C in photobacteria filtering gained filtrateExperiment 0And CBlank 1It respectively indicates laboratory sample and blank sample and directly filters institute Obtain the quantity of bacterium in filtrate.
Step 5: calculating the photobacteria luminous value after correction by formula (2):
LCorrection=LMeasurement×(1+RLoss) (2)
L in formula (2)CorrectionPhotobacteria luminous value after indicating correction, LMeasurementIndicate the luminous value of the photobacteria of measurement, RLossIndicate bacterium loss late.
Step 6: calculating the luminous value inhibiting rate after the correction by formula (3), toxicity is carried out to solid particle and is commented Estimate:
RCorrection=(La-Lb)/La× 100% (3)
R in formula (3)CorrectionLuminous value inhibiting rate after indicating correction, LaPhotobacteria hair after indicating blank sample correction Light value, LbPhotobacteria luminous value after indicating laboratory sample correction.
As shown in Figure 1, illustrating the fluidic cell in the present embodiment by taking dilution is 25% laboratory sample solution as an example The scattergram of analysis, wherein deposit 11, deposit 21, deposit 31With deposit 41Respectively indicate deposit 1, deposit 2, The filtrate filtered after Fei Shi orphan bacterium, deposit 1 are added in the laboratory sample of deposit 3 and deposit 40, deposit 20, deposit 30With deposit 40Respectively indicating the laboratory sample of deposit 1, deposit 2, deposit 3 and deposit 4, to be not added with Fei Shi orphan bacterium straight Take over the filtrate of filter, blank controlFilteringIt indicates to add the filtrate filtered after Fei Shi orphan bacterium, blank control in blank sampleIt does not filterIt indicates The solution not filtered after Fei Shi orphan bacterium is added in blank sample.As seen from the figure, Fei Shi orphan bacterium in the solution of blank sample filtering front and back Quantity between there is no difference, illustrate experiment used in filter membrane do not retain Fei Shi orphan bacterium, experimental result is not had an impact; In four groups of laboratory samples, the point added in the sample filtrate figure of Fei Shi orphan bacterium is less than its corresponding control group, illustrates laboratory sample Loss phenomenon occurs for the Fei Shi orphan bacterium in filtrate;Also occur less in addition, being not added in the laboratory sample filtrate figure of Fei Shi orphan bacterium Point is measured, illustrates that four laboratory samples have endogenous bacterium.
Fig. 2 is the bacterium loss late schematic diagram of laboratory sample, it can be seen that existing in the laboratory sample of various concentration The loss of Fei Shi orphan bacterium, and loss late increases with the increase of solid particle ratio in laboratory sample solution, deposit 4 sinks In product object 3, the corresponding laboratory sample of deposit 2 and deposit 1 loss late of bacterium be respectively 50.5%~93.6%, 66.7%~ 89.8%, 62.0%~83.7% and 30.2%~79.2%.
Fig. 3 is the relationship of the loss late and solid particle ratio in laboratory sample of Fei Shi orphan bacterium in various concentration laboratory sample Schematic diagram, it can be seen that Fei Shi is lonely in the laboratory sample of deposit 4,1 various concentration of deposit 3, deposit 2 and deposit Solid particle ratio in the loss ratio and laboratory sample of bacterium is directly related, shows significant log-log linear relationship, R2 0.830,0.959,0.965 and 0.837 is respectively reached, therefore, it is possible to use regression parameter is sent out to calculate the Fei Shi orphan bacterium of correction Luminous intensity, it can calculate the photobacteria luminous value after correction by formula (2).
Fig. 4 is that luminous value inhibiting rate schematic diagram is exposed to deposit 1, deposit 2, deposit 3 as seen from the figure after correcting Be respectively -484%~-175% with the luminous intensity inhibition ratio of the Fei Shi orphan bacterium after the various concentration laboratory sample of deposit 4, - 43.8%~2.36%, -31.2%~18.3% and 4.46%~42.0%.The solid particle of four sediments is to Fei Shi orphan bacterium The authentic virulence of luminous intensity are as follows: 1 solid particle of deposit promotes the luminous intensity of Fei Shi orphan bacterium, deposit 2 and deposit 3 Solid particle promotes the luminous intensity of Fei Shi orphan bacterium when concentration is less than 100% and 75% respectively, and 4 solid particle of deposit is dense Degree generates inhibition to the luminous intensity of Fei Shi orphan bacterium after being greater than 12.5%.
Comparative example-leaching liquor toxicity test
The laboratory sample and blank sample of each concentration obtained in Example step 1 are obtained with 0.45 μm of membrane filtration Fei Shi orphan bacterium bacterium solution is added into filtrate for filtrate (leaching liquor), after exposing 15min, takes the filtrate of same volume to be placed in water quality fast In fast toxicity detector, the luminous value of photobacteria is measured after 5min.
Inhibited by uncorrected luminous value inhibiting rate in formula (4) calculating embodiment and the luminous value in this comparative example Rate:
RMeasurement=(LBlank-LExperiment)/LBlank× 100% (4)
R in formula (4)MeasurementIndicate luminous value inhibiting rate, LBlankIndicate the bacterial luminescence value of blank sample measurement, LExperimentIt indicates The bacterial luminescence value of laboratory sample measurement.
Fig. 5,6,7,8 are respectively deposit 1, deposit 2, sediment 3 and 4 solid phase toxicity test of sediment and leaching liquor poison Property test in Fei Shi orphan's bacterium luminous intensity inhibition ratio schematic diagram.It can be seen that in solid phase toxicity test, 1 group of bacterium of deposit Luminosity be promoted, deposit 2,4 groups of bacterium of sediment 3 and sediment luminous intensity be suppressed.In leaching liquor toxicity In experiment, deposit 1 still shows facilitation to the luminous intensity of photobacteria, but the degree promoted is compared in solid phase experiment It is weak, illustrate that 1 solid particle of deposit is conducive to the luminous function of Fei Shi orphan bacterium;Deposit 2 is only just showed when concentration is 100% Inhibiting effect out;The inhibiting effect that shines, and inhibiting effect are shown under deposit 3 and each concentration experiment sample of deposit 4 Intensity is significantly lower than the intensity in corresponding solid phase toxicity test.
There is leaching liquor toxicity test result the main reason for difference to be in solid phase toxicity test and comparative example in embodiment Fei Shi orphan bacterium is mainly influenced by the material composition being dissolved in leaching liquor in leaching liquor toxicity test, and real in solid phase toxicity The ingredient for testing Fei Shi orphan bacterium while being exposed to the ingredient being fixed on solid particle and being dissolved in digestion agent.In addition, Fei Shi Lonely bacterium is fixed on fine particle and/or organic matter the luminous intensity that can reduce them.It is thin with streaming in the embodiment of the present invention Born of the same parents' analytical technology quantifies Fei Shi orphan bacterium, corrects photobacteria luminous value and meter by calculating the loss late of Fei Shi orphan bacterium True luminous value inhibiting rate is calculated, keeps result accurate and reliable, and then really assesses the toxicity of solid particle.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, several improvement and deformations can also be made, these improvement and deformations Also it should be regarded as protection scope of the present invention.

Claims (8)

1. a kind of method for carrying out solid particle toxicity evaluation using photobacteria, which comprises the following steps:
Solid deposits to be measured are acquired, dilute the laboratory sample for obtaining various concentration after pretreatment with solvent, used with dilution Solvent is blank sample;
Photobacteria bacterium solution is added in each laboratory sample and blank sample, is obtained after 15~20min of exposure with membrane filtration Filtrate takes the filtrate of same volume to be placed in water quality immediate toxicities detector, and the luminous value of photobacteria is measured after 5min;
The bacterium loss late of each laboratory sample and blank sample is calculated based on flow cytometry assay;
Photobacteria luminous value after calculating each laboratory sample and blank sample correction according to the bacterium loss late;
The luminous value inhibiting rate after correction is calculated according to the photobacteria luminous value after correction, toxicity is carried out to solid particle and is commented Estimate.
2. the method according to claim 1 for carrying out solid particle toxicity evaluation using photobacteria, which is characterized in that institute The preprocess method for stating solid deposits to be measured includes: to remove impurity with mesh screen, is air-dried, and is then crushed to diameter less than 2mm, obtains Obtain solid particle to be measured.
3. the method according to claim 2 for carrying out solid particle toxicity evaluation using photobacteria, which is characterized in that institute Stating the preparation method of laboratory sample includes: that the solid particle to be measured and 3% sodium chloride solution is mixed in the ratio of 1:10 (w/v) It closes uniformly, then dilutes the laboratory sample for obtaining various concentration as solvent using 3% sodium chloride solution.
4. the method according to claim 1 for carrying out solid particle toxicity evaluation using photobacteria, which is characterized in that institute State flow cytometry assay the following steps are included:
It filters to get filtrate after photobacteria is added into each laboratory sample and blank sample;
Another group of laboratory sample and blank sample are directly filtered to get filtrate;
Dyestuff Syto13 is added into gained filtrate, until its concentration is 5 μm of ol/L, cultivates 15min at dark at room temperature, then makes It is distinguished and is counted with bacterial cell of the flow cytometer to dyeing.
5. the method according to claim 4 for carrying out solid particle toxicity evaluation using photobacteria, which is characterized in that logical It crosses formula (1) and calculates the bacterium loss late:
RLoss=[1- (CExperiment 1-CExperiment 0)/(CBlank 1-CBlank 0)] × 100% (1)
R in formula (1)LossIndicate bacterium loss late, CExperiment 1And CBlank 1It respectively indicates laboratory sample and blank sample is added and shines carefully The quantity of bacterium, C in bacterium filtering gained filtrateExperiment 0And CBlank 0It respectively indicates laboratory sample and blank sample and directly filters gained filter The quantity of bacterium in liquid.
6. the method for carrying out solid particle toxicity evaluation using photobacteria according to claim 1 or 5, feature exist In passing through formula (2) and calculate the photobacteria luminous value after the correction:
LCorrection=LMeasurement×(1+RLoss) (2)
L in formula (2)CorrectionPhotobacteria luminous value after indicating correction, LMeasurementIndicate the luminous value of the photobacteria of measurement, RLoss Indicate bacterium loss late.
7. the method according to claim 1 for carrying out solid particle toxicity evaluation using photobacteria, which is characterized in that logical It crosses formula (3) and calculates the luminous value inhibiting rate after the correction:
RCorrection=(La-Lb)/La× 100% (3)
R in formula (3)CorrectionLuminous value inhibiting rate after indicating correction, LaPhotobacteria luminous value after indicating blank sample correction, LbPhotobacteria luminous value after indicating laboratory sample correction.
8. the method according to claim 1 for carrying out solid particle toxicity evaluation using photobacteria, which is characterized in that institute Stating photobacteria includes Fei Shi orphan bacterium.
CN201910623602.5A 2019-07-11 2019-07-11 Method for evaluating toxicity of solid particles by using luminous bacteria Pending CN110441223A (en)

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Application publication date: 20191112