CN117849292A - Method for detecting toxicity of water body - Google Patents
Method for detecting toxicity of water body Download PDFInfo
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- CN117849292A CN117849292A CN202211206818.XA CN202211206818A CN117849292A CN 117849292 A CN117849292 A CN 117849292A CN 202211206818 A CN202211206818 A CN 202211206818A CN 117849292 A CN117849292 A CN 117849292A
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 231100000419 toxicity Toxicity 0.000 title claims abstract description 51
- 230000001988 toxicity Effects 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000000523 sample Substances 0.000 claims abstract description 48
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- 238000010521 absorption reaction Methods 0.000 claims abstract description 32
- -1 potassium ferricyanide Chemical compound 0.000 claims abstract description 30
- 239000000725 suspension Substances 0.000 claims abstract description 26
- 239000003440 toxic substance Substances 0.000 claims abstract description 26
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- 241000556533 uncultured marine bacterium Species 0.000 claims description 4
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- FRHBOQMZUOWXQL-UHFFFAOYSA-L ammonium ferric citrate Chemical compound [NH4+].[Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FRHBOQMZUOWXQL-UHFFFAOYSA-L 0.000 claims description 3
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- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 claims description 3
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- DCYOBGZUOMKFPA-UHFFFAOYSA-N iron(2+);iron(3+);octadecacyanide Chemical compound [Fe+2].[Fe+2].[Fe+2].[Fe+3].[Fe+3].[Fe+3].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] DCYOBGZUOMKFPA-UHFFFAOYSA-N 0.000 abstract description 12
- 229960003351 prussian blue Drugs 0.000 abstract description 12
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- 239000000276 potassium ferrocyanide Substances 0.000 abstract description 9
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 abstract description 9
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- WUPHOULIZUERAE-UHFFFAOYSA-N 3-(oxolan-2-yl)propanoic acid Chemical compound OC(=O)CCC1CCCO1 WUPHOULIZUERAE-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- PANJMBIFGCKWBY-UHFFFAOYSA-N iron tricyanide Chemical compound N#C[Fe](C#N)C#N PANJMBIFGCKWBY-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A20/00—Water conservation; Efficient water supply; Efficient water use
- Y02A20/20—Controlling water pollution; Waste water treatment
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for detecting water toxicity, which comprises the following steps: a) Adding the marine bacteria suspension, potassium ferricyanide and the water body to be detected into an orifice plate, mixing, and reacting to obtain a sample to be detected; adding marine bacteria suspension, potassium ferricyanide and water into a pore plate, mixing, and reacting to obtain a blank sample; b) Testing ultraviolet absorption of the sample to be tested and the blank sample by using an enzyme-labeled instrument, recording ultraviolet absorption peak value, and calculating inhibition rate and/or IC 50 Values. The invention uses marine bacteria as a test body for toxicity detection, under the condition of no toxicity and toxicity, the quantity of reduced mediator potassium ferrocyanide generated by the reaction of microorganisms and potassium ferricyanide is different, and the potassium ferrocyanide is continuously released from Fe in suspension bacteria 3+ The amount of Prussian Blue (PB) is also different, and then the enzyme-labeled instrument is used for carrying out high-efficiency colorimetric detection on toxic substances according to the color difference of green solution obtained by compositing blue PB and yellow potassium ferricyanide.
Description
Technical Field
The invention relates to the technical field of detection methods, in particular to a detection method for water toxicity.
Background
With the continuous emphasis of the national environmental problems, the water pollution condition of China has been greatly improved, but in order to further optimize the water quality and enable the sewage treatment plant to reach the discharge standard, the water quality toxicity detection is necessary. Compared with a large-sized spinal animal water toxicity detection sensor, the water toxicity detection sensor based on the microorganism as a test body has wide bacterial sources, high sensitivity and small individual difference, and becomes a research hot spot in recent decades. Among them, colorimetric detection of water toxicity has received attention in recent years due to visual perception. However, the existing water toxicity colorimetric detection uses common microorganisms (some pathogenic bacteria) as mode bacteria, the types of the bacteria are not uniform and are inconvenient to compare, and the data are mainly acquired by single-channel ultraviolet detection, so that the efficiency is low, and the requirement of rapid detection in an emergency pollution event cannot be met.
The water toxicity detection method based on the luminescent bacteria test body is increasingly paid attention to because of simplicity, rapidness, sensitivity and lower cost. Luminous bacilli (Photobacterium phoshoreum T spp., T3) can reproduce in cadavers and meats of cattle and horses; it invades the human body to produce luminous urine. These bacteria are generally preferably low temperature, preferably about 18℃and not luminescent above 37 ℃. The luminescence phenomenon is enzymatic oxidation reaction, and FM-NH is necessary 2 ，O 2 Long chain saturated aldehydes, luciferase, and the like. It is generally considered that FMNH 2 Namely fluorescein. The luminous bacillus is used as a test body in national standard GB/T15441-1995 method for measuring acute toxicity of water quality.
Hendriwaiitto et al developed a fast immobilized bacterial biosensor for colorimetric detection of water toxicity. However, this method has the disadvantages: (1) manufacturing alginate beads, and the procedure is complex; (2) The use of high-concentration potassium ferricyanide is easy to cause environmental pollution; (3) Stepwise addition of FeCl 3 Complicated operation; (4) adding hydrochloric acid, and having potential safety hazard; (5) And the single-channel ultraviolet instrument is used for detection, so that the detection efficiency is low.
Therefore, it is very necessary to develop a simple, environment-friendly and macroscopic multichannel efficient colorimetric detection method for water toxicity.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a method for detecting water toxicity, which is simple, environment-friendly and visible to naked eyes, and has high detection efficiency.
The invention provides a method for detecting water toxicity, which comprises the following steps:
a) Adding the marine bacteria suspension, potassium ferricyanide and the water body to be detected into an orifice plate, mixing, and reacting to obtain a sample to be detected;
adding marine bacteria suspension, potassium ferricyanide and water into a pore plate, mixing, and reacting to obtain a blank sample;
b) Testing ultraviolet absorption of the sample to be tested and the blank sample by using an enzyme-labeled instrument, recording ultraviolet absorption peak value, and calculating inhibition rate and/or IC 50 Values.
Preferably, the preparation method of the marine bacteria suspension specifically comprises the following steps:
inoculating marine bacteria in a culture medium, culturing, centrifuging, harvesting the light-emitting bacilli in the logarithmic phase, and then suspending in NaCl solution again to obtain bacterial suspension;
the culture is carried out at 50-300 rpm and 15-40 ℃ for 10-40 h; the centrifugation is carried out at 2000-10000 rpm for 1-20 min; the NaCl concentration is 0-30%;
preferably, the marine bacteria are Pseudomonas, vibrio, achromobacter, flavobacterium, helicobacter, micrococcus, sarcina, bacillus, corynebacterium, cladosporium, nocardia and Streptomyces; preferably, the marine bacterium is a luminous bacillus or a vibrio freudenreichii.
Preferably, step A) the marine bacterial suspension OD 600 =0.1 to 20; the concentration of the potassium ferricyanide is 0.5-50 mM;
the reaction temperature is 4-40 ℃; the reaction time is 1 min-2 h.
Preferably, the culture medium is a culture medium containing ferric salt; the mass percentage of the ferric salt is 0.01-10.0%. .
The ferric salt is ferric citrate, ferric ammonium citrate, ferric sulfate or ferric chloride, etc.
Preferably, the culture medium is a Y culture medium;
the Y culture medium specifically comprises the following components: 1.0g of yeast powder, 5.0g of peptone, 0.1g of Fe (III) citrate, 19.45g of NaCl and MgCl 2 5.98g，KCl0.55g，Na 2 SO 4 3.24g，CaCl 2 1.80g，KCl 1.8g，Na 2 CO 3 0.16g，KBr 0.08g，SrCl 2 34.0mg，H 3 BO 3 22.0mg，NaSiO 3 4.0mg，NaF 2.4mg，NH 4 NO 3 1.6mg，Na 2 HPO 4 8mg, distilled water 1.0L, pH7.6.
Preferably, said step B) is recording an ultraviolet absorption peak at 690 nm.
Preferably, the inhibition rate calculation specifically includes: inhibition% = (1-Abs) tox /Abs con ) X 100 (1); wherein Abs tox Representing the ultraviolet absorption peak value, abs, of the sample to be tested at 690nm con Representing the ultraviolet absorption peak at 690nm for a blank sample;
the IC (integrated circuit) 50 The calculation of the values is specifically: drawing a curve by taking the concentration of toxic substances in the water body to be measured as an abscissa and the inhibition rate as an ordinate, and fitting to obtain the IC 50 。
Preferably, the water body to be detected contains 3, 5-dichlorophenol and Cd 2+ 、Hg 2+ 、Zn 2+ 、Cr 6+ 、U 6+ 、Te 3+ 、Co 3+ 、Se 6 + 、Pu 3+ 、Hg 2+ 、Mn 4+ 、Cd 2+ And the like, or a mixture obtained by compounding binary, ternary, multiple and the like, or an actual water sample;
the concentration of toxic substances in the water body to be detected is 0.001-200 mg L -1 ；
The pore plate is 2-200 pore plates.
Preferably, the step B) further comprises directly observing the color of the sample with naked eyes, and judging whether the sample has toxicity or the toxicity size by comparing the color difference of the toxic sample and the blank sample.
Compared with the prior art, the invention provides a method for detecting water toxicity, which comprises the following steps: a) Adding the marine bacteria suspension, potassium ferricyanide and the water body to be detected into an orifice plate, mixing, and reacting to obtain a sample to be detected; adding the luminous bacillus suspension, potassium ferricyanide and water into a pore plate, mixing, and reacting to obtain a blank sample; b) Testing ultraviolet absorption of the sample to be tested and the blank sample by using an enzyme-labeled instrument, recording ultraviolet absorption peak value, and calculating inhibition rate and/or IC 50 Values. The invention utilizes marine bacteria as toxicityUnder the non-toxic and toxic conditions, the detected test body has different amounts of reduced mediator potassium ferrocyanide generated by the reaction of microorganisms and potassium ferricyanide, and the potassium ferrocyanide is continuously released from Fe in suspension bacteria 3+ The amount of Prussian Blue (PB) is also different, and then the enzyme-labeled instrument is used for carrying out high-efficiency colorimetric detection on toxic substances according to the color difference of green solution obtained by compositing blue PB and yellow potassium ferricyanide.
Drawings
FIG. 1 is a schematic diagram of a toxicity detection principle;
FIG. 2, reaction temperature optimization, (A) spectral curves; (B) a ultraviolet absorption peak histogram; (C) inhibition curves at different temperatures;
FIG. 3, cd-containing 2+ Detecting toxicity of the wastewater;
FIG. 4, hg-containing 2+ Detecting toxicity of the wastewater;
FIG. 5, zn-containing 2+ Detecting toxicity of the wastewater;
FIG. 6, photo comparison of samples at different times of reaction, (A) 0.5min; (B) 20min;
FIG. 7, photographs of samples at different temperatures of reaction, (A) 26 ℃; (B) 45 ℃.
Detailed Description
The invention provides a method for detecting water toxicity, and a person skilled in the art can refer to the content of the water toxicity and properly improve the process parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and they are intended to be within the scope of the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The invention provides a method for detecting water toxicity, which comprises the following steps:
a) Adding the marine bacteria suspension, potassium ferricyanide and the water body to be detected into an orifice plate, mixing, and reacting to obtain a sample to be detected;
adding the luminous bacillus suspension, potassium ferricyanide and water into a pore plate, mixing, and reacting to obtain a blank sample;
b) Testing ultraviolet absorption of the sample to be tested and the blank sample by using an enzyme-labeled instrument, recording ultraviolet absorption peak value, and calculating inhibition rate and/or IC 50 Values.
The invention provides a method for detecting water toxicity, which comprises the steps of firstly preparing marine bacteria suspension.
The marine bacteria are Pseudomonas, vibrio, achromobacter, flavobacterium, helicobacter, micrococcus, sarcina, bacillus, corynebacterium, cladosporium, nocardia and Streptomyces; preferably, the marine bacterium is a luminous bacillus or a vibrio freudenreichii.
The strain preparation of the invention is preferably specifically as follows:
sterilizing the culture medium at 121deg.C under high temperature and high pressure of 101.325kPa for 20min, and cooling. The preparation method comprises the steps of activating the freeze-dried powder of the luminous bacillus for 1-3 generations, mixing the cultured luminous bacillus bacterial liquid with sterilized 10-70% glycerol in a volume ratio of 1-5:1, sub-packaging the mixed liquid in a strain tube, and finally storing the mixed liquid in a refrigerator at a temperature of between 80 ℃ below zero and 20 ℃ below zero for later use.
The strain can also be prepared by a flat-plate preservation method, or freeze-dried bacteria resuscitator.
According to the invention, the preparation method of the marine bacterium suspension comprises the following steps:
inoculating marine bacteria in a culture medium, culturing, centrifuging, harvesting the light-emitting bacilli in the logarithmic phase, and then suspending in NaCl solution again to obtain bacterial suspension;
the culture is carried out at 50-300 rpm and 15-40 ℃ for 10-40 h; the centrifugation is carried out at 2000-10000 rpm for 1-20 min;
the NaCl concentration is 0-30%; preferably 0.5 to 10%;
according to the invention, salinity experiments are carried out on all strains, and it is found that bacteria from marine sediment environment are distributed in 6 physiological groups under an artificial high-salinity environment, namely non-halophilic bacteria (the growth salinity range is 0-5%, the optimal growth salinity is 0-1.5%), halophilic bacteria (the growth salinity range is 0-20%, the optimal growth salinity is 0-1.5%), wide-area halophilic bacteria (the growth salinity range is 0-30%, the optimal growth salinity is 3-15%), low-level halophilic bacteria (the growth salinity range is 1.5-30%, the optimal growth salinity is 3-5%), moderate halophilic bacteria (the growth salinity range is 1.5-30%, the optimal growth salinity is 5-20%) and critical extreme halophilic bacteria (the growth salinity range is 5-30% and the optimal growth salinity is 15-20%).
In some embodiments, the bacterial culture of the present invention is more preferred, in particular:
inoculating the strain into the culture medium, and culturing for 10-40 h at 15-40 ℃ by using a shaking table at 50-300 rpm. The light-emitting bacilli harvested in the logarithmic growth phase were centrifuged at 2000-10000 rpm for 1-20 min at room temperature, and then washed and centrifuged twice with 0.1-30% NaCl solution to remove the medium. Finally, the luminous bacillus is resuspended in 0.1-30% NaCl solution and stored in the environment of 0-30 ℃ for subsequent experiments.
According to the invention, the medium is a medium containing an iron salt; the mass percentage of the ferric salt is 0.01-10.0%.
The ferric salt is ferric citrate, ferric ammonium citrate, ferric sulfate or ferric chloride, etc.
In some embodiments, the medium is a Y medium;
the Y culture medium specifically comprises the following components: 1.0g of yeast powder, 5.0g of peptone, 0.1g of Fe (III) citrate, 19.45g of NaCl and MgCl 2 5.98g，KCl0.55g，Na 2 SO 4 3.24g，CaCl 2 1.80g，KCl 1.8g，Na 2 CO 3 0.16g，KBr 0.08g，SrCl 2 34.0mg，H 3 BO 3 22.0mg，NaSiO 3 4.0mg，NaF 2.4mg，NH 4 NO 3 1.6mg，Na 2 HPO 4 8mg, distilled water 1.0L, pH7.6.
Adding the marine bacteria suspension, potassium ferricyanide and the water body to be detected into an orifice plate, mixing, and reacting to obtain a sample to be detected.
The water body to be measured contains 3, 5-dichlorophenol and Cd 2+ 、Hg 2+ 、Zn 2+ 、Cr 6+ 、U 6+ 、Te 3+ 、Co 3+ 、Se 6+ 、Pu 3+ 、Hg 2+ 、Mn 4+ 、Cd 2+ And the like, or a mixture obtained by compounding binary, ternary, multiple and the like, or an actual water sample;
the concentration of toxic substances in the water body to be detected is 0.001-200 mg L -1 ；
The pore plate is 2-200 pore plates; the invention preferably adopts 96-well plates, can simultaneously measure 96 samples, and has higher detection efficiency compared with the measurement of single sample in the prior art.
The marine bacterial suspension according to the invention is preferably OD 600 0.1 to 20; more preferably 0.1 to 15; most preferably 1 to 10; the concentration of the potassium ferricyanide is 0.5-50 mM; more preferably 2 to 20mM;
in one embodiment, the OD 600 Concentration of potassium ferricyanide was 8mM;
the reaction temperature is 4-40 ℃; preferably 20 to 30 ℃; the reaction time is 1 min-2 h; preferably 10min to 1h; more preferably from 10 minutes to 50 minutes.
In one embodiment, the reaction temperature is 26-27 ℃; the reaction time is 20-30 min.
In one embodiment, the OD 600 =4, potassium ferricyanide concentration was 20mm, nacl final concentration was 1.5%, reaction time was 10min, and temperature was 26.5 ℃.
Adding the marine bacteria suspension, potassium ferricyanide and water into an orifice plate, mixing, and reacting to obtain a blank sample.
To ensure simultaneous addition, multichannel pipettes were used for sample addition. After the reaction, directly observing the color of the sample by naked eyes, and judging whether the sample has toxicity or not or the toxicity size by comparing the color difference of the toxic sample and the blank sample.
Simultaneously, the ultraviolet absorption of each sample can be tested by using an enzyme-labeled instrument.
The method comprises the following steps: testing a sample to be tested by using an enzyme-labeled instrumentUltraviolet absorbance of blank sample and recording ultraviolet absorbance peak, and calculating inhibition rate and/or IC 50 Values.
The ultraviolet absorption peak at 690nm was recorded according to the present invention.
All samples were run in triplicate and all colorimetric and electrochemical experiments were performed at room temperature.
The inhibition rate is calculated specifically as follows: inhibition% = (1-Abs) tox /Abs con )×100 (1)；
Wherein Abs tox Representing the ultraviolet absorption peak value, abs, of the sample to be tested at 690nm con Representing the ultraviolet absorption peak at 690nm for a blank sample;
the IC (integrated circuit) 50 The calculation of the values is specifically: drawing a curve by taking the concentration of toxic substances in the water body to be detected as an abscissa and the inhibition rate as an ordinate, and fitting to obtain the IC 50 。
The toxicity detection principle of the invention is schematically shown in figure 1. When there is no toxic substance in water, the electrons generated by the respiration of the luminous bacilli reduce the mediator potassium ferricyanide to potassium ferrocyanide, and the sustained release of Fe in the potassium ferrocyanide and bacteria suspension 3+ The reaction produced Prussian Blue (PB). Blue PB and the remaining yellow potassium ferricyanide were mixed to form a green solution. Ultraviolet absorption peak Abs of PB at 690nm was measured using a microplate reader con . When toxic substances exist in the water body, the bacterial activity is inhibited, the respiration is weakened, the amount of potassium ferrocyanide obtained by reducing potassium ferricyanide is reduced, correspondingly, the amount of generated PB is also reduced, and the ultraviolet absorption peak Abs of PB at 690nm is reduced tox The color of the solution is reduced, and the toxicity of the water body can be semi-quantitatively detected by comparing the colors of the nontoxic and toxic water samples.
The invention provides a method for continuously releasing Fe by ferric salt in a marine culture medium 3+ The method for detecting the toxicity of the water body by colorimetry is simple, environment-friendly and macroscopic. In the water toxicity detection process, potassium ferrocyanide obtained by microbial reduction of potassium ferrocyanide and Fe released continuously in solution 3+ The PB is reacted with yellow iron cyanide in blueThe green solution of potassium chloride obtained by mixing the color principle is used as an indicator. The sensor can realize colorimetric detection of water toxicity by one-step mixing without adding FeCl step by step 3 (acidic) the detection procedure is greatly simplified. Meanwhile, 96-channel simultaneous high-efficiency colorimetric detection of water toxicity is realized by using a 96-well plate of the novel enzyme-labeled instrument.
In order to further illustrate the invention, the following describes in detail a method for detecting toxicity of water body provided by the invention by combining with the embodiment.
Example 1: cd-containing 2+ Waste water toxicity detection
(1) Bacterial preparation
Sterilizing the Y culture medium containing ferric citrate at 121deg.C under high temperature and high pressure of 101.325kPa for 20min, and cooling. The preparation method comprises the steps of activating the freeze-dried powder of the luminous bacillus for 2 generations, mixing the cultured luminous bacillus bacterial liquid with sterilized 50% glycerol in a volume ratio of 1:1, sub-packaging the mixed liquid in a strain tube, and finally storing the mixed liquid in a refrigerator at the temperature of minus 80 ℃ for standby.
(2) Bacterial culture
The luminous bacillus species was inoculated in the Y medium and cultured at 200rpm,30 ℃ for 20 hours using a shaker. The light-emitting bacilli harvested in the logarithmic growth phase were centrifuged at 7000rpm for 5min at room temperature, and then washed with 3% NaCl solution and centrifuged twice to remove the medium. Finally, the luminous bacillus was resuspended in 3% nacl solution and stored in a refrigerator at 4 ℃ for subsequent experiments.
(3) Optimization of detection conditions
The concentration of bacteria, potassium ferricyanide and NaCl, the reaction time and the reaction temperature are respectively OD after experimental optimization (the highest inhibition rate) 600 ＝4，20mM，1.5％，10min，26.5℃。
Wherein the temperature optimization experiment is shown in FIG. 2, OD 600 =4, nacl final concentration of 1.5%, potassium ferricyanide final concentration of 20mM, 10min of reaction. As is evident from FIG. 2C, the highest inhibition was obtained at a temperature of 26.6 ℃. FIG. 2, reaction temperature optimization, (A) spectral curve; (B) a ultraviolet absorption peak histogram; (C) inhibition curves at different temperatures.
(4) Toxicity detection
Preparing a sample to be tested: mu.L of the bacterial suspension and 50. Mu.L of Cd-containing solutions of various concentrations were added to wells of a 96-well plate, respectively 2+ 50 mu L of potassium ferricyanide with a certain concentration. Preparation of control samples: cd-containing 2+ The solution was replaced with an equal volume of deionized water. To add samples as simultaneously as possible, multichannel pipettes were used for sample addition. After reacting for 10min, comparing the color difference of the control sample and the detection sample solution to judge whether the water sample is toxic or not and the toxicity. Simultaneously, the enzyme-labeled instrument is used for simultaneously testing the ultraviolet absorption of each sample and recording the ultraviolet absorption peak value at 690nm, the toxicity inhibition rate is calculated, and the IC is calculated by drawing 50 Values. All samples were run in triplicate and all colorimetric experiments were performed at room temperature. Cd (cadmium sulfide) 2+ Final concentrations of 0.4,0.8,1.2,1.6,2.0,4.0 and 8.0mg L, respectively -1 。
FIG. 3A is a graph showing the ultraviolet absorption spectra of the respective samples, wherein the ultraviolet absorption intensity gradually decreases with the gradual increase of the concentration of the toxic substances. FIG. 3B is a graph of the inhibition ratio at Cd obtained by fitting the absorption peak at 690nm according to the ultraviolet absorption spectrum in FIG. 3A 2+ The concentration is 0.4-2.0 mg L -1 In the range, the inhibition rate gradually increases with the increase of the concentration of toxic substances, and the inhibition rate is 2.0-8.0 mg L -1 Within the range, the inhibition rate tends to stabilize as the concentration of toxic substances increases. Fig. 3C is a photograph of a sample under test, the color of the solution gradually transitioning from green to yellow as the concentration of toxic substances increases. The concentration of 1.6mg/LCd can be clearly distinguished by naked eyes 2+ Therefore, the detection limit was 1.6mg/L.
FIG. 3 Cd-containing 2+ And (5) detecting toxicity of the wastewater. (a) ultraviolet absorption spectra of each sample; (B) toxicity inhibition rate profile; (C) photographs of the respective samples. The final concentration of toxic substances is respectively as follows from top to bottom: 0,0.4,0.8,1.2,1.6,2.0,4.0 and 8.0mg L -1 。
Example 2: hg-containing 2+ Waste water toxicity detection
The procedure is similar to example 1, except that the toxic substance is derived from Cd 2+ Becomes Hg 2+ . FIG. 4A is a graph showing the ultraviolet absorption spectrum of each sample asThe concentration of toxic substances is gradually increased, and the ultraviolet absorption intensity is gradually reduced. FIG. 4B is a graph of the inhibition ratio at Hg of the ultraviolet absorption spectrum of FIG. 4A fitted to the absorption peak at 690nm 2+ The concentration is 0.4-1.2 mg L -1 In the range, the inhibition rate increases slowly with the increase of the concentration of toxic substances, and is 1.2-4.0 mg L -1 Within the range, the inhibition rate increases faster as the concentration of toxic substances increases. At 4.0mg L -1 After that, as the concentration of the toxic substance increases, the inhibition rate tends to be stable. Fig. 4C is a photograph of a sample to be tested, and the color of the solution gradually changes from green to yellow as the concentration of the concentration substance increases. The concentration of 2.0mg/LHg can be clearly distinguished by naked eyes 2+ Therefore, the detection limit was 2.0mg/L. FIG. 4 Hg-containing 2+ And (5) detecting toxicity of the wastewater. (a) ultraviolet absorption spectra of each sample; (B) toxicity inhibition rate profile; (C) photographs of the respective samples. The final concentration of toxic substances is respectively as follows from top to bottom: 0,0.4,0.8,1.2,1.6,2.0,4.0 and 8.0mg L -1 。
Example 3: zn-containing alloy 2+ Waste water toxicity detection
The procedure is similar to example 1, except that the toxic substance is derived from Cd 2+ Change to Zn 2+ . FIG. 5A is a graph showing the ultraviolet absorption spectra of the respective samples, wherein the ultraviolet absorption intensity gradually decreases with the gradual increase of the concentration of the toxic substances. FIG. 5B is a graph showing the inhibition ratio obtained by fitting the absorption peak at 690nm to the ultraviolet absorption spectrum in FIG. 5A, in Zn 2+ The concentration is 0.4-1.2 mg L -1 Within the range, the inhibition rate is rapidly increased with the increase of the concentration of toxic substances, and is 1.2-8.0 mg L -1 Within the range, the inhibition rate tends to stabilize as the concentration of toxic substances increases. Fig. 5C is a photograph of a sample to be tested, and the color of the solution gradually changes from green to yellow as the concentration of the concentration substance increases. The concentration of 1.2mg/LZn can be clearly distinguished by naked eyes 2+ Therefore, the detection limit was 1.2mg/L. FIG. 5 Zn-containing 2+ And (5) detecting toxicity of the wastewater. (a) ultraviolet absorption spectra of each sample; (B) toxicity inhibition rate profile; (C) photographs of the respective samples. The final concentration of toxic substances is respectively as follows from top to bottom: 0,0.4,0.8,1.2,1.6,2.0,4.0 and 8.0mg L -1 。
Comparative example 1: comparison of reaction time 0.5min and 20min
OD 600 =3, nacl final concentration of 1.0%, potassium ferricyanide final concentration of 20mm, hg 2+ The concentration was 3.0mg/L, and the reaction temperature was 30 ℃. At 0.5min of reaction, as is evident from fig. 6A, the color of both the blank sample (A1) and the sample to be tested (A2) was hardly changed, and it was not suitable for colorimetric detection. At 20min of reaction, as is evident from fig. 6B, the blank sample (B1) and the sample to be tested (B2) have a distinct color difference, and are suitable for colorimetric detection. FIG. 6 photo comparison of samples at different times of reaction, (A) 0.5min; (B) 20min.
Comparative example 2: comparison of reaction temperatures of 26℃and 45 ℃
OD 600 =5, nacl final concentration of 2%, potassium ferricyanide final concentration of 30mm, cd 2+ The concentration was 2.0mg/L, and the reaction was carried out for 30 minutes. As is evident from FIG. 7A, the microorganism activity is higher, the reaction is faster, the color difference between the blank sample and the sample to be detected is obvious, and the method is suitable for colorimetric detection at the temperature of 26 ℃. As is evident from FIG. 7B, the microorganism activity is higher, the reaction is faster, and the color difference between the blank sample and the sample to be detected is smaller, so that the sample is not suitable for colorimetric detection. FIG. 7 is a photograph of a sample at various temperatures, (A) 26 ℃; (B) 45 ℃.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The method for detecting the toxicity of the water body is characterized by comprising the following steps of:
a) Adding the marine bacteria suspension, potassium ferricyanide and the water body to be detected into an orifice plate, mixing, and reacting to obtain a sample to be detected;
adding marine bacteria suspension, potassium ferricyanide and water into a pore plate, mixing, and reacting to obtain a blank sample;
b) Testing sample to be tested and blank sample by using enzyme-labeled instrumentUltraviolet absorption of (2) and recording ultraviolet absorption peak value, and calculating inhibition rate and/or IC 50 Values.
2. The method according to claim 1, wherein the preparation method of the marine bacterial suspension comprises the following steps:
inoculating marine bacteria in a culture medium, culturing, centrifuging, washing, harvesting the light-emitting bacilli in the logarithmic phase, and then re-suspending in NaCl solution to obtain bacterial suspension;
the culture is carried out at 50-300 rpm and 15-40 ℃ for 10-40 h; the centrifugation is carried out at 2000-10000 rpm for 1-20 min; the NaCl concentration is 0-30%.
3. The method according to claim 2, wherein the marine bacteria are Pseudomonas, vibrio, achromobacter, flavobacterium, helicobacter, micrococcus, sarcina, bacillus, corynebacterium, cladosporium, nocardia, and Streptomyces; preferably, the marine bacterium is a luminous bacillus or a vibrio freudenreichii.
4. The method according to claim 1, wherein the marine bacterial suspension OD of step A) is 600 =0.1 to 20; the concentration of the potassium ferricyanide is 0.5-50 mM;
the reaction temperature is 4-40 ℃; the reaction time is 1 min-2 h.
5. The method according to claim 1, wherein the medium is a medium containing an iron salt; the mass percentage of the ferric salt is 0.01-10.0%;
the ferric salt is ferric citrate, ferric ammonium citrate, ferric sulfate or ferric chloride.
6. The method according to claim 5, wherein the medium is a Y medium;
the Y culture medium specifically comprises the following components:yeast powder 1.0g, peptone 5.0g, fe (III) citrate 0.1g,NaCl19.45g,MgCl 2 5.98g，KCl 0.55g，Na 2 SO 4 3.24g，CaCl 2 1.80g，KCl 1.8g，Na 2 CO 3 0.16g，KBr 0.08g，SrCl 2 34.0mg，H 3 BO 3 22.0mg，NaSiO 3 4.0mg，NaF 2.4mg，NH 4 NO 3 1.6mg，Na 2 HPO 4 8mg, distilled water 1.0L, pH7.6.
7. The method according to claim 1, wherein the step B) is recording the ultraviolet absorption peak at 690 nm.
8. The method according to claim 1, wherein the inhibition ratio calculation is specifically:
Inhibition％＝(1-Abs tox /Abs con )×100 (1)；
wherein Abs tox Representing the ultraviolet absorption peak value, abs, of the sample to be tested at 690nm con Representing the ultraviolet absorption peak at 690nm for a blank sample;
the IC (integrated circuit) 50 The calculation of the values is specifically: drawing a curve by taking the concentration of toxic substances in the water body to be measured as an abscissa and the inhibition rate as an ordinate, and fitting to obtain the IC 50 。
9. The detection method according to claim 1, wherein the water to be detected contains 3, 5-dichlorophenol, cd 2 + 、Hg 2+ 、Zn 2+ 、Cr 6+ 、U 6+ 、Te 3+ 、Co 3+ 、Se 6+ 、Pu 3+ 、Hg 2+ 、Mn 4+ 、Cd 2+ And the like, or a mixture obtained by binary, ternary and multielement combination of the two components, or an actual water sample;
the concentration of toxic substances in the water body to be detected is 0.001-200 mg L -1 ；
The pore plate is 2-200 pore plates.
10. The method according to claim 1, wherein the step B) further comprises directly observing the color of the sample with naked eyes, and judging whether the sample is toxic or not or the toxicity is large or not by comparing the color difference between the toxic sample and the blank sample;
or the inhibition rate is obtained by adopting RGB color taking, electrochemical measurement of current value and other modes, and further whether the sample has toxicity or not is judged.
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