CN118028418A - Method for detecting biotoxicity of soil in tanning site - Google Patents
Method for detecting biotoxicity of soil in tanning site Download PDFInfo
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- 239000002689 soil Substances 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 35
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 82
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- 231100001231 less toxic Toxicity 0.000 claims description 2
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- 231100000584 environmental toxicity Toxicity 0.000 abstract description 6
- 239000010985 leather Substances 0.000 abstract description 5
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- 229910052804 chromium Inorganic materials 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 5
- 239000002699 waste material Substances 0.000 description 4
- JOPOVCBBYLSVDA-UHFFFAOYSA-N chromium(6+) Chemical compound [Cr+6] JOPOVCBBYLSVDA-UHFFFAOYSA-N 0.000 description 3
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- 108090000363 Bacterial Luciferases Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
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- 239000002253 acid Substances 0.000 description 1
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- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
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Abstract
A method for detecting biotoxicity of the soil in the leather field by leaching the soil in the leather field with a special leaching agent is to leach the easily-migrated chemical substances in the soil in the leather field by using a NaCl and AEO mixed solution as the leaching agent to obtain a soil leaching solution. Adding NaCl to the leaching solution to make the concentration of the NaCl to be 2.5 percent by weight, and adjusting the pH to 7.0; or diluting the leaching solution with 2.5% wt NaCl solution to a proper concentration gradient, and adjusting pH to 7.0; samples were assayed for toxicity. Taking cultured luminous bacteria, adding a sample solution, uniformly mixing, standing, and measuring luminous intensity. Expression of sample toxicity: the relative luminous intensity and half relative luminous intensity concentration EC 50 of the mixture after standing were measured with a 2.5% wt NaCl solution as a blank. According to the invention, the mixed solution of sodium chloride and AEO is used as the leaching agent, toxic substances in the soil sample of the tanning site are leached, and the ecotoxicity of the leaching solution is directly detected by using a toxicity detector, so that a convenient method for detecting the ecotoxicity of the soil of the tanning site is constructed, and the defects of the traditional method are avoided.
Description
Technical Field
The invention relates to the field of a method for detecting the biotoxicity of soil in a tanning field, in particular to a method for detecting the biotoxicity of soil in a tanning field based on a special leaching agent.
Background
The pollution characteristic of the soil of the tanning site is the combined pollution of chromium and organic matters, wherein the total chromium content is high and can reach 59400mg/kg, and is mainly trivalent chromium, but also contains higher hexavalent chromium in certain areas and can reach 827mg/kg. More than 90% of tanning sections use trivalent chromium, which generates a large amount of chromium-containing waste liquid and chromium-containing waste after tanning and post-treatment; meanwhile, a large number of organic matters such as grease, resin, tannin, protein and the like are used in the tanning process; the waste liquid drips and solid waste is stored and drained out, which becomes a main source of site pollution. These contaminants are not only toxic, but also potentially toxic by mixing and interacting with various contaminants; thus, comprehensive toxicity assessment is indispensable. The existing detection technology of the tanning site soil is limited to the content measurement of partial harmful components such as ammonia nitrogen, chromium (VI) and sulfide in the soil, and the extraction mode is different for each harmful component. For example, ISO/TS 14256-1-2003 uses potassium chloride solution to extract nitrate, nitrite and ammonium in soil, HJ 833-2017 uses strong acid to convert sulfide into hydrogen sulfide to blow out and measure its content, ISO 15192-2010 uses strong alkaline extracting solution to extract chromium (VI) in sample, and the influence of chromium (III) on test result is not considered in the case of reducing or oxidizing waste substrate. These soil leaching methods do not adequately take into account the risk of further transformation of the various components of the soil during extraction under changes in ambient temperature, pH and soil matrix, thereby affecting the results of the soil toxicity test.
China is a large country of leather production, the adjustment of an industrial structure promotes the tanning industry to form an industrial cluster with matched upstream, middle and downstream products, and part of small and medium-sized tanning enterprises are gradually shut down. The soil pollution control action plan published in 2016 clearly proposes that the tanning industry is one of industries for key inspection of chromium pollution. Before the shut-down tanning sites are reused, risk assessment is required. Therefore, an accurate, reliable and high-sensitivity soil ecotoxicity detection technology for the tanning sites is established, the soil ecotoxicity detection capability in the environment of the tanning industry in China is improved on the whole, important theoretical basis and guidance opinion are provided for recycling green tanning chemicals and land resources in China, and very important theoretical and practical significance is achieved.
Disclosure of Invention
The invention aims to overcome the defects that Cr (III) and Cr (VI) contents cannot be accurately tested, and the ecological toxicity screening operation is complex, long in time and high in detection cost in the prior art, and provides a tanning site soil biotoxicity detection method based on luminous bacteria, which is easy to control in operation, economical and practical.
The invention provides a method for detecting the ecological toxicity of soil in a tanning site based on a special leaching agent, which comprises the following steps:
(1) Pretreatment: air-drying, crushing, screening and homogenizing soil, sampling and performing constant temperature and humidity treatment;
(2) Leaching: adding a leaching agent into the pretreated soil, wherein the leaching agent is a solution with a certain pH value and containing 0.1-0.3 wt% of NaCl and 1% of AEO, oscillating for a certain time at constant temperature and level, and filtering to obtain leaching solution;
(3) Toxicity detection: adding sodium chloride into the leaching solution to enable the concentration of the sodium chloride to be 2.5 wt%, adjusting the pH value to 7.0+/-0.2, then adding the cultured fresh luminous bacterial liquid with the bacterial density for testing, mixing uniformly, standing, and measuring the luminous intensity;
(4) Toxicity expression: the relative luminescence intensity of the leaching solution after standing was calculated with the luminescence intensity of a 2.5% wt NaCl solution as a control.
Further, the pretreatment method specifically comprises the following steps: the soil is air-dried, sieved to a 2mm screen, sampled, dried at 45 ℃ for 48 hours, placed in standard air, and kept at 20+/-2 ℃ and constant temperature and humidity with relative humidity of 65+/-5% for 48 hours for standby.
Further, the soil is the soil of the tanning site.
Further, in the step (2), the liquid-solid ratio (mL/g) of the leaching agent to the soil is 2: 1-10: 1, carrying out oscillation leaching for 18-48 hours at the constant temperature of 25-30 ℃ and the horizontal speed of 60-100 rpm, wherein the filtering method is to pass through a filter membrane of 0.45 um.
Further, the leaching agent is a mixed solution of 0.1-0.3 wt% of NaCl and 1% of AEO, and the pH is adjusted to 4-9 by using H 2SO4 and NaOH.
Further, the luminescent bacteria in the luminescent bacteria liquid is luminescent bacteria T3.
Further, the culture method of the luminous bacterial liquid comprises the following steps:
(1) Preparation of a luminescent bacterial culture medium: taking 5.0g/L of tryptone, 5.0g/L of yeast powder, 5.0g/L of Na 2HPO4 5.0g/L,K2HPO4, 30g/L of NaCl, 3.0 g/L of glycerol and deionized water for dissolution, and adjusting the pH to 7.0;
(2) The culture process comprises the following steps: inoculating the luminous bacillus T3 into a conical flask filled with a culture medium, placing the conical flask in a shaking table with oscillation at 20 ℃ and 200 rpm, culturing for 18-20 hours, immediately inoculating the strain with the best luminous effect, placing the strain in a shaking table with 200 rpm at 20 ℃ again, measuring the luminous intensity of fresh bacterial liquid every 2h, and culturing for 18-20 hours to reach a growth log stabilization period for testing.
Further, the method for determining the bacterial density for testing comprises the following steps: taking fresh bacterial liquid of luminous bacteria which grows for 18-20 h to a logarithmic stationary phase, diluting the bacterial liquid to different bacterial densities by using 2.5 wt% NaCl, sequentially adding 950 uL of 1.23 mg/L ZnSO 4 solution containing 2.5 wt% NaCl, standing the bacterial liquid for 15min, measuring the luminous intensity by using a LumiFox6000 biotoxicity tester, and carrying out parallel measurement for 3 times, wherein the bacterial liquid with the relative luminous intensity of 50+/-5% is made blank by using the 2.5 wt% NaCl solution, and is the bacterial density for testing.
Further, the step (4) calculates the relative luminous intensity R of the leaching stock solution according to R=I/I 0 multiplied by 100% by using the luminous intensity average value I 0 of the NaCl control sample and the luminous intensity average value I of the sample to be detected; the greater the relative luminous intensity R, the less toxic.
The invention has the following beneficial effects:
The ionic strength is regulated by sodium chloride, the pH value is regulated by sulfuric acid or sodium hydroxide, the surface activity is regulated by AEO, and the weakly-bound heavy metals and organic matters in the soil are leached, so that the reaction of a leaching agent and leachable components in the soil is effectively avoided, the toxic effects of the mobilizable organic matters and Cr in the soil are accurately evaluated, and the operation is simple, convenient and effective.
The luminous bacteria detection method adopted by the invention is based on the bacterial luminous biosensing technology. The luminous bacillus is marine bacteria, and the bacteria in the logarithmic phase of growth have strong luminous capability and stable luminescence in a certain salt concentration and pH range, and when the environment changes or toxic substances exist, bacterial luciferase is deactivated or cell respiration is inhibited, the luminescence is weakened, and the weakening degree is positively related to the toxicity of the toxic substances. The luminous bacteria method has the advantages of low cost, rapid detection, high sensitivity, strong adaptability and the like. Therefore, the method can be completely used for detecting the ecological toxicity of the soil in the tanning field.
The invention uses the standard toxicity reference substance 1.23 mg/L ZnSO 4 with low toxicity to determine the density of the fresh bacterial liquid to be tested, so that the detection result is stable and the repeatability is good.
The method utilizes the luminescent bacteria method to detect, has short reaction time of the sample and the luminescent bacteria, can rapidly obtain detection results, is suitable for rapid detection of a large number of samples, greatly shortens the detection time of comprehensive toxicity, and improves the detection efficiency and accuracy.
Drawings
FIG. 1 is a flow chart of the invention.
Figure 2 soil leaching agent dose-toxicity effect curve.
FIG. 3 shows a comparison of leaching effects of leaching agents.
Fig. 4 dose response curves for example 2 and comparative example.
Detailed Description
The present invention is described in further detail below with reference to the following examples, which are only for further illustration of the present invention and are not to be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial improvements and modifications to the present invention based on the above-described summary.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biological materials, etc. used in the examples described below are commercially available unless otherwise specified.
Example 1
A method for detecting the ecotoxicity of soil in a tanning field based on a special leaching agent, referring to fig. 1, the method comprises the following steps:
(1) Pretreating a soil sample; the soil sample is sourced from a certain fur company site in China, the soil is air-dried, sieved into a 2mm screen, sampled, dried at 45 ℃ for 48 hours, placed in standard air, and kept for later use at 20+/-2 ℃ under constant temperature and humidity of 65+/-5% relative humidity for 48 hours;
(2) Sample leaching: sample 5.0 g was weighed into a 100mL Erlenmeyer flask, 25: 25 mL was added to a mixed solution of 0.1% wt.NaCl and 0.1% wt.AEO with pH adjusted to 8.0 by 0.1: 0.1M H 2SO4, and the mixture was placed into a ZWY-2102C constant temperature shaker at 25℃and 28: 28 rpm rotational speed, and after 8 hours of horizontal shaking, filtered through a 0.45um filter. The filtrate was diluted with 2.5 wt% NaCl solution in 10 concentration gradients, each containing leachate (V/V) as follows: toxicity was evaluated immediately by adjusting all diluted samples to pH 7.0±0.2 with 0.1M NaOH at 6%, 12%, 18%, 24%, 30%, 36%, 42%, 48%, 54% and 60%;
(3) Toxicity detection of the leaching solution: the invention adopts the fresh bacteria liquid of the luminous bacillus T3 to measure the comprehensive toxicity of crust leather. The preparation of the luminescent bacterial medium is as follows: tryptone 5.0g/L, yeast powder 5.0g/L, na 2HPO4 5.0g/L,K2HPO4 5.0.0 g/L, naCl 30g/L, glycerol 3.0g/L, deionized water, and adjusting pH to 7.0. The culture operation flow is as follows: preparing 1L culture medium, packaging into 250 mL conical bottles according to 100 mL bottles, wrapping with cotton, gauze and newspaper, and sterilizing at 121deg.C in a high-temperature sterilizing pot for 20 min. And (3) taking the luminous bacillus T3 freeze-dried powder, adding a 2.5% sodium chloride solution, and resuscitating at 20-25 ℃ for 15 min. Inoculating 1mL resuscitated luminous bacillus to a conical flask, placing the conical flask in a shaking table oscillating at 20 ℃ and 200 rpm, and culturing for about 18-20 hours until the luminous intensity reaches the maximum value, thereby harvesting. 1.5 mL of the strain with the best luminous effect is inoculated for the second time and placed on a 200 rpm shaking table at 20 ℃ to measure the luminous intensity of bacteria every 2 h. The specific determination method comprises the following steps: 50 uL fresh bacterial liquid is added with 950 uL of 2.5 wt.% NaCl, and the mixture is placed in 15min,LumiFox6000 biotoxicity tester to measure the luminous intensity. Under the culture conditions of this example, the luminescent bacteria did not emit light during the culture stage of the preceding 11 h. The luminous intensity is rapidly increased after the growth period of the logarithm, and the luminous intensity tends to be maximum and stable when the culture is carried out for 18-20 hours. Therefore, the experiment adopts fresh bacterial liquid for 18-20 hours to carry out toxicity test;
(4) Taking 5 parts of luminous bacteria liquid of which 1.0 mL is cultured to 19 h, sequentially adding 2.0mL, 2.5mL, 3.0mL, 3.5mL and 4.0mL of 2.5 wt% NaCl solution, and uniformly mixing to obtain luminous bacteria diluents with different bacterial densities. Taking 50 uL luminous bacteria dilutions with different bacterial densities, sequentially adding 950 uL of 1.23 mg/L ZnSO 4 solution (containing 2.5 wt% NaCl), standing for 15min, measuring the luminous intensity by using a LumiFox6000 biotoxicity tester, measuring in parallel for 3 times, and calculating the relative luminous intensity by taking 2.5 wt% NaCl solution as a blank. Wherein, the bacterial liquid with the relative luminous intensity of 50+/-5 percent is the bacterial density for testing;
(5) Taking 50 uL luminescent bacterial liquid with bacterial density calibrated by ZnSO 4, sequentially adding 950 and uL serial concentration gradient samples of the leaching liquid obtained in the step (2), uniformly mixing, standing for 15 min, measuring the luminescence intensity by using a LumiFox6000 biotoxicity tester, measuring in parallel for 3 times, and taking the average value of the 3 times of measurement as a result;
(6) Various indexes of the sample leaching solution: leaching liquor pH:8.49, eh:38, TOC content: 4562mg/L, cr (III) content 0.285mg/L, no Cr (VI);
(7) Toxicity expression of samples: dose-response curves were made with the concentration of the leaching solution on the abscissa and the relative luminous intensity of the luminescent bacteria on the ordinate (see fig. 2). The raw liquid is not less than 50% and has low toxicity of luminous intensity.
Example 2
A method for detecting the ecotoxicity of soil in a tanning field based on a special leaching agent, referring to fig. 1, the method comprises the following steps:
(1) Pretreatment of soil samples: the soil is sourced from certain leather-making limited company in China, the soil is air-dried, sieved into a 2mm screen, sampled, dried at 45 ℃ for 48 hours, placed in standard air, and kept for standby at 20+/-2 ℃ and constant temperature and humidity with relative humidity of 65+/-5%;
(2) Sample leaching: sample 5.0 g was weighed into a 100mL Erlenmeyer flask, 25: 25 mL was added to a mixed solution of 0.1% wt.NaCl and 0.1% wt.AEO with pH adjusted to 8.0 by 0.1: 0.1M H 2SO4, and the mixture was placed into a ZWY-2102C constant temperature shaker at 25℃and 28: 28 rpm rotational speed, and after 8 hours of horizontal shaking, filtered through a 0.45um filter. The filtrate was diluted with 2.5 wt% NaCl solution in 10 concentration gradients, each containing leachate (V/V) as follows: toxicity was evaluated immediately by adjusting all diluted samples to pH 7.0±0.2 with 0.1M NaOH at 6%, 12%, 18%, 24%, 30%, 36%, 42%, 48%, 54% and 60%;
(3) Toxicity detection of the leaching solution: the invention adopts the fresh bacteria liquid of the luminous bacillus T3 to measure the soil ecological toxicity. The preparation of the luminescent bacterial liquid in this example is the same as in example 1. Taking bacterial liquid 50 uL with determined bacterial density, and sequentially adding 950 and uL steps; (4) The prepared serial leaching liquor dilution samples are uniformly mixed and then are kept stand for 15min, the luminescence intensity is measured by using a LumiFox biological toxicity tester, the luminescence intensity is measured in parallel for 3 times, the relative deviation is not more than 10%, and the result is taken as the average value of the 3 times of measurement;
(5) Toxicity expression of samples: the dose-response curve is plotted with the concentration of the leaching dilution on the abscissa and the relative luminescence intensity on the ordinate, with a 2.5% wt.nacl solution as reference, see fig. 3. The EC 50 value of the obtained soil leaching solution is 69.21 +/-0.56 percent, and the toxicity is extremely low in terms of EC 50(ppm)= EC50(%)* (5/25)*106 = 138420 +/-1120 ppm of the soil.
Comparative example
Sample 5.0 g of example 2 was weighed into a 100 mL Erlenmeyer flask, 25. 25 mL was added with 0.1% wt. NaCl solution adjusted to pH 8.0 by 0.1M H 2SO4, placed in a ZWY-2102C constant temperature shaker, constant temperature 25℃and rotational speed 28 rpm, and after 8h horizontal shaking, filtered through a 0.45um filter. The filtrate was diluted with 2.5 wt% NaCl solution in 10 concentration gradients, each containing leachate (V/V) as follows: toxicity was evaluated immediately by adjusting all diluted samples to pH 7.0±0.2 with 0.1M NaOH at 6%, 12%, 18%, 24%, 30%, 36%, 42%, 48%, 54% and 60%. The dose-toxicity effect curve of the leachate is shown in the comparative example of figure 3. The rest of the procedure is the same as in example 2;
The respective leaching effect tests were carried out on example 2 and comparative example, and the test results are shown in table 1. It can be seen that the leaching agent provided by the present invention (example 2) has a greater difference in total leached material and biotoxicity than deionized water (comparative) and a deviation of greater than 10%.
Claims (10)
1. The method for detecting the biotoxicity of the soil of the tanning site is characterized by comprising the following steps of:
(1) Pretreatment: air-drying, crushing, screening and homogenizing soil, sampling and performing constant temperature and humidity treatment;
(2) Leaching: adding a leaching agent into the pretreated soil, horizontally oscillating for a certain time at constant temperature, and filtering to obtain leaching solution;
(3) Toxicity detection: adding sodium chloride into the leaching solution to enable the concentration of the sodium chloride to be 2.5 wt%, adjusting the pH value to 7.0+/-0.2, then adding the cultured fresh luminous bacterial liquid with the bacterial density for testing, mixing uniformly, standing, and measuring the luminous intensity;
(4) Toxicity expression: the relative luminescence intensity of the leaching solution after standing was calculated with the luminescence intensity of a 2.5% wt NaCl solution as a control.
2. The method for detecting the comprehensive toxicity of the soil of the tanning site is characterized by comprising the following steps of:
(1) Pretreatment: air-drying, crushing, screening and homogenizing soil, sampling and performing constant temperature and humidity treatment;
(2) Leaching: adding a leaching agent into the pretreated soil, regulating the pH value of the leaching agent by H 2SO4 and NaOH, adding NaCl with the weight of 0.1-0.3% and AEO with the weight of 1% to extract organic matters in the soil, oscillating for a certain time at constant temperature and level, and filtering to obtain leaching liquor;
(3) Diluting the leaching solution by using 2.5 wt% NaCl to form a concentration gradient, so that the relative luminous intensity range is 0% -100%, and regulating the pH value to 7.0+/-0.2; then adding fresh luminous bacterial liquid with the cultured bacterial density for testing, uniformly mixing, standing and measuring luminous intensity;
(4) Toxicity expression: the relative luminescence intensity of the leaching solution after standing was calculated with the luminescence intensity of a 2.5% wt NaCl solution as a control.
3. The detection method according to claim 1 or 2, wherein the pretreatment method specifically comprises: the soil is air-dried, sieved to a 2mm screen, sampled, dried at 45 ℃ for 48 hours, placed in standard air, and kept at 20+/-2 ℃ and constant temperature and humidity with relative humidity of 65+/-5% for 48 hours for standby.
4. The detection method according to claim 1 or 2, wherein in the step (2), the leaching agent is adjusted to have a pH value of 5.5 to 10.5 by H 2SO4 and NaOH, and a liquid-solid ratio (mL/g) of soil is 2: 1-10: 1, carrying out oscillation leaching for 18-48 hours at the constant temperature of 25-30 ℃ and the horizontal speed of 60-100 rpm, wherein the filtering method is to pass through a filter membrane of 0.45 um.
5. The method according to claim 1 or 2, wherein the leaching agent is a NaCl solution of 0.1 to 0.3% by weight and contains 1% AEO.
6. The method according to claim 1 or 2, wherein the luminescent bacteria in the luminescent bacteria liquid is luminescent bacteria T3.
7. The method according to claim 1, 2 or 7, wherein the method for culturing the luminescent bacterial liquid comprises:
(1) Preparation of a luminescent bacterial culture medium: taking 5.0g/L of tryptone, 5.0g/L of yeast powder, 5.0g/L of Na 2HPO4 5.0g/L,K2HPO4, 30g/L of NaCl, 3.0 g/L of glycerol and deionized water for dissolution, and adjusting the pH to 7.0;
(2) The culture process comprises the following steps: inoculating the luminous bacillus T3 into a conical flask filled with a culture medium, placing the conical flask in a shaking table with oscillation at 20 ℃ and 200 rpm, culturing for 18-20 hours, immediately inoculating the strain with the best luminous effect, placing the strain in a shaking table with 200 rpm at 20 ℃ again, measuring the luminous intensity of fresh bacterial liquid every 2h, and culturing for 18-20 hours to reach a growth log stabilization period for testing.
8. The method according to claim 1,2 or 8, wherein the method for determining the bacterial density for testing is: taking fresh bacterial liquid of luminous bacteria which grows for 18-20 h to a logarithmic stationary phase, diluting the bacterial liquid to different bacterial densities by using 2.5 wt% NaCl, sequentially adding 950 uL of 1.23mg/L ZnSO 4 solution containing 2.5 wt% NaCl, standing the bacterial liquid for 15min, measuring the luminous intensity by using a LumiFox6000 biotoxicity tester, and carrying out parallel measurement for 3 times, wherein the bacterial liquid with the relative luminous intensity of 50+/-5% is made blank by using the 2.5 wt% NaCl solution, and is the bacterial density for testing.
9. The method according to claim 1, wherein the step (4) calculates the relative luminescence intensity R of the leaching stock solution as r=i/I 0 x 100% with the luminescence intensity average I 0 of the 2.5% wt. NaCl control sample and the luminescence intensity average I of the sample to be detected; the greater the relative luminous intensity R, the less toxic.
10. The method according to claim 2, wherein the step (4) is to make a dose-response curve with the serial concentration of the sample to be tested diluted as an abscissa and the relative luminous intensity R as an ordinate, and to read the corresponding concentration from the curve when the relative luminous intensity is 50%, i.e. the greater the half relative luminous intensity concentration EC 50;EC50, the smaller the toxicity.
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