CN114324310A - Method for detecting acute toxicity of arsenic-polluted soil by using luminescent bacteria - Google Patents
Method for detecting acute toxicity of arsenic-polluted soil by using luminescent bacteria Download PDFInfo
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Abstract
The invention discloses a method for detecting acute toxicity of arsenic-polluted soil by utilizing luminescent bacteria, which comprises the following steps: s101, reviving the luminescent bacterium freeze-dried powder to obtain a revival bacterium liquid; s102, reading the luminous intensity of the resuscitation bacteria liquid by a toxicity detector to be more than 100 million photons, and detecting the acute toxicity of the arsenic-polluted soil; s103, preparing a leached water sample of the soil sample to be detected as arsenic pollution; s104, taking a leaching water sample, adding osmotic pressure regulating liquid, and uniformly mixing to obtain a leaching water sample with the osmotic pressure regulated; and S105, detecting by using a toxicity detector, reading the initial luminous intensity of the resuscitation bacteria liquid, adding the leaching water sample with the osmotic pressure adjusted into the resuscitation bacteria liquid, detecting the luminous intensity after sample addition, and calculating the relative luminosity. The invention utilizes the luminescent bacteria to carry out acute toxicity detection, can rapidly analyze the environmental safety of the soil polluted by arsenic to be detected through relative luminosity, and provides effective detection analysis for the remediation treatment effect of the soil polluted by arsenic.
Description
Technical Field
The invention relates to the technical field of soil detection, in particular to a method for detecting acute toxicity of arsenic-polluted soil by using luminescent bacteria.
Background
Elemental arsenic is extremely toxic, but arsenides are all more toxic, trivalent arsenic compounds are more toxic than other arsenic compounds, and arsenic accumulates in soil and thus enters crop tissues, which can generally enter the human body through water, atmosphere, food and other pathways, causing harm. Along with industrial promotion, pesticide application and the like, the pollution of arsenic to soil is continuously aggravated, which not only causes pollution to the environment, but also harms human health; it is often necessary to perform acute toxicity tests on leachate from arsenic contaminated soils in order to determine the environmental safety of arsenic contaminated soils.
Acute toxicity refers to the toxic effect caused by the body after contacting with the foreign compound once (or more than once within 24 hours), even causing death, but it should be pointed out that the speed and the intensity of the toxic effect of the compound on the experimental animal can be different according to the quality and the quantity of the contacted compound, and toxic symptoms and even death can occur when some compounds are within minutes of the lethal dose of the experimental animal.
The luminous bacteria biotoxicity test has the characteristics of rapidness, simplicity, sensitivity, low price and the like, but the traditional method for carrying out acute toxicity test by using luminous bacteria has low accuracy and is easy to cause the situations of detection deviation and the like; therefore, a new method for detecting the acute toxicity of arsenic-contaminated soil by using luminescent bacteria is needed to solve the above problems.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for detecting acute toxicity of arsenic-polluted soil by using luminescent bacteria.
The technical scheme of the invention is as follows: a method for detecting acute toxicity of arsenic-polluted soil by using luminescent bacteria comprises the following steps:
s101, reviving the luminescent bacteria freeze-dried powder stored at-22 ℃ to obtain reviving bacteria liquid;
s102, taking 0.1mL of resuscitation bacteria liquid, reading the luminous intensity of the resuscitation bacteria liquid by using a toxicity detector, wherein the luminous intensity is more than 100 ten thousand photons, and then the resuscitation bacteria liquid can be used for detecting the acute toxicity of the arsenic-polluted soil;
s103, taking a soil sample to be detected as arsenic pollution, and leaching the soil sample to obtain a leached water sample;
s104, taking 1mL of the leached water sample, adding 0.1mL of osmotic pressure regulating solution, and uniformly mixing to obtain the leached water sample after the osmotic pressure is regulated;
and S105, detecting by using a toxicity detector, reading the initial luminous intensity of 0.1mL of resuscitation bacteria liquid, adding 0.9mL of leached water sample after osmotic pressure adjustment into 0.1mL of resuscitation bacteria liquid, uniformly mixing, keeping the temperature for 3-8 min, detecting the luminous intensity after sample addition, and calculating the relative luminosity.
Further, the luminescent bacteria is vibrio fischeri; the vibrio fischeri is a deep-sea (with osmotic pressure) luminescent bacterium and can well meet the method for detecting the acute toxicity of the arsenic-polluted soil, so that the acute toxicity of the arsenic-polluted soil can be judged according to the relative luminosity.
As a technical solution of the present invention, the method for resuscitation in step S101 is: the luminescent bacteria freeze-dried powder is revived for 15min by using 1mL of Microtox diluent stored at 4 ℃.
As another technical solution of the present invention, the resuscitation method in step S101 is divided into a pre-resuscitation stage and a magnetic bead resuscitation stage, and includes the following steps:
1) pre-resuscitation phase
Adding the luminescent bacteria freeze-dried powder into 1mL of Microtox diluent stored at 4 ℃ for resuscitation for 1-3 min;
2) magnetic bead recovery phase
Then, magnetic beads with the temperature of 15 ℃, 21 ℃ and 28 ℃ are sequentially added for resuscitation for 5-8 min, the adding amount of the magnetic beads each time is 10-15 particles/mL, the particle size of the magnetic beads is 0.5-1 mm, an alternating magnetic field is continuously applied during the resuscitation period of the magnetic beads, the magnetic field strength of the alternating magnetic field is 0.1-0.5 mT, and the magnetic field frequency is 10-15 Hz;
wherein, the adding conditions of the magnetic beads are as follows: when the temperature difference between the magnetic beads and the Microtox diluent is less than 1 ℃ after the magnetic beads are added, adding the magnetic beads for the next time;
quantitative magnetic beads are added in a stepped mode, the initial temperatures of different magnetic beads are utilized to slowly raise the temperature of Microtox diluent, and meanwhile, an alternating magnetic field is matched for auxiliary recovery, so that the recovered luminous bacteria liquid is rapidly obtained, the recovery success rate of the luminous bacteria with the luminous intensity larger than 100 thousands of photons is high, and the detection efficiency of detecting the acute toxicity of the arsenic-polluted soil can be remarkably improved.
Further, in the step S102, the luminous intensity of the resuscitation liquid is read by using the ATP mode of the toxicity detector; in step S105, the B-Tox mode of the toxicity detector is used for detection.
Further, the leaching process in step S103 includes the steps of:
1) weighing a soil sample to be detected as arsenic pollution, placing the soil sample into a grinding pot, adding ceramic hollow spheres according to 30% of the mass of the soil sample to be detected as arsenic pollution, and adding 8-12L of the ceramic hollow spheres per 1kg of the soil sample to be detected to calculate the volume of the needed leaching agent for later use;
2) grinding a soil sample to be detected with arsenic pollution by using a grinding rod, dripping 10-20% by volume of a leaching agent for wet grinding, and continuously performing infrared illumination irradiation on the ground soil sample to be detected with arsenic pollution to obtain a leaching mixture;
3) uniformly mixing the leaching mixture obtained in the step 2) with the rest leaching agent, placing the mixture in an oscillating device, and oscillating for 8-12 hours at the rotating speed of 40 +/-5 r/min and the temperature of 20 +/-5 ℃;
4) standing for 1-2 h, filtering on a high-pressure filter, and storing filtrate at 4 ℃ to obtain a leached water sample;
by the leaching treatment method, the ceramic hollow spheres and infrared light irradiation can be utilized, and the leaching extraction of the leaching agent for arsenic in the arsenic-polluted soil can be enhanced by grinding, so that more accurate detection result analysis is provided for detecting the acute toxicity of the arsenic-polluted soil by using the luminescent bacteria.
Further, the lixiviant is 0.1mol/L hydrochloric acid lixiviant; graphite and silicon carbide are filled in the ceramic hollow ball according to the ratio of 3-5: 2, mixing and pressing the mixture according to the mass ratio to form a filled sphere, soaking the ceramic hollow sphere in 10-15% dopamine aqueous solution for 10-15 min before use, and then washing the filled sphere for 2-3 times by using deionized water; the infrared illumination power is 500-1200W, and the wavelength range is 30-50 mu m; the ceramic hollow spheres prepared according to the proportion can well assist infrared light irradiation to leach and extract arsenic in arsenic-polluted soil while not affecting grinding of the soil to be detected for arsenic pollution.
Further, the method for calculating the relative luminosity in step S105 includes:
wherein, the relative luminosity is more than or equal to 90 percent and is low-toxicity or non-toxicity; poisoning is caused when the relative luminosity is more than or equal to 40% and less than 90%; the relative luminosity is more than or equal to 20 percent and less than 40 percent, and the toxicity is high; the relative luminosity is less than 20%, which is extremely toxic.
The invention has the beneficial effects that:
(1) according to the method for detecting the acute toxicity of the arsenic-polluted soil, disclosed by the invention, the acute toxicity is detected by using the luminescent bacteria, and the environmental safety of the arsenic-polluted soil to be detected can be quickly analyzed through the relative luminosity, so that effective detection and analysis are provided for the remediation treatment effect of the arsenic-polluted soil.
(2) According to the method for detecting the acute toxicity of the arsenic-polluted soil, the luminescent bacteria with the luminous intensity of more than 100 million photons can be rapidly obtained through the provided luminescent bacteria recovery method, so that the detection efficiency for detecting the acute toxicity of the arsenic-polluted soil is remarkably improved.
(3) According to the method for detecting the acute toxicity of the arsenic-polluted soil, the ceramic hollow spheres and infrared light are used for irradiation, and the grinding is used for assisting in enhancing the leaching and extraction of the leaching agent for the arsenic in the arsenic-polluted soil, so that a more accurate detection result analysis is provided for detecting the acute toxicity of the arsenic-polluted soil by using luminescent bacteria.
Drawings
FIG. 1 shows the relative luminosity of the luminescent bacteria in the invention for detecting the acute toxicity of the arsenic-contaminated soil leaching water sample.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments thereof for better understanding the advantages of the invention.
Example 1
A method for detecting acute toxicity of arsenic-polluted soil by using luminescent bacteria comprises the following steps:
s101, reviving the luminescent bacteria freeze-dried powder stored at the temperature of-22 ℃ for 15min by using 1mL of Microtox diluent stored at the temperature of 4 ℃ to obtain revival bacteria liquid; the luminescent bacteria is vibrio fischeri; the vibrio fischeri is a deep-sea (with osmotic pressure) luminescent bacterium and can well meet the method for detecting the acute toxicity of the arsenic-polluted soil, so that the acute toxicity of the arsenic-polluted soil can be judged according to the relative luminosity;
s102, taking 0.1mL of resuscitation bacteria liquid, reading the luminous intensity of the resuscitation bacteria liquid in an ATP mode of a toxicity detector, and detecting the acute toxicity of the arsenic-polluted soil if the luminous intensity is more than 100 ten thousand photons;
s103, taking a soil sample to be detected as arsenic pollution, and leaching the soil sample to obtain a leached water sample; the leaching treatment comprises the following steps:
1) weighing a soil sample to be detected as arsenic pollution, placing the soil sample into a grinding pot, adding ceramic hollow spheres according to 30% of the mass of the soil sample to be detected as arsenic pollution, and calculating the volume of a required leaching agent according to 10L of the soil sample to be detected as arsenic pollution per 1kg for later use; the lixiviant is 0.1mol/L hydrochloric acid lixiviant; the ceramic hollow ball is filled with graphite and silicon carbide according to the weight ratio of 2: 1, mixing and pressing the mixture into a filled sphere, soaking the ceramic hollow sphere in 12% dopamine aqueous solution for 13min before use, and then washing the filled sphere for 3 times by using deionized water;
2) grinding a soil sample to be detected with arsenic pollution by using a grinding rod, dripping 15% by volume of a leaching agent for wet grinding, and continuously performing infrared illumination irradiation on the ground soil sample to be detected with arsenic pollution to obtain a leaching mixture; the infrared illumination power is 850W, and the wavelength range is 45 μm; the ceramic hollow spheres prepared according to the proportion can well assist infrared light irradiation to leach and extract arsenic in arsenic-polluted soil while grinding the arsenic-polluted soil to be detected;
3) uniformly mixing the leaching mixture obtained in the step 2) with the rest leaching agent, placing the mixture in an oscillating device, and oscillating for 10 hours at the rotating speed of 40r/min and the temperature of 20 ℃;
4) standing for 1.5h, filtering on a high-pressure filter, and storing the filtrate at 4 ℃ to obtain a leached water sample;
by the leaching treatment method, the ceramic hollow spheres and infrared light irradiation can be utilized, and the leaching extraction of arsenic in the arsenic-polluted soil can be enhanced by grinding with the leaching agent, so that more accurate detection result analysis is provided for detecting the acute toxicity of the arsenic-polluted soil by using luminescent bacteria;
s104, taking 1mL of the leached water sample, adding 0.1mL of osmotic pressure regulating solution, and uniformly mixing to obtain the leached water sample after the osmotic pressure is regulated;
s105, detecting by using a B-Tox mode of a toxicity detector, reading the initial luminous intensity of 0.1mL of resuscitation bacteria liquid, adding 0.9mL of leached water sample after osmotic pressure adjustment into 0.1mL of resuscitation bacteria liquid, uniformly mixing, keeping the temperature for 5min, detecting the luminous intensity after sample addition, and calculating the relative luminosity, wherein the calculation method of the relative luminosity comprises the following steps:
wherein, the relative luminosity is more than or equal to 90 percent and is low-toxicity or non-toxicity; poisoning is caused when the relative luminosity is more than or equal to 40% and less than 90%; the relative luminosity is more than or equal to 20 percent and less than 40 percent, and the toxicity is high; the relative luminosity is less than 20%, which is extremely toxic.
Example 2
This embodiment is substantially the same as embodiment 1, except that the method for resuscitation in step S101 is divided into a pre-resuscitation phase and a magnetic bead resuscitation phase, and includes the following steps:
1) pre-resuscitation phase
Adding the luminescent bacteria freeze-dried powder into 1mL of Microtox diluent stored at 4 ℃ for resuscitation for 2 min;
2) magnetic bead recovery phase
Then, magnetic beads with the temperature of 15 ℃, 21 ℃ and 28 ℃ are sequentially added for resuscitation for 7min, the adding amount of the magnetic beads each time is 12 particles/mL, the particle size of the magnetic beads is 0.8mm, an alternating magnetic field is continuously applied during the resuscitation period of the magnetic beads, the magnetic field intensity of the alternating magnetic field is 0.3mT, and the magnetic field frequency is 12 Hz;
wherein, the adding conditions of the magnetic beads are as follows: when the temperature difference between the magnetic beads and the Microtox diluent is less than 1 ℃ after the magnetic beads are added, adding the magnetic beads for the next time;
quantitative magnetic beads are added in a stepped mode, the initial temperatures of different magnetic beads are utilized to slowly raise the temperature of Microtox diluent, and meanwhile, an alternating magnetic field is matched for auxiliary recovery, so that the recovered luminous bacteria liquid is rapidly obtained, the recovery success rate of the luminous bacteria with the luminous intensity larger than 100 thousands of photons is high, and the detection efficiency of detecting the acute toxicity of the arsenic-polluted soil can be remarkably improved.
Example 3
The present embodiment is substantially the same as embodiment 1, except that the ceramic hollow spheres are filled with graphite and silicon carbide according to a ratio of 3: 2 mass ratio, and soaking the ceramic hollow spheres in 10% dopamine aqueous solution for 10min before use, and then washing 3 times with deionized water.
Example 4
The present embodiment is substantially the same as embodiment 1, except that the ceramic hollow spheres are filled with graphite and silicon carbide according to a ratio of 5: 2 mass ratio, and soaking the ceramic hollow spheres in 15% dopamine aqueous solution for 15min before use, and then washing 3 times with deionized water.
Example 5
The embodiment is basically the same as the embodiment 1, except that 2) a grinding rod is used for grinding a soil sample to be detected with arsenic pollution, meanwhile, 10 volume percent of leaching agent is dripped for wet grinding, and meanwhile, infrared illumination irradiation is continuously carried out on the ground soil sample to be detected with arsenic pollution to obtain a leaching mixture; the infrared illumination power is 500W, and the wavelength range is 30 μm; the ceramic hollow spheres prepared according to the proportion can well assist infrared light irradiation to leach and extract arsenic in arsenic-polluted soil while not affecting grinding of the soil to be detected for arsenic pollution.
Example 6
The embodiment is basically the same as the embodiment 1, except that 2) a grinding rod is used for grinding a soil sample to be detected with arsenic pollution, 20% by volume of leaching agent is dripped for wet grinding, and infrared illumination irradiation is continuously carried out on the ground soil sample to be detected with arsenic pollution to obtain a leaching mixture; the infrared illumination power is 1200W, and the wavelength range is 50 μm; the ceramic hollow spheres prepared according to the proportion can well assist infrared light irradiation to leach and extract arsenic in arsenic-polluted soil while not affecting grinding of the soil to be detected for arsenic pollution.
Example 7
The embodiment is basically the same as the embodiment 2, and the difference is that 1) in the pre-recovery stage, the luminescent bacteria freeze-dried powder is added into 1mL of Microtox diluent stored at 4 ℃ for recovery for 1 min.
Example 8
The embodiment is basically the same as the embodiment 2, and the difference is that 1) in the pre-recovery stage, the luminescent bacteria freeze-dried powder is added into 1mL of Microtox diluent stored at 4 ℃ for recovery for 3 min.
Example 9
The present example is substantially the same as example 2, except that, 2) a magnetic bead recovery stage, and then magnetic beads with the temperature of 15 ℃, 21 ℃ and 28 ℃ are added in sequence for 5min for recovery, the adding amount of the magnetic beads is 10 particles/mL each time, the particle size of the magnetic beads is 0.8mm, and an alternating magnetic field with the magnetic field intensity of 0.3mT and the magnetic field frequency of 12Hz is continuously applied during the magnetic bead recovery period.
Example 10
The present example is substantially the same as example 2, except that, 2) a magnetic bead recovery stage, and then magnetic beads with the temperature of 15 ℃, 21 ℃ and 28 ℃ are added in sequence for recovery for 8min, wherein the adding amount of the magnetic beads is 15 particles/mL each time, the particle size of the magnetic beads is 0.8mm, and an alternating magnetic field with the magnetic field intensity of 0.3mT and the magnetic field frequency of 12Hz is continuously applied during the magnetic bead recovery period.
Example 11
The present example is substantially the same as example 2, except that, 2) a magnetic bead recovery stage, and then magnetic beads with the temperature of 15 ℃, 21 ℃ and 28 ℃ are added in sequence for recovery for 7min, the adding amount of the magnetic beads is 12 particles/mL each time, the particle size of the magnetic beads is 0.8mm, and an alternating magnetic field with the magnetic field intensity of 0.1mT and the magnetic field frequency of 10Hz is continuously applied during the magnetic bead recovery period.
Example 12
The present example is substantially the same as example 2, except that, 2) a magnetic bead recovery stage, and then magnetic beads with the temperature of 15 ℃, 21 ℃ and 28 ℃ are added in sequence for recovery for 7min, the adding amount of the magnetic beads is 12 particles/mL each time, the particle size of the magnetic beads is 0.8mm, and an alternating magnetic field with the magnetic field intensity of 0.5mT and the magnetic field frequency of 15Hz is continuously applied during the magnetic bead recovery period.
Examples of the experiments
The method is characterized in that a certain polluted site in the great west of the river is taken as a test site, the heavy metal content of soil is shown in table 1, the pH value of the soil to be tested is alkaline, and the arsenic content in the soil to be tested is higher as can be seen from table 1;
TABLE 1 soil heavy metal content
The determination of the acute toxicity of arsenic contamination of soil sampled from the test site was carried out as in example 1, and fig. 1 shows the acute toxicity level of soil leachate to luminophores before and after stabilization. The reference luminescent bacteria acute toxicity grading method comprises the following steps: l is more than 90, and is nontoxic; l is more than 70 and less than or equal to 90, and the toxicity is low; l is more than 50 and less than or equal to 70, and poisoning; l is more than 30 and less than or equal to 50, and the toxicity is serious; l is more than 0 and less than or equal to 30, and the toxicity is high; l is 0, virulent;
blank controls are respectively set, the zeolite-based mineral restoration material and the pyrite are added according to the addition amounts of 4% and 1.5%, and the zeolite-based mineral restoration material and the pyrite are added according to the addition amounts of 8% and 3% to restore a comparison group of arsenic-contaminated soil;
as can be seen from fig. 1, after 2, 7, 15 and 28 days of stabilization by the zeolite-based mineral remediation material and the pyrite, the toxicity level of the soil leachate to the luminescent bacteria (vibrio fischeri) is reduced, because the concentration of As in the soil leachate is reduced compared with that of the control group along with the increase of the reaction time and the addition amount, so that the toxicity level is reduced compared with that of the original soil before stabilization, and As can be seen from comparison, the acute toxicity level of the arsenic-contaminated soil can be well reflected by the luminescent bacteria by adopting the method of example 1, so that the detection analysis can be effectively provided for the remediation treatment effect of the arsenic-contaminated soil by adopting the method for acute toxicity of arsenic-contaminated soil;
meanwhile, in order to further investigate the effect of each example on the detection of acute toxicity of arsenic-contaminated soil, the test was repeated 10 times, and the relative luminosity of example 1 was averaged to 95.7%, and as a control group similar to other examples, the following were investigated:
explore one
Taking examples 1 and 2 as examples, the relative luminosity was measured by stabilizing the zeolite-based mineral repair material and pyrite at 8% and 3% addition levels for 28 days, and example 2 was set up with two sets of ordinary tests, and after taking the average value, the results were compared and are shown in table 2 below:
table 2 relative luminosity of the tests for acute toxicity of arsenic contaminated soils in examples 1 and 2
| Group of | Relative luminosity (%) |
| Example 1 | 95.7 |
| Example 2 | 95.6 |
As can be seen from the results in Table 2, the relative luminosity of the acute toxicity detection of arsenic-contaminated soil is not greatly influenced by different recovery methods, wherein the relative luminosity of the example 2 is basically the same as that of the example 1, but the recovery time of the luminescent bacteria of the example 2 is 9min, while the recovery time of the luminescent bacteria of the example 1 is 15min, the recovery time of the luminescent bacteria is obviously shortened by the example 2, and the relative luminosity of the acute toxicity detection of arsenic-contaminated soil is basically the same as that of the example 1, so that the recovery method of the example 2 is relatively better.
Study on
Taking examples 1, 3 and 4 as examples, the zeolite-based mineral restoration material and the pyrite were added at 8% and 3%, and the stabilization was carried out for 28 days as relative luminosity measurement, and examples 3 and 4 were provided with two sets of ordinary tests, and after measuring the average value, the measurement results were compared, and the results are shown in the following table 3:
table 3 relative luminosity of the tests for acute toxicity of arsenic contaminated soils in examples 1, 3 and 4
| Group of | Relative luminosity (%) |
| Example 1 | 95.7 |
| Example 3 | 99.8 |
| Example 4 | 99.5 |
As can be seen from the results in table 3, different ceramic hollow spheres have certain influence on the relative luminosity of the detection of the acute toxicity of the arsenic-contaminated soil, wherein the relative luminosity of examples 3 and 4 on the detection of the acute toxicity of the arsenic-contaminated soil is higher than that of example 1, and considering that the leaching treatment method is insufficient for leaching the arsenic-contaminated soil, so that the arsenic content in examples 3 and 4 is lower than that in example 1, the leaching treatment method for the arsenic-contaminated soil to be detected is insufficient compared with that in example 1, so that the detection results of examples 3 and 4 are biased, and therefore, the leaching treatment method in example 1 is relatively better.
Study three
Taking examples 1, 5 and 6 as examples, the zeolite-based mineral restoration material and the pyrite were added at 8% and 3%, and the stabilization was carried out for 28 days as relative luminosity measurement, and examples 5 and 6 were provided with two sets of ordinary tests, and after measuring the average value, the measurement results were compared, and the results are shown in the following table 4:
table 4 relative luminosity of the tests for acute toxicity of examples 1, 5, 6 on arsenic contaminated soils
| Group of | Relative luminosity (%) |
| Example 1 | 95.7 |
| Example 5 | 99.3 |
| Example 6 | 98.4 |
As can be seen from the results in table 4, different infrared irradiation has certain influence on the relative luminosity of the detection of the acute toxicity of the arsenic-contaminated soil, wherein the relative luminosity of the detection of the acute toxicity of the arsenic-contaminated soil in examples 5 and 6 is higher than that in example 1, and the leaching treatment method in examples 5 and 6 is insufficient compared with that in example 1 because the arsenic content in the arsenic-contaminated soil in examples 5 and 6 is lower than that in example 1 due to insufficient leaching of the arsenic-contaminated soil in the leaching treatment method, so that the detection results in examples 5 and 6 are deviated, and therefore, the leaching treatment method in example 1 is relatively better.
Explore four
Taking examples 2 and 7, 8 as examples, the zeolite-based mineral restoration material and the pyrite were added at 8% and 3%, and the stabilization was carried out for 28 days as relative luminosity measurement, and examples 7 and 8 were provided with two sets of ordinary tests, and after measuring the average value, the measurement results were compared, and the results are shown in the following table 5:
TABLE 5 relative luminosity of the detection of acute toxicity of examples 2, 7, 8 on arsenic contaminated soils
| Group of | Relative luminosity (%) |
| Example 2 | 95.6 |
| Example 7 | 91.8 |
| Example 8 | 95.7 |
As can be seen from the results of table 5, the relative luminosity detected by the different pre-resuscitation phases on the arsenic contaminated soil has certain influence, wherein the relative luminosity detected by the examples 2 and 8 on the arsenic contaminated soil is higher than that detected by the example 7, but the relative luminosity detected by the example 8 has no influence on the example 2 from the economic point of view, but the pre-resuscitation phase time is increased, so that the resuscitation method of the example 2 is relatively better.
Study on five
Taking examples 2, 9 and 10 as examples, the zeolite-based mineral restoration material and the pyrite were added at 8% and 3%, and the stabilization was carried out for 28 days as relative luminosity measurement, and examples 9 and 10 were provided with two sets of ordinary tests, and after measuring the average value, the measurement results were compared, and the results are shown in the following table 6:
TABLE 6 relative luminosity of the tests for acute toxicity of examples 2, 9, 10 on arsenic contaminated soils
| Group of | Relative luminosity (%) |
| Example 2 | 95.6 |
| Example 9 | 90.2 |
| Example 10 | 95.7 |
From the results in table 6, it can be seen that different magnetic bead recovery stages have certain influence on the relative luminosity of the arsenic-contaminated soil acute toxicity test, wherein the relative luminosity of examples 2 and 10 on the arsenic-contaminated soil acute toxicity test is higher than that of example 9, but from the economic point of view, example 10 has substantially no influence on example 2, but the magnetic bead recovery stage time and the magnetic bead dosage per time are increased, so that the recovery method of example 2 is relatively better.
Explore six
Taking examples 2 and 11, 12 as examples, the zeolite-based mineral restoration material and pyrite were added at 8% and 3%, and stabilized for 28 days as relative luminosity measurement, and examples 11, 12 were set up with two sets of ordinary tests, and after taking average values, the measurement results were compared, and the results are shown in table 7 below:
TABLE 7 relative luminosity of the tests for acute toxicity of examples 2, 11, 12 on arsenic contaminated soils
| Group of | Relative luminosity (%) |
| Example 2 | 95.6 |
| Example 11 | 92.3 |
| Example 12 | 92.9 |
As can be seen from the results in Table 6, different stages of magnetic bead recovery have certain influence on the relative luminosity of the detection of the acute toxicity of the arsenic-contaminated soil, wherein the relative luminosity of examples 11 and 12 on the detection of the acute toxicity of the arsenic-contaminated soil is lower than that of example 2, so that the recovery method of example 2 is relatively better.
Claims (9)
1. A method for detecting acute toxicity of arsenic-polluted soil by using luminescent bacteria is characterized by comprising the following steps:
s101, reviving the luminescent bacteria freeze-dried powder stored at-22 ℃ to obtain reviving bacteria liquid;
s102, taking 0.1mL of resuscitation bacteria liquid, reading the luminous intensity of the resuscitation bacteria liquid by using a toxicity detector, wherein the luminous intensity is more than 100 ten thousand photons, and then the resuscitation bacteria liquid can be used for detecting the acute toxicity of the arsenic-polluted soil;
s103, taking a soil sample to be detected as arsenic pollution, and leaching the soil sample to obtain a leached water sample;
s104, taking 1mL of the leached water sample, adding 0.1mL of osmotic pressure regulating solution, and uniformly mixing to obtain the leached water sample after the osmotic pressure is regulated;
and S105, detecting by using a toxicity detector, reading the initial luminous intensity of 0.1mL of resuscitation bacteria liquid, adding 0.9mL of leached water sample after osmotic pressure adjustment into 0.1mL of resuscitation bacteria liquid, uniformly mixing, keeping the temperature for 3-8 min, detecting the luminous intensity after sample addition, and calculating the relative luminosity.
2. The method as claimed in claim 1, wherein the luminescent bacteria is Vibrio fischeri.
3. The method for detecting the acute toxicity of the arsenic-contaminated soil by using the luminescent bacteria as claimed in claim 1, wherein the method for recovering in step S101 is as follows: the luminescent bacteria freeze-dried powder is revived for 15min by using 1mL of Microtox diluent stored at 4 ℃.
4. The method for detecting the acute toxicity of arsenic-contaminated soil by using luminescent bacteria as claimed in claim 1, wherein the recovery method in step S101 is divided into a pre-recovery stage and a magnetic bead recovery stage, and comprises the following steps:
1) pre-resuscitation phase
Adding the luminescent bacteria freeze-dried powder into 1mL of Microtox diluent stored at 4 ℃ for resuscitation for 1-3 min;
2) magnetic bead recovery phase
Then, magnetic beads with the temperature of 15 ℃, 21 ℃ and 28 ℃ are sequentially added for resuscitation for 5-8 min, the adding amount of the magnetic beads each time is 10-15 particles/mL, the particle size of the magnetic beads is 0.5-1 mm, an alternating magnetic field is continuously applied during the resuscitation period of the magnetic beads, the magnetic field strength of the alternating magnetic field is 0.1-0.5 mT, and the magnetic field frequency is 10-15 Hz;
wherein, the adding conditions of the magnetic beads are as follows: and when the temperature difference between the magnetic beads and the Microtox diluent is less than 1 ℃ after the magnetic beads are added, adding the magnetic beads for the next time.
5. The method for detecting the acute toxicity of arsenic-contaminated soil by using luminescent bacteria as claimed in claim 1, wherein the ATP mode of the toxicity detector is used to read the luminescence intensity of the resuscitating bacteria liquid in step S102; in step S105, the B-Tox mode of the toxicity detector is used for detection.
6. The method for detecting the acute toxicity of the arsenic-polluted soil by using the luminescent bacteria as claimed in claim 1, wherein the detection of the acute toxicity of the arsenic-polluted soil can be performed only when the luminescence intensity of the resuscitation bacteria solution is greater than 100 million photons.
7. The method for detecting the acute toxicity of the arsenic-polluted soil by using the luminescent bacteria as claimed in claim 1, wherein the leaching treatment in the step S103 comprises the following steps:
1) weighing a soil sample to be detected as arsenic pollution, placing the soil sample into a grinding pot, adding ceramic hollow spheres according to 30% of the mass of the soil sample to be detected as arsenic pollution, and adding 8-12L of the ceramic hollow spheres per 1kg of the soil sample to be detected to calculate the volume of the needed leaching agent for later use;
2) grinding a soil sample to be detected with arsenic pollution by using a grinding rod, dripping 10-20% by volume of a leaching agent for wet grinding, and continuously performing infrared illumination irradiation on the ground soil sample to be detected with arsenic pollution to obtain a leaching mixture;
3) uniformly mixing the leaching mixture obtained in the step 2) with the rest leaching agent, placing the mixture in an oscillating device, and oscillating for 8-12 hours at the rotating speed of 40 +/-5 r/min and the temperature of 20 +/-5 ℃;
4) and standing for 1-2 h, filtering on a high-pressure filter, and storing the filtrate at 4 ℃ to obtain a leached water sample.
8. The method for detecting the acute toxicity of arsenic-contaminated soil by using luminescent bacteria as claimed in claim 7, wherein said leaching agent is 0.1mol/L hydrochloric acid leaching agent; graphite and silicon carbide are filled in the ceramic hollow ball according to the ratio of 3-5: 2, mixing and pressing the mixture according to the mass ratio to form a filled sphere, soaking the ceramic hollow sphere in 10-15% dopamine aqueous solution for 10-15 min before use, and then washing the filled sphere for 2-3 times by using deionized water; the infrared illumination power is 500-1200W, and the wavelength range is 30-50 mu m.
9. The method for detecting the acute toxicity of arsenic-contaminated soil by using luminescent bacteria as claimed in claim 1, wherein the calculating method of the relative luminosity in step S105 is as follows:
wherein, the relative luminosity is more than or equal to 90 percent and is low-toxicity or non-toxicity; poisoning is caused when the relative luminosity is more than or equal to 40% and less than 90%; the relative luminosity is more than or equal to 20 percent and less than 40 percent, and the toxicity is high; the relative luminosity is less than 20%, which is extremely toxic.
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