CN112029813A - Method for evaluating leaching toxicity of municipal waste incineration fly ash - Google Patents

Method for evaluating leaching toxicity of municipal waste incineration fly ash Download PDF

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CN112029813A
CN112029813A CN202010872460.9A CN202010872460A CN112029813A CN 112029813 A CN112029813 A CN 112029813A CN 202010872460 A CN202010872460 A CN 202010872460A CN 112029813 A CN112029813 A CN 112029813A
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邓怡
杨为中
韩秋阳
赵建奎
陈勇
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Abstract

The invention discloses a method for evaluating the leaching toxicity of municipal waste incineration fly ash, which comprises the following steps: s1, preparing a waste incineration fly ash sample; s2, preparing a leaching agent; s3, adding the sample prepared in the step S1 into the leaching agent prepared in the step S2 to prepare leaching liquid; s4, preparing a bacterial culture solution; s5, culturing bacteria, and evaluating the activity of the bacteria; s6, if the bacterial activity detection in the step S5 is qualified, further preparing a cell culture solution, and if the bacterial activity detection is not qualified, performing comet assay and western blot detection; s7, culturing cells; s8, taking the cells cultured in the step S7 to carry out a multi-cell toxicity test, and evaluating the activity of the cells; s9, if the cell activity detection in the step S8 is qualified, ending the experiment, and if the cell activity detection is unqualified, performing a comet assay; s10, carrying out western blot analysis on the step S7 by using various cells. The method directly utilizes the toxic reaction of bacteria and biological cells to the leaching solution, and can carry out scientific evaluation on the toxicity detection of the waste incineration fly ash.

Description

Method for evaluating leaching toxicity of municipal waste incineration fly ash
Technical Field
The invention relates to the technical field of environmental analysis and detection, in particular to a method for evaluating leaching toxicity of municipal waste incineration fly ash.
Background
The incineration fly ash is a solid byproduct generated in the municipal refuse incineration power generation technology, the main components of the incineration fly ash comprise silicate, carbonate, chloride, a small amount of organic matters, heavy metals and other substances, and among the components, particularly the heavy metals such as Pb, Cr, Cd, Cu, Zn and the like have serious harm effects on the natural environment and the biological safety. Therefore, the fly ash from waste incineration is classified as a hazardous waste by governments of various countries, and is also a main research object for eliminating secondary pollution in the municipal waste incineration technology.
The hazard of the incineration fly ash to the environment is essentially the result that toxic pollutants (such as heavy metals) are migrated and transformed to be leached into the nature when the incineration fly ash interacts with liquid phases such as surface water, underground water and rainwater in the natural environment. In recent years, environmental safety scientists have developed a series of researches on treatment technologies such as fly ash detoxification, stable solidification, separation and purification and the like aiming at substances such as toxic heavy metals and the like which can be leached from incineration fly ash. Many research results show that after the incineration fly ash is subjected to a proper treatment process, according to a Toxicity detection method widely adopted locally or internationally, namely a TCLP Toxicity Leaching program (Toxicity Characteristic Leaching Procedure) or other Toxicity evaluation methods based on TCLP, the environment safety of the treated incineration fly ash product meets relevant requirements, and the incineration fly ash can be directly subjected to final landfill disposal or can be used as a secondary building material for resource utilization in an environment-friendly manner. However, in practice, the leaching of incineration fly ash in nature is a very slow and influenced process by many factors, and TCLP is an evaluation method for evaluating the acute toxicity and static toxicity of solid waste from the program itself. Therefore, the evaluation of fly ash incineration by TCLP toxicity leaching procedures or other toxicity evaluation methods based on TCLP has resulted in inaccurate results, and the leaching toxicity of fly ash incineration, such as TCLP, does not guarantee the biological safety of fly ash or its treated products even within safety thresholds, since toxic substances can pass through the food chain and ultimately produce bio-amplification in animals and humans. Therefore, a method for scientifically detecting and evaluating the toxicity of the fly ash is lacked at present.
Moreover, different organisms respond differently to toxic substances due to the large difference in stress response of cells of different organisms or different tissues and organs in the organisms to exogenous substances, particularly due to the difference in physiological function, structure and expression of related genes of cell lines. The toxicity of a substance is detected by using a single individual or cell, and the result is obviously different according to the individual and the position; and the biological safety of the sample can be comprehensively judged by the multi-cellular reaction toxicity results from different sources, and the mechanism of toxic action of the substance on the cells is determined by further researching the changes of gene expression, physiological function and the like of the cells, so that the method has important guiding significance for optimizing the treatment technology of the incineration fly ash, resource utilization and other related researches.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a method for evaluating the leaching toxicity of the fly ash generated by burning the municipal refuse, which is used for scientifically detecting the toxicity of the fly ash, detecting the toxic reactions of different bacteria and cells and exploring the toxicity mechanism of a leaching solution to human bodies and organisms.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for evaluating the leaching toxicity of municipal waste incineration fly ash comprises the following steps:
s1, preparing a waste incineration fly ash sample;
s2, preparing a leaching agent;
s3, adding the sample prepared in the step S1 into the leaching agent prepared in the step S2, uniformly mixing to obtain a suspension, and filtering the suspension through a microporous filter membrane to obtain a leaching solution;
s4, preparing a bacterial culture solution: taking the leachate prepared in the step S3, adjusting the pH value to 7-8, taking deionized water, adding beef extract, peptone and NaCl into the deionized water to prepare a liquid culture medium, preparing the leachate after the pH value is adjusted and the liquid culture medium according to the proportion of 30-60% (v/v) to obtain a mixed bacterial culture solution, and refrigerating and storing the bacterial culture solution;
s5, culturing bacteria: taking the original bacterial liquid, diluting the original bacterial liquid in a gradient manner, then measuring the absorbance of the diluted bacterial liquid, mixing and culturing the diluted bacterial liquid and the bacterial culture solution prepared in the step S4 for a preset time, and then measuring the absorbance (OD value) of the bacterial liquid, thereby evaluating the bacterial activity;
s6, if the bacterial activity detection in the step S5 is qualified, further preparing a cell culture solution, and if the bacterial activity detection is not qualified, performing comet assay and western blot detection: taking a low-sugar medium (DMEM), adding Fetal Bovine Serum (FBS), penicillin and streptomycin into the DMEM to prepare a cell culture medium, taking the leachate prepared in the step S3, adjusting the pH value to 7-8, filtering out microorganisms in the leachate through a microfiltration membrane, mixing the filtered leachate with the cell culture medium according to the proportion of 30-60% (v/v) to prepare a cell culture solution, and refrigerating and storing the cell culture solution;
s7, culturing cells: incubating a plurality of cells with the cell culture medium prepared in the step S6, digesting the cells in the exponential growth cycle by trypsin to prepare a cell suspension, inoculating the cell suspension into a cell culture plate, placing the cell culture plate in a cell culture box for incubation, replacing the cell culture medium in the cell culture plate with the cell culture solution prepared in the step S6, and simultaneously setting a group of comparison groups, wherein the comparison groups replace the cell culture medium in the cell culture plate with the same volume of physiological saline;
s8, taking the cells cultured in the step S7 to perform a multi-cell toxicity test, measuring the absorbance of the cells, and evaluating the activity of the cells;
s9, if the cell activity detection in the step S8 is qualified, ending the experiment, if the cell activity detection is unqualified, taking the cell suspension prepared in the step S7, inoculating the cell suspension into a cell culture plate, placing the cell culture plate into a cell culture box for incubation, then replacing a culture medium in the cell culture plate with the bacterial culture solution prepared in the step S4, continuing the incubation, and carrying out a comet assay on the cells after the incubation is finished;
s10, carrying out western blot analysis on the step S7 by using various cells.
Further, in the step S1, the waste incineration fly ash is dried at 90-110 ℃ for 1 day to prepare a sample; the lixiviant in the step S2 is prepared by mixing concentrated sulfuric acid and concentrated nitric acid in a volume ratio of 2:1, adding the prepared mixed solution into deionized water, and adjusting the pH value to 3-5; the content of the sample added in the step S3 is 10g, the content of the leaching agent is 100mL, the sample and the leaching agent are sealed after being mixed, the mixture is overturned and oscillated at the speed of 20-50rpm for 18h at room temperature, the aperture of the microporous filter membrane is 0.3-0.5 μm, the leachate is alkaline, and the pH value is 11-13; the pH value of the leachate obtained in the step S4 is adjusted to 7.4, the content of deionized water is 100mL, the content of beef extract is 0.3g, the content of peptone is 1g, the content of NaCl is 0.5g, and the refrigeration temperature is 4 ℃.
Further, the dilution factor of the step S5 is 1-1000, the diluted bacterial liquid and the bacterial culture solution prepared in the step S4 are respectively cultured for 3h, 6h, 9h and 12h at 37 ℃, the absorbance of the bacterial liquid at the wavelength of 600nm is measured by an enzyme labeling instrument, and the type of the bacterial liquid is at least one of escherichia coli and staphylococcus aureus.
Further, the fetal bovine serum added in the step S6 contains 10% of low-sugar medium, 1% of penicillin and 1% of streptomycin, the pH of the leachate is adjusted to 7.4, the pore size of the microfiltration membrane is 0.22 μm, and the refrigeration temperature is 4 ℃.
Further, the plurality of cells of step S7 includes mouse embryonic osteogenic precursor cells (MC3T3-E1), mouse fibroblasts (L929), Human Umbilical Vein Endothelial Cells (HUVECs), mouse breast cancer cells (4T1), and human osteosarcoma cells (MG 63).
Further, the step S8 takes out the cells cultured in the step S7, washes 2 times with Phosphate Buffered Saline (PBS), and simultaneously takes out the CCK-8 reagent and the cell culture medium in the step S7 at a ratio of 10:1, adding a CCK-8 reagent into the wells of the cell culture plate in the step S7, placing the cell culture plate in a cell culture box for incubation for 2h, placing the CCK-8 solution in the wells of the cell culture plate in a microplate reader for measuring the absorbance value at 450nm, wherein the detection is carried out after the cells are cultured for 1 day, 3 days and 5 days in the step S7 respectively.
Specifically, in step S9, the cell suspension prepared in step S7 is inoculated into a cell culture plate, the culture medium in the cell culture plate is replaced by saline with the same volume ratio and set as a control group, the cell culture plate and the control group are placed in a cell culture box to be incubated for 1 day, the cells in the cell culture plate and the control group are digested by trypsin, the cells in the cell culture plate and the control group are mixed with 1% low-melting-point agarose and added onto a glass slide coated with 1% agarose in advance, electrophoresis is performed, and then comet is observed after electrophoresis.
Specifically, the western blot analysis of step S10 is to culture a plurality of cells for 1 day using the cells prepared in step S6, replace the culture medium in the cell culture plate with the bacterial culture solution prepared in step S4, incubate the cell culture plate in a cell incubator for 1 day, wash off the residual cell culture solution in the cell culture plate, add trypsin to the cell culture solution for digestion to prepare a cell suspension, centrifuge the cell culture suspension for 5-15 min, add lysis buffer (200 μ L of RIPA buffer, 1 μ L of protease inhibitor and 1 μ L of phenylmethylsulfonyl fluoride) to the cell culture suspension for 16-18 h, centrifuge for 10-20 min, take the supernatant, evaluate the protein content of the supernatant with BCA protein kit, and electrophorese the supernatant by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 100V for 1-3 h, protein was separated, transferred to nitrocellulose membrane, washed with TBST buffer for 10min, and repeated 3 times. Placing the nitrocellulose membrane on a flat dish, adding a confining liquid containing 5% skimmed milk powder, shaking for 2h, after blocking was completed, TBST buffer was added, the membrane was washed for 10min, repeated 3 times, and after the membrane was placed in a dish containing the primary antibody, culturing in a shaking table at 4 ℃ for 16-18 h, taking out, oscillating for 20-40 min at room temperature, sucking out a first antibody, washing for 10min by using TBST buffer solution, repeating for 3 times, diluting a second antibody by using skimmed milk powder confining liquid containing 5%, carrying out shaking table reaction at room temperature for 2h, sucking out a second antibody solution after the reaction is finished, washing for 10min by using TBST buffer solution, repeating for 3 times, mixing an enhanced luminescent agent and a stabilizing agent in the chemiluminescence immunoblot detection kit according to the equal volume of 1:1 to prepare a working reagent, dripping the working reagent on a washed nitrocellulose membrane containing protein, and visualizing immunoreaction bands by using a high-performance chemiluminescence instrument.
Specifically, the step S5 evaluates the bacterial activity using the following calculation formula:
Figure BDA0002651543900000051
wherein A iseAverage absorbance determined by the experimental group (different ratios of leachates); a. thec-average absorbance determined for the control group (saline); v-bacterial Activity.
Specifically, the step S8 evaluates the activity of the cell using the following calculation formula:
Figure BDA0002651543900000052
wherein: a. theeAverage absorbance determined by the experimental group (different ratios of leachates); a. thec-average absorbance determined for the control group (saline); a. the0-average absorbance determined for the blank (CCK-8 solution only); v-cellular Activity.
Compared with the prior art, the invention has the following beneficial effects:
(1) firstly, performing bacterial culture to preliminarily evaluate the toxicity of the fly ash leachate, determining the damage of the fly ash toxicity to organisms by further performing a cell experiment if the toxicity detection of the bacterial culture passes, determining the damage of the fly ash to the human body without performing the cell toxicity detection if the toxicity detection of the bacterial culture does not pass, analyzing the toxicity mechanism of the fly ash by performing a comet assay and a western blot detection, exploring the toxicity mechanism of the fly ash of the leachate to the organisms from the gene and protein level, and further determining the damage of the fly ash of the leachate; the damage of the leachate to the cell layer can be determined from the cytotoxicity detection, and if the cell experiment determines that the leachate has no toxicity, the leachate can be proved to have the condition of being capable of being processed, so that the mechanism of the toxicity to the human body does not need to be further analyzed.
(2) The bacteria toxicity detection is carried out by mixing the waste incineration fly ash with a leaching agent to prepare a leaching solution, preparing the leaching solution into a bacteria culture solution, culturing the bacteria in the bacteria culture solution, and the absorbance detection is carried out on the cultured bacteria, the activity of the bacteria is calculated according to a formula, the higher the absorbance of the bacteria in an experimental group is, the higher the activity of the bacteria is, the lower the toxicity of the leachate is, the lower the toxicity of the waste incineration fly ash can be obtained, the less harmful to the environment, the more than 90% of the activity of the bacteria was detected at each time point in step S5, then, a cytotoxicity detection test can be further performed on the fly ash sample, the leaching toxicity of the waste incineration fly ash to be detected sample prepared in the step S1 on cells is judged, the detection of the toxicity of the waste incineration fly ash is qualified, the evaluation is finished, and the detection of the genotoxicity is continued by performing the cytotoxicity test and the comet assay on the unqualified waste incineration fly ash; if the toxicity is less than the toxicity, the genotoxicity can be detected by a cytotoxicity test and a comet assay, the toxicity harm condition of the waste incineration fly ash reflecting human bodies and organisms is further tested, and compared with the prior art, the toxicity detection and evaluation of the waste incineration fly ash is more scientific.
(3) In order to ensure the effectiveness and the scientificity of a fly ash leaching toxicity test, bacteria or cell co-culture is introduced into the fly ash leaching toxicity test, but the bacteria co-culture method cannot completely simulate the microenvironment in an organism, so that the cell culture method is necessarily introduced after the preliminary evaluation of the bacteria co-culture, the leaching solution toxicity is more scientifically evaluated through the cooperative evaluation, and the damage of the fly ash to a human body is determined; in the bacterial culture process, because heavy metal ions exist in the fly ash leachate, the heavy metals act on enzymes in bacterial cells to cause the enzymes to lose activity, so that the growth of bacteria is inhibited, the higher the concentration of the heavy metals related to leaching capacity is, the stronger the inhibition effect is, the activity of the bacteria cultured in incineration ash can be calculated through the OD value of the bacterial liquid, the growth condition of the bacteria after treatment in the incineration ash is further judged, the influence of the incineration ash on the growth of the bacteria is further judged, and the toxicity of the fly ash leachate is further preliminarily evaluated; in the cytotoxicity detection, the toxicity effect of the incineration fly ash leachate is evaluated by detecting the proliferation and growth states of cells in the culture process in step S8, and the higher the OD value in the CCK-8 solution is, the higher the cell activity is, the higher the proportion of living cells is, the lower the leaching toxicity of the corresponding fly ash is, otherwise, the higher the leaching toxicity of the fly ash is.
(4) Compared with the traditional toxicity leaching test of the incineration fly ash, the method provided by the invention directly utilizes the toxic reaction of biological cells to the leaching solution, detects the multi-cell toxicity of the incineration fly ash from a cell level, and reveals the toxic action mechanism from a gene level and a protein level.
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FIG. 1 is a technical scheme for detecting toxicity of fly ash leachate from waste incineration.
FIG. 2 is a bacterial toxicity test chart of fly ash leachate from incineration of refuse according to example 1 of the present invention.
FIG. 3 is a cytotoxicity test chart of leachate of fly ash from incineration of refuse according to example 1 of the present invention.
FIG. 4 is a bacterial toxicity test chart of fly ash leachate from waste incineration in example 2 of the present invention.
FIG. 5 is a cytotoxicity test chart of leachate of fly ash from incineration of refuse in example 2 of the present invention.
FIG. 6 is a graph showing the bacterial toxicity of the fly ash leachate from incineration of refuse according to example 3 of the present invention.
FIG. 7 is a cytotoxicity test chart of leachate of fly ash from incineration of refuse according to example 3 of the present invention.
Detailed Description
The present invention will be further described with reference to the following description and examples, which include but are not limited to the following examples.
Example 1
S1, drying the waste incineration fly ash patterns of the same batch from the waste incineration power plant at 105 ℃ for 24 hours, sealing and storing for later use, wherein TCLP detection results are shown in Table 1;
s2, taking concentrated sulfuric acid and concentrated nitric acid, and mixing the concentrated sulfuric acid and the concentrated nitric acid with a mixture of 2:1, after uniformly mixing, dropwise adding the mixed solution into deionized water to make the pH value reach 4 to prepare an extracting agent;
s3, weighing 10g of the sample obtained in the step S1 and 100mL of the leaching agent obtained in the step S2, uniformly mixing the materials in a PE barrel, sealing the mixture, fixing the mixture on a turnover type oscillation device, carrying out turnover oscillation at the room temperature at the speed of 30rpm for 18 hours, and filtering the obtained suspension through a 0.3-micron microporous filter membrane to obtain a leaching solution, wherein the leaching solution is alkaline and has the pH value of 11;
s4, acidifying the leachate obtained in the step S3 to pH 7.4, adding 0.3g of beef extract, 1g of peptone and 0.5g of NaCl into 100ml of deionized water to prepare a bacterial culture medium, preparing the leachate and the bacterial culture medium in a ratio of 40% (v/v) to obtain a mixed bacterial culture solution, simulating the toxicity leaching of incineration fly ash in the nature, transferring the incineration fly ash into a centrifugal tube, and storing the incineration fly ash at 4 ℃ for later use;
s5, taking the original bacterial liquid for gradient dilution (1-1000 times), determining the absorbance of the diluted bacterial liquid by using an enzyme-labeling instrument to draw a standard curve so as to determine the concentration of the bacterial liquid, mixing and culturing the bacterial liquid and the bacterial culture liquid prepared in the step S4 in a humid environment at 37 ℃ for predetermined test time (3h, 6h, 9h and 12h), transferring the treated bacterial liquid into an enzyme-labeled plate, measuring the OD value of the bacterial liquid at the wavelength of 600nm, calculating the average value according to the measured absorbance value, and further determining the activity of bacteria so as to preliminarily evaluate the toxicity of the incineration fly ash leachate, wherein the experiment group is shown; meanwhile, putting the bacterial liquid with the same concentration into physiological saline with the same volume ratio, co-culturing for preset test time (3h, 6h, 9h and 12h) in a humid environment at 37 ℃, transferring the treated bacterial liquid into an enzyme label plate, measuring the OD value of the bacterial liquid at the wavelength of 600nm, and calculating the average value according to the measured absorbance value, wherein the average value is a control group; the bacterial activity was calculated from the average OD values measured in the experimental group and the average OD values measured in the control group according to the following formula:
Figure BDA0002651543900000081
wherein A iseExperimental group (a)Different proportions of leachates) measured average absorbance; a. thec-average absorbance determined for the control group (saline); v-bacterial Activity; the bacterial liquid is at least one of escherichia coli and staphylococcus aureus;
the results of the bacterial toxicity test are shown in FIG. 2.
In the process of detecting the bacteria, if the bacterial activity is higher, the toxicity of the leachate to the bacteria is lower, otherwise, the toxicity of the leachate to the bacteria is higher; if the activity of the two kinds of bacteria is not more than 90% at each time point, the toxicity of the fly ash sample can not meet the requirement, the comet assay and the western blot detection are required to be carried out continuously, if the activity of the bacteria is more than 90% at each time point, the toxicity of the fly ash sample can meet the requirement, and the cytotoxicity detection can be carried out to further determine the influence of the toxicity on the human body;
s6, adding 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin into a low-sugar medium (DMEM) to prepare a cell culture medium, acidifying the leachate obtained in the step S3 to pH 7.4 to be suitable for the growth of biological cells, filtering the leachate through a 0.22-micron microporous filter membrane to remove bacteria and other microorganisms in the leachate, preparing the leachate subjected to bacterial filtration and the cell culture medium in a ratio of 40% (v/v) to obtain a mixed cell culture solution, transferring the mixed cell culture solution into PE, and storing the mixed cell culture solution in a centrifuge tube at 4 ℃ for later use;
s7, frozen mouse embryo osteogenic precursor cells (MC3T3-E1), mouse fibroblasts (L929), Human Umbilical Vein Endothelial Cells (HUVECs), mouse breast cancer cells (4T1) and human osteosarcoma cells (MG63) were removed from a liquid nitrogen tank and transferred to a cell culture flask using a cell culture medium containing 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin under standard culture conditions (5% CO)2Incubation at 37 ℃ C., fresh cell culture medium was changed every 2 days, cells in exponential growth phase were trypsinized under microscopic observation to prepare a cell suspension, which was seeded in 48-well cell culture plates at a cell suspension volume of 500. mu.L/well and a cell density of 4X 104One well per well, place well plate in cell incubator (5% CO)2Incubation at 37 ℃ for 24h, at which time cells in the normal state will adhere to the wallGrowing the cells at the bottoms of the wells, replacing the culture medium in the 48-well cell culture plate with the cell culture solution obtained in the step S5, replacing the culture medium in the 48-well cell culture plate with normal saline with the same volume ratio as a control group, replacing the culture medium in the 48-well cell culture plate with CCK-8 solution with the same volume ratio as a blank group, and continuously culturing the experimental group, the control group and the blank group in an incubator for 1, 3 and 5 days, wherein each experimental group is provided with three repeated samples;
s8, after the culture plate of the step S7 is incubated for each preset test time (1d, 3d and 5d), sucking out the mixed culture solution in the holes, washing the mixed culture solution for 2 times by Phosphate Buffered Saline (PBS), mixing a CCK-8 reagent and a cell culture medium according to the volume ratio of 10:1, adding 330 mu L of a pre-prepared CCK-8 solution into each hole, putting the mixed culture solution into a cell incubator for incubation for 2h, sucking 100 mu L of the CCK-8 solution in each hole, placing the mixed culture solution in a microplate reader for measuring the absorbance value at 450nm, removing the CCK-8 solution in the hole after the measurement is finished, washing the mixed culture solution for 2 times by using the PBS, replacing the mixed culture solution with a new mixed culture solution, continuing the culture in the incubator, and repeating the toxicity detection step at the next test time point; the bacterial activity was calculated from the average OD values measured in the experimental group and the average OD values measured in the control group according to the following formula:
Figure BDA0002651543900000091
wherein: a. theeAverage absorbance determined by the experimental group (different ratios of leachates); a. thec-average absorbance determined for the control group (saline); a. the0-average absorbance determined for the blank (CCK-8 solution only); v-cellular activity;
the cytotoxicity test results are shown in fig. 3.
Calculating an average value according to the measured OD values, and obtaining cell activity through a calculation formula, wherein the higher the activity is, the more the number of cells in a corresponding hole is, the lower the toxicity of the leachate to the cells is, otherwise, the higher the toxicity of the leachate to the cells is, if the activity of the five cells (MC3T3, L929, HUVECs, 4T1 and MG63) at each time point is not more than 90%, the toxicity of the fly ash sample can not be met, so that a comet assay is continued, and the toxicity action mechanism of the fly ash leachate is researched;
s9, inoculating the cell suspension prepared in the step S7 into a 6-well cell culture plate, wherein the volume of the suspension is 2 mL/well, and the cell density is 1 multiplied by 105And each well, placing the pore plate in an incubator, incubating for 24h, then allowing cells to grow adherently, replacing the culture medium in the 6-well cell culture plate with the bacterial culture solution obtained in step S4, replacing the culture medium in the 6-well cell culture plate with physiological saline with the same volume ratio, setting the culture medium as a control group, further incubating for 24h in the incubator, separating the cells by trypsinization, mixing the cells with 1% low-melting agarose, adding the mixture onto a slide coated with 1% agarose in advance, treating the cells with lysis buffer (2.5M NaCl, 0.1M Na2EDTA, 10 mM-HCl, pH 10) for 16h before electrophoresis, treating in alkaline electrophoresis solution (10M NaOH, 0.2M EDTA, pH 13) for 20min, then performing electrophoresis at 25V for 15min, placing the slide in 70% ethanol for 15min, infiltrating with ethidium bromide (2mg/mL, 100mL) and retrogradely stained, comet was observed under a fluorescence microscope at 518/605nm excitation/emission wavelength;
s10, pre-culturing cells from each organ and tissue in different organisms in a cell culture medium for 24 hours, adding the bacterial culture solution prepared in the step S4 into the cell culture plate to replace the culture medium in the cell culture plate, continuously incubating the cells in an incubator for 24 hours, sucking the culture medium, adding 10ml PBS buffer solution into the cells twice, shaking the cells uniformly, washing off the residual cell culture solution in the culture plate, adding trypsin into the cells for digestion to prepare a cell suspension, transferring the cell culture solution into a centrifuge tube, centrifuging the cell culture solution for 10 minutes at 1000r/min, transferring the cell culture solution into a precooled centrifuge tube, adding a lysis buffer solution (200 mu L of RIPA buffer solution, 1 mu L of protease inhibitor and 1 mu L of phenylmethylsulfonyl fluoride) into the centrifuge tube, and treating the cell culture solution for 16 hours. Centrifuging at 14000r/min for 15min, and collecting supernatant as protein extract. Protein content was assessed using the BCA protein kit, proteins were separated by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) after 2h of 100V electrophoresis, proteins were transferred to NC membrane, membrane washing was performed with TBST for 10min, repeated 3 times, NC membrane was placed on a flat dish, blocking solution containing 5% skim milk powder was added, and shaking table was performed for 2 h. After sealing is finished, adding TBST, washing the membrane for 10min, repeating for 3 times, putting the membrane into a flat dish containing a first antibody, culturing overnight in a shaking table at 4 ℃, taking out, shaking for 30min at room temperature, sucking out the first antibody, washing for 10min by TBST, repeating for 3 times, diluting a second antibody by using 5% skimmed milk powder sealing solution, reacting for 2h in the shaking table at room temperature, sucking out the second antibody solution after the reaction is finished, washing for 10min by TBST, repeating for 3 times, mixing two reagents 1:1 in the chemiluminescence immunoblotting detection kit in equal volume to prepare a working reagent, dripping the working reagent on a washed NC membrane containing protein, and visualizing immunoreaction bands by using a high-performance chemiluminescence instrument.
And (3) jointly exploring a toxicity mechanism of the leachate fly ash to organisms by combining a comet experiment result and a western blot analysis result, and further determining the harm of the leachate fly ash.
TABLE 1 EXAMPLE 1 toxicity test results (unit: mg/L) of waste incineration fly ash TCLP
Cd Cr Cu Pb Zn
Example 1 0.0033 0.1659 0.0012 0.4255 0.3145
Threshold value 1 15 100 5 100
Comparing and analyzing the graph 2, according to the bacterial activity in the graph 2, the bacterial activity of the escherichia coli and the staphylococcus aureus which are detected in 3h, 6h, 9h and 12h is less than 90%, the leaching toxicity of the waste incineration fly ash sample cannot be judged to be beyond the standard, further detection is needed, and the release of toxic substances in the incineration ash to the environment poses great threat to organisms is revealed. During the bacterial culture process, heavy metal ions exist in the fly ash leaching liquid, and the heavy metals act on enzymes in bacterial cells to cause the enzymes to lose activity, so that the growth of bacteria is inhibited. The higher the heavy metal concentration associated with leaching ability, the stronger the inhibitory effect. The activity of bacteria cultured in incineration ash can be calculated through the OD value of the bacteria liquid, and then the growth condition of the bacteria treated in the incineration ash is judged, so that the influence of the incineration fly ash on the growth of the bacteria is judged, and the toxicity of the fly ash leachate is preliminarily evaluated.
The comparative analysis of figure 3 shows that mouse embryonic osteogenic precursor cells, mouse breast cancer cells and human osteosarcoma cells are sensitive to the toxic reaction of the leachate, which is closely related to the cell structure and gene expression of the cells. Moreover, the cell activities of the five cells which are examined at 1d, 3d and 5d are all less than 90%, the results in bacterial toxicity detection (the fly ash sample is harmful to human bodies) can be verified, the toxicity mechanism analysis is carried out when the bacterial toxicity detection is unqualified, and the release of toxic substances in the incineration ash to the environment poses great threat to organisms. Meanwhile, the synergistic evaluation of the toxicity of the incineration fly ash leachate by using various cells is an efficient and accurate mode, the toxic reactions of different cells to the leachate show great difference, and the incineration fly ash is treated and recycled to avoid the leachate from contacting sensitive parts in animals and human bodies, so that the possible biosafety hazard is reduced.
Comparing and analyzing the table 1, the fig. 2 and the fig. 3, when the TCLP detection meets the requirement, the toxicity detection of the evaluation method does not meet the requirement, i.e., the evaluation method has a stricter toxicity detection standard than the traditional toxicity detection method, the evaluation method can be more strict and definite for the toxicity detection, and the evaluation of the method is more scientific compared with the existing TCLP detection method.
Example 2
S1, drying the waste incineration fly ash patterns of the same batch from the waste incineration power plant at 105 ℃ for 24 hours, sealing and storing for later use, wherein the toxicity detection results of the waste incineration fly ash TCLP are shown in Table 2;
s2, taking concentrated sulfuric acid and concentrated nitric acid, and mixing the concentrated sulfuric acid and the concentrated nitric acid with a mixture of 2:1, after uniformly mixing, dropwise adding the mixed solution into deionized water to make the pH value reach 3 to prepare an extracting agent;
s3, weighing 10g of the sample obtained in the step S1 and 100mL of the leaching agent obtained in the step S2, uniformly mixing the materials in a PE barrel, sealing the mixture, fixing the mixture on a turnover type oscillation device, carrying out turnover oscillation at the room temperature at the speed of 40rpm for 18 hours, and filtering the obtained suspension through a 0.4-micron microporous filter membrane to obtain a leaching solution, wherein the leaching solution is alkaline and has the pH value of 12;
s4, acidifying the leachate obtained in the step S3 to pH 7.4, adding 0.3g of beef extract, 1g of peptone and 0.5g of NaCl into 100ml of deionized water to prepare a bacterial culture medium, preparing the leachate and the bacterial culture medium in a ratio of 50% (v/v) to obtain a mixed bacterial culture solution, simulating the toxicity leaching of incineration fly ash in the nature, transferring the incineration fly ash into a centrifugal tube, and storing the incineration fly ash at 4 ℃ for later use;
s5, taking the original bacterial liquid for gradient dilution (1-1000 times), determining the absorbance of the diluted bacterial liquid by using an enzyme-labeling instrument to draw a standard curve so as to determine the concentration of the bacterial liquid, mixing and culturing the bacterial liquid and the bacterial culture liquid prepared in the step S4 in a humid environment at 37 ℃ for predetermined test time (3h, 6h, 9h and 12h), transferring the treated bacterial liquid into an enzyme-labeled plate, measuring the OD value of the bacterial liquid at the wavelength of 600nm, calculating the average value according to the measured absorbance value, and further determining the activity of bacteria so as to preliminarily evaluate the toxicity of the incineration fly ash leachate, wherein the experiment group is shown; meanwhile, putting the bacterial liquid with the same concentration into physiological saline with the same volume ratio, co-culturing for preset test time (3h, 6h, 9h and 12h) in a humid environment at 37 ℃, transferring the treated bacterial liquid into an enzyme label plate, measuring the OD value of the bacterial liquid at the wavelength of 600nm, and calculating the average value according to the measured absorbance value, wherein the average value is a control group; the bacterial activity was calculated from the average OD values measured in the experimental group and the average OD values measured in the control group according to the following formula:
Figure BDA0002651543900000121
wherein A iseAverage absorbance determined by the experimental group (different ratios of leachates); a. thec-average absorbance determined for the control group (saline); v-bacterial Activity; the bacterial liquid is at least one of escherichia coli and staphylococcus aureus;
the results of the bacterial toxicity test are shown in FIG. 4.
In the process of detecting the bacteria, if the bacterial activity is higher, the toxicity of the leachate to the bacteria is lower, otherwise, the toxicity of the leachate to the bacteria is higher; if the activity of the two kinds of bacteria is not more than 90% at each time point, the toxicity of the fly ash sample can not meet the requirement, the comet assay and the western blot detection are required to be carried out continuously, if the activity of the bacteria is more than 90% at each time point, the toxicity of the fly ash sample can meet the requirement, and the cytotoxicity detection can be carried out to further determine the influence of the toxicity on the human body;
s6, adding 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin into a low-sugar medium (DMEM) to prepare a cell culture medium, acidifying the leachate obtained in the step S3 to pH 7.4 to be suitable for the growth of biological cells, filtering the leachate through a 0.22-micron microporous filter membrane to remove bacteria and other microorganisms in the leachate, preparing the leachate subjected to bacterial filtration and the cell culture medium in a ratio of 50% (v/v) to obtain a mixed cell culture solution, transferring the mixed cell culture solution into PE, and storing the mixed cell culture solution in a centrifuge tube at 4 ℃ for later use;
s7, frozen mouse embryo osteogenic precursor cells (MC3T3-E1), mouse fibroblasts (L929), Human Umbilical Vein Endothelial Cells (HUVECs), mouse breast cancer cells (4T1) and human osteosarcoma cells (MG63) were removed from a liquid nitrogen tank and transferred to a cell culture flask using a cell culture medium containing 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin under standard culture conditions (5% CO)2Incubation at 37 ℃ C., fresh cell culture medium was changed every 2 days, cells in exponential growth phase were trypsinized under microscopic observation to prepare a cell suspension, which was seeded in 48-well cell culture plates at a cell suspension volume of 500. mu.L/well and a cell density of 4X 104One well per well, place well plate in cell incubator (5% CO)2Incubating for 24 hours at 37 ℃), wherein cells in a normal state grow on the bottom of the wells in an adherent manner, replacing the culture medium in the 48-well cell culture plate with the cell culture solution obtained in step S5, replacing the culture medium in the 48-well cell culture plate with normal saline with the same volume ratio as a control group, replacing the culture medium in the 48-well cell culture plate with CCK-8 solution with the same volume ratio as a blank group, and continuously culturing the experiment group, the control group and the blank group in an incubator for 1, 3 and 5 days, wherein each experiment group is provided with three repeat samples;
s8, after the culture plate of the step S7 is incubated for each preset test time (1d, 3d and 5d), sucking out the mixed culture solution in the holes, washing the mixed culture solution for 2 times by Phosphate Buffered Saline (PBS), mixing a CCK-8 reagent and a cell culture medium according to the volume ratio of 10:1, adding 330 mu L of a pre-prepared CCK-8 solution into each hole, putting the mixed culture solution into a cell incubator for incubation for 2h, sucking 100 mu L of the CCK-8 solution in each hole, placing the mixed culture solution in a microplate reader for measuring the absorbance value at 450nm, removing the CCK-8 solution in the hole after the measurement is finished, washing the mixed culture solution for 2 times by using the PBS, replacing the mixed culture solution with a new mixed culture solution, continuing the culture in the incubator, and repeating the toxicity detection step at the next test time point; the bacterial activity was calculated from the average OD values measured in the experimental group and the average OD values measured in the control group according to the following formula:
Figure BDA0002651543900000141
wherein: a. theeAverage absorbance determined by the experimental group (different ratios of leachates); a. thec-average absorbance determined for the control group (saline); a. the0-average absorbance determined for the blank (CCK-8 solution only); v-cellular activity;
the results of the bacterial toxicity test are shown in FIG. 5.
Calculating an average value according to the measured OD values, and obtaining cell activity through a calculation formula, wherein the higher the activity is, the more the number of cells in a corresponding hole is, the lower the toxicity of the leachate to the cells is, otherwise, the higher the toxicity of the leachate to the cells is, if the activity of the five cells (MC3T3, L929, HUVECs, 4T1 and MG63) at each time point is not more than 90%, the toxicity of the fly ash sample can not be met, so that a comet assay is continued, and the toxicity action mechanism of the fly ash leachate is researched;
s9, inoculating the cell suspension prepared in the step S7 into a 6-well cell culture plate, wherein the volume of the suspension is 2 mL/well, and the cell density is 1 multiplied by 105Culturing the pore plate in an incubator for 24h, then allowing cells to grow in an adherent manner, replacing the culture medium in the 6-well cell culture plate with the bacterial culture solution obtained in step S4, replacing the culture medium in the 6-well cell culture plate with physiological saline in the same volume ratio, setting the culture medium as a control group, continuously incubating for 24h in the incubator, separating the cells by trypsinization, mixing the cells with 1% low-melting agarose, adding the mixture onto a slide coated with 1% agarose in advance, treating the cells with lysis buffer (2.5M NaCl, 0.1M Na2EDTA, 10 mM-HCl, pH 10) for 17h before electrophoresis, treating the cells in alkaline electrophoresis solution (10M NaOH, 0.2M EDTA, pH 13) for 20min, and then treating the cells in Tris-HCl solution (10M NaOH, 0.2M EDTA, pH 13) for 20minCarrying out electrophoresis at 25V for 15min, soaking the glass slide in 70% ethanol for 15min, staining with ethidium bromide (2mg/mL, 100mL) in a reverse direction, and observing comet at 518/605nm excitation/emission wavelength under a fluorescence microscope;
s10, pre-culturing cells from each organ and tissue in different organisms in a cell culture medium for 24 hours, adding the bacterial culture solution prepared in the step S4 into the cell culture plate to replace the culture medium in the cell culture plate, continuously incubating the cells in an incubator for 24 hours, sucking the culture medium, adding 10ml PBS buffer solution into the cells twice, shaking the cells uniformly, washing off the residual cell culture solution in the culture plate, adding trypsin into the cells for digestion to prepare a cell suspension, transferring the cell culture solution into a centrifuge tube, centrifuging the cell culture solution for 10 minutes at 1000r/min, transferring the cell culture solution into a precooled centrifuge tube, adding a lysis buffer solution (200 mu L of RIPA buffer solution, 1 mu L of protease inhibitor and 1 mu L of phenylmethylsulfonyl fluoride) into the centrifuge tube, and treating the cell culture solution for 18 hours. Centrifuging at 14000r/min for 15min, and collecting supernatant as protein extract. Protein content was assessed using the BCA protein kit, proteins were separated by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) after 2h of 100V electrophoresis, proteins were transferred to NC membrane, membrane washing was performed with TBST for 10min, repeated 3 times, NC membrane was placed on a flat dish, blocking solution containing 5% skim milk powder was added, and shaking table was performed for 2 h. After sealing is finished, adding TBST, washing the membrane for 10min, repeating for 3 times, putting the membrane into a flat dish containing a first antibody, culturing overnight in a shaking table at 4 ℃, taking out, shaking for 30min at room temperature, sucking out the first antibody, washing for 10min by TBST, repeating for 3 times, diluting a second antibody by using 5% skimmed milk powder sealing solution, reacting for 2h in the shaking table at room temperature, sucking out the second antibody solution after the reaction is finished, washing for 10min by TBST, repeating for 3 times, mixing two reagents 1:1 in the chemiluminescence immunoblotting detection kit in equal volume to prepare a working reagent, dripping the working reagent on a washed NC membrane containing protein, and visualizing immunoreaction bands by using a high-performance chemiluminescence instrument.
And (3) jointly exploring a toxicity mechanism of the leachate fly ash to organisms by combining a comet experiment result and a western blot analysis result, and further determining the harm of the leachate fly ash.
TABLE 2 example 2 toxicity test results (unit: mg/L) of fly ash from incineration of refuse TCLP
Cd Cr Cu Pb Zn
Example 1 0.0019 0.0911 0.0010 0.2845 0.2164
Threshold value 1 15 100 5 100
Comparing and analyzing the graph 4, according to the bacterial activity in the graph 4, the bacterial activity of the escherichia coli and the staphylococcus aureus which are detected in 3h, 6h, 9h and 12h is larger than 90%, the leaching toxicity of the waste incineration fly ash sample can be judged to be qualified in bacterial detection, cell detection can be carried out, and the influence of the fly ash on cells can be determined.
Comparing and analyzing the figure 5, it is found that the mouse embryo osteogenic precursor cells and the human umbilical vein endothelial cells are sensitive to the toxic reaction of the leaching solution, which is closely related to the cell structure and the gene expression condition of the cells. Furthermore, the cell activities of the five cells examined at 1d, 3d and 5d are less than 90%, the existence of harm to human body can be confirmed, and the toxic mechanism analysis should be carried out, which reveals that the release of toxic substances in the incineration ash to the environment poses a great threat to organisms. Meanwhile, the synergistic evaluation of the toxicity of the incineration fly ash leachate by using various cells is an efficient and accurate mode, the toxic reactions of different cells to the leachate show great difference, and the incineration fly ash is treated and recycled to avoid the leachate from contacting sensitive parts in animals and human bodies, so that the possible biosafety hazard is reduced. And cell detection is continued after the bacteria detection is qualified, so that the harm condition of the fly ash to the human body can be more accurately determined.
Comparing and analyzing the table 2, the fig. 4 and the fig. 5, when the TCLP detection meets the requirement, the toxicity detection of the evaluation method does not meet the requirement, i.e., the evaluation method has a stricter toxicity detection standard than the traditional toxicity detection method, the evaluation method can be more strict and definite for the toxicity detection, and the evaluation of the method is more scientific compared with the existing TCLP detection method.
Example 3
S1, drying the waste incineration fly ash patterns of the same batch from the waste incineration power plant at 105 ℃ for 24 hours, sealing and storing for later use, wherein TCLP detection results are shown in Table 3;
s2, taking concentrated sulfuric acid and concentrated nitric acid, and mixing the concentrated sulfuric acid and the concentrated nitric acid with a mixture of 2:1, after uniformly mixing, dropwise adding the mixed solution into deionized water to make the pH value reach 5 to prepare an extracting agent;
s3, weighing 10g of the sample obtained in the step S1 and 100mL of the leaching agent obtained in the step S2, uniformly mixing the materials in a PE barrel, sealing the mixture, fixing the mixture on a turnover type oscillation device, carrying out turnover oscillation at the room temperature at the speed of 30rpm for 18 hours, and filtering the obtained suspension through a 0.5-micron microporous filter membrane to obtain a leaching solution, wherein the leaching solution is alkaline and has the pH value of 12;
s4, acidifying the leachate obtained in the step S3 to pH 7.4, adding 0.3g of beef extract, 1g of peptone and 0.5g of NaCl into 100ml of deionized water to prepare a bacterial culture medium, preparing the leachate and the bacterial culture medium in a ratio of 60% (v/v) to obtain a mixed bacterial culture solution, simulating the toxicity leaching of incineration fly ash in the nature, transferring the incineration fly ash into a centrifugal tube, and storing the incineration fly ash at 4 ℃ for later use;
s5, taking the original bacterial liquid for gradient dilution (1-1000 times), determining the absorbance of the diluted bacterial liquid by using an enzyme-labeling instrument to draw a standard curve so as to determine the concentration of the bacterial liquid, mixing and culturing the bacterial liquid and the bacterial culture liquid prepared in the step S4 in a humid environment at 37 ℃ for predetermined test time (3h, 6h, 9h and 12h), transferring the treated bacterial liquid into an enzyme-labeled plate, measuring the OD value of the bacterial liquid at the wavelength of 600nm, calculating the average value according to the measured absorbance value, and further determining the activity of bacteria so as to preliminarily evaluate the toxicity of the incineration fly ash leachate, wherein the experiment group is shown; meanwhile, putting the bacterial liquid with the same concentration into physiological saline with the same volume ratio, co-culturing for preset test time (3h, 6h, 9h and 12h) in a humid environment at 37 ℃, transferring the treated bacterial liquid into an enzyme label plate, measuring the OD value of the bacterial liquid at the wavelength of 600nm, and calculating the average value according to the measured absorbance value, wherein the average value is a control group; the bacterial activity was calculated from the average OD values measured in the experimental group and the average OD values measured in the control group according to the following formula:
Figure BDA0002651543900000171
wherein A iseAverage absorbance determined by the experimental group (different ratios of leachates); a. thec-average absorbance determined for the control group (saline); v-bacterial Activity; the bacterial liquid is at least one of escherichia coli and staphylococcus aureus;
the results of the bacterial tests are shown in FIG. 6.
In the process of detecting the bacteria, if the bacterial activity is higher, the toxicity of the leachate to the bacteria is lower, otherwise, the toxicity of the leachate to the bacteria is higher; if the activity of the two kinds of bacteria is not more than 90% at each time point, the toxicity of the fly ash sample can not meet the requirement, the comet assay and the western blot detection are required to be carried out continuously, if the activity of the bacteria is more than 90% at each time point, the toxicity of the fly ash sample can meet the requirement, and the cytotoxicity detection can be carried out to further determine the influence of the toxicity on the human body;
s6, adding 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin into a low-sugar medium (DMEM) to prepare a cell culture medium, acidifying the leachate obtained in the step S3 to pH 7.4 to be suitable for the growth of biological cells, filtering the leachate through a 0.22-micron microporous filter membrane to remove bacteria and other microorganisms in the leachate, preparing the leachate subjected to bacterial filtration and the cell culture medium in a ratio of 60% (v/v) to obtain a mixed cell culture solution, transferring the mixed cell culture solution into PE, and storing the mixed cell culture solution in a centrifuge tube at 4 ℃ for later use;
s7, frozen mouse embryo osteogenic precursor cells (MC3T3-E1), mouse fibroblasts (L929), Human Umbilical Vein Endothelial Cells (HUVECs), mouse breast cancer cells (4T1) and human osteosarcoma cells (MG63) were removed from a liquid nitrogen tank and transferred to a cell culture flask using a cell culture medium containing 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin under standard culture conditions (5% CO)2Incubation at 37 ℃ C., fresh cell culture medium was changed every 2 days, cells in exponential growth phase were trypsinized under microscopic observation to prepare a cell suspension, which was seeded in 48-well cell culture plates at a cell suspension volume of 500. mu.L/well and a cell density of 4X 104One well per well, place well plate in cell incubator (5% CO)2And 37 ℃) for 24 hours, wherein cells in a normal state grow adherently at the bottom of the hole, the culture medium in the 48-hole cell culture plate is replaced by the cell culture solution obtained in the step S5, the culture medium in the 48-hole cell culture plate is replaced by normal saline with the same volume ratio as a control group, and the culture medium in the 48-hole cell culture plate is replaced by CCK-8 solution with the same volume ratio as a blankGroup, experimental group, control group and blank group are continuously cultured in incubator for 1, 3 and 5 days, each experimental group is provided with three repeat samples;
s8, after the culture plate of the step S7 is incubated for each preset test time (1d, 3d and 5d), sucking out the mixed culture solution in the holes, washing the mixed culture solution for 2 times by Phosphate Buffered Saline (PBS), mixing a CCK-8 reagent and a cell culture medium according to the volume ratio of 10:1, adding 330 mu L of a pre-prepared CCK-8 solution into each hole, putting the mixed culture solution into a cell incubator for incubation for 2h, sucking 100 mu L of the CCK-8 solution in each hole, placing the mixed culture solution in a microplate reader for measuring the absorbance value at 450nm, removing the CCK-8 solution in the hole after the measurement is finished, washing the mixed culture solution for 2 times by using the PBS, replacing the mixed culture solution with a new mixed culture solution, continuing the culture in the incubator, and repeating the toxicity detection step at the next test time point; the bacterial activity was calculated from the average OD values measured in the experimental group and the average OD values measured in the control group according to the following formula:
Figure BDA0002651543900000181
wherein: a. theeAverage absorbance determined by the experimental group (different ratios of leachates); a. thec-average absorbance determined for the control group (saline); a. the0-average absorbance determined for the blank (CCK-8 solution only); v-cellular activity;
the results of the bacterial assay are shown in FIG. 7.
Calculating an average value according to the measured OD values, and obtaining cell activity through a calculation formula, wherein the higher the activity is, the more the number of cells in a corresponding hole is, the lower the toxicity of the leachate to the cells is, otherwise, the higher the toxicity of the leachate to the cells is, if the activity of the five cells (MC3T3, L929, HUVECs, 4T1 and MG63) at each time point is not more than 90%, the toxicity of the fly ash sample can not be met, so that a comet assay is continued, and the toxicity action mechanism of the fly ash leachate is researched;
s9, taking the cell suspension prepared in the step S7, inoculating the cell suspension into a 6-hole cell culture plate, wherein the volume of the suspension is 2 mL/hole, and the cell density is highIs 1 × 105And each well, placing the pore plate in an incubator, incubating for 24h, then allowing cells to grow adherently, replacing the culture medium in the 6-well cell culture plate with the bacterial culture solution obtained in step S4, replacing the culture medium in the 6-well cell culture plate with physiological saline with the same volume ratio, setting the culture medium as a control group, further incubating for 24h in the incubator, separating the cells by trypsinization, mixing the cells with 1% low-melting agarose, adding the mixture onto a slide coated with 1% agarose in advance, treating the cells with lysis buffer (2.5M NaCl, 0.1M Na2EDTA, 10 mM-HCl, pH 10) for 17h before electrophoresis, treating in alkaline electrophoresis solution (10M NaOH, 0.2M EDTA, pH 13) for 20min, then performing electrophoresis at 25V for 15min, placing the slide in 70% ethanol for 15min, infiltrating with ethidium bromide (2mg/mL, 100mL) and retrogradely stained, comet was observed under a fluorescence microscope at 518/605nm excitation/emission wavelength;
s10, pre-culturing cells from each organ and tissue in different organisms in a cell culture medium for 24 hours, adding the bacterial culture solution prepared in the step S4 into the cell culture plate to replace the culture medium in the cell culture plate, continuously incubating the cells in an incubator for 24 hours, sucking the culture medium, adding 10ml PBS buffer solution into the cells twice, shaking the cells uniformly, washing off the residual cell culture solution in the culture plate, adding trypsin into the cells for digestion to prepare a cell suspension, transferring the cell culture solution into a centrifuge tube, centrifuging the cell culture solution for 10 minutes at 1000r/min, transferring the cell culture solution into a precooled centrifuge tube, adding a lysis buffer solution (200 mu L of RIPA buffer solution, 1 mu L of protease inhibitor and 1 mu L of phenylmethylsulfonyl fluoride) into the centrifuge tube, and treating the cell culture solution for 18 hours. Centrifuging at 14000r/min for 15min, and collecting supernatant as protein extract. Protein content was assessed using the BCA protein kit, proteins were separated by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) after 2h of 100V electrophoresis, proteins were transferred to NC membrane, membrane washing was performed with TBST for 10min, repeated 3 times, NC membrane was placed on a flat dish, blocking solution containing 5% skim milk powder was added, and shaking table was performed for 2 h. After sealing is finished, adding TBST, washing the membrane for 10min, repeating for 3 times, putting the membrane into a flat dish containing a first antibody, culturing overnight in a shaking table at 4 ℃, taking out, shaking for 30min at room temperature, sucking out the first antibody, washing for 10min by TBST, repeating for 3 times, diluting a second antibody by using 5% skimmed milk powder sealing solution, reacting for 2h in the shaking table at room temperature, sucking out the second antibody solution after the reaction is finished, washing for 10min by TBST, repeating for 3 times, mixing two reagents 1:1 in the chemiluminescence immunoblotting detection kit in equal volume to prepare a working reagent, dripping the working reagent on a washed NC membrane containing protein, and visualizing immunoreaction bands by using a high-performance chemiluminescence instrument.
And (3) jointly exploring a toxicity mechanism of the leachate fly ash to organisms by combining a comet experiment result and a western blot analysis result, and further determining the harm of the leachate fly ash.
TABLE 3 example 3 toxicity test results (unit: mg/L) of fly ash from incineration of refuse TCLP
Cd Cr Cu Pb Zn
Example 1 0.0061 0.2348 0.0033 0.6197 0.2075
Threshold value 1 15 100 5 100
Comparing and analyzing the graph 6, according to the bacterial activity of the graph 6, the bacterial activity of the escherichia coli and the staphylococcus aureus which are detected in 3h, 6h, 9h and 12h is less than 90%, the fact that the leaching toxicity of the waste incineration fly ash sample exceeds the standard can be judged, further detection is needed, and the fact that the release of toxic substances in the incineration ash to the environment poses great threat to organisms is revealed.
Comparing and analyzing the figure 7, the mouse embryo osteogenesis precursor cells, the mouse breast cancer cells and the toxic reaction to the leachate are more sensitive, which is closely related to the cell structure and the gene expression condition of the mouse embryo osteogenesis precursor cells and the mouse breast cancer cells. Moreover, the cell activities of the five cells which are examined at 1d, 3d and 5d are all less than 90%, the results in bacterial toxicity detection (the fly ash sample is harmful to human bodies) can be verified, the toxicity mechanism analysis is carried out when the bacterial toxicity detection is unqualified, and the release of toxic substances in the incineration ash to the environment poses great threat to organisms. Meanwhile, the synergistic evaluation of the toxicity of the incineration fly ash leachate by using various cells is an efficient and accurate mode, the toxic reactions of different cells to the leachate show great difference, and the incineration fly ash is treated and recycled to avoid the leachate from contacting sensitive parts in animals and human bodies, so that the possible biosafety hazard is reduced.
Comparing and analyzing the table 3, fig. 6 and fig. 7, when the TCLP detection meets the requirement, the toxicity detection of the evaluation method does not meet the requirement, i.e. the evaluation method has a stricter toxicity detection standard than the traditional toxicity detection method.
By integrating the analysis of the embodiments 1 to 3, under the condition that the TCLP detection result is qualified, the bacteria toxicity detection and the cytotoxicity detection are both unqualified, so that the bacteria toxicity detection and the cytotoxicity detection are determined to be more qualified compared with the TCLP detection, and when one of the bacteria toxicity detection and the cytotoxicity detection is unqualified, the toxicity mechanism analysis (comet assay result and western blot analysis) is carried out, so that the generation reason of the fly ash toxicity can be determined.
The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or changes made within the spirit and scope of the main design of the present invention, which still solve the technical problems consistent with the present invention, should be included in the scope of the present invention.

Claims (10)

1. A method for evaluating the leaching toxicity of municipal waste incineration fly ash is characterized by comprising the following steps:
s1, preparing a waste incineration fly ash sample;
s2, preparing a leaching agent;
s3, adding the sample prepared in the step S1 into the leaching agent prepared in the step S2, uniformly mixing to obtain a suspension, and filtering the suspension through a microporous filter membrane to obtain a leaching solution;
s4, preparing a bacterial culture solution: taking the leachate prepared in the step S3, adjusting the pH value to 7-8, taking deionized water, adding beef extract, peptone and NaCl into the deionized water to prepare a liquid culture medium, preparing the leachate after the pH value is adjusted and the liquid culture medium according to the proportion of 30-60% (v/v) to obtain a mixed bacterial culture solution, and refrigerating and storing the bacterial culture solution;
s5, culturing bacteria: taking the original bacterial liquid, diluting the original bacterial liquid in a gradient manner, measuring the absorbance of the diluted bacterial liquid, carrying out mixed culture on the diluted bacterial liquid and the bacterial culture liquid prepared in the step S4 for a preset time, measuring the absorbance of the bacterial liquid, and further evaluating the bacterial activity;
s6, if the bacterial activity detection in the step S5 is qualified, further preparing a cell culture solution, and if the bacterial activity detection is not qualified, performing comet assay and western blot detection: taking a low-sugar culture medium, adding fetal calf serum, penicillin and streptomycin into the low-sugar culture medium to prepare a cell culture medium, taking the leachate prepared in the step S3, adjusting the pH value to 7-8, filtering out microorganisms in the leachate through a microporous filtering membrane, mixing the filtered leachate with the cell culture medium in a ratio of 30-60% (v/v) to prepare a cell culture solution, and refrigerating and storing the cell culture solution;
s7, culturing cells: incubating a plurality of cells with the cell culture medium prepared in the step S6, digesting the cells in the exponential growth cycle by trypsin to prepare a cell suspension, inoculating the cell suspension into a cell culture plate, placing the cell culture plate in a cell culture box for incubation, replacing the cell culture medium in the cell culture plate with the cell culture solution prepared in the step S6, and simultaneously setting a group of comparison groups, wherein the comparison groups replace the cell culture medium in the cell culture plate with the same volume of physiological saline;
s8, taking the cells cultured in the step S7 to perform a multi-cell toxicity test, measuring the absorbance of the cells, and evaluating the activity of the cells;
s9, if the cell activity detection in the step S8 is qualified, ending the experiment, if the cell activity detection is unqualified, taking the cell suspension prepared in the step S7, inoculating the cell suspension into a cell culture plate, placing the cell culture plate into a cell culture box for incubation, then replacing a culture medium in the cell culture plate with the bacterial culture solution prepared in the step S4, continuing the incubation, and carrying out a comet assay on the cells after the incubation is finished;
s10, carrying out western blot analysis on the step S7 by using various cells.
2. The method for evaluating the leaching toxicity of the municipal solid waste incineration fly ash according to claim 1, wherein the step S1 is to dry the municipal solid waste incineration fly ash at 90-110 ℃ for 1 day to obtain a sample; the lixiviant in the step S2 is prepared by mixing concentrated sulfuric acid and concentrated nitric acid in a volume ratio of 2:1, adding the prepared mixed solution into deionized water, and adjusting the pH value to 3-5; the content of the sample added in the step S3 is 10g, the content of the leaching agent is 100mL, the sample and the leaching agent are sealed after being mixed, the mixture is overturned and oscillated at the speed of 20-50rpm for 18h at room temperature, the aperture of the microporous filter membrane is 0.3-0.5 μm, and the leaching solution is alkaline; the pH value of the leachate obtained in the step S4 is adjusted to 7.4, the content of deionized water is 100mL, the content of beef extract is 0.3g, the content of peptone is 1g, the content of NaCl is 0.5g, and the refrigeration temperature is 4 ℃.
3. The method for evaluating the leaching toxicity of the municipal solid waste incineration fly ash according to claim 1, wherein the dilution factor of step S5 is 1-1000, the diluted bacterial solution and the bacterial culture solution prepared in step S4 are respectively cultured at 37 ℃ for 3h, 6h, 9h and 12h, the absorbance of the bacterial solution at the wavelength of 600nm is measured by a microplate reader, and the bacterial solution is at least one of Escherichia coli and Staphylococcus aureus.
4. The method as claimed in claim 1, wherein the fetal bovine serum content added in step S6 is 10% of low-sugar medium, penicillin is 1% of low-sugar medium, and streptomycin is 1% of low-sugar medium, the pH of the leachate is adjusted to 7.4, the pore size of the microfiltration membrane is 0.22 μm, and the refrigeration temperature is 4 ℃.
5. The method of claim 1, wherein the plurality of cells of step S7 includes mouse embryonic osteogenic precursor cells, mouse fibroblasts, human umbilical vein endothelial cells, mouse breast cancer cells, and human osteosarcoma cells.
6. The method of claim 5, wherein the step S8 is to take out the cells cultured in step S7, wash the cells with phosphate buffer 2 times, and take out the CCK-8 reagent and the cell culture medium in step S7 at a ratio of 10:1, adding a CCK-8 reagent into the wells of the cell culture plate in the step S7, placing the cell culture plate in a cell culture box for incubation for 2h, placing the CCK-8 solution in the wells of the cell culture plate in a microplate reader for measuring the absorbance value at 450nm, wherein the detection is carried out after the cells are cultured for 1 day, 3 days and 5 days in the step S7 respectively.
7. The method of claim 6, wherein the step S9 comprises inoculating the cell suspension obtained in step S7 into a cell culture plate, replacing the culture medium in the cell culture plate with physiological saline at the same volume ratio to obtain a control group, incubating the cell culture plate and the control group in a cell incubator for 1 day, digesting the cells in the cell culture plate and the control group with trypsin, mixing the cells in the cell culture plate and the control group with 1% low melting point agarose, loading the mixture onto a glass slide coated with 1% agarose, performing electrophoresis, and observing comet after electrophoresis.
8. The method of claim 7, wherein the Western blot analysis of step S10 is performed by culturing a plurality of cells for 1 day using the cells prepared in step S6, replacing the culture medium in the cell culture plate with the bacterial culture solution prepared in step S4, incubating the cell culture plate in a cell incubator for 1 day, washing off the residual cell culture solution in the cell culture plate, adding trypsin for digestion, preparing a cell suspension, centrifuging the cell suspension for 5-15 min, adding lysis buffer for treatment for 16-18 h, centrifuging for 10-20 min, collecting the supernatant, evaluating the protein content of the supernatant with a BCA protein kit, and separating the protein after electrophoresis at 100V for 1-3 h by sodium dodecyl sulfate polyacrylamide electrophoresis, proteins were transferred to nitrocellulose membranes and membranes were washed with TBST buffer for 10min and repeated 3 times. Placing the nitrocellulose membrane on a flat dish, adding a confining liquid containing 5% skimmed milk powder, shaking for 2h, after blocking was completed, TBST buffer was added, the membrane was washed for 10min, repeated 3 times, and after the membrane was placed in a dish containing the primary antibody, culturing in a shaking table at 4 ℃ for 16-18 h, taking out, oscillating for 20-40 min at room temperature, sucking out a first antibody, washing for 10min by using TBST buffer solution, repeating for 3 times, diluting a second antibody by using skimmed milk powder confining liquid containing 5%, carrying out shaking table reaction at room temperature for 2h, sucking out a second antibody solution after the reaction is finished, washing for 10min by using TBST, repeating for 3 times, mixing an enhanced luminescent agent and a stabilizing agent in the chemiluminescence immunoblotting detection kit according to the equal volume of 1:1 to prepare a working reagent, dripping the working reagent on a washed nitrocellulose membrane containing protein, and visualizing immunoreaction bands by using a high-performance chemiluminescence instrument.
9. The method for evaluating fly ash leaching toxicity of municipal refuse incineration according to claim 3, wherein said step S5 of evaluating bacterial activity employs the following calculation formula:
Figure FDA0002651543890000041
wherein A iseAverage absorbance determined by the experimental group (different ratios of leachates); a. thecAverage absorbance determined for the control group (mixed with the same volume of saline and bacteria); v-bacterial Activity.
10. The method for evaluating fly ash leaching toxicity of municipal refuse incineration according to claim 6, wherein said step S8 of evaluating cell activity employs the following calculation formula:
Figure FDA0002651543890000042
wherein: a. theeAverage absorbance determined by the experimental group (different ratios of leachates); a. thec-average absorbance determined for the control group (saline); a. the0-average absorbance determined for the blank (CCK-8 solution only); v-cellular Activity.
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