Methods for testing the biodegradability of perfluoro- and polyfluorinated compounds

Abstract

This invention relates to the field of perfluorinated and polyfluorinated compound (PFPC) degradation, and discloses a method for testing the biodegradability of PFPCs. The method includes: (1) mixing PFPCs with a first culture medium to obtain a concentrated solution; (2) inoculating a bacterial culture for degrading PFPCs into a second culture medium to obtain a culture medium containing the bacterial culture; (3) placing the concentrated solution in the culture medium containing the bacterial culture for degradation, followed by mixing the supernatant, alizarin red S, lanthanum salt, and buffer solution for reaction; (4) mixing the reactants, dimethyl sulfoxide, and polyethylene glycol octylphenyl ether, and testing the mixture using ultraviolet-visible spectrophotometry, determining the biodegradability of PFPCs based on the absorbance obtained from the test. The method described in this invention is simple and improves the efficiency of detecting the biodegradability of PFPCs.

Description

Technical Field

[0001] This invention relates to the field of perfluorinated and polyfluorinated compound degradation, and more specifically to a method for testing the biodegradability of perfluorinated and polyfluorinated compounds. Background Technology

[0002] Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic chemicals encompassing more than 10,000 substances. Due to the presence of high-energy carbon-fluorine bonds (CF), they possess strong thermal and chemical stability. The introduction of fluorine atoms gives PFAS excellent surface activity and hydrophobic and oleophobic properties, thus making them widely used in industrial fields such as textiles, leather, electroplating, petroleum, mining, aerospace, and semiconductors, as well as in commercial products such as water-based film-forming foams, pesticides, jackets, personal care products, paints, non-stick cookware, and food contact materials.

[0003] Due to their persistence, long-distance migration, and bioaccumulation, PFAS are widely detected in environmental media such as soil, atmosphere, surface water, sediments, and oceans, as well as in biological tissues such as plants, animals, and humans. Toxicological studies have shown that PFAS possess various biotoxicities, including hepatotoxicity, neurotoxicity, reproductive and developmental toxicity, endocrine-disrupting effects, and carcinogenicity, posing a serious threat to human and other biological health.

[0004] Currently, green alternatives are the main method to address PFAS environmental pollution at its source. Perfluorooctanoic acid (PFOA) and perfluorooctanoic acid (PFOA) are the most widely used and impactful PFAS, also known as traditional PFAS. With the banning of traditional PFAS, a large number of new PFAS with diverse structures have been applied to industrial production as replacements. Currently, there are over 10,000 registered PFAS types. PFAS replacements are mainly divided into two categories: fluorine-free replacements and new PFAS with structures similar to traditional PFAS. Although fluorine-free replacements have applications in areas such as fire-fighting foam, PFAS remains the best choice in many fields due to its superior performance. Therefore, a large number of new PFAS are widely used in various industrial and commercial manufacturing processes. However, there is still a lack of research on the degradation performance and ecotoxicity of these new PFAS. Using new PFAS for production in a hasty manner may lead to a vicious cycle of "pollution-substitution-re-pollution". Therefore, it is urgent to conduct high-throughput screening of the biodegradability and toxicity of a large number of new PFAS, and to screen out green new PFAS with low toxicity and biodegradability to replace traditional PFAS.

[0005] In studies on the degradability of PFAS, the amount of fluoride ions produced is an important indicator for characterizing the degree of PFAS degradation. The more fluoride ions produced, the more organic fluorine is removed, and the more likely its persistence, bioaccumulation, and toxicity are to be reduced. In addition, quantitative analysis of fluoride ions also helps to elucidate the degradation mechanism of PFAS.

[0006] Current research on PFAS degradation mainly uses serum bottles or glass bottles as reaction vessels to culture single or mixed bacterial species, adding PFAS as substrates or co-metabolites. Samples are taken during the culture process, and fluoride ions are quantitatively analyzed using ion chromatography or selective fluoride ion electrodes. This method is cumbersome and has low detection efficiency. With nearly ten thousand novel PFAS existing, there is an urgent need for a rapid and convenient method to test the biodegradability of perfluorinated and polyfluorinated compounds, efficiently screen for degradable green PFAS, and provide scientific support for the green alternatives to PFAS. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems of low efficiency and cumbersome operation in the existing technology for testing the biodegradability of perfluorinated and polyfluorinated compounds. This invention provides a method for testing the biodegradability of perfluorinated and polyfluorinated compounds. This method involves a colorimetric reaction between Alizarin Red S and lanthanum salt and the F ions generated during the degradation of the perfluorinated and polyfluorinated compounds. The compounds can then be detected using ultraviolet-visible spectrophotometry. This method is convenient and simple, and can simultaneously detect hundreds of perfluorinated and polyfluorinated compounds, greatly improving the efficiency of detecting the biodegradability of perfluorinated and polyfluorinated compounds.

[0008] To achieve the above objectives, the present invention provides a method for testing the biodegradability of perfluorinated and polyfluorinated compounds, the method comprising the following steps:

[0009] (1) A concentrated solution was obtained by mixing perfluorinated and polyfluorinated compounds with the first culture medium;

[0010] (2) Inoculate the bacterial culture containing the strains used to degrade perfluorinated and polyfluorinated compounds into the second culture medium, add a carbon source and culture to obtain a culture medium containing the bacterial culture.

[0011] (3) The concentrated solution is placed in the culture medium containing the bacterial solution for degradation. The supernatant after degradation, Alizarin Red S, Lanthanum salt and buffer solution are mixed and reacted, and the pH of the reaction system is controlled to be 5-6.

[0012] (4) Mix the reactants obtained in step (3), dimethyl sulfoxide and polyethylene glycol octylphenyl ether, and test the mixture by ultraviolet-visible spectrophotometry. Determine the biodegradability of perfluorinated and polyfluorinated compounds based on the absorbance obtained from the test.

[0013] The first and second culture media contain 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, alkali metal hydrochloride, alkali metal nitrate, calcium salt and magnesium salt.

[0014] Preferably, in step (4), the specific process of determining the biodegradability of perfluorinated and polyfluorinated compounds based on the absorbance obtained from the test includes:

[0015] S1. Sodium fluoride is mixed with the first culture medium, and then the mixture is diluted to obtain standard solutions with different fluoride ion concentrations;

[0016] S2. Mix the standard solution, alizarin red S, lanthanum salt and buffer solution and react them, controlling the pH of the reaction system to be 5-6. Then mix the obtained reaction material, dimethyl sulfoxide and polyethylene glycol octylphenyl ether, and test the absorbance of the solution by ultraviolet-visible spectrophotometry.

[0017] S3. Using the fluoride ion concentration of the standard solution as the abscissa and the absorbance of the solution obtained in step S3 as the ordinate, a standard curve is plotted. Then, the absorbance obtained in step (4) is substituted into the standard curve to calculate the fluoride ion concentration in the solution after the reaction, and the degradation rate of the perfluorinated and polyfluorinated compounds is calculated based on the fluoride ion concentration.

[0018] S4. Determine the biodegradability of the perfluorinated and polyfluorinated compounds based on the defluorination rate.

[0019] Preferably, in the first culture medium and the second culture medium, the concentration of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid is 8-12 mmol / L, the concentration of alkali metal hydrochloride is 12-18 mmol / L, the concentration of alkali metal nitrate is 4-6 mmol / L, the concentration of magnesium salt is 0.8-1.5 mmol / L, and the concentration of calcium salt is 0.8-1.5 mmol / L.

[0020] Preferably, in the first culture medium and the second culture medium, the alkali metal hydrochloride is NaCl and / or KCl, the alkali metal nitrate is NaNO3 and / or KNO3, the calcium salt is calcium chloride, and the magnesium salt is magnesium chloride;

[0021] Preferably, the first culture medium and the second culture medium contain 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, NaCl, KCl, NaNO3, calcium salt and magnesium salt.

[0022] Preferably, in the first culture medium and the second culture medium, the molar ratio of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, alkali metal hydrochloride, alkali metal nitrate, calcium salt and magnesium salt is 8-12:12-17:3-6:0.8-1.2:1.

[0023] Preferably, the bacterial strains used to degrade perfluorinated and polyfluorinated compounds are selected from one or more of the following: *Pseudomonas fluorescens*, *Pseudomonas putida*, *Gordonella*, strain NB4–1Y, *Phanerochaete chrysosporium*, *Paragonimococcus pantrophicus*, Gram-negative bacteria, and *Bacillus subtilis*.

[0024] Preferably, the volume ratio of the degraded supernatant, dimethyl sulfoxide, and polyethylene glycol octylphenyl ether is (93-98):(3-7):0.3.

[0025] Preferably, the buffer solution is sodium acetate buffer solution.

[0026] Preferably, the pH value of the buffer solution is 5-6.

[0027] Preferably, the molar ratio of alizarin red S to the lanthanum salt is 0.8-1.5:1.

[0028] Preferably, the alizarin red S is added in the form of an alizarin red S solution;

[0029] Preferably, the pH value of the alizarin red S solution is 5-6.

[0030] Preferably, the lanthanum salt is added in the form of a lanthanum salt solution;

[0031] Preferably, the lanthanum salt is at least one selected from lanthanum nitrate hexahydrate, lanthanum chloride heptahydrate, and lanthanum carbonate trihydrate.

[0032] Preferably, the pH value of the lanthanum salt solution is 5-6.

[0033] Preferably, the test wavelength of the ultraviolet-visible spectrophotometry is 600-650 nm.

[0034] Preferably, the specific process of step (2) includes: inoculating the bacterial solution containing the strain for degrading perfluorinated and polyfluorinated compounds into the third culture medium, adding a carbon source for constant temperature culture, taking the bacterial solution every 7-10 hours and testing it with a UV-Vis spectrophotometer to read the absorbance at a wavelength of 590-610 nm, and when the absorbance is >0.4, inoculating the corresponding bacterial solution containing the strain for degrading perfluorinated and polyfluorinated compounds into the second culture medium, adding a carbon source for culture, and obtaining a culture medium containing the bacterial solution.

[0035] Preferably, the composition of the third culture medium is the same as that of the second culture medium.

[0036] Preferably, the carbon source is selected from one or more of glucose, lactose, sodium propionate, sodium acetate, and straight-chain alkanes.

[0037] Preferably, the concentration of perfluorinated and polyfluorinated compounds in the concentrated solution is 0.8-1.5 mmol / L.

[0038] Preferably, the concentration of the carbon source is 1-3 g / L.

[0039] The method described in this invention involves reacting the supernatant from the biodegradation of perfluorinated and polyfluorinated compounds with Alizarin Red S and lanthanum salt for colorimetric analysis, followed by UV-Vis spectrophotometry to calculate the concentration of fluoride ions in the supernatant. This fluoride ion concentration is then used to calculate the degree of biodegradability of the perfluorinated and polyfluorinated compounds, thus determining their biodegradability. This method is simple to operate and eliminates the need for complex instruments such as ion chromatography; a microplate reader is sufficient for the entire detection process, significantly reducing testing time. Furthermore, this method allows for simultaneous, high-throughput detection, further improving efficiency. It effectively enables high-throughput detection and evaluation of the biodegradability of perfluorinated and polyfluorinated compounds, providing a theoretical basis for the subsequent screening of novel, green perfluorinated and polyfluorinated compounds. Attached Figure Description

[0040] Figure 1 This is a layout diagram of the 96-hole plate used in Example 1;

[0041] Figure 2 This is the test result graph for test example 2;

[0042] Figure 3 This is a graph showing the sensitivity of fluoride ion detection at different test wavelengths;

[0043] Figure 4 This is a graph showing the effect of culture medium components on the absorbance of the solution. Detailed Implementation

[0044] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0045] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0046] The method for testing the biodegradability of perfluorinated and polyfluorinated compounds according to the present invention includes the following steps:

[0047] (1) A concentrated solution was obtained by mixing perfluorinated and polyfluorinated compounds with the first culture medium;

[0048] (2) Inoculate the bacterial culture containing the strains used to degrade perfluorinated and polyfluorinated compounds into the second culture medium, add a carbon source and culture to obtain a culture medium containing the bacterial culture.

[0049] (3) The concentrated solution is placed in the culture medium containing the bacterial solution for degradation. The supernatant after degradation, Alizarin Red S, Lanthanum salt and buffer solution are mixed and reacted, and the pH of the reaction system is controlled to be 5-6.

[0050] (4) Mix the reactants obtained in step (3), dimethyl sulfoxide and polyethylene glycol octylphenyl ether, and test the mixture by ultraviolet-visible spectrophotometry. Determine the biodegradability of perfluorinated and polyfluorinated compounds based on the absorbance obtained from the test.

[0051] In the method described in this invention, the composition of the first and second culture media is particularly important for the implementation and accuracy of the method. Therefore, in the method described in this invention, the composition of the first and second culture media has been adaptively adjusted to ensure that the strains used to degrade perfluorinated and polyfluorinated compounds can reproduce and grow in the culture media, and to ensure that the composition of the culture media does not affect the accuracy of the spectrophotometric detection of the colorimetric reaction between the degraded supernatant and Alizarin Red S and lanthanum salt. This avoids the influence of the composition of the culture media on the accuracy of fluoride ion content detection, thereby enabling the method described in this invention to more accurately and conveniently detect the biodegradability of perfluorinated and polyfluorinated compounds.

[0052] In a specific embodiment, the first and second culture media contain 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, alkali metal hydrochloride, alkali metal nitrate, calcium salt, and magnesium salt. Specifically, the 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid is a buffer salt used to adjust the pH value of the system in the culture medium, preventing acidification of the system due to acidic products generated during microbial growth, thereby inhibiting microbial growth and the degradation reaction of perfluorinated and polyfluorinated compounds. The alkali metal hydrochloride, alkali metal nitrate, calcium salt, and magnesium salt provide the necessary salts for the growth of the microorganisms without interfering with subsequent detection processes, thus avoiding affecting the accuracy of the test method.

[0053] In a preferred embodiment, in the first culture medium and the second culture medium, the concentration of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid is 8-12 mmol / L, the concentration of the alkali metal hydrochloride is 12-18 mmol / L, the concentration of the alkali metal nitrate is 4-6 mmol / L, the concentration of the magnesium salt is 0.8-1.5 mmol / L, and the concentration of the calcium salt is 0.8-1.5 mmol / L.

[0054] In a preferred embodiment, the alkali metal hydrochloride is NaCl and / or KCl, the alkali metal nitrate is NaNO3 and / or KNO3, the calcium salt is calcium chloride, and the magnesium salt is magnesium chloride;

[0055] In a further preferred embodiment, the first culture medium and the second culture medium contain 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, NaCl, KCl, NaNO3, calcium salts, and magnesium salts.

[0056] In the method described in this invention, in order to ensure that the composition of the culture medium used does not have a negative impact on the testing process, the molar ratio of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, alkali metal hydrochloride, alkali metal nitrate, calcium salt and magnesium salt in the first culture medium and the second culture medium is 8-12:12-17:3-6:0.8-1.2:1, preferably 9-11:14-16:4-6:0.9-1.1:1.

[0057] In a specific embodiment, the concentration of perfluorinated and polyfluorinated compounds in the concentrated solution is 0.8-1.5 mmol / L, preferably 0.9-1.2 mmol / L. Specifically, the concentration of perfluorinated and polyfluorinated compounds in the concentrated solution can be 0.8 mmol / L, 0.9 mmol / L, 1 mmol / L, 1.1 mmol / L, or 1.2 mmol / L.

[0058] In some specific embodiments, the perfluorinated and polyfluorinated compounds to be tested are dissolved in a first culture medium. For perfluorinated and polyfluorinated compounds to be tested that are insoluble in water or have poor water solubility, dimethyl sulfoxide can be added to the first culture medium to dissolve them and prepare a concentrated solution. The volume ratio of dimethyl sulfoxide to the first culture medium is 8-15:100.

[0059] In the method described in this invention, the specific species of degrading bacteria used is not limited, as long as they are bacteria capable of degrading perfluorinated and polyfluorinated compounds. In a preferred embodiment, to further improve the efficiency of the test, the bacterial species used to degrade perfluorinated and polyfluorinated compounds are selected from one or more of the following: *Pseudomonas fluorescens*, *Pseudomonas putida*, *Goldenella*, strain NB4–1Y, *Phanerochaete chrysosporium*, *Paragonimella pantrophiformis*, Gram-negative bacteria, and *Bacillus subtilis*. All of these bacterial species are common and can be obtained through commercial channels.

[0060] In a specific implementation, the bacterial strains used to degrade perfluorinated and polyfluorinated compounds can be obtained from fresh activated sludge in the biochemical unit of a municipal wastewater treatment plant.

[0061] In a specific implementation, step (2) includes: inoculating the bacterial culture of bacteria used to degrade perfluorinated and polyfluorinated compounds into a third culture medium, adding a carbon source for constant temperature culture, taking samples of the bacterial culture every 7-10 hours and testing them with a UV-Vis spectrophotometer to read the absorbance at a wavelength of 590-610 nm, and when the absorbance is >0.4, inoculating the corresponding bacterial culture containing bacteria used to degrade perfluorinated and polyfluorinated compounds into a second culture medium, adding a carbon source for culture, and obtaining a culture medium containing bacterial culture.

[0062] In some specific embodiments, the specific process of step (2) includes: taking 1L of fresh activated sludge from the biochemical unit of the municipal sewage treatment plant, allowing it to settle naturally for 1 hour, then inoculating the supernatant of the activated sludge into 500mL of the third culture medium at a volume ratio of 10%, and adding a carbon source with a final concentration of 1-3g / L. The mixture is then cultured in an incubator at a rotation speed of 100rpm and a constant temperature of 28℃. Every 8 hours, the bacterial solution is taken and tested with a UV-Vis spectrophotometer to read the absorbance at a wavelength of 600nm. When the absorbance is >0.4, the bacterial solution used to degrade perfluorinated and polyfluorinated compounds is inoculated into the second culture medium, and a carbon source is added for further culture to obtain a culture medium containing the bacterial solution.

[0063] In a preferred embodiment, the concentration of the carbon source is 1-3 g / L. Specifically, the concentration of the carbon source can be 1 g / L, 2 g / L, or 3 g / L.

[0064] In a preferred embodiment, the carbon source is selected from one or more of glucose, lactose, sodium propionate, sodium acetate, and straight-chain alkanes, more preferably glucose.

[0065] In the method described in this invention, bacterial culture of bacteria used to degrade perfluorinated and polyfluorinated compounds (PFOCs) is used to degrade PFOCs. The supernatant after degradation is then mixed with Alizarin Red S and lanthanum salt for a colorimetric reaction to determine whether the added PFOCs have been biodegraded. Specifically, when the added PFOCs are biodegradable, fluoride ions are generated in the supernatant after degradation. The concentration of fluoride ions generated during degradation in the supernatant can be further determined by the colorimetric reaction of fluoride ions with Alizarin Red S and lanthanum salt, allowing for the calculation of the degree of degradation of the added PFOCs and thus assessing their biodegradability. The method described in this invention provides a convenient and efficient way to test the biodegradability of various PFOCs without the need for cumbersome instruments such as ion chromatography; a microplate reader is sufficient. Therefore, compared to traditional methods, this significantly improves detection efficiency and provides strong support for the biodegradability of novel PFOCs. More importantly, the method described in this invention, by adaptively altering the composition of the culture medium and further adjusting the detection method, can further improve the detection of the biodegradability of perfluorinated and polyfluorinated compounds, and ensure the accuracy of the test results.

[0066] In the method described in this invention, in step (3), the concentrated solution is placed in the culture medium containing the bacterial solution for degradation. The supernatant after degradation, Alizarin Red S, lanthanum salt and buffer solution are mixed and reacted, and the pH value of the reaction system is controlled to be 5-6. By controlling the pH value of the system, the colorimetric detection of the reaction between the fluoride ions in the supernatant after degradation and Alizarin Red S can be made most sensitive, thereby further improving the accuracy of the test.

[0067] In a specific embodiment, the buffer solution is a sodium acetate buffer solution, and preferably, the pH value of the buffer solution is 5-6.

[0068] In one specific embodiment, the pH of the sodium acetate buffer solution is adjusted to 5-6 using acetic acid before use.

[0069] In a specific embodiment, the concentration of sodium acetate in the sodium acetate buffer solution is 70-90 mmol / L. In the mixture obtained by mixing the degraded supernatant, Alizarin Red S, lanthanum salt, and buffer solution, the concentration of sodium acetate is 70-90 μmol / L.

[0070] In a preferred embodiment, to further improve the accuracy of the test, the molar ratio of alizarin red S to the lanthanum salt is 0.8-1.5:1, preferably 0.9-1.2:1. Specifically, the molar ratio of alizarin red S to the lanthanum salt can be 0.8:1, 0.9:1, 1:1, 1.2:1, 1.4:1, or 1.5:1.

[0071] In a specific embodiment, Alizarin Red S is added in the form of an Alizarin Red S solution. Preferably, the concentration of Alizarin Red S in the Alizarin Red S solution is 400-550 μmol / L. In the mixture obtained by mixing the degradation supernatant, Alizarin Red S, lanthanum salt, and buffer solution, the concentration of Alizarin Red S is 40-70 μmol / L.

[0072] In a preferred embodiment, to further control the pH of the reaction system stably at 5-6, the pH of the alizarin red S solution is limited to 5-6. Specifically, the pH of the alizarin red S solution can be adjusted using hydrochloric acid solution.

[0073] In a specific embodiment, the lanthanum salt is added in the form of a lanthanum salt solution. Preferably, the concentration of lanthanum salt in the lanthanum salt solution is 400-550 μmol / L. In the mixture obtained by mixing the degradation supernatant, Alizarin Red S, lanthanum salt, and buffer solution, the concentration of lanthanum salt is 40-70 μmol / L.

[0074] In a specific embodiment, the lanthanum salt is at least one of lanthanum nitrate hexahydrate, lanthanum chloride heptahydrate, and lanthanum carbonate trihydrate.

[0075] In a preferred embodiment, the pH value of the lanthanum salt solution is 5-6. Specifically, the pH value of the lanthanum salt solution can be adjusted using a 1 mol / L sodium acetate solution.

[0076] In the method described in this invention, in step (4), the reactants obtained in step (3) are mixed with dimethyl sulfoxide and polyethylene glycol octylphenyl ether. During the mixing process, the colorimetric reactions of fluoride ions, alizarin red S, and lanthanum salt in the supernatant continue. The added dimethyl sulfoxide and polyethylene glycol octylphenyl ether can further enhance the colorimetric reaction by improving the solubility of the reactants, the polarity of the reaction medium, and the homogeneity of the reaction system, making the test results more accurate. Furthermore, the method described in this invention, by optimizing and replacing acetone used in the standard method with dimethyl sulfoxide, can better reduce the toxicity of organic solvents in the culture medium, protect the safety of experimental personnel, and make the solution after detection have low biotoxicity, so that it can continue to be used for microbiology-related experiments.

[0077] In a preferred embodiment, the volume ratio of the degraded supernatant, dimethyl sulfoxide, and polyethylene glycol octylphenyl ether is (93-98):(3-7):0.3, more preferably (94-96):(4-6):0.3, and even more preferably 95:5:0.3.

[0078] In a specific implementation, the reaction time in step (3) is 8-15 minutes. Specifically, the reaction time can be 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, or 15 minutes.

[0079] In a specific implementation, the mixing time in step (4) is 25-40 minutes. Specifically, the mixing time can be 25 minutes, 30 minutes, 35 minutes, or 40 minutes.

[0080] In the method described in this invention, the specific process of determining the biodegradability of perfluorinated and polyfluorinated compounds based on the absorbance obtained from the test in step (4) includes:

[0081] S1 mixes sodium fluoride with the first culture medium, and then dilutes the mixture to obtain standard solutions with different fluoride ion concentrations;

[0082] S2 mixes the standard solution, alizarin red S, lanthanum salt and buffer solution to react, and controls the pH of the reaction system to 5-6. Then, the obtained reaction material, dimethyl sulfoxide and polyethylene glycol octylphenyl ether are mixed, and the absorbance of the mixed material is tested by ultraviolet-visible spectrophotometry.

[0083] S3 uses the fluoride ion concentration of the standard solution as the abscissa and the absorbance of the solution obtained in step S2 as the ordinate to plot a standard curve. Then, the absorbance obtained in step (4) is substituted into the standard curve to calculate the fluoride ion concentration in the solution after the reaction, and the amount of defluorination of the perfluorinated and polyfluorinated compounds is calculated based on the fluoride ion concentration.

[0084] S4 determines the biodegradability of the perfluorinated and polyfluorinated compounds based on the amount of defluorination.

[0085] In a specific implementation, in step S4, the defluorination rate is calculated using the formula: Defluorination rate = Molar amount of fluoride ions in the supernatant after degradation / Molar amount of fluorine in perfluorinated and polyfluorinated compounds * 100%. When the calculated defluorination rate is ≥ 50%, the corresponding perfluorinated and polyfluorinated compounds are considered to have high biodegradability; when the calculated defluorination rate is between 10-50%, the corresponding perfluorinated and polyfluorinated compounds are considered to have moderate biodegradability; when the calculated defluorination rate is ≤ 10%, the corresponding perfluorinated and polyfluorinated compounds are considered to have low biodegradability.

[0086] In a specific embodiment, sodium fluoride is used as a standard substance. Sodium fluoride is mixed with the first culture medium and then diluted to different concentrations to obtain standard solutions. The standard solutions are then subjected to a complexation colorimetric reaction with Alizarin Red S and lanthanum salt. The reactants are then mixed with dimethyl sulfoxide and polyethylene glycol octylphenyl ether. The absorbance of the mixed solution is then measured. A standard curve is then plotted with the concentration of fluoride ions in the standard solution as the abscissa and the absorbance of the measured solution as the ordinate. The fluoride ion content in the supernatant after degradation is determined using the standard curve. The amount of defluorination of the perfluorinated and polyfluorinated compounds is further calculated based on the measured fluoride ion content. The biodegradability of the corresponding perfluorinated and polyfluorinated compounds is determined based on the calculated defluorination rate.

[0087] In some specific embodiments, sodium fluoride is mixed with the first culture medium, and then the mixture is diluted to obtain standard solutions with fluoride ion concentrations of 20 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L and 100 μmol / L, respectively.

[0088] In the method described in this invention, the test wavelength when using ultraviolet-visible spectrophotometry is 600-650 nm, preferably 610-630 nm, and more preferably 620 nm. It can be understood that in steps (4) and S2, the test wavelength of the ultraviolet-visible spectrophotometry used is 600-650 nm.

[0089] In some specific embodiments, the method described in this invention can be tested using 96-well or 120-well plates. Specifically, multiple perfluorinated and polyfluorinated compounds of different compositions can be simultaneously mixed with culture medium containing microorganisms in a 96-well plate for degradation, and the fluoride ion content in the various supernatants obtained after degradation can be detected simultaneously. This also allows for the determination of the biodegradability of the added perfluorinated and polyfluorinated compounds, significantly improving testing efficiency. Understandably, the method described in this invention can also be tested using plates with even more wells. Furthermore, the method described in this invention does not use highly toxic solvents, making the detection method environmentally friendly. After detecting the fluoride ion content in the supernatant, the solution in the microwells can still be used for subsequent related microbiological experimental research.

[0090] It should be noted that, due to the large number of degrading bacteria disclosed in the prior art, the method described in this invention provides a method for testing whether the perfluorinated and polyfluorinated compounds (PFOCs) to be tested possess degrading properties relative to the strain used. The test results indicate that the PFOCs to be tested possess degrading properties relative to that specific strain, but do not indicate that the PFOCs to be tested can be degraded by any known strain. In the method described in this invention, the PFOCs to be tested are deemed to possess biodegradable properties as long as they can be degraded by microorganisms. The method described in this invention provides a more convenient way to obtain the biodegradability of the PFOCs to be tested, and has high accuracy.

[0091] The method described in this invention can simultaneously test the biodegradability of dozens, hundreds, or even thousands of perfluorinated and polyfluorinated compounds, avoiding the cumbersome steps and extensive labor involved in previous studies on the degradation performance of single PFAS using glass bottles. Using serum bottles or glass bottles as reaction vessels, at least 50 mL of solution system is required for a single experiment. Considering the time spent on sampling, sample processing, and sample detection, a single batch of experiments can conduct up to 40-50 single-bottle experiments to detect the degradation performance of PFAS with different structures or carbon sources. Using the high-throughput testing method of this invention, up to 96 reactions can be performed on a single deep-well plate, greatly reducing reagent consumption and the tedious sampling and testing process. In laboratories with suitable experimental conditions, multiple deep-well plates can be used simultaneously for detection, allowing for the study and detection of multiple influencing factors at once, and enabling the detection of the degradability of a large number of PFAS. This provides a more efficient assessment of the biodegradability of various novel PFAS with high accuracy, offering a scientific basis for the risk management and control of new pollutants.

[0092] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.

[0093] Example 1

[0094] (1) Preparation of fluoride ion standard solution: Dissolve 0.42g of sodium fluoride reagent in 10mL of the first culture medium (containing 10mmol / L of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, 10mmol / L of NaCl, 5mmol / L of KCl, 5mmol / L of NaNO3, 1mmol / L of CaCl2 and 1mmol / L of MgCl2) to prepare a 1mol / L fluoride ion solution. Then dilute with deionized water to prepare solutions with fluoride ion concentrations of 20μmol / L, 40μmol / L, 60μmol / L, 80μmol / L and 100μmol / L as standard solutions for later use.

[0095] (2) A series of three-carbon carboxylic acid PFAS were selected, including six types (2-fluoropropionic acid, 2,2-difluoropropionic acid, 3,3,3-trifluoropropionic acid, 2,3,3,3-tetrafluoropropionic acid, 2,2,3,3-tetrafluoropropionic acid and perfluoropropionic acid). The PFAS samples to be tested were dissolved in the first culture medium (containing 10 mmol / L of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 10 mmol / L of NaCl, 5 mmol / L of KCl, 5 mmol / L of NaNO3, 1 mmol / L of CaCl2 and 1 mmol / L of MgCl2) to obtain a concentrated solution with a concentration of 1 mmol / L of perfluorinated and polyfluorinated compounds for later use. After preparation, the solution was sealed and stored for later use.

[0096] (3) Preparation of sodium acetate buffer: Weigh 10.9g of sodium acetate trihydrate and dissolve it in deionized water, then add 11mL of glacial acetic acid and add deionized water to make up to 1L to obtain 80mmol / L sodium acetate buffer. Then, use acetic acid to adjust the pH of the sodium acetate buffer to 5, and dilute the concentration of sodium acetate in the sodium acetate solution to 160μmol / L before use.

[0097] (4) Preparation of colorimetric reagent stock solution: Weigh 0.342 g of alizarin red S and 0.433 g of lanthanum nitrate hexahydrate and dissolve them in 100 mL of deionized water. Then dilute to prepare a 500 μmol / L alizarin red S solution and a 500 μmol / L lanthanum nitrate solution. Then adjust the pH of the lanthanum nitrate solution to 5 with hydrochloric acid and store it in the dark for later use. Adjust the pH of the alizarin red S solution to 5 with a 1 mol / L sodium acetate solution and store it in the dark for later use.

[0098] (5) Fresh activated sludge was taken from the biochemical unit of a sewage treatment plant in Qingdao, and degrading bacteria (mainly Pseudomonas spp., with Pseudomonas putida as the main species) were isolated and inoculated at a volume ratio of 10% into 500 mL of the third culture medium (containing 10 mmol / L of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, 10 mmol / L of NaCl, 5 mmol / L of KCl, 5 mmol / L of NaNO3, 1 mmol / L of CaCl2 and 1 mmol / L of MgCl2). Glucose with a final concentration of 2 g / L was added as a carbon source and cultured in an incubator at a speed of 100 rpm and a constant temperature of 28 °C. The absorbance was measured every 8 hours using a UV-Vis spectrophotometer and read at a wavelength of 600 nm. Inoculation was carried out when the absorbance was >0.4.

[0099] (6) Next, the culture medium containing bacterial solution was measured using an Agilent Cary 3500 dual-beam UV-Vis spectrophotometer, and the absorbance at a wavelength of 600 nm was read to characterize the degree of microbial growth.

[0100] (7) In a 96-well deep-well plate with a pore volume of 2 mL (the well plate is arranged as follows) Figure 1 Inoculate 0.9 mL of the bacterial culture from step (5) into the culture medium (as shown). Centrifuge at 2000 rpm for 20 minutes and remove the supernatant using a pipette. Then add an equal volume of the second culture medium (containing 10 mmol / L of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, 10 mmol / L of NaCl, 5 mmol / L of KCl, 5 mmol / L of NaNO3, 1 mmol / L of CaCl2 and 1 mmol / L of MgCl2) and add glucose at a final concentration of 2 g / L as a carbon source. Gently shake to resuspend the microorganisms.

[0101] (8) Add different types of PFAS to the deep wells inoculated with bacterial solution. Add 100 μL of concentrated PFAS solution to each deep well for degradation. Place the deep well plate on a microplate shaker and shake at 50 rpm for 48 hours. Then centrifuge again at 2000 rpm for 20 minutes to obtain the supernatant after degradation.

[0102] (9) Add 100 μL of the degraded supernatant to the deep well plate, and add 100 μL of Alizarin Red S solution and 500 μL of sodium acetate solution. Then add 100 μL of Lanthanum nitrate solution (the concentration of Alizarin Red S in the final solution after mixing is 50 μmol / L, the concentration of Lanthanum nitrate is 50 μmol / L, and the concentration of sodium acetate is 80 μmol / L) to carry out the reaction, and control the pH of the reaction system to 6 and the reaction time to 10 min.

[0103] (10) After the reaction is complete, dimethyl sulfoxide and polyethylene glycol octylphenyl ether are added to the microwell (the volume ratio of the supernatant after degradation, dimethyl sulfoxide and polyethylene glycol octylphenyl ether is 95:5:0.3). After the mixture is reacted for 30 minutes, the solution is detected at a wavelength of 620 nm using an ELISA reader (SpectraMax 384plus) to obtain the absorbance of the solution after the reaction.

[0104] (11) Add 1 mL of fluoride ion standard solutions with concentrations of 20 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L, and 100 μmol / L to a deep-well plate. Then add 100 μL of Alizarin Red S solution and 500 μL of sodium acetate solution. Next, add 100 μL of lanthanum nitrate solution to the standard solutions with different fluoride ion concentrations (the final solution has Alizarin Red S concentration of 50 μmol / L, lanthanum nitrate concentration of 50 μmol / L, and sodium acetate concentration of 80 μmol / L) and react for 10 min. Then add dimethyl sulfoxide and polyethylene glycol octylphenyl ether (the volume ratio of standard solution, dimethyl sulfoxide, and polyethylene glycol octylphenyl ether is 95:5:0.3). After mixing and reacting for 30 min, use a microplate reader (SpectraMax) to read the solution. The absorbance of the solution after the reaction was obtained by spectrophotometric detection at a wavelength of 620 nm using a 384plus microscope. Then, a standard curve was plotted using the fluoride ion concentration of the standard solution as the x-axis and the absorbance of the solution after the reaction as the y-axis. The standard curve was y = 0.0075x + 0.4857, R0. 2 =0.9986;

[0105] (12) The absorbance of the solution obtained in step (10) is substituted into the standard curve to calculate the concentration of fluoride ions in the solution after the reaction, and the defluorination rate of the perfluorinated and polyfluorinated compounds to be tested is calculated based on the obtained fluoride ion concentration. The results are shown in Table 1.

[0106] Table 1. Defluorination detection and defluorination rate of C3 PFAS

[0107] PFAS name CAS number Molecular formula Number of fluorine atoms Defluorination amount (μM) Defluorination rate % 2-Fluoropropionic acid 6087-13-4 <![CDATA[C3H5FO2]]> 1 16 16％ 2,2-Difluoropropionic acid 373-96-6 <![CDATA[C3H4F2O2]]> 2 10 5％ 3,3,3-Trifluoropropionic acid 2516-99-6 <![CDATA[C3H3F3O2]]> 3 267 89％ 2,3,3,3-Tetrafluoropropionic acid 359-49-9 <![CDATA[C3H2F4O2]]> 4 0 0 2,2,3,3-Tetrafluoropropionic acid 756-09-2 <![CDATA[C3H2F4O2]]> 4 0 0 Perfluoropropionic acid 422-64-0 <![CDATA[C3HF5O2]]> 5 0 0

[0108] As shown in Table 1, 3,3,3-trifluoropropionic acid has high biodegradability, 2-fluoropropionic acid has moderate biodegradability, 2,2-difluoropropionic acid has low biodegradability, and the other three PFAS have virtually no biodegradability.

[0109] Example 2

[0110] The method of Example 1 was followed, except that the PFAS substances to be tested were perfluorooctanoic acid, perfluorooctanoic acid sulfonic acid, 6:2 fluoropolymer alcohol and 6:2 fluoropolymer sulfonic acid, and the defluorination results of the perfluorinated and polyfluorinated compounds to be tested are shown in Table 2.

[0111] Table 2. Defluorination detection and defluorination rate of C8 PFAS

[0112] PFAS name CAS number Molecular formula Number of fluorine atoms Defluorination amount (μM) Defluorination rate % Perfluorooctanoic acid 335-67-1 <![CDATA[C8HF 15 O2]]> 15 0 0％ Perfluorooctanoic acid 1763-23-1 <![CDATA[C8HF 17 O3S]]> 17 0 0％ 6:2 Fluoropolymer 647-42-7 <![CDATA[C8H5F 13 O]]> 13 22 1.7％ 6:2 Fluoropolymer Sulfonic Acid 27619-94-9 <![CDATA[C8H5F 13 O3S]]> 13 14 1.1％

[0113] As shown in Table 2, both perfluorooctanoic acid (PFOA) and perfluorooctanoic acid (PFOS), two octane-3-carbon PFAS, cannot be degraded by microorganisms under aerobic conditions, exhibiting low biodegradability. However, 6:2 fluoropolymer alcohol and 6:2 fluoropolymer sulfonic acid undergo minor defluorination and can be partially degraded by microorganisms.

[0114] Example 3

[0115] The method was implemented according to Example 1, except that the PFAS substances to be tested were perfluoro-3,5,7,8-tetrachlorooctanoic acid, 9-chlorohexadecanoic acid, perfluoro-3,5,6-trichlorohexanoic acid, and 3,4-dichloropentafluorobutyric acid. The defluorination results of the perfluorinated and polyfluorinated compounds to be tested are shown in Table 3.

[0116] Table 3. Defluorination detection and defluorination rate of chlorinated PFAS

[0117]

[0118]

[0119] As shown in Table 3, the four chlorinated PFAS cannot be degraded by microorganisms, and their biodegradability is low.

[0120] Example 4

[0121] The method was implemented according to Example 1, except that the PFAS substances to be tested were 4,4,4-trifluorobutyric acid, 4,4-difluorobutyric acid, 4,4,4-trifluorobutenoic acid, 2,2-difluorobutyric acid, and 2-fluoroisobutyric acid. The defluorination results of the perfluorinated and polyfluorinated compounds to be tested are shown in Table 4.

[0122] Table 4. Defluorination detection and defluorination rate of C4 PFAS

[0123] PFAS name CAS# Molecular formula Number of fluorine atoms Defluorination amount (μM) Defluorination rate 4,4,4-Trifluorobutyric acid 406-93-9 <![CDATA[C4H5F3O2]]> 3 0 0％ 4,4-Difluorobutyric acid 944328-71-6 <![CDATA[C4H4F2O2]]> 2 0 0％ 4,4,4-Trifluorobutenoic acid 71027-02-6 <![CDATA[C4H3F3O2]]> 3 22 7.3％ 2,2-Difluorobutyric acid 2366-62-3 <![CDATA[C4H6F2O2]]> 2 0 0％ 2-Fluoroisobutyric acid 63812-15-7 <![CDATA[C4H7FO2]]> 1 0 0％

[0124] As shown in Table 4, the above five PFAS cannot be degraded by microorganisms and have low biodegradability. Among them, only 4,4,4-trifluorobutenoic acid can be degraded by microorganisms in small amounts.

[0125] Example 5

[0126] The method was implemented according to Example 1, except that the PFAS substances to be tested were 5,5,5-trifluorovaleric acid, 4,5,5-trifluoro-4-pentenoic acid, 4,4,5,5,5-pentafluorovaleric acid, and perfluorovaleric acid. The defluorination results of the perfluorinated and polyfluorinated compounds to be tested are shown in Table 5.

[0127] Table 5. Defluorination detection and defluorination rate of chlorinated PFAS

[0128] PFAS name CAS# Molecular formula Number of fluorine atoms Defluorination amount (μM) Defluorination rate 5,5,5-Trifluorovaleric acid 407-62-5 <![CDATA[C5H7F3O2]]> 3 132 44％ 4,5,5-Trifluoro-4-pentenoic acid 110003-22-0 <![CDATA[C5H5F3O2]]> 3 88 29.3％ 4,4,5,5,5-Pentafluoropentanoic acid 3637-31-8 <![CDATA[C5H5F5O2]]> 5 0 0％ Perfluorovalerate 2706-90-3 <![CDATA[C5HF9O2]]> 5 0 0％

[0129] As shown in Table 5, among the four chlorinated PFAS, 5,5,5-trifluorovaleric acid and 4,5,5-trifluoro-4-pentenoic acid have moderate biodegradability, while the remaining two substances are almost non-biodegradable and do not possess biodegradable properties.

[0130] Example 6

[0131] The method was implemented according to Example 1, except that the PFAS substances to be tested were 1,2,3-trifluorobenzene, 1,2,4-trifluorobenzene, 1,3,5-trifluorobenzene, 1,2,3,4-tetrafluorobenzene, 1,2,3,5-tetrafluorobenzene, and 1,2,4,5-tetrafluorobenzene. The defluorination results of the perfluorinated and polyfluorinated compounds to be tested are shown in Table 6.

[0132] Table 6. Defluorination Detection and Defluorination Rate of Benzene Series PFAS

[0133] PFAS name CAS# Molecular formula Number of fluorine atoms Defluorination amount (μM) Defluorination rate 1,2,3-Trifluorobenzene 1489-53-8 <![CDATA[C6H3F3]]> 3 12 4％ 1,2,4-Trifluorobenzene 367-23-7 <![CDATA[C6H3F3]]> 3 15 5％ 1,3,5-Trifluorobenzene 372-38-3 <![CDATA[C6H3F3]]> 3 5 1.6％ 1,2,3,4-Tetrafluorobenzene 551-62-2 <![CDATA[C6H2F4]]> 4 0 0％ 1,2,3,5-Tetrafluorobenzene 2367-82-0 <![CDATA[C6H2F4]]> 4 6 1.5％ 1,2,4,5-Tetrafluorobenzene 327-54-8 <![CDATA[C6H2F4]]> 4 9 2.2％

[0134] As shown in Table 6, the above six benzene-based PFAS have low biodegradability.

[0135] Example 7

[0136] The method was implemented according to Example 1, except that the PFAS substances to be tested were 4:2 fluoropolymer, perfluorohexanoic acid, 4:2 fluoropolymer sulfonic acid, and 5,5,6,6,6-pentafluorohexanoic acid. The defluorination results of the perfluorinated and polyfluorinated compounds to be tested are shown in Table 7.

[0137] Table 7. Defluorination Detection and Defluorination Rate of C6 PFAS

[0138] PFAS name CAS# Molecular formula Number of fluorine atoms Defluorination amount (μM) Defluorination rate 4:2 Fluoropolymer 679-03-8 <![CDATA[C6H5F7O]]> 7 176 25％ Perfluorohexanoic acid 2706-90-3 <![CDATA[C5HF9O2]]> 9 0 0％ 4:2 Fluoropolymer Sulfonic Acid 757124-72-4 <![CDATA[C6H5F9O3S]]> 9 52 5.7％ 5,5,6,6,6-Pentafluorohexanoic acid 148043-70-3 <![CDATA[C6H7F5O2]]> 5 0 0％

[0139] As shown in Table 7, among the four six-carbon PFAS, only 4:2 fluoropolymer has moderate biodegradability, while the other three have low biodegradability.

[0140] Example 8

[0141] The method of Example 1 was implemented, except that in step (9), the pH value of the reaction system was limited to 5.

[0142] Example 9

[0143] The method of Example 1 was implemented, except that in step (9), the pH value of the reaction system was limited to 5.5.

[0144] Comparative Example 1

[0145] The method of Example 1 was implemented, except that the first and second culture media did not contain 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, and 0.2 mol / L 3-(N-morpholino)propanesulfonic acid was used as a buffer salt. The results of the three-carbon PFAS defluorination detection and defluorination rate are shown in Table 8.

[0146] Table 8

[0147]

[0148]

[0149] Comparative Example 2

[0150] The method of Example 1 was implemented, except that the first and second culture media did not contain 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, and a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate with a sodium ion concentration of 0.1 mol / L was used as a buffer salt.

[0151] When using an ELISA reader for testing, the absorbance of the mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate is high at 620 nm, making it impossible to obtain the absorbance of the solution after the reaction. Therefore, the test results cannot be obtained, and the test fails.

[0152] Comparative Example 3

[0153] The method of Example 1 was implemented, except that the first and second culture media did not contain 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, and 0.2 mol / L tris(hydroxymethyl)aminomethane was used as a buffer salt. The results of the three-carbon PFAS defluorination detection and defluorination rate obtained by the test are shown in Table 9.

[0154] Table 9

[0155] PFAS name CAS number Molecular formula Number of fluorine atoms Defluorination amount (μM) Defluorination rate % 2-Fluoropropionic acid 6087-13-4 <![CDATA[C3H5FO2]]> 1 7 7％ 2,2-Difluoropropionic acid 373-96-6 <![CDATA[C3H4F2O2]]> 2 8 4％ 3,3,3-Trifluoropropionic acid 2516-99-6 <![CDATA[C3H3F3O2]]> 3 219 73％ 2,3,3,3-Tetrafluoropropionic acid 359-49-9 <![CDATA[C3H2F4O2]]> 4 0 0 2,2,3,3-Tetrafluoropropionic acid 756-09-2 <![CDATA[C3H2F4O2]]> 4 0 0 Perfluoropropionic acid 422-64-0 <![CDATA[C3HF5O2]]> 5 0 0

[0156] Comparative Example 4

[0157] The method of Example 1 was implemented, except that the first and second culture media did not contain 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid; the results of the three-carbon PFAS defluorination detection and defluorination rate obtained by the test are shown in Table 10.

[0158] Table 10

[0159] PFAS name CAS number Molecular formula Number of fluorine atoms Defluorination amount (μM) Defluorination rate % 2-Fluoropropionic acid 6087-13-4 <![CDATA[C3H5FO2]]> 1 3 3％ 2,2-Difluoropropionic acid 373-96-6 <![CDATA[C3H4F2O2]]> 2 12 6％ 3,3,3-Trifluoropropionic acid 2516-99-6 <![CDATA[C3H3F3O2]]> 3 96 32％ 2,3,3,3-Tetrafluoropropionic acid 359-49-9 <![CDATA[C3H2F4O2]]> 4 0 0 2,2,3,3-Tetrafluoropropionic acid 756-09-2 <![CDATA[C3H2F4O2]]> 4 0 0 Perfluoropropionic acid 422-64-0 <![CDATA[C3HF5O2]]> 5 0 0

[0160] Comparative Example 5

[0161] The method of Example 1 was implemented, except that in step (9), the pH value of the reaction system was limited to 3.5. The results of the three-carbon PFAS defluorination detection and defluorination rate obtained by the test are shown in Table 11.

[0162] Table 11

[0163] PFAS name CAS number Molecular formula Number of fluorine atoms Defluorination amount (μM) Defluorination rate % 2-Fluoropropionic acid 6087-13-4 <![CDATA[C3H5FO2]]> 1 12 12％ 2,2-Difluoropropionic acid 373-96-6 <![CDATA[C3H4F2O2]]> 2 6 3％ 3,3,3-Trifluoropropionic acid 2516-99-6 <![CDATA[C3H3F3O2]]> 3 178 59.3％ 2,3,3,3-Tetrafluoropropionic acid 359-49-9 <![CDATA[C3H2F4O2]]> 4 0 0 2,2,3,3-Tetrafluoropropionic acid 756-09-2 <![CDATA[C3H2F4O2]]> 4 0 0 Perfluoropropionic acid 422-64-0 <![CDATA[C3HF5O2]]> 5 0 0

[0164] Comparative Example 6

[0165] The method of Example 1 was implemented, except that in step (9), the pH value of the reaction system was limited to 4; the results of the three-carbon PFAS defluorination detection and defluorination rate obtained by the test are shown in Table 12.

[0166] Table 12

[0167] PFAS name CAS number Molecular formula Number of fluorine atoms Defluorination amount (μM) Defluorination rate % 2-Fluoropropionic acid 6087-13-4 <![CDATA[C3H5FO2]]> 1 8 8％ 2,2-Difluoropropionic acid 373-96-6 <![CDATA[C3H4F2O2]]> 2 5 2.5％ 3,3,3-Trifluoropropionic acid 2516-99-6 <![CDATA[C3H3F3O2]]> 3 202 67.3％ 2,3,3,3-Tetrafluoropropionic acid 359-49-9 <![CDATA[C3H2F4O2]]> 4 0 0 2,2,3,3-Tetrafluoropropionic acid 756-09-2 <![CDATA[C3H2F4O2]]> 4 0 0 Perfluoropropionic acid 422-64-0 <![CDATA[C3HF5O2]]> 5 0 0

[0168] Comparative Example 7

[0169] The method of Example 1 was implemented, except that in step (9), the pH value of the reaction system was limited to 4.5; the results of the three-carbon PFAS defluorination detection and defluorination rate obtained by the test are shown in Table 13.

[0170] Table 13

[0171] PFAS name CAS number Molecular formula Number of fluorine atoms Defluorination amount (μM) Defluorination rate % 2-Fluoropropionic acid 6087-13-4 <![CDATA[C3H5FO2]]> 1 8 8％ 2,2-Difluoropropionic acid 373-96-6 <![CDATA[C3H4F2O2]]> 2 8 4％ 3,3,3-Trifluoropropionic acid 2516-99-6 <![CDATA[C3H3F3O2]]> 3 233 77.6％ 2,3,3,3-Tetrafluoropropionic acid 359-49-9 <![CDATA[C3H2F4O2]]> 4 0 0 2,2,3,3-Tetrafluoropropionic acid 756-09-2 <![CDATA[C3H2F4O2]]> 4 0 0 Perfluoropropionic acid 422-64-0 <![CDATA[C3HF5O2]]> 5 0 0

[0172] Comparative Example 8

[0173] The method of Example 1 was implemented, except that the 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid contained in the first and second culture media was replaced with sodium acetate of the same concentration.

[0174] Test case

[0175] Test Example 1

[0176] The fluoride ion content in the supernatant obtained after degradation in step (8) of Examples 1, Comparative Examples 1 and Comparative Examples 3-7 was tested using a fluoride ion selective electrode to verify the accuracy of the method described in this invention.

[0177] The test method is as follows: 2 mL of the sample to be tested was added to 0.1 g of fluoride ion enhancer (HACH) and shaken to dissolve. The fluoride ion content in the sample was detected using a multi-parameter water quality analyzer of model HQ440D (HACH) and a fluoride ion selective electrode. Before use, the electrode was calibrated with fluoride ion standards of 0.5 mg / L, 1 mg / L and 2 mg / L respectively. The test results are shown in Table 14.

[0178] Table 14

[0179]

[0180] As shown in Table 14, the method described in this invention provides a more accurate and less relative error for measuring fluoride ion concentration, making it well-suited for testing the biodegradability of perfluorinated and polyfluorinated compounds. Ion chromatography, on the other hand, struggles to accurately quantify fluoride ion concentrations due to the overlap between the culture medium components and the fluoride ion peaks.

[0181] Test Example 2

[0182] The absorbance of the solutions obtained in step (10) of Examples 1, 8, 9 and Comparative Examples 5-7 at different wavelengths was tested, and the test results are as follows: Figure 2 As shown.

[0183] according to Figure 2 It is evident that the method employed in this invention enhances the sensitivity of fluoride ion colorimetric analysis and improves the accuracy of the test, enabling more accurate and rapid testing of the biodegradability of perfluorinated and polyfluorinated compounds. Furthermore, from Figure 2 It can be seen that although the absorbance of the test solution is high at a wavelength of 550 nm, the sensitivity for fluoride ion detection is weak at this wavelength, and the test accuracy is lower than that at a detection wavelength of 620 nm.

[0184] Test Example 3

[0185] The sensitivity of fluoride ion detection was tested at different test wavelengths.

[0186] Test method: Standard solutions with fluoride ion concentrations of 2.5 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, and 40 μmol / L were measured at wavelengths of 550 nm and 620 nm, respectively, and the absorbance was plotted. The results are shown below. Figure 3 As shown.

[0187] according to Figure 3 It is known that when the detection wavelength is 550nm, the change in the absorbance of the solution is small when the concentration of fluoride ions in the solution changes. Its sensitivity to changes in the concentration of fluoride ions is much lower than that of the wavelength 620nm. Therefore, in the method described in this invention, the selected test wavelength is 600-650nm, and the optimal test wavelength is 620nm.

[0188] Test Example 4

[0189] The effect of the culture medium used in Example 1, Comparative Examples 1-4 and Comparative Example 8 on the absorbance of the solution was tested.

[0190] Test method: The test culture medium was mixed with standard solutions containing different concentrations of fluoride ions, and the absorbance of the resulting mixed solution was measured at 620 nm. The concentrations of fluoride ions in the standard solutions were 2.5 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, and 40 μmol / L, respectively. The test results are as follows: Figure 4 As shown.

[0191] according to Figure 4 The results show that the culture medium components used in the method described in this invention do not affect the detection of fluoride ion absorbance in the solution. Furthermore, when the concentration of fluoride ions in the solution changes, the absorbance of the solution changes significantly, further confirming that the culture medium components used in this invention do not reduce the detection of fluoride ion absorbance, thereby further ensuring the testing accuracy of the method described in this invention.

[0192] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for testing the biodegradability of perfluorinated and polyfluorinated compounds, characterized in that, The method includes the following steps: (1) A concentrated solution was obtained by mixing perfluorinated and polyfluorinated compounds with the first culture medium; (2) Inoculate the bacterial culture containing the strains used to degrade perfluorinated and polyfluorinated compounds into the second culture medium, add a carbon source and culture to obtain a culture medium containing the bacterial culture. (3) The concentrated solution is placed in the culture medium containing the bacterial solution for degradation. The supernatant after degradation, Alizarin Red S, Lanthanum salt and buffer solution are mixed and reacted, and the pH of the reaction system is controlled to be 5-6. (4) Mix the reactants obtained in step (3), dimethyl sulfoxide and polyethylene glycol octylphenyl ether, and test the mixture by ultraviolet-visible spectrophotometry. Determine the biodegradability of perfluorinated and polyfluorinated compounds based on the absorbance obtained from the test. The first and second culture media contain 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, alkali metal hydrochloride, alkali metal nitrate, calcium salt and magnesium salt.

2. The method according to claim 1, characterized in that, In step (4), the specific process of determining the biodegradability of perfluorinated and polyfluorinated compounds based on the absorbance obtained from the test includes: S1. Sodium fluoride is mixed with the first culture medium, and then the mixture is diluted to obtain standard solutions with different fluoride ion concentrations; S2. Mix the standard solution, alizarin red S, lanthanum salt and buffer solution and react them, controlling the pH of the reaction system to be 5-6. Then mix the obtained reaction material, dimethyl sulfoxide and polyethylene glycol octylphenyl ether, and test the absorbance of the solution by ultraviolet-visible spectrophotometry. S3. Using the fluoride ion concentration of the standard solution as the abscissa and the absorbance of the solution obtained in step S2 as the ordinate, a standard curve is plotted. Then, the absorbance obtained in step (4) is substituted into the standard curve to calculate the fluoride ion concentration in the solution after the reaction, and the defluorination rate of the perfluorinated and polyfluorinated compounds is calculated based on the fluoride ion concentration. S4. Determine the biodegradability of the perfluorinated and polyfluorinated compounds based on the defluorination rate.

3. The method according to claim 1, characterized in that, In the first and second culture media, the concentration of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid is 8-12 mmol / L, the concentration of alkali metal hydrochloride is 12-18 mmol / L, the concentration of alkali metal nitrate is 4-6 mmol / L, the concentration of magnesium salt is 0.8-1.5 mmol / L, and the concentration of calcium salt is 0.8-1.5 mmol / L.

4. The method according to claim 1 or 3, characterized in that, In the first and second culture media, the alkali metal hydrochloride is NaCl and / or KCl, the alkali metal nitrate is NaNO3 and / or KNO3, the calcium salt is calcium chloride, and the magnesium salt is magnesium chloride; and / or The first and second culture media contain 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, NaCl, KCl, NaNO3, calcium salts, and magnesium salts; Preferably, the first culture medium and the second culture medium have the same composition.

5. The method according to claim 1, 3, or 4, characterized in that, In the first and second culture media, the molar ratio of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, alkali metal hydrochloride, alkali metal nitrate, calcium salt and magnesium salt is 8-12:12-17:3-6:0.8-1.2:

1.

6. The method according to claim 1, characterized in that, The bacterial strains used to degrade perfluorinated and polyfluorinated compounds are selected from one or more of the following: *Pseudomonas fluorescens*, *Pseudomonas putida*, *Gordonella*, strain NB4–1Y, *Phanerochaete chrysosporium*, *Paragonimococcus pantrophicus*, Gram-negative bacteria, and *Bacillus subtilis*.

7. The method according to claim 1, characterized in that, The volume ratio of the degraded supernatant, dimethyl sulfoxide, and polyethylene glycol octylphenyl ether is (93-98):(3-7):0.

3.

8. The method according to claim 1, characterized in that, The buffer solution is sodium acetate buffer solution; Preferably, the pH value of the buffer solution is 5-6.

9. The method according to claim 1 or 2, characterized in that, The molar ratio of the amount of Alizarin Red S to the amount of the lanthanum salt is 0.8-1.5:

1.

10. The method according to claim 1 or 2, characterized in that, The alizarin red S was added in the form of an alizarin red S solution; Preferably, the pH value of the alizarin red S solution is 5-6.

11. The method according to claim 1 or 2, characterized in that, The lanthanum salt is added in the form of a lanthanum salt solution; Preferably, the lanthanum salt is at least one selected from lanthanum nitrate hexahydrate, lanthanum chloride heptahydrate, and lanthanum carbonate trihydrate; Preferably, the pH value of the lanthanum salt solution is 5-6.

12. The method according to claim 1 or 2, characterized in that, The test wavelength for the ultraviolet-visible spectrophotometry is 600-650 nm.

13. The method according to claim 1, characterized in that, The specific process of step (2) includes: inoculating the bacterial solution containing the strains for degrading perfluorinated and polyfluorinated compounds into the third culture medium, adding a carbon source and culturing at a constant temperature, taking the bacterial solution every 7-10 hours and testing it with a UV-Vis spectrophotometer, reading the absorbance at a wavelength of 590-610 nm, when the absorbance is >0.4, inoculating the corresponding bacterial solution containing the strains for degrading perfluorinated and polyfluorinated compounds into the second culture medium, adding a carbon source and culturing to obtain a culture medium containing bacterial solution; Preferably, the composition of the third culture medium is the same as that of the second culture medium.

14. The method according to claim 1 or 13, characterized in that, The carbon source is selected from one or more of glucose, lactose, sodium propionate, sodium acetate, and straight-chain alkanes.

15. The method according to claim 1, characterized in that, The concentration of perfluorinated and polyfluorinated compounds in the concentrated solution is 0.8-1.5 mmol / L.

16. The method according to any one of claims 1, 13, and 14, characterized in that, The concentration of the carbon source is 1-3 g / L.

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