A method for constructing and evaluating an assessment model for the ecological risk of environmental bisphenol pollutants.

Abstract

This invention provides a method for constructing and evaluating an assessment model for the ecological risks of environmental bisphenol pollutants, belonging to the field of ecological risk assessment technology. Based on the similar chemical structures between environmental bisphenol pollutants and pheromones, this invention establishes functions to determine the influence of pheromone concentration on binding transfer frequency and the influence of different bisphenol structures on binding transfer frequency, thereby obtaining the relationship between the ability of bisphenols to influence binding transfer and the ability of pheromones to influence binding transfer, and clarifying the biotoxic effects of environmental bisphenol pollutants with different structures. The results show that: bisphenol A > tetrabromobisphenol A > hexafluorobisphenol A > bisphenol F > pheromone cCF10 > bisphenol S, providing a theoretical basis for the selection of bisphenol A substitutes.

Description

Technical Field

[0001] This invention belongs to the field of ecological risk assessment technology, specifically relating to a method for constructing and evaluating an assessment model for the ecological risk of environmental bisphenol pollutants. Background Technology

[0002] Bisphenol compounds (BPs) are a series of compounds formed by bridging two hydroxyphenyl groups with carbon or sulfur atoms. BPs have similar molecular structures and can be classified into bisphenol A (BPA), bisphenol F (BPF), and bisphenol S (BPS) based on the different groups linking the hydroxyphenyl groups. BPA is the most representative compound and one of the most widely produced and used chemical substances in the world. It is used in the manufacture of polycarbonate plastics and epoxy resins and is extensively used in food and beverage packaging, including baby bottles, food can linings, food packaging, and dental sealants. The migration of BPA from these materials leads to its bioaccumulation in the human body, causing a series of health hazards. Studies have confirmed that endocrine system diseases, reproductive damage, neurological disorders, and behavioral dysfunctions are all related to BPA exposure. Various countries have successively promulgated policies and regulations restricting or prohibiting the use of BPA, leading to the emergence of "BPA-free" products on the market and the development of BPA analogues in an attempt to replace BPA.

[0003] BPS and BPF, as structural analogs of BPA, are widely used BPA substitutes in the market in recent years. BPS is commonly used as a color developer in thermal paper, an electroplating solvent, and a component of phenolic resins, while BPF is mainly used in the manufacture of epoxy resins and coatings. With their widespread use in daily life, BPS and BPF have been detected in surface water, sediments, sewage, and human biological samples, with detection frequencies and concentrations similar to BPA. Increasing research indicates that BPS and BPF also possess endocrine-disrupting activities similar to BPA. Furthermore, there is evidence that BPS can cross the placental barrier and cause reproductive and developmental toxicity. Whether existing "BPA analogs" on the market can safely replace BPA remains inconclusive. Currently, research results on the health effects of BPs are contradictory. Studies on the health risks of BPs are limited to acute / chronic developmental toxicity and endocrine toxicity derived from animal / in vitro experiments, while the ecological risks of environmental concentrations of BPs are still unclear. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for constructing and evaluating an assessment model for the ecological risk of environmental bisphenol pollutants. The assessment model and method of this invention can clearly identify and compare the ecotoxicity of BPA and its alternatives.

[0005] This invention provides a method for constructing an assessment model for the ecological risk of environmental bisphenol pollutants, comprising the following steps:

[0006] Recombinant Enterococcus faecalis was used as the donor bacteria, and streptomycin-resistant Enterococcus faecalis was used as the recipient bacteria; the recombinant Enterococcus faecalis contained a pheromone regulatory plasmid.

[0007] The donor and recipient bacteria were first mixed to obtain a first mixture. Different concentrations of pheromones were added to the first mixture, and the mixture was subjected to a first static culture. The number of conjugates was measured, and the first conjugation transfer frequency was calculated. A function f, which describes the effect of pheromone concentration on the conjugation transfer frequency, was established with pheromone concentration as the independent variable and the first conjugation transfer frequency as the dependent variable. 信息素 (y);

[0008] The donor and recipient bacteria were mixed to obtain a second mixture. Different concentrations of bisphenol A were added to this second mixture, followed by a second static incubation. The number of conjugates was measured, and the second conjugation transfer frequency was calculated. A function fi was established, with the concentration of environmental bisphenol A as the independent variable and the second conjugation transfer frequency as the dependent variable, to determine the effect of bisphenol A concentration on the conjugation transfer frequency. BPs (x);

[0009] According to the f 信息素 (y) and f BPs (x) Establish the functional equation between the influence of environmental bisphenols on binding and transfer capacity and the influence of pheromones on binding and transfer capacity, and construct an evaluation model for the ecological risk of bisphenol pollutants.

[0010] The assessment model for the ecological risk of environmental bisphenol pollutants is f. BPs (x)=f 信息素 (y);

[0011] x represents the concentration of BPs, and y represents the concentration of pheromones.

[0012] After simplification, we get the following equation: y = kx + b.

[0013] Where k and b are constants;

[0014] The ratio of the number of viable donor bacteria to the number of viable recipient bacteria in the first or second mixture is 1:1.

[0015] Preferably, the pheromone regulatory plasmid is the pCF10 plasmid.

[0016] Preferably, the original strain of the donor bacteria includes Enterococcus faecalis OG1RF.

[0017] Preferably, the concentration of the donor or recipient bacteria is (1–9) × 10⁻⁶. 8 CFU / mL.

[0018] Preferably, the temperature of the first static incubation or the second static incubation is 25-37°C; and the time of the first static incubation or the second static incubation is 4 hours.

[0019] Preferably, the pheromone includes cCF10.

[0020] Preferably, the effective concentration of the pheromone includes 1, 3, 5, 7, 10, 13, 15, 17 and 20 nmol / L.

[0021] Preferably, the environmental bisphenol pollutants are selected from one or more of bisphenol A, bisphenol F, bisphenol S, tetrabromobisphenol A, and hexafluorobisphenol A.

[0022] Preferably, the effective concentration of the environmental bisphenol pollutant is 0.01–50 nmol / L.

[0023] This invention also provides a method for assessing the ecological risk of environmental bisphenol pollutants, comprising the following steps:

[0024] An evaluation model for the ecological risk of bisphenol pollutants in the environment to be tested is constructed using the construction method described above. The k value is calculated, and the larger the k value, the greater the ecological risk of the corresponding bisphenol pollutants in the environment.

[0025] This invention provides a method for constructing an assessment model for the ecological risk of environmental bisphenol pollutants, comprising the following steps: using recombinant Enterococcus faecalis as the donor bacterium and streptomycin-resistant Enterococcus faecalis as the recipient bacterium; the recombinant Enterococcus faecalis contains a pheromone regulatory plasmid; mixing the donor and recipient bacteria, adding different concentrations of pheromone, performing static culture, measuring the number of conjugates, calculating the first conjugation transfer frequency, and establishing a function f(x) of the effect of pheromone concentration on the conjugation transfer frequency, with pheromone concentration as the independent variable and the first conjugation transfer frequency as the dependent variable. 信息素 (y); The donor and recipient bacteria were mixed with environmental bisphenols of different known effective concentrations, and then statically cultured. The number of conjugates was measured, and the second conjugation transfer frequency was calculated. A function f of the effect of bisphenol concentration on the conjugation transfer frequency was established, with the concentration of environmental bisphenols as the independent variable and the second conjugation transfer frequency as the dependent variable. BPs (x); according to the f 信息素 (y) and f BPs (x) Establish a functional equation relating the influence of environmental bisphenols on conjugation and transfer capacity to that of pheromones, and construct an evaluation model for the ecological risk of bisphenol pollutants; the evaluation model for the ecological risk of environmental bisphenol pollutants is f. BPs (x)=f 信息素(y). This invention, based on the similar chemical structures between environmental bisphenol pollutants and pheromones, establishes functions to determine the influence of pheromone concentration on binding transfer frequency and the influence of different bisphenol structures on binding transfer frequency. This yields a functional equation relating the binding transfer capacity of bisphenols to that of pheromones, clarifying the biotoxic effects of environmental bisphenol pollutants with different structures. Results show that the order is: bisphenol A > tetrabromobisphenol A > hexafluorobisphenol A > bisphenol F > pheromone cCF10 > bisphenol S, providing theoretical and technical support for the selection of bisphenol A substitutes. Attached Figure Description

[0026] Figure 1 To construct the donor bacteria in the conjugation system, the pCF10 plasmid was successfully introduced into the donor bacteria OG1RF, and the donor bacteria could express the pCF10 plasmid-related specific genes normally.

[0027] Figure 2 To construct the recipient bacteria in the conjugation system, the induced OG1RS successfully expressed the str gene (A), and its growth was unaffected (B).

[0028] Figure 3 To verify the conjugate results, conjugates were successfully screened on the double-antibiotic medium. The conjugates expressed the pCF10 plasmid-related specific gene. A: Conjugate screening on double-antibiotic plates; B: Conjugate plasmid verification; C: pCF10 specific gene verification after plasmid extraction from conjugates and donor bacteria.

[0029] Figure 4 The correlation between pheromone cCF10 concentration and bisphenol A concentration and conjugation transfer frequency was investigated. The concentrations of both bisphenol A and pheromones showed similar trends in their effects on conjugation transfer frequency, promoting the occurrence of conjugation transfer within a certain concentration range. A: Effect of pheromone cCF10 concentration on conjugation transfer frequency; B: Effect of bisphenol A concentration on conjugation transfer frequency; C: Effect of bisphenol S concentration on conjugation transfer frequency; D: Effect of bisphenol F concentration on conjugation transfer frequency; E: Effect of tetrabromobisphenol A concentration on conjugation transfer frequency; F: Effect of bisphenol AF concentration on conjugation transfer frequency.

[0030] Figure 5 The effect of pheromone concentration on binding transfer frequency;

[0031] Figure 6 The following equations represent the effects of bisphenol A concentration on conjugation transfer frequency: A: Functional equation of bisphenol A concentration on conjugation transfer frequency; B: Functional equation of bisphenol S concentration on conjugation transfer frequency; C: Functional equation of bisphenol AF concentration on conjugation transfer frequency; D: Functional equation of bisphenol F concentration on conjugation transfer frequency; E: Functional equation of tetrabromobisphenol A concentration on conjugation transfer frequency. Detailed Implementation

[0032] This invention provides a method for constructing an assessment model for the ecological risk of environmental bisphenol pollutants, comprising the following steps:

[0033] Recombinant Enterococcus faecalis was used as the donor bacteria, and streptomycin-resistant Enterococcus faecalis was used as the recipient bacteria; the recombinant Enterococcus faecalis contained a pheromone regulatory plasmid.

[0034] The donor and recipient bacteria were first mixed to obtain a first mixture. Different concentrations of pheromones were added to the first mixture, and the mixture was subjected to a first static culture. The number of conjugates was measured, and the first conjugation transfer frequency was calculated. A function f, which describes the effect of pheromone concentration on the conjugation transfer frequency, was established with pheromone concentration as the independent variable and the first conjugation transfer frequency as the dependent variable. 信息素 (y);

[0035] The donor and recipient bacteria were mixed to obtain a second mixture. Different concentrations of bisphenol A were added to this second mixture, followed by a second static incubation. The number of conjugates was measured, and the second conjugation transfer frequency was calculated. A function fi was established, with the concentration of environmental bisphenol A as the independent variable and the second conjugation transfer frequency as the dependent variable, to determine the effect of bisphenol A concentration on the conjugation transfer frequency. BPs (x);

[0036] According to the f 信息素 (y) and f BPs (x) Establish the functional equation between the influence of environmental bisphenols on binding and transfer capacity and the influence of pheromones on binding and transfer capacity, and construct an evaluation model for the ecological risk of bisphenol pollutants.

[0037] The assessment model for the ecological risk of environmental bisphenol pollutants is f. BPs (x)=f 信息素 (y);

[0038] x represents the concentration of BPs, and y represents the concentration of pheromones.

[0039] After simplification, we get the following equation: y = kx + b.

[0040] Where k and b are constants;

[0041] The ratio of the number of viable donor bacteria to the number of viable recipient bacteria in the first or second mixture is 1:1.

[0042] This invention uses recombinant Enterococcus faecalis as the donor bacterium and Enterococcus faecalis induced to develop streptomycin resistance as the recipient bacterium; the recombinant Enterococcus faecalis contains a pheromone-regulating plasmid. In this invention, the pheromone-regulating plasmid is preferably the pCF10 plasmid. In this invention, the original strain of the donor or recipient bacterium preferably includes Enterococcus faecalis OG1RF. In this invention, the donor bacterium is preferably OG1RF carrying the pCF10 plasmid, exhibiting tetracycline resistance, and the pCF10 plasmid is a conjugation transfer plasmid; the recipient bacterium is preferably OG1RS, obtained by inducing streptomycin resistance with OG1RF.

[0043] In this invention, the bacterial concentration of the donor or recipient bacteria is preferably (1-9) × 10⁻⁶. 8 CFU / mL.

[0044] This invention involves first mixing the donor and recipient bacteria to obtain a first mixture, adding different concentrations of pheromones to the first mixture, performing a first static culture, measuring the number of conjugates, and calculating the first conjugation transfer frequency. Using pheromone concentration as the independent variable and the first conjugation transfer frequency as the dependent variable, a function f is established to determine the effect of pheromone concentration on the conjugation transfer frequency. 信息素 (y).

[0045] In this invention, the pheromone preferably includes cCF10, with the amino acid sequence shown in SEQ ID NO.1, specifically: LVTLVFV. In this invention, the effective concentration of the pheromone preferably includes 1, 3, 5, 7, 10, 13, 15, 17, and 20 nmol / L.

[0046] In this invention, the preferred formula for calculating the conjugation transfer frequency is: conjugation transfer frequency = number of conjugates / number of recipient bacteria.

[0047] This invention involves a second mixing of the donor and recipient bacteria to obtain a second mixture. Different concentrations of bisphenol A are added to this second mixture, followed by a second static incubation. The number of conjugates is measured, and the second conjugation transfer frequency is calculated. A function fi is established, with the concentration of environmental bisphenol A as the independent variable and the second conjugation transfer frequency as the dependent variable, to determine the effect of bisphenol A concentration on the conjugation transfer frequency. BPs (x).

[0048] In this invention, the environmental bisphenol pollutants are preferably selected from one or more of bisphenol A (BPA), bisphenol F (BPF), bisphenol S (BPS), tetrabromobisphenol A (ATBBPA), and hexafluorobisphenol A (BPAF); the effective concentration of the environmental bisphenol pollutants is preferably 0.01 to 50 nmol / L.

[0049] In this invention, the temperature of the first static culture or the second static culture is preferably 25-37°C; the time of the first static culture or the second static culture is preferably 4 hours.

[0050] In this invention, the determination of the number of conjugates preferably includes the following steps: placing the conjugate bacterial culture after a first static culture or a second static culture on a double-antibiotic (tetracycline and streptomycin) medium for screening, culturing for 36 hours, and counting the colonies; the number of colonies is the number of conjugates.

[0051] After the first static culture or the second static culture, the present invention preferably further includes verifying the conjugate to confirm that the screened conjugate contains the pCF10 plasmid.

[0052] According to the f 信息素 (y) and f BPs (x) Establish the functional equation between the influence of environmental bisphenols on binding and transfer capacity and the influence of pheromones on binding and transfer capacity, and construct an evaluation model for the ecological risk of bisphenol pollutants.

[0053] The assessment model for the ecological risk of environmental bisphenol pollutants is f. BPs (x)=f 信息素 (y);

[0054] x represents the concentration of BPs, and y represents the concentration of pheromones.

[0055] After simplification, we get the following equation: y = kx + b, where k and b are constants.

[0056] This invention also provides a method for assessing the ecological risk of environmental bisphenol pollutants, comprising the following steps:

[0057] Using the construction method described above, an evaluation model for the ecological risk of bisphenol pollutants in the environment under test was constructed. The k-value was calculated; the larger the k-value, the greater the ecological risk of the corresponding bisphenol pollutant. Ultimately, it can be determined that the biological effects of bisphenol substances are several times greater than those of other bisphenol substances or pheromones.

[0058] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0059] Example 1

[0060] Example 1: Establishment of a pheromone-mediated conjugation transfer model (see Appendix). Figure 1-3 ;

[0061] (1) Strains and plasmids: Enterococcus faecalis OG1RF (ATCC47077, NCBI: txid474186) was purchased from the American Type Culture Collection (ATCC). The pheromone-inducing plasmid pCF10, which encodes a tetracycline resistance gene, was kindly provided by Professor Gary Dunny of the University of Minnesota. The pCF10 plasmid was introduced into OG1RF to obtain the donor strain OG1RF (pCF10). The recipient strain was Enterococcus faecalis OG1RS, which was induced to develop streptomycin resistance and named OG1RS. Tetracycline concentration: 10 mg / L, streptomycin concentration: 3 g / L.

[0062] (2) Preparation of donor bacteria: Enterococcus faecalis OG1RF was cultured to the logarithmic phase and washed 4 times with pre-cooled sucrose solution (0.5M) (4℃, 6000rpm). The precipitate was centrifuged and resuspended in 200μL of pre-cooled sucrose solution to obtain OG1RF competent cells. 90μL of competent cells were mixed with 10μL of pCF10 plasmid and placed in a pre-cooled electroporation cuvette. The mixture was electroporated 3 times at 1.7kV. After the mixed bacterial solution was cultured in fresh BHI medium at 37℃ for 1h, 100μL of the bacterial solution was spread on BHI agar medium containing 10μg / mL tetracycline for screening to obtain donor bacteria OG1RF (pCF10).

[0063] (3) Validation of donor bacteria: After the donor bacteria OG1RF(pCF10) were cultured to the logarithmic phase, the plasmid was extracted and electrophoresed on a 1% agarose gel. The plasmid bands were observed after 30 min.

[0064] (4) Preparation of recipient strain: OG1RF was cultured in BHI liquid medium to the logarithmic phase, and streptomycin was added to a final concentration of 1 g / L. After culturing for another 8 h, the culture was transferred to fresh BHI liquid medium containing 1 g / L streptomycin. After culturing for another 8 h, 100 μL of the bacterial culture was spread on BHI agar medium containing 1 g / L streptomycin and cultured at 37 °C for 12 h. Then, single colonies were picked and cultured. The above steps were repeated by increasing the concentration of streptomycin to 2 g / L and 3 g / L, and finally streptomycin-resistant Enterococcus faecalis was induced and named OG1RS as the recipient strain.

[0065] (5) Recipient bacterial verification: OG1RS were cultured in BHI broth containing streptomycin for 8 hours and counted, and the bacterial count was compared with that of OG1RF diluted to the same gradient and grown on ordinary BHI medium. After the recipient bacteria OG1RS were cultured to the logarithmic growth phase, bacterial genomes were extracted, and specific primers were designed for PCR amplification. The obtained PCR products were electrophoresed on a 1% agarose gel at 100V, and the PCR product bands were observed after 30 minutes.

[0066] (6) Conjugate selection: Donor bacteria OG1RF (pCF10) and recipient bacteria OG1RS were inoculated into BHI broth containing the corresponding antibiotic concentrations and cultured at 37°C until the logarithmic growth phase. The cultures were then washed three times with PBS buffer containing 2 mmol / L EDTA (4°C, 6000 rpm, 5 min), and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL. The donor and recipient bacteria were mixed at a 1:1 ratio to form a conjugation transfer system. The system was incubated statically in a constant temperature and humidity incubator at 37°C for 4 hours. 10 μL of conjugant bacterial solution with appropriate dilution gradient was dropped onto double-antibiotic medium and incubated at 37°C for 36 hours to screen conjugants. At the same time, donor and recipient bacteria with appropriate dilution gradient were dropped onto their respective antibiotic mediums as controls to exclude spontaneous mutations in the donor and recipient bacteria.

[0067] (7) Conjugate verification: Conjugates were randomly selected and inoculated into liquid culture medium containing double antibodies. They were cultured at 37°C to the logarithmic phase. Plasmids and genomes were extracted and PCR amplified using specific primers. The obtained plasmids and PCR products were electrophoresed on a 1% agarose gel. The bands of plasmids and PCR products were observed after 30 min.

[0068] Example 2: Establishment of a method for evaluating the biological effects of bisphenol compounds

[0069] See Figures 4-6 Table 1;

[0070] (1) Reagents: Pheromones cCF10 were synthesized by GenScript and prepared into a stock solution of 100 mg / mL pheromone cCF10 using a mixed solution of 0.3% acetic acid and 0.7% acetonitrile as the solvent. The solution was stored at -20℃ for later use. Bisphenols included bisphenol A (BPA), bisphenol F (BPF), bisphenol S (BPS), tetrabromobisphenol A (TBBPA), and hexafluorobisphenol A (BPAF), all of which were purchased from Maclean Company. The solvent was dimethyl sulfoxide (DMSO).

[0071] (2) 10 8 OG1RF (pCF10) and OG1RS were mixed at a 1:1 ratio (CFU / mL), and pheromone cCF10 was added to final concentrations of 1, 3, 5, 7, 10, 13, 15, 17, and 20 ng / mL (corresponding to molar concentrations of 1, 3, 5, 7, 10, 13, 15, 17, and 20 nmol / L, respectively). The mixture was incubated at 37℃ for 4 h, and the binding transfer frequency variation function f based on pheromone cCF10 concentration was calculated. cCF10 (y).

[0072] (3) 10 8OG1RF (pCF10) and OG1RS were mixed at a 1:1 ratio (CFU / mL), and different concentrations of BPA, BPF, BPS, TBBPA, and BPAF were added. The mixtures were incubated at 37°C for 4 hours. The concentration range of bisphenols was 0–50 nmol / L (environmental pollution concentration, which had no effect on normal bacterial growth). The conjugation frequency and the function f of the effect of bisphenol concentration on the conjugation transfer frequency were calculated. BPs (x).

[0073] (4) Based on the results of steps (2) and (3), calculate the relationship between the ability of different bisphenol substances to affect conjugation transfer and the ability of pheromone cCF10 to affect conjugation transfer. BPs (x)=f cCF10 (y). After simplification, we obtain the following equation: y = kx + b, where k and b are constants. The biological effects of bisphenol A on binding transfer can be compared by the magnitude of the k value.

[0074] Table 1. Evaluation Model of Bisphenolic Effects

[0075]

[0076] The biological effects of bisphenols are ranked as follows: bisphenol A > tetrabromobisphenol A > hexafluorobisphenol A > bisphenol F > pheromone cCF10 > bisphenol S.

[0077] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing an assessment model for the ecological risk of environmental bisphenol pollutants, comprising the following steps: Recombinant Enterococcus faecalis was used as the donor bacteria, and streptomycin-resistant Enterococcus faecalis was used as the recipient bacteria; The recombinant Enterococcus faecalis contains a pheromone regulatory plasmid; The donor and recipient bacteria were first mixed to obtain a first mixture. Different concentrations of pheromones were added to the first mixture, and the mixture was subjected to a first static culture. The number of conjugates was measured, and the first conjugation transfer frequency was calculated. A function was established to determine the effect of pheromone concentration on the conjugation transfer frequency, with pheromone concentration as the independent variable and the first conjugation transfer frequency as the dependent variable. f 信息素 (y); The donor and recipient bacteria were mixed to obtain a second mixture. Different concentrations of bisphenol A were added to this second mixture, followed by a second static incubation. The number of conjugates was measured, and the second conjugation transfer frequency was calculated. A function was established to determine the effect of bisphenol A concentration on the conjugation transfer frequency, with the concentration of environmental bisphenol A as the independent variable and the second conjugation transfer frequency as the dependent variable. f BPs (x); According to the above f 信息素 (y) and f BPs (x) Establish the functional equation between the influence of environmental bisphenols on binding and transfer capacity and the influence of pheromones on binding and transfer capacity, and construct an evaluation model for the ecological risk of bisphenol pollutants. The assessment model for the ecological risk of environmental bisphenol pollutants is as follows: f BPs (x)= f 信息素 (y); x is the concentration of BPs, and y is the concentration of pheromones; after simplification, we get the following equation: y = kx + b, Where k and b are constants; The ratio of the number of viable donor bacteria to the number of viable recipient bacteria in the first mixture or the second mixture is 1:1; The effective concentration of the environmental bisphenol pollutants is 0.01~50 nmol / L; The pheromone is cCF10; the effective concentration of the pheromone is 1, 3, 5, 7, 10, 13, 15, 17 and 20 nmol / L; The temperature for the first static incubation or the second static incubation is 25~37℃; the time for the first static incubation or the second static incubation is 4h.

2. The construction method according to claim 1, characterized in that, The pheromone regulation plasmid is the pCF10 plasmid.

3. The construction method according to claim 1, characterized in that, The original strains of the donor and recipient bacteria were Enterococcus faecalis OG1RF.

4. The construction method according to claim 1, characterized in that, The concentration of the donor or recipient bacteria is (1~9)×10⁻⁶. 8 CFU / mL.

5. The construction method according to claim 1, characterized in that, The bisphenol contaminants are one or more of bisphenol A, bisphenol F, bisphenol S, tetrabromobisphenol A, and hexafluorobisphenol A.

6. A method for assessing the ecological risk of environmental bisphenol pollutants, comprising the following steps: An evaluation model for the ecological risk of bisphenol pollutants in the environment to be tested is constructed using the construction method described in any one of claims 1 to 5. The k value is calculated, and the larger the k value, the greater the ecological risk of the corresponding bisphenol pollutants in the environment.

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