CN112352741B - Biological model for bisphenol S and lead combined pollution and establishment method thereof - Google Patents
Biological model for bisphenol S and lead combined pollution and establishment method thereof Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A20/00—Water conservation; Efficient water supply; Efficient water use
- Y02A20/20—Controlling water pollution; Waste water treatment
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Abstract
The invention provides a biological model of bisphenol S and lead combined pollution and an establishment method thereof, belonging to the technical field of food safety detection; in the invention, a biological model of bisphenol S and lead combined pollution is constructed based on bisphenol S and lead with different concentrations, so that the toxicity of the bisphenol S and lead combined pollution is verified, and a test basis is provided for the safety of the wide application of the bisphenol S and lead; the biological model has the advantages of fast propagation, low cost, simple and efficient operation and the like.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a biological model for bisphenol S and lead combined pollution and an establishment method thereof.
Background
Bisphenol S (BPS) is a substitute of bisphenol A and is widely applied to production and life. It was found that bisphenol S causes problems with dyskinesia, growth and dysplasia, and fat accumulation. At present, the researches on bisphenol substances and metal substances focus on the degradation of the content of bisphenol substances in water by magnetic materials, and the combined toxicity is not reported too much.
In recent years, lead has been widely used in industry as an industrial raw material, but lead ions have high toxicity and strong accumulation in the living body. Currently, only toxicity reports on the single action of bisphenol S and lead and related researches on the combined toxicity of bisphenol S and lead are lacked.
Although the development pathway of mammals such as mice is highly similar to that of human beings, the mammals such as mice consume more materials and have higher cost when being used for constructing models, and the requirements on culture environment are strict. Caenorhabditis elegans (C. elegans) has short life cycle, less culture consumables and high safety, and has great advantages in the field of toxicology research.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a biological model for bisphenol S and lead combined pollution and an establishment method thereof. In the invention, the toxicity of the bisphenol S and lead combined pollution is verified by establishing a biological model of the bisphenol S and lead combined pollution, thereby providing a test basis for the safety of the bisphenol S and lead in wide application.
The invention provides a biological model of bisphenol S and lead combined pollution, which is constructed on the basis of bisphenol S and lead with different concentrations.
The invention also provides a construction method of the biological model for the joint pollution of the bisphenol S and the lead, which comprises the following steps:
(1) preparation of bisphenol S and lead containing NGM medium:
dissolving bisphenol S in an ethanol solution, respectively preparing bisphenol S solution and lead solution with different concentrations, mixing the bisphenol S solution and the lead solution, mixing the mixed solution with different concentrations and E.coli OP50 again, and respectively coating the mixed solution and the E.coli OP50 on a blank NGM culture medium;
(2) establishing a biological model of bisphenol S and lead combined pollution:
c. elegans in L1 stage was equally distributed to NGM medium containing bisphenol S and lead, and when cultured to L4 stage, M9 was used to wash the C.elegans on the medium into the tubes, to obtain a biological model of C.elegans exposed to different concentrations of bisphenol S and lead.
Further, in the step (1), the final concentration of the bisphenol S in the mixed solution is 0.001-10 mu M.
Further, in the step (1), the final concentration of lead in the mixed solution is 0.001-10 μ M.
Further, in the step (1), the final concentration of the ethanol in the mixed solution is 0.1%.
Further, in the step (1), the volume ratio of the mixed solution to the E.coli OP50 is 1: 1.
Further, in the step (1), the total amount of the mixed solution and e.coli OP50 was coated at 50 μ L.
Further, in the step (2), the culture method of the caenorhabditis elegans of L1 stage comprises the following steps: e, coli OP50 is coated on the surface of an NGM culture medium, and then the caenorhabditis elegans is inoculated in the culture medium for expanded culture; then flushing, precipitating and washing the cultured caenorhabditis elegans; and sequentially adding M9, NaClO and NaOH into the precipitate, shaking to obtain caenorhabditis elegans eggs, and incubating the eggs to obtain the caenorhabditis elegans in the L1 stage.
Further, in the step (2), the culture conditions are as follows: the culture was carried out at 20 ℃ for 48 h.
Compared with the prior art, the invention has the beneficial effects that:
the caenorhabditis elegans is selected to construct the model, has the advantages of easy culture, quick propagation, low cost and the like, and can reflect the damage of substances to human bodies to a great extent when being used as a model organism for toxicological research.
In the invention, the caenorhabditis elegans is utilized to construct the biological model of the bisphenol S and lead combined pollution, and the biological model has the advantages of fast propagation, low cost, simple and efficient operation and the like. A series of tests are carried out on the biological model, and the results show that the lethal action degree of the caenorhabditis elegans is different under the condition that bisphenol S and lead are independently exposed, and the toxicity of the lead is far greater than that of the bisphenol S; when both are exposed simultaneously, more time is spent on antagonism, but when both are at low concentrations, the toxicity of both is additive. Moreover, the combined exposure also antagonizes both the locomotor activity and neurotoxicity of caenorhabditis elegans, as compared to the separate exposure of the two substances.
Drawings
FIG. 1 is a comparison of the actual and theoretical mortality rates for C.elegans for different mixed concentrations of bisphenol S and lead, where A is BPS and the concentration is 1/5LC 50 Combined toxicity of Pb to caenorhabditis elegans; b is BPS and the concentration is 1/2LC 50 Combined toxicity of Pb to caenorhabditis elegans; c is BPS and concentration is LC 50 Combined toxicity of Pb to caenorhabditis elegans.
FIG. 2 is a comparison of actual and theoretical values of the locomotor activity of C.elegans by the combined exposure of varying concentrations of bisphenol S and lead, wherein A is head swing; and B is body curvature.
FIG. 3 is a comparison of actual and theoretical values for body length and body surface area of C.elegans for combined exposure to varying concentrations of bisphenol S and lead, where A is body length and B is body surface area.
FIG. 4 is a comparison of actual and theoretical values for the combined exposure of different concentrations of bisphenol S and lead to C.elegans progeny.
Detailed Description
The invention will be further described with reference to the following figures and specific examples, but the scope of the invention is not limited thereto.
Example 1:
(1) preparation of C.elegans of stage L1:
configuration of NGM medium: weighing 3g of sodium chloride and 2.5g of tryptone in a 1L big beaker, adding 1L of distilled water, uniformly stirring to fully dissolve the sodium chloride and the tryptone, and then uniformly subpackaging into 4 conical bottles with 500mL, wherein each conical bottle contains 250mL of solution. Since the agar powder is insoluble at low temperature, after the prepared solution is subpackaged, respectively weighing 4.25g of agar powder and adding the agar powder into four conical flasks, and finally carrying out autoclaving at 121 ℃ for 30 min. After the autoclaving, the mixture is cooled to 65 ℃, and 1M calcium chloride and magnesium sulfate solutions, 5mg/mL cholesterol solutions 250. mu.L and 1M potassium phosphate buffer solutions 6.25mL, which are prepared for sterilization by a filter membrane, are added to each Erlenmeyer flask in a clean bench.
Configuration of M9: 3g of potassium dihydrogen phosphate, 6g of disodium hydrogen phosphate, 5g of sodium chloride and 1mL of 1M magnesium sulfate are weighed in a 1L big beaker, 1L of distilled water is added, the mixture is stirred and fully dissolved and then is subpackaged in a conical flask, and the mixture is sterilized by high-pressure steam at 121 ℃ for 30 min.
200 μ L of E-coli OP50 (university of southeast King of Adam professor laboratories) was spread on the surface of 9cm NGM medium, and then the medium was inoculated with C.elegans for scale-up culture; washing the cultured caenorhabditis elegans in the oviposition stage from the culture medium by using M9, naturally settling to obtain a precipitate, and washing by using M9; then adding the mixture into the precipitate according to the volume ratio of 2: 1: 2, sequentially adding M9, 1M NaClO and 1M NaOH, shaking to obtain caenorhabditis elegans eggs, washing the eggs with M9, and then placing the eggs in a 3.5cm culture dish to incubate at 20 ℃ overnight to obtain the caenorhabditis elegans in the L1 stage.
(2) Preparing an NGM culture medium containing a bisphenol S solution and lead:
BPS was dissolved in ethanol to obtain a 1M bisphenol S mother liquor, and a lead nitrate solution was dissolved in distilled water to obtain a 1mM lead mother liquor. Then, the solutions were diluted with M9 to obtain bisphenol S solutions and lead solutions having concentrations of 0 (Control), 0.002, 0.02, 0.2, 2 and 20. mu.M, respectively, and the two solutions were mixed. The final concentration of ethanol in the mixed solution is 0.1 percent, and the final concentrations of the bisphenol S solution and the lead solution are respectively 0 (Control), 0.001, 0.01, 0.1, 1 and 10 mu M; the diluted mixed solution containing bisphenol S and lead with different concentrations is respectively and uniformly mixed with E.coli OP50, and 50 mu L of the mixed solution is respectively coated on the surface of NGM medium with the thickness of 3.5 cm.
(3) Construction of a biological model of bisphenol S and lead combined contamination:
the NGM culture medium containing bisphenol S solution and lead is divided into three groups which are marked as a1-a3, b1-b3, C1-C3, d1-d3, e1-e3 and f1-f3 respectively, the caenorhabditis elegans in the L1 stage are transferred to the culture medium and placed in a 20-degree C incubator for continuous culture, when the caenorhabditis elegans grow to the L4 stage, M9 is used for washing the caenorhabditis elegans on the culture medium a1-a3, b1-b3, C1-C3, d1-d 1, e1-e 1 and f1-f 1 to be corresponding to 1.5ml of EP tubes, the caenorhabditis elegans obtained from the EP tubes are biological models exposed under different bisphenol S and lead concentrations and are marked as a1-a 1, b1-b 1, C72-C72, d 1-f 72-f 1, f 72-f 1, f 72-f-1 is adopted as a biological models.
The C.elegans used for the establishment of the above biological model was wild-type N2 C.elegans (purchased from Caenorhabditis Genetics Center University of Minnesota, MN, USA).
Example 2:
in this example, the extent of lethal effect on caenorhabditis elegans from combined bisphenol S and lead contamination was determined.
(1) Caenorhabditis elegans LC under single substance exposure 50 The determination of (1):
bisphenol S solution: dissolve BPS with ethanol to obtain 1M BPS mother liquor, and dilute with M9 to obtain 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 5.0 mM, ethanol concentration of each concentration is 0.1%.
Lead solution: lead was dissolved in deionized water to obtain 1mM lead mother liquor, which was diluted with deionized water to 500. mu.M, 300. mu.M, 250. mu.M, 200. mu.M, 125. mu.M, 67.5. mu.M.
Preparing S base liquid: using 50mL of K + Dissolving 5.8g of sodium chloride in the solution (1M), adjusting the pH to be =6.0, adding 950mL of distilled water and 1.0mL of cholesterol solution (5 mg/mL), uniformly mixing, then carrying out autoclaving at 121 ℃ for 30min, and cooling to the normal temperature for later use.
K + Preparation of solution (1M): weighing 10.83g of monopotassium phosphate and 3.56g of dipotassium phosphate, adding distilled water to a constant volume of 100mL, subpackaging into a spiral-sealed 50mL test tube, and placing in a sterilizing pot to sterilize at 121 ℃ for 30min under high pressure for later use.
Nematode liquid medium: dissolving 10mL of 1M potassium citrate solution, 10mL of trace metal solution, 3mL of 1M calcium chloride solution and 3mL of 1M magnesium sulfate solution in the S base solution, and metering the volume to 1L by using the S base solution, wherein all operations are carried out in a super clean bench without carrying out secondary sterilization.
The C.elegans of stage L1 was cultured to stage L4 in a medium coated with E. coli OP50 only, and the exposed C.elegans 20 at the corresponding concentration were randomly picked up with a platinum wire needle into 24-well plates containing liquid medium (200. mu.L), and after 24 hours, the number of nematodes surviving was recorded under a stereomicroscope.
TABLE 1 Effect of bisphenol S acute exposure on the number of lethal caenorhabditis elegans
| Concentration (mM) | Total number (only) | Number of deaths (one) | Mortality rate |
| 2.0 | 20 | 1 | 0.05 |
| 2.5 | 20 | 3 | 0.15 |
| 3.0 | 20 | 6 | 0.30 |
| 3.5 | 20 | 8 | 0.40 |
| 4.0 | 20 | 10 | 0.50 |
| 5.0 | 20 | 16 | 0.80 |
TABLE 2 Effect of acute exposure to lead on the number of lethal caenorhabditis elegans
| Concentration (μ M) | Total number (only) | Number of deaths (one) | Mortality rate |
| 500 | 20 | 18 | 0.90 |
| 300 | 20 | 11 | 0.55 |
| 250 | 20 | 8 | 0.40 |
| 200 | 20 | 6 | 0.30 |
| 125 | 20 | 3 | 0.15 |
| 67.5 | 20 | 1 | 0.05 |
Tables 1 and 2 show the effect on the number of caenorhabditis elegans deaths under acute exposure to BPS and Pb, respectively. The data shown in the table are displayed by bodyUnder microscopic observation, the survival number of 20 C.elegans (stage L4) exposed to each concentration for 24 hours was recorded to calculate the mortality of nematodes contaminated with that concentration, and the half-Lethal Concentration (LC) was calculated using the PROBIT module of SPSS software 50 ). The resulting simulated linear equation for BPS is probit (p) =0.759x-2.950, and Sig =0.00 <0.05, indicating a good fit, when probit (p) =0.5, the simulated equation is substituted, i.e. LC is obtained 50 =3.884mM, on the basis of which no lethal effect on C.elegans occurs when the BPS concentration is diluted 100-fold. Similarly, the simulation equation for lead is probit (p) =0.007x +1.882, and Sig =0.00 <0.05, calculated to obtain LC 50 =289.075 μ M, which combines the daily exposure of lead with the lethality of caenorhabditis elegans at 24 hour exposure, to provide a reference for the subsequent impact of mortality of nematodes at combined concentration contamination and determination of various indicators.
(2) Determination of the mortality of caenorhabditis elegans under mixed exposure of bisphenol S and lead:
the concentrations of bisphenol S and lead were set to 1/5LC, respectively 50 、1/2LC 50 、LC 50 The three concentrations were combined with each other to determine the actual mortality of C.elegans.
FIG. 1 is a comparison of the actual and theoretical mortality rates for C.elegans for various mixed concentrations of bisphenol S and lead. As can be seen from FIG. 1A, three different concentrations of BPS were compared with 1/5LC 50 The observed mortality rate of caenorhabditis elegans is substantially less than the theoretical value under the combined action of Pb. When 1/5LC 50 BPS and 1/5LC 50 The final result is shown to be additive when Pb is combined. The remaining two concentrations are indicated as antagonism. At 1/2LC 50 、LC 50 The toxicity of Pb is obviously reduced under the action of BPS with concentration, so that the death rate caused by the combined toxicity of the two is about 20 percent.
FIG. 1B shows BPS at a concentration of 1/2LC 50 The combined toxicity of Pb on caenorhabditis elegans is actually measured to be obviously reduced under three different concentrations of BPS, and the higher the concentration of BPS is, the more obvious the antagonism is. Finally, the combined toxic effect of the two is shown as antagonistic phaseHas no side effect.
FIG. 1C shows that BPS and concentration are LC 50 The combined toxicity of Pb on caenorhabditis elegans is also obviously reduced under three different concentrations of BPS, and the higher the concentration of BPS is, the more obvious the antagonism is. When the concentrations of bisphenol S and lead are both half lethal, the mortality of the combined toxicity remains around 40%.
All data are presented as Mean ± standard error (Mean ± SEM), plotted using GraphPad Primer7, and analyzed for significance using SPSS19.0, with significance levels set at P <0.05 and P < 0.01.
As can be seen, only 1/5 bisphenol S LC 50 (776.8. mu. mol/L) and 1/5 lead LC 50 (57.8. mu. mol/L) in combination, the toxic effect appears to be an additive effect; the remaining combinations showed antagonism.
Example 3:
in this example, a platinum wire needle was used to randomly pick a caenorhabditis elegans model exposed to different concentrations, and the motor ability of caenorhabditis elegans under bisphenol S and lead combined pollution was verified by measuring the head swing frequency and body bending frequency of caenorhabditis elegans and comparing the difference between the actual value and the theoretical value. Wherein the theoretical value is the superposition of corresponding index measurement of the caenorhabditis elegans under the single substance exposure.
(1) Frequency of head swing
20-30 mu L of M9 buffer solution is dripped on a glass slide cleaned by alcohol to form a small water drop, 1 piece of the elegans after acute exposure is picked into the small water drop by a platinum wire needle, and the recovery is carried out for 1 minute. The number of swings of the caenorhabditis elegans head from one side to the other side was observed under a stereomicroscope. Timing for 1min, and recording the head swing times of caenorhabditis elegans.
(2) Frequency of body bending
The caenorhabditis elegans model exposed to the colophony concentration was randomly picked with a platinum wire needle, placed on a blank NGM medium plate, and after recovering for a little 1 minute, the measurement was carried out. Observations were counted as one walk forward through a sinusoidal wavelength, using a point in the middle of the caenorhabditis elegans body as a reference standard. The sinusoidal course followed by C.elegans was recorded for 30 s.
The results are shown in FIG. 2, and the behavior of C.elegans after acute exposure to BPS in combination with Pb at each concentration was determined. Overall, the locomotor activity of caenorhabditis elegans is significantly inhibited with increasing toxicant concentration.
As shown in fig. 2A, when the concentration of the combination group was 0.1 μmol/L, the head oscillation frequency was significantly suppressed (p < 0.05) compared to both the control group and the theoretical value, and when the concentration of the combination group was 10 μmol/L, the head oscillation frequency was extremely significantly suppressed (p < 0.01). At concentrations as high as 10 μmol/L, the head swing frequency decreased by 7.8%, 5.1%, 6.4% respectively compared to the blank.
As shown in fig. 2B, body bending frequency was significantly suppressed (p < 0.05) at the combined group concentration of 0.1 μmol/L; when the concentration of the combination group exceeds 1 mu mol/L, the inhibition of the body bending frequency is weakened compared with the theoretical value. At concentrations as high as 10 μmol/L, the body bending frequency decreased by 13.5%, 12.7%, 7.7% compared to the blank.
It can be seen that the combined action of bisphenol S and lead has a reduced effect on the motility of C.elegans compared with the theoretical value.
All data are expressed as Mean ± standard error (Mean ± SEM), plotted using GraphPad Primer7, and analyzed for significance using SPSS19.0, with significance levels set at P <0.05 and P < 0.01.
Example 4:
the following method was used in this example to examine the growth and development of C.elegans under combined exposure to different concentrations of bisphenol S and lead. The pollution concentration of the required mixed solution is 0.001, 0.01, 0.1, 1 and 10 mu M to control, wherein the ratio of BPS to lead is 1:1, final concentration was control, 0.001, 0.01, 0.1, 1, 10. mu.M.
The C.elegans model was rinsed from the medium into 1.5ml EP tubes with M9 and labeled a, b, c, d, e, f, centrifuged three times to remove E.coil from the C.elegans surface, followed by addition of a suitable concentration of 2mmol/L levorotatory anesthetic hydrochloride to keep the C.elegans static. And then, dripping 100 mu L of caenorhabditis elegans liquid on a glass slide, covering a cover glass, observing and photographing under a fluorescence inverted microscope, selecting a clear visual field containing 2-5 pieces of caenorhabditis elegans at each concentration, and measuring the body length and the body surface area of the caenorhabditis elegans by means of a microscope scale and Image J Image software.
FIG. 3 shows the effect of BPS in combination with Pb on the growth and development of C.elegans in the current generation at various concentrations. Both body length and body surface area were significantly inhibited compared to the blank group. When the combined group concentration is 0.1 mu mol/L, the body length and the body surface area are remarkably inhibited (p < 0.05); the body surface area was very significantly inhibited (p < 0.01) at the combined group concentration of 1 μmol/L. When the concentration of each component is 10 mu mol/L, the body length of the caenorhabditis elegans is respectively reduced by 27.2 percent, 33.7 percent and 22.9 percent; when the concentration of BPS is 10. mu. mol/L, the body surface area of the caenorhabditis elegans is reduced by 47%, 49% and 46%, so that the negative effect on the growth and development of the caenorhabditis elegans under the combined action of bisphenol S and lead is weakened by the relative ratio of the theoretical value
All data are expressed as Mean ± standard error (Mean ± SEM), plotted using GraphPad Primer7, and analyzed for significance using SPSS19.0, with significance levels set at P <0.05 and P < 0.01.
Example 5:
in this example, the sub-algebraic quantities of caenorhabditis elegans in the biological model described in example 1 were examined: one maternal worm was randomly picked with a platinum wire needle from the medium labeled a1-a3, b1-b3, c1-c3, d1-d3, e1-e3, f1-f3 and inoculated onto a fresh blank NGM that was not coated with any caenorhabditis elegans diet. The transfer is picked once a day in the oviposition period. The day after transfer, the C.elegans plates of the previous day were counted. And (3) adding all plates of the caenorhabditis elegans at the same concentration to obtain the number of filial generation of the caenorhabditis elegans at the concentration.
FIG. 4 is a graph showing the number of progeny of C.elegans under co-contamination with BPS and Pb. The figure shows that the number of caenorhabditis elegans progeny at the combined concentration is slightly higher than the theoretical value under the combined condition, and the overall result is consistent with the combined toxicity effect of mutual antagonism.
All data are presented as mean ± standard error (mean ± SEM), plotted using GraphPad Primer7, and analyzed for significance using SPSS19.0, with significance levels set at P <0.05 and P < 0.01.
The present invention is not limited to the above-described embodiments, and any obvious improvements, substitutions or modifications can be made by those skilled in the art without departing from the spirit of the present invention.
Claims (6)
1. A method for establishing a biological model of bisphenol S and lead combined pollution is characterized by comprising the following steps:
(1) preparation of NGM medium containing bisphenol S and lead:
dissolving bisphenol S in an ethanol solution, diluting a lead nitrate solution with distilled water, respectively preparing a bisphenol S solution and a lead solution with different concentrations, mixing the two solutions, mixing the mixed solution with different concentrations and E. coli OP50 again, and respectively coating the mixed solution and the E. coli OP50 on a blank NGM culture medium;
(2) establishing a biological model of bisphenol S and lead combined pollution:
c, equally distributing the caenorhabditis elegans in the L1 stage to NGM culture medium containing bisphenol S and lead, and flushing the caenorhabditis elegans on the culture medium into a tube by using M9 when the caenorhabditis elegans is cultured to the L4 stage to obtain a biological model of the caenorhabditis elegans exposed to bisphenol S and lead with different concentrations;
in the biological model of the bisphenol S and lead combined pollution, the analog linear equation of the bisphenol S is PROBIT (p) =0.759x-2.950, and the half-lethal concentration LC of the bisphenol S 50 =3.884 mM; the final concentration of the bisphenol S in the mixed solution is 1/2 half-lethal concentration LC50 of the bisphenol S and half-lethal concentration LC50 of the bisphenol S;
simulation equation for lead is PRObit (p) =0.007x +1.882, lead semilethal concentration LC 50 =289.075 μ M; the final lead concentration in the mixed solution is 1/2 lead half-lethal concentration LC50 and lead half-lethal concentration LC 50;
the biological model of bisphenol S and lead combined pollution plays an antagonistic role on the movement behavior and the neurotoxicity of caenorhabditis elegans.
2. The method for constructing a biological model of combined bisphenol S and lead contamination according to claim 1, wherein in step (1), the final concentration of ethanol in the mixed solution is 0.1%.
3. The method for establishing a biological model of combined bisphenol S and lead contamination according to claim 1, wherein in step (1), the volume ratio of the mixed solution to E. coli OP50 is 1: 1.
4. The method for establishing a biological model of bisphenol S and lead combined contamination according to claim 1, wherein the total coating amount of the mixed solution and the mixed solution of E. coli OP50 in step (1) is 50. mu.L.
5. The method for establishing a biological model of bisphenol S and lead combined pollution according to claim 1, wherein in the step (2), the culture method of C.elegans of L1 stage comprises the following steps: e, coli OP50 is coated on the surface of the NGM culture medium, and then the caenorhabditis elegans is inoculated in the culture medium for amplification culture; then flushing, precipitating and washing the cultured caenorhabditis elegans; and sequentially adding M9, NaClO and NaOH into the precipitate, shaking to obtain caenorhabditis elegans eggs, and incubating the eggs to obtain the caenorhabditis elegans in the L1 stage.
6. The method for constructing a biological model of bisphenol S and lead combined contamination according to claim 1, wherein in the step (2), the culture conditions are as follows: the culture was carried out at 20 ℃ for 48 h.
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