A method for evaluating the transgenerational biological effects of water pollutants based on a multi-level biomarker response method

Abstract

The present invention discloses a method for evaluating the transgenerational biological effects of pollutants in water based on a multi-level biomarker response method. The method for evaluating the transgenerational biological effects of pollutants in the present invention uses the pollutants in water as the target compounds, conducts continuous two-generation exposure and recovery experiments on Daphnia magna, and quantitatively determines the cumulative levels of the target substances in the parental and offspring generations through mass spectrometry technology; analyzes the swimming and feeding behavior vitality, antioxidant enzyme activity, and neurotoxic enzyme activity of organisms, and evaluates the transgenerational biological effects of pollutants through a multi-level biomarker response method. The standards of biomarkers at different levels can be integrated and normalized, which can be used to evaluate the transgenerational biological effects of new pollutants and achieve an intuitive and quantitative evaluation of the long-term ecological risks of pollutants at environmentally relevant concentrations.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the transgenerational biological effects of pollutants in water based on a multi-level biomarker response method, belonging to the field of ecological risk assessment of emerging pollutants. Background Art

[0002] Emerging pollutants refer to toxic and harmful chemical substances that are discharged into the environment and have characteristics such as biotoxicity, environmental persistence, and bioaccumulation, posing a relatively high risk to the ecological environment or human health, but have not been included in management or the existing management measures are insufficient. The "List of Key Controlled Emerging Pollutants (2023 Edition)" has included perfluorooctane sulfonic acid, perfluorooctanoic acid, nonylphenol, antibiotics, etc. in the list of key controlled pollutants. Although the list of controlled emerging pollutants is constantly being enriched, the concern about their aquatic ecological risks, especially the multi-generation toxicity to aquatic organisms, is still in its infancy.

[0003] In recent years, molecular biomarkers have been applied to the monitoring and early warning of pollutants due to their characteristics such as short detection period, high efficiency, practicality, and high sensitivity. The stress effects of pollutants on multiple levels of aquatic organisms have important indicative significance for comprehensively evaluating the ecological risks of substances. However, how to summarize multiple levels of biomarkers and comprehensively evaluate the transgenerational effects of trace pollutants still needs to be further improved. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide a method for evaluating the transgenerational biological effects of emerging pollutants based on multi-level biomarker responses.

[0005] Technical Solution: A method for evaluating the transgenerational biological effects of pollutants based on multi-level biomarker responses provided by the present invention includes the following steps:

[0006] (1) Daphnia magna transgenerational exposure experiment: Select multiple pollutants as target pollutants, the number of target pollutants ≥ 2, detect the relevant concentrations of the target pollutants in the water environment, and select at least one relevant concentration as the exposure concentration of the target pollutant; Select one Daphnia magna larva born within ≤ 24 h after more than three generations of reproduction as the parental generation, add it to the target pollutant exposure solution, and culture it in a light incubator for 21 days; Observe and record the growth and development indicators; Expose and culture one newly born juvenile Daphnia magna in the offspring separately and culture it in clean water for recovery for 21 days; Observe and record the growth and development indicators; Set a blank control group for culturing in clean water;

[0007] (2) After the culture of the parental generation and the offspring is completed, take out all the Daphnia magna, and respectively conduct the determination of the cumulative concentration of the target pollutant, biomarker test, swimming and feeding behavior test; and calculate the intrinsic growth rate r of the parental generation and the offspring m and the 21-day survival rate;

[0008] (3) Using the intrinsic growth rate r of parental, offspring exposure, and offspring clean water recovery m , survival rate, growth and development indicators, cumulative concentration of target pollutants, biomarkers, swimming and feeding behavior data to draw a star chart, and calculate the IBR value. Evaluate the transgenerational biological effects of pollutants by comparing the IBR values of parental, offspring exposure, and offspring clean water recovery.

[0009] Among them, the conditions of the light incubator in step (1) are temperature 21±1°C, light-dark ratio 16h:8h; the exposure solution is changed every two days during exposure, and Chlorella is fed every day, and the feeding density is 0.16mg C / individual / day.

[0010] Among them, the growth and development indicators described in step (1) include the time of first oviposition, the time of first daphnia production, the number of first daphnia produced, and the time and number of daphnia produced during the whole process.

[0011] Among them, the exposure concentration of the target pollutant in step (1) is to select the lowest relevant concentration in the target pollutant as the exposure concentration. If the target pollutant > 2, multiple experimental exposure concentrations can be set according to the relevant concentration.

[0012] Among them, the biomarker test described in step (2) includes acetylcholinesterase, superoxide dismutase, and malondialdehyde activity tests.

[0013] Among them, the pretreatment method of the daphnia magna sample for determining the cumulative concentration of the target pollutant in step (2) is: adding the known extraction solution and internal standard to the pre-weighed biological sample, vortexing for 3 cycles at 4°C, ultrasonic extraction for 1h at 20°C, centrifuging at 13000rpm for 15min, separating the supernatant, repeating three times, then drying the supernatant by nitrogen blowing, and making the volume constant to 1mL with methanol for mass spectrometry quantitative analysis.

[0014] Among them, the liquid chromatography-mass spectrometry quantitative analysis is completed by high performance liquid chromatography-tandem mass spectrometry analysis, and the specific analysis conditions are determined according to the properties of the target pollutant.

[0015] Among them, the detection steps of the swimming behavior described in step (2) are: taking the live daphnia magna and placing it in a culture plate, one in each well; adding 2 cm high of the corresponding exposure solution to each well, after 5 min, using a digital camera to record the movement trajectory of the daphnia magna in each well for 1 min, through image processing software Analysis, after setting the scale and coordinate origin, capturing the coordinates of the daphnia magna frame by frame, and obtaining the average speed and average acceleration according to the movement trajectory.

[0016] Among them, the intrinsic growth rate r described in step (2) m The calculation formula is as follows:

[0017]

[0018] Among them, x is the age in days of Daphnia magna; l x is the survival rate of Daphnia magna on the x-th day; m x is the number of eggs laid by each Daphnia magna on the x-th day;

[0019] The formula for calculating the survival rate is the number of individuals surviving after 21 days of exposure / the total number of individuals at the initial stage of exposure.

[0020] Among them, the steps for calculating the IBR value in step (3) are as follows:

[0021] (1) Compare the data X of each treatment group with the same type of data X 0 of the blank control group respectively, and take the logarithm to reduce the deviation: Y = log X / X 0 ; the treatment groups are the parental exposure group, the offspring exposure group, and the offspring clean water recovery group; the data includes the intrinsic growth rate r m , survival rate, growth and development indicators, cumulative concentration of target pollutants, biomarkers, swimming and feeding behavior data;

[0022] (2) Calculate the mean m and standard deviation s of the data, and let Z = (Y – m) / s, where Z is the value after normalization;

[0023] (3) Calculate the data deviation index A, A = Z - Z 0 , where Z is the treatment group and Z 0 is the blank control group;

[0024] (4) Draw a star chart based on the A values of each item of data, and calculate the IBR values of the parental generation, offspring exposure, and offspring clean water recovery under continuous exposure respectively. The calculation formula is IBR = Σ|A|.

[0025] The present invention also provides an application of the above method in evaluating the transgenerational biological effects of new pollutants in the water environment.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The method for evaluating the transgenerational biological effects of pollutants in the present invention uses the pollutants in water as the target compounds, conducts continuous two-generation exposure and recovery experiments on Daphnia magna, and quantitatively determines the cumulative levels of the target substances in the parental generation and offspring through mass spectrometry technology; analyzes the swimming and feeding behavior activities, antioxidant enzyme activities, and neurotoxic enzyme activities of organisms, and evaluates the transgenerational biological effects of pollutants through the multi-level biomarker response method. The standards of biomarkers at different levels are integrated and normalized, which can be used to evaluate the transgenerational biological effects of new pollutants and realize the intuitive and quantitative evaluation of the long-term ecological risks of pollutants at environmentally relevant concentrations. Description of the Drawings

[0027] Figure 1IBR star charts of generations, offspring exposure, and offspring recovery in clean water. Detailed implementation manners

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0029] Example 1

[0030] (1) Multi-generation exposure experiment of Daphnia magna: The test organism, adult Daphnia magna, was purchased from the Institute of Hydrobiology, Wuhan, and had been continuously cultured in the laboratory for more than three generations. Taking bisphenol A (BPA), a phenolic pollutant with high current attention, and structurally similar phenolic pollutants tetrabromobisphenol A (TBBPA) and bisphenol S (BPS) as target pollutants, multi-generation exposure experiments were carried out by setting the environmentally relevant concentration (1 μg / L) as the exposure concentration. Add 50 mL of exposure solution to a 100 mL beaker, randomly select Daphnia magna juveniles (≤24 h) that have reproduced for more than three generations as the parental generation F0, add one to each beaker, set 10 parallels as the parental exposure group (F0); another blank control group was cultured in clean water. The culture conditions of the parental exposure group and the blank control group were the same: placed in a light incubator, temperature 21 ± 1 °C, light-dark ratio 16 h:8 h, the exposure solution was changed every two days during the exposure period, and Chlorella was fed every day, and the feeding density was 0.16 mg C / individual / day.

[0031] The culture period of the parental generation (F0) was 21 days. During this period, the newly born juveniles (F1) were taken out, and the offspring exposure group (F1-exposure, cultured in the exposure solution) and the offspring clean water recovery group (F1-recovery, cultured in clean water) were set respectively, with 10 parallels in each group and 1 in each parallel, and they were also cultured for 21 days. Under the same culture conditions as the parental exposure group, observations were made once every 24 h during the experiment, and the reproductive ability indexes of the F0 and F1 generations were recorded, including the time of the first egg laying, the time of the first production of juveniles, the number of times of producing juveniles, and the number of juveniles. After 21 days of exposure of the parental and offspring generations, all Daphnia magna were placed on filter paper to absorb their moisture, and then quickly frozen with liquid nitrogen and stored in a -80 °C ultra-low temperature refrigerator for cumulative concentration determination and enzyme activity testing.

[0032] (2) Evaluating the impact of exogenous pollutants on the Daphnia magna population through the intrinsic rate of increase (r m ). The intrinsic rate of increase (r m ) of Daphnia magna was calculated using the following equation:

[0033]

[0034] Where: x is the age of Daphnia magna in days; l x is the survival rate of Daphnia magna on the x-th day; m x is the number of eggs laid by each Daphnia magna on the x-th day, and the exact value of r m was obtained by the method of successive approximation on the basis of rough calculation.

[0035] (3) Biomarker test: Daphnia magna was homogenized with pre-cooled normal saline at a ratio of 9:1 at 4°C. The obtained homogenate was placed in a 1.5 mL centrifuge tube, and then centrifuged at a centrifugal force of 4000×g for 15 min. The supernatant was taken and stored at -80°C for enzyme activity determination. The determination of acetylcholinesterase (AChE), superoxide dismutase (SOD), malondialdehyde (MDA), and protein content was carried out batchwise using kits, and all kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0036] (4) Determination of cumulative concentration: The weighed biological individuals were placed in a 5 mL centrifuge tube, and 2 mL of a mixed solution of ice methanol / acetonitrile / ultrapure water (2:2:1, v / v / v) was added. Isotope internal standard 13 C-BPA, BPAS-d 6 ) 10 ng was added, and homogenized 3 times, 10 s each time. Then the homogenate was transferred to a test tube, sonicated at a frequency of 50 kHz for 30 min, centrifuged at 4°C for 5 min at a rotation speed of 4000×g, and the same steps were repeated twice. The supernatant was dried by nitrogen blowing and re-dissolved in acetonitrile:water (1:1) solvent for liquid chromatography-mass spectrometry analysis.

[0037] Specific operation of liquid chromatography-mass spectrometry analysis: An Agilent 1260 high performance liquid system and a 6550 Q-TOF mass spectrometer were used to analyze samples of BPA, TBBPA, BPS and their metabolites. The samples were analyzed by negative ion mode mass spectrometry using an Atlantis T3 chromatographic column (3 mm, 2.1×100 mm) in electrospray ionization (ESI - ). The mobile phase was water + 2 mM ammonium acetate (A) - acetonitrile (B), with gradient elution, a flow rate of 0.2 mL / min, and the gradient settings were: 0 - 3 min: 95% A; increased to 95% B at 14 min and remained unchanged at 17 min; 17 - 18 min: decreased from 95% B to 5% B. Compounds were identified using credible standards or accurate mass and MS / MS fragments. The mass spectrometry analysis parameters of the compounds are shown in Table 1.

[0038] All samples in this study were in triplicate. For each batch, a procedural and laboratory blank was run alongside during sample preparation and analysis to correct for background levels. During sample analysis, every 6 samples were pooled to check the instrument stability. Calibration standards were also run before and after the worksheet, with a large dynamic linear range to cover the concentration changes in the samples.

[0039] Table 1 Mass spectrometry analysis parameters of compounds

[0040]

[0041] Note: *Quantitative ion

[0042] (5) Swimming behavior: After the cultivation of the parental and offspring generations was completed, live Daphnia magna from the F0, F1-exposed, and F1-recovery groups were separately placed in a 6-well culture plate, one in each well. The exposure solution in each well was 2 cm high to ensure that the organisms only moved in a two-dimensional plane. After the organisms were allowed to acclimatize for 5 min, a digital camera with a frame rate of 30 frames per second was used to record the movement trajectories of the Daphnia magna in each well for 1 min. The recorded videos were analyzed using image processing software . After setting the scale and the origin of the coordinates, the coordinates of the Daphnia magna were captured frame by frame to obtain the movement trajectory of the organism, and its average speed and average acceleration were analyzed. To prevent individual differences, 10 organisms were selected from each parallel exposure group, and the mean value was used as the final result of that exposure group.

[0043] (6) Feeding behavior: At the end of the cultivation, 10 randomly selected 21-day-old Daphnia magna from each group were transferred to a solution with an initial concentration of 1×10 6 cells / mL of Chlorella. To prevent algal growth, the beakers were exposed to complete darkness for 5 h. At the end of the experiment, the test organisms were removed, and the algal concentration was detected using a hemocytometer under an optical microscope (×400). Each treatment was repeated three times. The ingestion rate (I) of the organisms was calculated separately as an index of food intake. Among them Among them, A = (lnC 0 - lnC′ t ) / t, where C 0 and C t are the initial and final food concentrations (cells / μL), respectively; t is the measurement duration in hours; n is the number of individuals in volume V (μL); A is the correlation factor related to the change of the control substance and the final concentration Ct′ after time t. The formula √(C 0 · C t ) is the geometric mean of the Chlorella concentration during time t.

[0044] (7) The above data on growth and development indicators, cumulative concentrations, biomarkers, swimming, and feeding behaviors were analyzed using the integrated biomarker response method. The specific calculation method is as follows:

[0045] ① The data (X) of each treatment group were compared with the same type of data (X 0 ) of the blank control group, and logarithms were taken to reduce the deviation: Y = log X / X 0 . The treatment groups were the parental exposure, offspring exposure, and offspring clean water recovery groups; the data included the intrinsic growth rate r m , survival rate, growth and development indicators, cumulative concentration, biomarker, swimming, and feeding behavior data;

[0046] ② Calculate the mean (m) and standard deviation (s) of the data. Let Z = (Y – m) / s, where Z is the value after normalization.

[0047] ③ Calculate the biomarker deviation index A, where A = Z - Z 0 (Z is the treatment group, and Z 0 is the control group).

[0048] ④ Draw a star plot. The length of the radial lines in the star plot represents the magnitude of the data deviation index A for each treatment group. The area above 0 indicates that the biomarker is induced, and the area below 0 indicates that the biomarker is inhibited.

[0049] ⑤ Calculate the IBR values for parental exposure, offspring exposure, and offspring recovery in clean water under continuous exposure respectively. IBR = Σ|A|, and compare the transgenerational effects of the new pollutants.

[0050] Table 2. IBR scores of bisphenol A, tetrabromobisphenol A, and bisphenol S under continuous exposure

[0051]

[0052] The results are as Figure 1 shown in and Table 2. For parental exposure, the biotic stress effect of bisphenol S > bisphenol A > tetrabromobisphenol A. After continuous exposure of offspring, the biotic stress effect of bisphenol S > bisphenol A > tetrabromobisphenol A. After recovery in clean water, the recovery ability of bisphenol A > bisphenol S > tetrabromobisphenol A.

Claims

1. A method for evaluating the transgenerational biological effects of pollutants based on multi-level biomarker responses, characterized in that, it includes the following steps: (1) Daphnia magna transgenerational exposure experiment: Select multiple pollutants as target pollutants, the number of target pollutants ≥ 2, detect the relevant concentrations of the target pollutants in the water environment, and select at least one relevant concentration as the exposure concentration of the target pollutants; Select one Daphnia magna larva born within 24 hours after more than three generations of reproduction as the parental generation, add it to the target pollutant exposure solution, and culture it in a light incubator for 21 days; Observe and record the growth and development indicators; Expose and culture and recover in clean water one newly born Daphnia magna larva of the offspring respectively, and the culture time is 21 days; Observe and record the growth and development indicators; Set a blank control group for culturing in clean water; The growth and development indicators include the time of first egg-bearing, the time of first Daphnia production, the number of first Daphnia produced, and the time and number of Daphnia produced during the whole process; (2) After the culture of the parental and offspring Daphnia magna is completed, all Daphnia magna are taken out and subjected to the determination of the cumulative concentration of the target pollutant, biomarker tests, swimming and feeding behavior tests respectively; and the intrinsic growth rate r of the parental and offspring is calculated. m and the 21-day survival rate; The biomarker tests include acetylcholinesterase, superoxide dismutase, and malondialdehyde activity tests; (3) Using the intrinsic growth rate r of parental generation, offspring exposure, and offspring clean water recovery m , survival rate, growth and development indicators, cumulative concentration of target pollutants, biomarkers, swimming and feeding behavior data to draw a star chart, calculate the IBR value, and evaluate the transgenerational biological effects of pollutants by comparing the IBR values of parental generation, offspring exposure, and offspring clean water recovery.

2. The method according to claim 1, characterized in that, In step (1), the conditions of the light incubator are temperature 21 ± 1 °C, light-dark ratio 16h:8h; During the exposure period, the exposure solution is changed every two days, and Chlorella is fed every day, and the feeding density is 0.16 mg C / individual / day.

3. The method according to claim 1, characterized in that, The pretreatment method of the Daphnia magna sample for the determination of the cumulative concentration of the target pollutant in step (2) is: Add the known extract and internal standard to the pre-weighed biological sample, vortex, ultrasonically extract, centrifuge, separate the supernatant, blow-dry the supernatant with liquid nitrogen, and make up the volume with methanol for quantitative analysis by mass spectrometry.

4. The method according to claim 3, characterized in that, The liquid chromatography-mass spectrometry quantitative analysis is completed by high performance liquid chromatography-tandem mass spectrometry analysis, and the specific analysis conditions are determined according to the properties of the target pollutants.

5. The method according to claim 1, characterized in that, The detection steps of the swimming behavior described in step (2) are as follows: Place a live Daphnia magna in a culture plate, one in each well; add 2 cm high corresponding exposure solution to each well. After 5 minutes, use a digital camera to record the movement trajectory of Daphnia magna in each well for 1 minute. After analysis by an image processing software and setting the scale and coordinate origin, capture the coordinates of Daphnia magna frame by frame, and obtain the average velocity and average acceleration based on the movement trajectory.

6. The method according to claim 1, characterized in that, The intrinsic growth rate r described in step (2) m The calculation formula is as follows: where x is the age in days of Daphnia magna; l x is the survival rate of Daphnia magna on the x-th day; m x is the number of eggs laid by each Daphnia magna on the x-th day; The survival rate calculation formula is the number of individuals surviving after 21 days of exposure / the total number of individuals at the initial stage of exposure.

7. The method according to claim 1, characterized in that, The calculation steps of the IBR value in step (3) are as follows: (1) Compare the data X of each treatment group with the same type of data X of the blank control group respectively, and take the logarithm to reduce the deviation: Y = log X / X 0 ; The treatment groups are the parental exposure group, the offspring exposure group, and the offspring clean water recovery group; The data includes the intrinsic growth rate r 0 , survival rate, growth and development indicators, cumulative concentration of target pollutants, biomarkers, swimming and feeding behavior data; m ​ (2) Calculate the mean m and standard deviation s of the data, and let Z = (Y–m) / s, where Z is the normalized value; (3) Calculate the data deviation index A, where A = Z - Z 0 , where Z is the treatment group and Z 0 is the blank control group; (4) Draw a star chart according to the A values of each item of data, and calculate the IBR values of the parental generation, offspring exposure and offspring clean water recovery under continuous exposure respectively. The calculation formula is IBR = Σ|A|.

8. Application of the method according to any one of claims 1 to 7 in evaluating the transgenerational biological effects of new pollutants in the water environment.

Images