CN114324627A - Method for detecting pollution of trace estrogen endocrine disrupting compounds in reclaimed water by biological-chemical combination - Google Patents
Method for detecting pollution of trace estrogen endocrine disrupting compounds in reclaimed water by biological-chemical combination Download PDFInfo
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Abstract
The invention belongs to the field of environmental detection, and particularly relates to a method for detecting pollution of trace estrogen endocrine disrupting compounds in reclaimed water by a biological-chemical combination method; the invention is based on biological detection to analyze the combined effect of a plurality of trace endocrine disrupting compounds in the reclaimed water, develops a detection technology based on a biological living body model, and directly evaluates the health hazard degree of the endocrine disrupting compound pollution in the reclaimed water from the comprehensive biological and toxicological activities of the reclaimed water. Can be effectively applied to the pollution detection of the estrogen endocrine disrupting compounds in the reclaimed water environment, and provides a monitoring and early warning technology for reclaimed water recycling safety evaluation.
Description
Technical Field
The invention belongs to the field of environmental detection, and particularly relates to a method for detecting pollution of trace estrogen endocrine disrupting compounds in reclaimed water by a biological-chemical combination method.
Background
At present, researches on water bodies polluted by trace or trace organic pollutants, such as surface water, effluent of sewage treatment plants or reclaimed water, on estrogen endocrine disrupting compounds are mostly focused on qualitative and quantitative analysis of specific endocrine disrupting compounds by adopting a chemical analysis means. The concentration of endocrine disruptors in the reclaimed water is low, the accuracy of the conventional analysis method is not high, and an analysis instrument is expensive and has very high cost, so that the method is difficult to popularize and popularize. The regenerated water sample is different from a common sample, and has the characteristics of various compounds, low content, complex sample composition, flowability, instability and the like. When the concentration of the pollutant to be measured is lower than the detection limit of the existing method or the sample is complex and the organism interference is serious, the direct measurement is impossible. The biological method is a very promising method for judging whether the compound is an endocrine disruptor and the endocrine disrupting activity of the compound, and the sensitivity and the accuracy of the mass spectrometry are high in the aspect of quantitative analysis of the endocrine disruptor. The zebra fish has a series of characteristics of short reproduction period, high egg laying amount and transparent eggs, so that the zebra fish is widely applied to the field of environmental toxicology. The gene similarity of the zebra fish reaches 87% with the human gene, and the zebra fish has similar physiological characteristics with the human, for example, the zebra fish endocrine system is similar with the human. Thus, zebrafish have been widely used in testing or screening for endocrine disruptors for the last decade.
The interaction of various environmental endocrine disrupting compounds is less studied. The influence of a single environmental endocrine disrupting compound on organisms is very weak, but the synergistic effect of the effect is generated after the compound is combined with other environmental endocrine disrupting compounds, and the synergistic effect has very important significance. Environmental endocrine disrupting compounds are often present in a mixed state in the environment, and the determination of the activity of a single environmental endocrine disrupting compound is difficult to reflect the actual activity of an endocrine disrupting compound in the environment.
The prior patent document with publication number CN102660644B discloses a method for regulating mRNA level of vitellogenin of zebra fish, which is used for extracting total RNA of liver of aquatic organism to be detected and detecting the mRNA expression level of VTG1 and/or VTG3 of the vitellogenin and can be applied to the field of detection of estrogen-like interferents of the aquatic organism.
In the literature, the study on the characteristics of vitellogenin mRNA of zebra fish (Danio rerio) and Odontobutis potamophila (Odontobutis potamophila) and the application of the vitellogenin mRNA in environmental monitoring are carried out, the induction of the mRNA expression of male fish vitellogenin by environmental estrogen is detected by using a semi-quantitative R T-PC R method, and the VTG expression gene of experimental fish can be used as a molecular model to be applied to environmental estrogen detection.
The prior art at present basically uses an estrogenic compound such as PFOS or E2 to stimulate zebra fish or other fish bodies, does not consider that pollutants in the environment do not exist independently, but exist together with other compounds, and the combined existence of various compounds has toxicity enhancing or antagonizing effects, namely E2 does exist in water environment, but the antagonism of other compounds does not cause VTG induction, so that no estrogenic compound can not be inferred according to the absence of VTG induction, and the practical application has limitation.
Disclosure of Invention
The invention provides a method for detecting the pollution of trace estrogen endocrine disrupting compounds in the reclaimed water by combining biology and chemistry to solve the problems.
The method is realized by the following technical scheme:
a method for detecting trace estrogen endocrine disrupting compound pollution in reclaimed water by biological-chemical combination comprises the following steps:
1. chemical analysis: carrying out pollution detection on the estrogen endocrine disrupting compounds in the reclaimed water environment by adopting a GC-MS method, and carrying out pretreatment on a water sample before detection;
further, the pretreatment includes phthalate compounds analysis water sample pretreatment and sterol and phenolic compounds analysis water sample pretreatment.
Further, the pretreatment of the phthalate ester compound for analyzing the water sample comprises the following specific steps: adding sodium chloride into a water sample: sodium chloride 50 ml: 1g, shaking uniformly, adding ethyl acetate, and adding a water sample: ethyl acetate 25 ml: 1ml, repeatedly extracting for 3-4 times, combining the extract, blowing nitrogen to constant volume and concentrating 500 times.
Further, the sterol and phenolic compounds are subjected to pretreatment of the analyzed water sample, and the specific method comprises the following steps: adding bisphenol A-d16 into a water sample, wherein the adding amount is 200ng.L-1, and uniformly mixing.
Further, the GC-MS method comprises the following specific analysis method: before loading, sequentially activating an HLB (hydrophile-lipophile balance) small column by using methanol and ultrapure water, loading a water sample at the flow rate of 5mL/min, eluting the small column by using 10-20mL of methanol, blowing nitrogen to a constant volume of 1mL, adding 30 mu L of heptafluorobutyric anhydride, deriving for 30min at 30 ℃, blowing nitrogen to be dry, adding 1mL of n-hexane, concentrating by 500 times, and then performing chromatographic analysis, wherein the chromatographic column adopts Rtx-5ms (30 multiplied by 0.25).
Further, the chromatographic analysis conditions are as follows: temperature rising procedure: the initial temperature is 50 ℃, and the temperature is kept for 1 min; raising the temperature to 200 ℃ at a speed of 10 ℃/min, and keeping the temperature for 1 min; raising the temperature to 280 ℃ at the speed of 10 ℃/min, and keeping the temperature for 2 min; the temperature was increased to 310 ℃ at a rate of 30 ℃/min and maintained for 5 min.
2. Biological analysis: the method comprises the steps of detecting the expression of sex mature zebra fish vitellogenin (VTG1) by a real-time quantitative PCR method, establishing a VTG1 transcription level detection system by taking beta-actin as an internal reference gene, and analyzing the pollution degree of a regenerated water estrogen endocrine disrupting compound by an exposure experiment.
Further, the biological analysis method specifically comprises the following steps:
(1) sample collection and pretreatment
Clarifying the collected reclaimed water sample overnight to obtain clarified reclaimed water, and discarding the solid sediment at the bottom;
(2) exposure experiments
Healthy male zebra fish of 3-4 months old is selected as an exposure object, and a fully aerated dechlorinated tap water sample is used as negative control water. Three replicates were set up, each containing 5 randomly harvested males. Samples were collected and exposed to the control for 14 days with 2 penaeus vannamei daily changes of water during the exposure period. After exposing the mature male fish, killing the fish, dissecting to obtain liver tissues, and rapidly storing in a refrigerator at the temperature of-80 ℃;
(3) fish sample processing
Extracting RNA by using an RNA extraction kit, carrying out reverse transcription on the RNA by using a reverse transcription kit to obtain cDNA, and carrying out fluorescent quantitative PCR by using a fluorescent quantitative PCR kit to detect the expression level of Vtg1 of adult male zebra fish vitellogenin so as to indicate the pollution of environmental estrogen endocrine disrupting compounds; the fluorescent quantitative PCR reaction system
qPCR Master Mix 10. mu. L, cDNA template 1. mu. L, Nuclease-free Water 7. mu.L and upstream and downstream primers 1. mu.L each; the reaction is carried out in a CFX96TM Real-Time-System Real-Time quantitative PCR instrument;
the Vtg1 primer sequence is: 5'-CCTTGGAGAAAATTGAGGCTATC-3' is used as a reference material; 5'-CTGAATGAACTCGGGAGTGGTA-3' is the ratio of R to R;
the sequence of the beta-actin primer is as follows: 5'-TCTGGCATCACACCTTCTACAAT-3' is used as a reference material; 5'-TGTTGGCTTTGGGATTCAGG-3' is added.
Further, the PCR temperature program is pre-denaturation: 3min at 95 ℃; denaturation: 95 ℃ for 30s, annealing: 60 ℃ 30s, extension: 30s at 72 ℃ for 40 cycles; and (3) melting curve analysis: 60 to 95 ℃.
The amounts of phenolic and steroidal estrogenic compounds sampled and the corresponding peak areas are as follows: the average peak area was 449.00 at a concentration of 10.000. mu.g/l, 1774.00 at a concentration of 20.000. mu.g/l, 6003.00 at a concentration of 50.000. mu.g/l, 13340.00 at a concentration of 100.000. mu.g/l and 27222.00 at a concentration of 200.000. mu.g/l.
The average peak area was 6829.00 at a nonyl phenol concentration of 40.000. mu.g/l, 13969.00 at a 80.000. mu.g/l, 35411.00 at a 200.000. mu.g/l, 72141.00 at a 400.000. mu.g/l and 152344.00 at a 800.000. mu.g/l.
The average peak area was 3676.00 at a concentration of 10.000. mu.g/l of BPA-D16, 7830.00 at a concentration of 20.000. mu.g/l, 19582.00 at a concentration of 50.000. mu.g/l, 40344.00 at a concentration of 100.000. mu.g/l and 80288.00 at a concentration of 200.000. mu.g/l.
The average peak area was 3299.00 at a concentration of 10.000. mu.g/l of bisphenol A, 7205.00 at a concentration of 20.000. mu.g/l, 18146.00 at a concentration of 50.000. mu.g/l, 36956.00 at a concentration of 100.000. mu.g/l and 74727.00 at a concentration of 200.000. mu.g/l.
17 α -ethinyl estradiol (EE2) had a mean peak area of 556.00 at a concentration of 10.000 μ g/l, of 1095.00 at a concentration of 20.000 μ g/l, of 3015.00 at a concentration of 50.000 μ g/l, of 6618.00 at a concentration of 100.000 μ g/l and of 13572.00 at a concentration of 200.000 μ g/l.
The average peak area was 2160.00 at a concentration of 10.000. mu.g/l, 5836.00 at a concentration of 20.000. mu.g/l, 15903.00 at a concentration of 50.000. mu.g/l, 32926.00 at a concentration of 100.000. mu.g/l and 66794.00 at a concentration of 200.000. mu.g/l.
The standard curves for phenolic and steroidal estrogenic compounds are shown in figure 1.
The sampling amount of the phthalate ester hormone compound and the corresponding peak area are as follows: the average peak area was 25373.00 at a concentration of 20.000. mu.g/l DMP, 52592.00 at a concentration of 40.000. mu.g/l, 102341.00 at a concentration of 80.000. mu.g/l, 210819.00 at a concentration of 160.000. mu.g/l, 407063.00 at a concentration of 300.000. mu.g/l and 650512.00 at a concentration of 500.000. mu.g/l.
The average peak area was 23215.00 at a DEP concentration of 20.000. mu.g/l, 47255.00 at a 40.000. mu.g/l, 93138.00 at a 80.000. mu.g/l, 192085.00 at a 160.000. mu.g/l, 373204.00 at a 300.000. mu.g/l and 602775.00 at a 500.000. mu.g/l.
The average peak area was 43356.00 at a concentration of 20.000. mu.g/l of DBP, 73472.00 at a concentration of 40.000. mu.g/l, 142162.00 at a concentration of 80.000. mu.g/l, 297239.00 at a concentration of 160.000. mu.g/l, 598069.00 at a concentration of 300.000. mu.g/l and 1001280.00 at a concentration of 500.000. mu.g/l.
The average peak area was 10840.00 at a concentration of 20.000. mu.g/l of BBP, 22625.00 at a concentration of 40.000. mu.g/l, 46391.00 at a concentration of 80.000. mu.g/l, 105054.00 at a concentration of 160.000. mu.g/l, 223900.00 at a concentration of 300.000. mu.g/l and 374175.00 at a concentration of 500.000. mu.g/l.
The average peak area was 28258.00 at a concentration of DEHP of 20.000. mu.g/l, 50765.00 at a concentration of 40.000. mu.g/l, 99003.00 at a concentration of 80.000. mu.g/l, 220082.00 at a concentration of 160.000. mu.g/l, 459435.00 at a concentration of 300.000. mu.g/l and 776241.00 at a concentration of 500.000. mu.g/l.
The average peak area was 21485.00 at a DNOP concentration of 20.000. mu.g/l, 47786.00 at a DNOP concentration of 40.000. mu.g/l, 109116.00 at a 80.000. mu.g/l, 259787.00 at a 160.000. mu.g/l, 558528.00 at a 300.000. mu.g/l and 945860.00 at a 500.000. mu.g/l.
The standard curve of the phthalate hormone compound is shown in FIG. 2.
In conclusion, the beneficial effects of the invention are as follows: the invention is based on biological detection to analyze the combined effect of a plurality of trace endocrine disrupting compounds in the reclaimed water, develops a detection technology based on a biological living body model, and directly evaluates the health hazard degree of the endocrine disrupting compound pollution in the reclaimed water from the comprehensive biological and toxicological activities of the reclaimed water. Can be effectively applied to the pollution detection of the estrogen endocrine disrupting compounds in the reclaimed water environment, and provides a monitoring and early warning technology for reclaimed water recycling safety evaluation.
The actual environmental pollutants are not present independently, but are present in combination with other compounds, each compound having an enhanced or antagonistic toxic effect. When the detection object is some clean water bodies with lower pollutant concentration, such as regenerated water, source water and the like, the pollutant types in the water bodies are low in concentration, and combined effect effects of antagonism, enhancement, addition and the like exist, so that the estrogen compound is possibly detected by GCMS, but the induction of VTG cannot be caused due to the antagonism, or the induction of VTG1 is possibly caused due to the undetected GC-MS but the synergistic enhancement effect exists in low-dose estrogen endocrine disruptors, so that the problem exists in the simple vitellogenin induction effect or the direct GCMS measurement.
The fish vitellogenin is specifically generated in the liver of a mature female animal, under normal conditions, the expression of the fish vitellogenin in male fishes or juvenile fishes is difficult to detect, but under the exposure of estradiol and environmental estrogen, the fish eggs, the juvenile fishes and males also express the vitellogenin in a large amount. Thus, induction of vitellogenin in roe, juvenile fish and male fish may be indicative of estrogen contamination in the environment. Mature male zebra fish is selected as a living model, and a living environment estrogen detection technology aiming at vitellogenin is established. Compared with a chemical method, the biological detection can avoid component detection and directly analyze the harm degree of environmental estrogen to health. However, at present, the detection of trace estrogen endocrine disrupting compounds in the regenerated water by using small-scale model water body organisms is rarely studied and applied. Therefore, it is necessary to develop a detection technology based on a biological living body model to directly evaluate the health hazard degree of endocrine disrupting compound pollution in the reclaimed water from the comprehensive biological and toxicological activities of the reclaimed water. The method provided by the invention combines the use of an estrogen endocrine disrupting compound chemical analysis method GC-MS to analyze the trace or trace organic pollutant polluted water body, can directly evaluate the health hazard degree of endocrine disrupting compound pollution in the reclaimed water, provides a monitoring and early warning technology for reclaimed water reuse safety evaluation, and is successfully applied to trace environment estrogen endocrine disrupting compound pollution detection in the reclaimed water of Guiyang sewage treatment plants.
Drawings
FIG. 1 is a standard graph of phenolic and steroidal estrogenic compounds; wherein a is 4-tert-Octylphenol (OP), b is nonylphenol, c is bisphenol A-D16(BPA-D16), D is bisphenol A, e is 17 alpha-ethinyl estradiol (EE2), and f is 17 beta-estradiol.
FIG. 2 is a standard curve of phthalate hormone compounds; wherein a is dimethyl phthalate (DMP), b is diethyl phthalate (DEP), c is di-n-butyl phthalate (DBP), d is Butyl Benzyl Phthalate (BBP), e is di-2-ethyl-hexyl phthalate (DEHP), and f is di-n-octyl phthalate (DNOP).
FIG. 3 is a mass spectrum of an estrogen endocrine disrupting compound; wherein, (1) is phenolic and steroidal estrogenic compounds; (2) is a phthalate ester hormone compound.
FIG. 4 shows the effect of regeneration water treatment on the transcriptional water of VTG1 gene of male zebra fish liver.
Detailed Description
The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
Example 1
A method for detecting trace estrogen endocrine disrupting compound pollution in reclaimed water by biological-chemical combination comprises the following steps:
1. reagents used for the experiment:
17 β -Estradiol (17 α -Estradiol, E2), Ethinylestradiol (17 α -Ethinylestradiol, EE2), octylphenol (Octyl phenol, OP), Nonylphenol (Nonylphenol, NP), Bisphenol a (Bisphenol a, BPA) and Phthalates (Phthalates, PAEs), including Dimethyl Phthalate (DMP), Diethyl Phthalate (DEP), Di-n-butyl Phthalate (DBP), Benzyl Phthalate (Benzyl Phthalate, BBP), Di-n-Octyl Phthalate (Di-n-Octyl Phthalate, OP) and Di-2-ethylhexyl Phthalate (Di (2-ethylhexyl) Phthalate, DEHP).
The solvent used was: methanol (shanghai an spectrum, chromatographically pure), ethyl acetate (shanghai an spectrum, chromatographically pure) and acetone (shanghai an spectrum, chromatographically pure). Derivatization reagents heptafluorobutyric anhydride (MTBSTFA), E2, EE2, OP, NP, BPA) and BPA-d16 were purchased from Supelco, and DMP, DEP, DBP, BBP, DNOP and DEHP were purchased from Sigma-Aldrich.
Preparing a standard solution: accurately weighing the solid alcohol and the phenolic compound standard, preparing 10 mg/mL standard storage solution by using methanol, respectively measuring the phthalate compound standard, diluting the phthalate compound standard with ethyl acetate to 100mg/mL standard storage solution, and storing all the standard solutions at 4 ℃ before use.
The standard curves for phenolic and steroidal estrogenic compounds are shown in FIG. 1; the standard curve of the phthalate hormone compound is shown in FIG. 2.
2. Chemical analysis: and respectively arranging sampling points S1 and S2 on the second-level water sample and the third-level water sample of the Guiyang regenerated water plant, wherein 1L of water samples are respectively sampled, and the water samples are stored at 4 ℃. Carrying out pollution detection on the estrogen endocrine disrupting compounds in the reclaimed water environment by adopting a GC-MS method, and carrying out pretreatment on a water sample before detection;
further, the pretreatment includes phthalate compounds analysis water sample pretreatment and sterol and phenolic compounds analysis water sample pretreatment.
Further, the pretreatment of the phthalate ester compound for analyzing the water sample comprises the following specific steps:
adding 10g of sodium chloride into 500mL of water sample, shaking up, adding 20mL of ethyl acetate, shaking for 10min, standing for 10min, repeatedly extracting for 3 times, combining extract liquor, blowing nitrogen to fix the volume to 1mL, concentrating a target analyte by 500 times, and performing on-machine analysis.
Further, the sterol and phenolic compounds are subjected to pretreatment of the analyzed water sample, and the specific method comprises the following steps:
500mL of water sample was taken and internal standard (bisphenol A-d16, 200ng. L) was added-1) And (5) uniformly mixing.
Further, the GC-MS method comprises the following specific analysis method:
before loading, sequentially using methanol and ultrapure water to activate an HLB (hydrophile company, 500mg/6mL) small column, loading a water sample at the flow rate of 5mL/min, eluting the small column by using 10-20mL of methanol, blowing nitrogen to a constant volume of 1mL, adding 30 mu L of heptafluorobutyric anhydride, deriving for 30min at the temperature of 30 ℃, blowing nitrogen to be dry, adding 1mL of n-hexane, concentrating a target analyte by 500 times, and loading on a machine for analysis.
The column was run on Rtx-5MS (30X 0.25) by directly injecting 1. mu.L of the extract into GC-MS2020NX (Shimadzu).
Temperature rising procedure: maintaining the initial temperature at 50 deg.C for 1 min; raising the temperature to 200 ℃ at the speed of 10 ℃/min, and keeping the temperature for 1 min; raising the temperature to 280 ℃ at the speed of 10 ℃/min, and keeping the temperature for 2 min; the temperature was increased to 310 ℃ at a rate of 30 ℃/min and maintained for 5 min.
Other conditions are as follows: temperature of the gasification chamber: 250 ℃; carrier gas: he gas; carrier gas flow: 1.0 mL/min; no flow split.
Mass spectrum conditions: an EI source; electron energy: 70 eV; ion source temperature: 240 ℃; scanning mode: and a SIM.
The target compound has a limit of detection (LOD) of 12.24, 25.98, 29.04, 2.64, 20.82, 2.7ng.L for DMP, DBP, DEP, BBP, DEHP, and DNOP-1LOD of OP, NP, BPA, EE2 and E2 were 5.66, 6.97, 8.27, 9.55, 12.35ng.L, respectively-1. The average recovery rate of the phthalate ester compounds is between 70 and 110 percent. The recovery rate of the samples is detected by adding the solid alcohol and the phenolic compounds into the BPA-d16, and the standard recovery rate of the samples is between 75 and 100 percent. The mass spectrometry conditions of the estrogen endocrine disrupting compounds are shown in table 1, and the mass spectrogram of the estrogen endocrine disrupting compounds is shown in fig. 3.
TABLE 1 Mass Spectrometry conditions for Estrogen endocrine disrupting Compounds
3. Biological analysis: the method comprises the steps of detecting the expression of sex mature zebra fish vitellogenin (VTG1) by a real-time quantitative PCR method, establishing a VTG1 transcription level detection system by taking beta-actin as an internal reference gene, and analyzing the pollution degree of a regenerated water estrogen endocrine disrupting compound by an exposure experiment. The method specifically comprises the following steps:
(1) sample collection and pretreatment
Clarifying the collected reclaimed water sample overnight to obtain clarified reclaimed water, and discarding the solid sediment at the bottom;
(2) exposure experiments
Healthy male zebra fish of 3-4 months old is selected as an exposure object, and a fully aerated dechlorinated tap water sample is used as negative control water. Three replicates were set up, each containing 5 randomly harvested males. Samples were collected and exposed to the control for 14 days with 2 penaeus vannamei daily changes of water during the exposure period. After exposing the mature male fish, killing the fish, dissecting to obtain liver tissues, and rapidly storing in a refrigerator at the temperature of-80 ℃;
(3) fish sample processing
Extracting RNA by using an RNA extraction kit (Chinese treasure bioengineering Co., Ltd.), carrying out reverse transcription on the RNA by using a reverse transcription kit (Beijing all-type gold biotechnology Co., Ltd.) to obtain cDNA, and carrying out fluorescence quantitative PCR (polymerase chain reaction) by using a fluorescence quantitative PCR kit (Beijing all-type gold biotechnology Co., Ltd.) to detect the expression level of Vtg1 of adult male zebra fish vitellogenin so as to indicate the pollution of an environmental estrogen endocrine disrupting compound; the fluorescent quantitative PCR reaction system
qPCR Master Mix 10. mu. L, cDNA template 1. mu. L, Nuclease-free Water 7. mu.L and upstream and downstream primers 1. mu.L each; the reaction is carried out in a CFX96TM Real-Time-System Real-Time quantitative PCR instrument;
the Vtg1 primer sequence is: 5'-CCTTGGAGAAAATTGAGGCTATC-3' is used as a reference material; 5'-CTGAATGAACTCGGGAGTGGTA-3' is the ratio of R to R;
the sequence of the beta-actin primer is as follows: 5'-TCTGGCATCACACCTTCTACAAT-3' is used as a reference material; 5'-TGTTGGCTTTGGGATTCAGG-3' is added.
The reaction was carried out in a CFX96TM Real-Time-System Real-Time quantitative PCR instrument (BIO-RAD, USA) with a PCR temperature program of pre-denaturation: 3min at 95 ℃; denaturation: 95 ℃ for 30s, annealing: 60 ℃ 30s, extension: 30s at 72 ℃ for 40 cycles; and (3) melting curve analysis: 60 to 95 ℃. Beta-actin is used as an internal reference gene
The method analyzes the relative expression level of Vtg 1. Collecting a water sample, taking fully aerated dechlorinated tap water as a negative control group, respectively carrying out fully aerated dechlorinated tap water and regenerated water exposure on adult male zebra fish, carrying out three parallel exposures for 14 days, extracting liver RNA of the adult male fish, detecting the expression quantity of VTG1, and analyzing the pollution degree of the regenerated water estrogen endocrine disrupting compounds compared with the negative control group.
And calculating the expression level of the gene exposed to the sample relative to the negative control group by taking the gene expression level of the negative control group as a reference value. The positive control group is used for confirming the normal operation of the system and providing qualitative cognition for the pollution degree of the estrogen endocrine disrupting compounds in the reclaimed water environment of the sample. The determination results of the estrogen compounds in the regenerated water are shown in table 2; the effect of the regeneration water treatment on the transcriptional water of the VTG1 gene of the male zebra fish liver is shown in FIG. 4, and the results are expressed as the mean value. + -. standard deviation (n-3) (. SP). ltoreq.0.05,. SP. ltoreq.0.01, which indicates that the experimental group is significantly different from the control group.
TABLE 2 determination of estrogenic Compounds in the regenerated Water
Note that nd means below the detection limit.
Claims (8)
1. A method for detecting trace estrogen endocrine disrupting compound pollution in reclaimed water by biological-chemical combination is characterized by comprising the following steps:
(1) chemical analysis: carrying out pollution detection on the estrogen endocrine disrupting compounds in the reclaimed water environment by adopting a GC-MS method, and carrying out pretreatment on a water sample before detection;
(2) biological analysis: the method comprises the steps of detecting the expression of sex mature zebra fish vitellogenin (VTG1) by a real-time quantitative PCR method, establishing a VTG1 transcription level detection system by taking beta-actin as an internal reference gene, and analyzing the pollution degree of a regenerated water estrogen endocrine disrupting compound by an exposure experiment.
2. The method for bio-chemical joint detection of trace estrogen endocrine disrupting compound contamination in reclaimed water as claimed in claim 1, wherein the pre-treatment comprises pre-treatment of phthalate compound analysis water sample and pre-treatment of sterol and phenolic compound analysis water sample.
3. The method for detecting the pollution of the trace estrogen endocrine disrupting compounds in the reclaimed water by the combination of biology and chemistry as claimed in claim 2, wherein the method for analyzing the pretreatment of the water sample by the phthalate ester compounds comprises the following specific steps: adding sodium chloride into a water sample: sodium chloride 50 ml: 1g, shaking uniformly, adding ethyl acetate, and adding a water sample: ethyl acetate 25 ml: 1ml, repeatedly extracting for 3-4 times, combining the extract, blowing nitrogen to constant volume and concentrating 500 times.
4. The method for detecting the pollution of the trace estrogen endocrine disrupting compounds in the reclaimed water by the combination of biology and chemistry as claimed in claim 2, wherein the sterol and phenolic compounds are analyzed in the pretreatment of the water sample, and the specific method is as follows: adding bisphenol A-d16 into water sample in an amount of 200ng-1And (4) uniformly mixing.
5. The method for detecting the pollution of the trace estrogen endocrine disrupting compounds in the reclaimed water by the combination of biology and chemistry as claimed in claim 1, wherein the GC-MS method comprises the following specific analysis methods: before loading, sequentially activating an HLB (hydrophile-lipophile balance) small column by using methanol and ultrapure water, loading a water sample at the flow rate of 5mL/min, eluting the small column by using 10-20mL of methanol, blowing nitrogen to a constant volume of 1mL, adding 30 mu L of heptafluorobutyric anhydride, deriving for 30min at 30 ℃, blowing nitrogen to be dry, adding 1mL of n-hexane, concentrating by 500 times, and then performing chromatographic analysis, wherein the chromatographic column adopts Rtx-5ms (30 multiplied by 0.25).
6. The method of claim 5, wherein the chromatographic analysis is performed under the following conditions: temperature rising procedure: the initial temperature is 50 ℃, and the temperature is kept for 1 min; raising the temperature to 200 ℃ at a speed of 10 ℃/min, and keeping the temperature for 1 min; raising the temperature to 280 ℃ at the speed of 10 ℃/min, and keeping the temperature for 2 min; the temperature was increased to 310 ℃ at a rate of 30 ℃/min and maintained for 5 min.
7. The method for bio-chemical combination detection of trace estrogen endocrine disrupting compound contamination in reclaimed water as claimed in claim 1, wherein the bioanalytical method specifically comprises the following steps:
(1) sample collection and pretreatment
Clarifying the collected reclaimed water sample overnight to obtain clarified reclaimed water, and discarding the solid sediment at the bottom;
(2) exposure experiments
Selecting healthy male zebra fish with the age of 3-4 months as an exposed object, and fully aerating a dechlorinated tap water sample as negative control water; setting three parallel samples, wherein each parallel sample comprises 5 randomly obtained male fishes; collecting samples and controls, exposing at the same time for 14 days, feeding brine shrimp 2 times a day during the exposure period, and changing water every day; after exposing the mature male fish, killing the fish, dissecting to obtain liver tissues, and rapidly storing in a refrigerator at the temperature of-80 ℃;
(3) fish sample processing
Extracting RNA by using an RNA extraction kit, carrying out reverse transcription on the RNA by using a reverse transcription kit to obtain cDNA, and carrying out fluorescent quantitative PCR by using a fluorescent quantitative PCR kit to detect the expression level of Vtg1 of adult male zebra fish vitellogenin so as to indicate the pollution of environmental estrogen endocrine disrupting compounds;the fluorescent quantitative PCR reaction system
qPCR Master Mix 10. mu. L, cDNA template 1. mu. L, Nuclease-free Water 7. mu.L and upstream and downstream primers 1. mu.L each; the reaction is carried out in a CFX96TM Real-Time-System Real-Time quantitative PCR instrument;
the Vtg1 primer sequence is: 5'-CCTTGGAGAAAATTGAGGCTATC-3' is used as a reference material; 5'-CTGAATGAACTCGGGAGTGGTA-3' is the ratio of R to R;
the sequence of the beta-actin primer is as follows: 5'-TCTGGCATCACACCTTCTACAAT-3' is used as a reference material; 5'-TGTTGGCTTTGGGATTCAGG-3' is added.
8. The method for bio-chemical combination detection of trace estrogen endocrine disrupting compound contamination in reclaimed water according to claim 7, wherein the PCR temperature program is pre-denaturation: 3min at 95 ℃; denaturation: 95 ℃ for 30s, annealing: 60 ℃ 30s, extension: 30s at 72 ℃ for 40 cycles; and (3) melting curve analysis: 60 to 95 ℃.
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