Refining wastewater biotoxicity detection method based on dual levels of zebra fish embryos

Abstract

The invention discloses a method for detecting biotoxicity of refinery wastewater based on dual levels of zebra fish embryos. The method comprises the following steps: S1, analyzing a death rate; s2, oxidative stress level analysis: respectively determining the ROS content and the SOD content of the zebrafish embryos by adopting an ROS kit and an SOD kit so as to determine the oxidative stress level of the zebrafish embryos; and S3, carrying out gene transcriptomics analysis. The invention provides a detection method for comprehensively and effectively determining the biotoxicity of the refinery wastewater by taking omics analysis as a technical means and combining determination of death rate of zebra fish embryos, morphological deformity occurrence condition and oxidative stress level, and a most scientific and reasonable detection result is finally obtained layer by layer progressively. The experimental process has the characteristics of short time period, intuitive and comprehensive experimental result and the like, and can truly reflect the biotoxicity of the refinery wastewater.

Description

Technical Field

[0001] This invention relates to the field of biotoxicity testing technology for petroleum refining wastewater, specifically to a method for detecting biotoxicity of refining wastewater based on a dual-level zebrafish embryo method. Background Technology

[0002] According to information released by the Ministry of Industry and Information Technology of the People's Republic of China, the water consumption of the refining and chemical industry in 2023 was approximately 8.31 billion cubic meters. 3 This accounts for approximately 8.6% of industrial water consumption. Refining wastewater is complex in quality and highly toxic, with significant differences in toxicity between different treatment processes and stages. Therefore, there is an urgent need to establish an efficient and feasible method to assess the toxicity of refining wastewater at each stage, in order to select appropriate processes for its treatment and achieve efficient reuse.

[0003] Currently, the main methods for assessing the toxicity of refining wastewater in China include biological and chemical testing methods. Among biological testing methods, the most commonly used is the luminescent bacteria method. Its advantage lies in the fact that changes in bacterial luminescence precede changes in metabolism, making it more sensitive to toxicity and able to quickly reflect the presence of toxic substances in the water sample. Furthermore, its luminescence intensity is significantly negatively correlated with the total concentration of toxic components in the water sample; the acute toxicity level of the water sample can be assessed by measuring the luminescence intensity. However, certain pollutants (including endocrine disruptors, polycyclic aromatic hydrocarbons, heavy metals, etc.) may have an excitatory effect on luminescent bacteria at low concentrations, i.e., promoting luminescence intensity at low concentrations but exhibiting an inhibitory effect at high concentrations. This phenomenon may lead to inaccurate toxicity assessment results and underestimate the actual toxicity of pollutants. The core of chemical analysis is to quantitatively detect the concentration of pollutants, rather than directly measuring their actual toxic effects on organisms. When the actual toxicity of certain substances is not completely positively correlated with their concentration due to their different forms of existence, it may lead to misjudgments of "low concentration but high toxicity". Environmental samples often contain multiple pollutants, and chemical analysis can only measure the content of each substance individually. It cannot reflect the direct synergistic or antagonistic effects of pollutants, and therefore it is difficult to assess the overall toxicity of the sample.

[0004] Among existing water quality characterization technologies for refining wastewater, Chinese patent CN117849294A discloses a method for characterizing refining wastewater based on biotoxicity indicators. This method not only tests the physicochemical indicators of the wastewater, but also uses luminescent bacteria and the SOS / umu method based on Salmonella Typhimurium strain TA1535 to detect acute toxicity and genotoxicity indicators. It analyzes the correlation between sensitive toxic environmental factors and the microbial community structure in the water sample, and can identify core toxic environmental factors and their impact on the microbial community in the water sample to more comprehensively assess the toxicity of the wastewater. However, the luminescent bacteria used in this method are easily affected by some pollutants in refining wastewater, and may produce an excitatory effect at low concentrations to enhance luminescence intensity, leading to inaccurate results. Chinese patent CN119001044A discloses a rapid determination method for comprehensive toxicity of wastewater. This method uses traditional toxicity indicators as target variables and zebrafish behavioral toxicity indicators as features to establish a comprehensive toxicity prediction model for wastewater based on different target variables, which is suitable for rapid detection of comprehensive toxicity of large batches of actual wastewater samples. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for detecting the biotoxicity of refining wastewater based on a dual-level approach using zebrafish embryos.

[0006] The technical solution of this invention is: A method for detecting the biotoxicity of refining wastewater based on a dual-level approach using zebrafish embryos includes the following steps: S1. Mortality analysis: Zebrafish fertilized eggs were placed in the water sample of the refining wastewater to be tested for exposure culture. After 120 hpf, zebrafish embryos were obtained. The mortality rate and morphological malformation rate of the zebrafish embryos were determined. During the culture period, a portion of the zebrafish embryos cultured to 72 hpf and 96 hpf were retained for later use. S2. Analysis of oxidative stress level: Zebrafish embryos cultured to 72 hpf and 96 hpf retained in S1 were taken, and the ROS and SOD contents of the zebrafish embryos were measured by ROS and SOD kits, respectively, to determine the oxidative stress level of the zebrafish embryos. S3. Transcriptomic analysis: Zebrafish embryos that were not exposed to the wastewater sample to be tested were used as a control group. Total RNA was extracted from the two types of zebrafish embryos and subjected to comparative transcriptomic analysis. RNA sequences were compared and gene expression levels were quantified. Genes with significant differences were screened out. By analyzing the differential expression of affected genes and signaling pathways, the biotoxicity of the wastewater from a gene perspective was determined. S4. The overall biotoxicity of the refining wastewater is determined by weighting the mortality rate and morphological malformation rate measured in S1, the ROS and SOD content measured in S2, and the biotoxicity of the refining wastewater from a genetic perspective determined in S3.

[0007] Further, in S1, the container used for the exposure culture is a 6-well plate. 2-6 mL of culture medium is added to each well of the 6-well plate. The culture medium is a mixture of embryo culture water and the refined wastewater sample to be tested at a volume ratio of 1:1. The pH of the embryo culture water is 6.8-7.5. The density of the exposure culture is 30-50 embryos / well, and several parallel groups are set up. The 6-well plate is placed in a light incubator, and the culture conditions are: temperature 28±1 ℃, light-dark ratio 14:10, and the culture medium is changed every 24 h.

[0008] Note: This method accurately determines the mortality and deformity rates of zebrafish exposed to refining wastewater.

[0009] Furthermore, in S1, zebrafish fertilized eggs are fertilized eggs produced by adult zebrafish within 1 hour after disinfection and in good condition.

[0010] Note: The accuracy of the test is ensured by screening out qualified fertilized eggs.

[0011] Further, in S2, the ROS content determination method was as follows: using the S0033 kit, zebrafish embryos cultured at 72 hpf were washed 2-3 times with PBS solution, each time soaking for 1-2 min. The washed embryos were then added to a 6-well plate containing 2 ml of reactive oxygen species (ROS) fluorescent probe working solution and incubated in a constant temperature incubator at 26-28℃ in the dark for 30-60 min. After that, the embryos were washed 3 times with PBS solution to remove free probes that had not entered the embryos. The washed embryos were then placed in a black 96-well plate, with 3-5 embryos and 100 μL of PBS solution added to each well. Each group was set up with 3 replicates. The fluorescence intensity was detected using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and the fluorescence intensity data were recorded.

[0012] Note: The results obtained by determining ROS content using the above method are relatively accurate.

[0013] Further, in S2, the SOD content determination method is as follows: using the S0109 kit, zebrafish embryos exposed to culture at 96 hpf were washed 2-3 times with PBS solution at 4℃, each time soaking for 1-2 min. The washed embryos were then added to PBS solution at 4℃ and centrifuged at 12000 rpm for 10-15 min. The supernatant was used as the test sample. The test sample was placed in a 96-well plate for detection. 20 μL of supernatant was taken from each well, and 20 μL of reaction starter solution and 160 μL of working solution were added. The plate was incubated at 37℃ for 20-30 min, and the absorbance value at 560 nm was read on a microplate reader.

[0014] Note: The results obtained by SOD determination using the above method are relatively accurate.

[0015] Furthermore, in S3, the method for extracting total RNA from the two types of zebrafish embryos was as follows: Zebrafish embryos were homogenized using a tissue homogenizer at room temperature (26-28℃). Total RNA was extracted using the Trizol method in a clean bench. The OD260 / OD280 ratio was first detected using a UV spectrophotometer, and the integrity of the 28S and 18S rRNA bands was checked by agarose gel electrophoresis. Finally, the RNA concentration was accurately determined using a Qubit quantitative PCR instrument. An mRNA-Seq library was constructed based on the Library Prep Kit for Illumina and analyzed using Illumina. The HiSeq™ platform generates raw short sequence fragments from RNA sequencing. After filtering with FASTP software, high-quality sequences are obtained from the raw data by removing sequences containing primer / adapter contamination, low-quality sequences, or sequences with more than 10% unidentified nucleotides. The obtained sequences are then compared and gene expression levels are quantified. The R package is used for analysis to screen genes with significantly different expression levels between the treatment group and the control group in the exposed culture, and functional enrichment analysis is performed to interpret the biological functions and pathways of the differentially expressed genes.

[0016] Note: The results of gene transcriptomics analysis using the above methods are relatively accurate.

[0017] Furthermore, in S1, the evaluation indicators for the biotoxicity of refining wastewater based on mortality rate are as follows: 0%~10% is considered low biotoxicity; 10%~20% is considered relatively low biotoxicity; 20%~30% is considered moderate biotoxicity; 30%~50% is considered relatively high biotoxicity; and above 50% is considered highly biotoxic. The evaluation indicators for the biological toxicity of refining wastewater based on the deformity rate are as follows: 0%~5% is considered low biotoxicity; 5%~10% is considered relatively low biotoxicity; 15%~20% is considered moderate biotoxicity; 25%~30% is considered relatively high biotoxicity; and above 30% is considered very high biotoxicity. If at least one of the evaluation indicators of mortality rate and deformity rate is high, then the biotoxicity of the tested refining wastewater sample is directly determined to be high without proceeding to S2~S3. If both the evaluation indicators of mortality rate and deformity rate are relatively high, then the biotoxicity of the tested refining wastewater sample is directly determined to be relatively high without proceeding to S2~S3. If at least one of the evaluation indicators of mortality rate and deformity rate is medium or below, then the biotoxicity is pending and S2~S3 should be continued.

[0018] Furthermore, in S2, the evaluation indicators for oxidative stress levels are divided as follows: ROS: 0~0.2 μmol / ml indicates low biotoxicity; 0.2~0.4 μmol / ml indicates relatively low biotoxicity; 0.4~0.6 μmol / ml indicates moderate biotoxicity; 0.6~0.8 μmol / ml indicates relatively high biotoxicity; and above 0.8 μmol / ml indicates very high biotoxicity. SOD: 0.00~0.05umol / ml is low biotoxicity; 0.05~0.10umol / ml is relatively low biotoxicity; 0.10~0.15umol / ml is moderate biotoxicity; 0.15~0.20umol / ml is relatively high biotoxicity; above 0.20umol / ml is highly biotoxic. If at least one of the evaluation indicators of ROS and SOD is high, then S3 is not required and the biotoxicity of the tested refining wastewater sample is directly determined to be high. If both the evaluation indicators of ROS and SOD are relatively high, then S3 is not required and the biotoxicity of the tested refining wastewater sample is directly determined to be high. If at least one of the evaluation indicators of mortality rate and deformity rate is medium or below, then the biotoxicity is pending and S3 is continued.

[0019] Preferably, in S3, the percentage of genes with significantly different expression levels between the treatment group and the control group in the exposed culture is X. If X < 2%, the biotoxicity is low; if 2% ≤ X < 5%, the biotoxicity is relatively low; if 5% ≤ X < 10%, the biotoxicity is moderate; if 10% ≤ X < 20%, the biotoxicity is relatively high; if X ≥ 20%, the biotoxicity is high. The evaluation results in S1 to S3 are summarized. Among them, low biotoxicity is scored as 0, low biotoxicity as 25, medium biotoxicity as 50, high biotoxicity as 75, and high biotoxicity as 100. The mortality rate score is a1, with a weight b1 of 0.22, and the deformity rate score is a2, with a weight b2 of 0.15. The score for ROS is a3, with a weight of b4 of 0.18; the score for SOD is a4, with a weight of b4 of 0.18. The score of the differentially expressed gene X is recorded as a5, and the weight b5 is 0.27; The final overall biotoxicity evaluation standard for the refining wastewater samples to be tested is as follows: L=a1*b1+a2*b2+a3*b3+a4*b4+a5*b5 If 0 ≤ L < 20, the biological toxicity of the refining wastewater sample is low; if 20 ≤ L < 40, the biological toxicity of the refining wastewater sample is relatively low; if 40 ≤ L < 60, the biological toxicity of the refining wastewater sample is moderate; if 60 ≤ L < 80, the biological toxicity of the refining wastewater sample is relatively high; and if 80 ≤ L < 100, the biological toxicity of the refining wastewater sample is very high.

[0020] The beneficial effects of this invention are: (1) The method for detecting the biotoxicity of refining wastewater based on the dual level of zebrafish embryos of the present invention uses omics analysis as a technical means, combined with the determination of zebrafish embryo mortality rate, morphological malformation occurrence and oxidative stress level, to provide a comprehensive and effective method for detecting the biotoxicity of refining wastewater. The method is progressive and ultimately obtains the most scientific and reasonable detection results. The experimental process has the characteristics of short time cycle, intuitive and comprehensive experimental results, and can truly reflect the biotoxicity of refining wastewater.

[0021] (2) In the selection of experimental test species, zebrafish embryos are used as experimental test objects in the dual-level biotoxicity detection method of refining wastewater based on zebrafish embryos of the present invention. Zebrafish embryos have the characteristics of being sensitive to pollutants, having a short development cycle and being transparent to individuals, which facilitates efficient dual-level assessment of the biotoxicity of refining wastewater. The dual level refers to the phenotypic level and the molecular level. The phenotypic level is the mortality rate and the incidence of malformations, and the molecular level is the oxidative stress level and the genetic perspective. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the mortality rate of zebrafish embryos caused by water samples from various stages of the chemical wastewater treatment unit in the experimental example. Figure 2 This is a schematic diagram illustrating the malformation rate of zebrafish embryos caused by water samples from various sections of the chemical wastewater treatment unit in the experimental example. Figure 3 This is a heat map showing the morphological deformities of zebrafish embryos caused by water samples from various stages of the chemical wastewater treatment unit in the experimental example. Figure 4 This is a schematic diagram illustrating the mortality rate of zebrafish embryos caused by water samples from various sections of the oil refinery wastewater treatment unit in the experimental example. Figure 5 This is a schematic diagram showing the malformation rate of zebrafish embryos caused by water samples from each section of the oil refinery wastewater treatment unit in the experimental example. Figure 6 This is a heat map showing the morphological deformities of zebrafish embryos caused by water samples from various sections of the oil refinery wastewater treatment unit in the experimental example. Figure 7This is a schematic diagram illustrating the mortality rate of zebrafish embryos caused by water samples from various sections of the recycled wastewater treatment unit in the experimental example. Figure 8 This is a schematic diagram illustrating the malformation rate of zebrafish embryos caused by water samples from each section of the recycled wastewater treatment unit in the experimental example. Figure 9 This is a heat map showing the morphological deformities of zebrafish embryos caused by water samples from various sections of the recycled wastewater treatment unit in the experimental example. Figure 10 This is a schematic diagram illustrating the mortality rate of zebrafish embryos caused by water samples from various sections of the concentrated wastewater treatment unit in the experimental example. Figure 11 This is a schematic diagram showing the malformation rate of zebrafish embryos caused by water samples from each section of the concentrated wastewater treatment unit in the experimental example. Figure 12 This is a heat map showing the morphological deformities of zebrafish embryos caused by water samples from various stages of the concentrated wastewater treatment unit in the experimental example. Detailed Implementation

[0023] Example 1 A method for detecting the biotoxicity of refining wastewater based on a dual-level approach using zebrafish embryos includes the following steps: S1. Mortality Analysis: Zebrafish fertilized eggs (those produced by adult zebrafish within 1 hour of sterilization and in good development) were collected and placed in the refined wastewater sample to be tested for exposure culture for 120 hpf. The exposure culture was carried out in 6-well plates, with 4 mL of culture medium added to each well. The culture medium was a 1:1 volume mixture of embryo culture water and the refined wastewater sample to be tested, with a pH of 7. The density of exposure culture was 40 zebrafish / well, and several parallel groups were set up. The 6-well plates were placed in a light incubator, and the culture conditions were: temperature 28 ℃, light-dark ratio 14:10. The culture medium was changed every 24 h to obtain zebrafish embryos. The mortality rate and morphological malformation rate of the zebrafish embryos were determined. During the culture period, a portion of the zebrafish embryos cultured to 72 hpf and 96 hpf were retained for later use. The criteria for a well-developed fertilized egg are as follows: intact chorion: the outer chorion of the fertilized egg should be smooth, without damage, depressions or deformation; uniform cytoplasm distribution: within 1 hour after fertilization, the cytoplasm should be evenly gathered at the animal pole (the future embryonic end) to form a distinct "blastodisc"; symmetrical cleavage: early cleavage (2-cell stage → 4-cell stage → 8-cell stage, etc.) should be regular and symmetrical, with clear cleavage furrows and no fragments or cytoplasmic leakage.

[0024] The evaluation indicators for the biotoxicity of refining wastewater based on mortality rate are as follows: 0%~10% is considered low biotoxicity; 10%~20% is considered relatively low biotoxicity; 20%~30% is considered moderate biotoxicity; 30%~50% is considered relatively high biotoxicity; and above 50% is considered highly biotoxic. The evaluation indicators for the biological toxicity of refining wastewater based on the deformity rate are as follows: 0%~5% is considered low biotoxicity; 5%~10% is considered relatively low biotoxicity; 15%~20% is considered moderate biotoxicity; 25%~30% is considered relatively high biotoxicity; and above 30% is considered very high biotoxicity. If at least one of the evaluation indicators of mortality rate and deformity rate is high, then there is no need to proceed to S2~S3 and the biotoxicity of the tested refining wastewater sample is directly determined to be high. If both the evaluation indicators of mortality rate and deformity rate are relatively high, then there is no need to proceed to S2~S3 and the biotoxicity of the tested refining wastewater sample is directly determined to be relatively high. If at least one of the evaluation indicators of mortality rate and deformity rate is medium or below, then the biotoxicity is pending and S2~S3 should be continued. S2. Analysis of oxidative stress level: Zebrafish embryos cultured to 72 hpf and 96 hpf retained in S1 were taken, and the ROS and SOD contents of the zebrafish embryos were measured by ROS and SOD kits, respectively, to determine the oxidative stress level of the zebrafish embryos. The method for determining ROS content was as follows: Using the Beyotime S0033 kit, zebrafish embryos cultured at 72 hpf were washed twice with PBS solution for 2 min each time. The washed embryos were then added to a 6-well plate containing 2 ml of the reactive oxygen species (ROS) fluorescent probe DCFH-DA working solution and incubated in a 27°C incubator for 40 min in the dark. After that, the embryos were washed three times with PBS solution to remove free probes that had not entered the embryos. The washed embryos were then placed in a black 96-well plate with 4 embryos and 100 μL of PBS solution in each well. Each group was set up with 3 replicates. The fluorescence intensity was detected using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and the fluorescence intensity data were recorded. The method for determining SOD content is as follows: Using the Beyotime S0109 kit, zebrafish embryos cultured at 96 hpf were washed three times with PBS solution at 4℃, each time soaked for 1 min. The washed embryos were then added to PBS solution at 4℃ and centrifuged at 12000 rpm for 12 min. The supernatant was used as the test sample. The test sample was placed in a 96-well plate for detection. 20 μL of supernatant was taken from each well, and 20 μL of reaction initiation solution (xanthine oxidase) and 160 μL of working solution (water-soluble tetrazolium salt) were added. The plate was incubated at 37℃ for 25 min, and the absorbance value at 560 nm was read on a microplate reader. The evaluation indicators for oxidative stress levels are divided as follows: ROS: 0~0.2 μmol / ml indicates low biotoxicity; 0.2~0.4 μmol / ml indicates relatively low biotoxicity; 0.4~0.6 μmol / ml indicates moderate biotoxicity; 0.6~0.8 μmol / ml indicates relatively high biotoxicity; and above 0.8 μmol / ml indicates very high biotoxicity. SOD: 0.00~0.05umol / ml is low biotoxicity; 0.05~0.10umol / ml is relatively low biotoxicity; 0.10~0.15umol / ml is moderate biotoxicity; 0.15~0.20umol / ml is relatively high biotoxicity; above 0.20umol / ml is highly biotoxic. If at least one of the evaluation indicators of ROS and SOD is high, then S3 is not required and the biotoxicity of the tested refining wastewater sample is directly determined to be high. If both the evaluation indicators of ROS and SOD are relatively high, then S3 is not required and the biotoxicity of the tested refining wastewater sample is directly determined to be high. If at least one of the evaluation indicators of mortality rate and deformity rate is medium or below, then the biotoxicity is pending and S3 is continued. S3. Transcriptomic analysis: Zebrafish embryos that were not exposed to the wastewater sample to be tested were used as a control group. Total RNA was extracted from the two types of zebrafish embryos and subjected to comparative transcriptomic analysis. RNA sequences were compared and gene expression levels were quantified. Genes with significant differences were screened out. By analyzing the differential expression of affected genes and signaling pathways, the biotoxicity of the wastewater from a gene perspective was determined. The method for extracting total RNA from two types of zebrafish embryos was as follows: Zebrafish embryos were homogenized using a tissue homogenizer at room temperature (28℃). Total RNA was extracted using the Trizol method in a clean bench. The OD260 / OD280 ratio was first measured using a UV spectrophotometer; the ideal value was 1.8–2.0. A value below 1.8 may indicate protein contamination, while a value above 2.0 may indicate RNA degradation products. The integrity of the 28S and 18S rRNA bands was then assessed by observing them using agarose gel electrophoresis. Finally, the RNA concentration was accurately determined using a Qubit quantitative PCR instrument. An mRNA-Seq library was constructed using the Library Prep Kit for Illumina. The HiSeq™ platform generates raw short sequence fragments from RNA sequencing. After filtering with FASTP software, sequences containing primer / adapter contamination, low-quality sequences, or unidentified nucleotides exceeding 10% are removed, resulting in high-quality sequences. These sequences are then aligned, and gene expression levels are quantified. Analysis is performed using the R package to screen for genes with significantly different expression levels between the treatment and control groups in the exposed culture environment. Functional enrichment analysis was conducted on these genes. Significantly different expression levels were found between the treatment and control groups for: NF-κB inflammatory pathway (related genes: tnf-α, il-1β, il-6); metallothionein (MT) genes (mt1, mt2); and apoptosis-related genes (caspase-3a, bcl2a). The biological functions and pathways of these differentially expressed genes are explained. The percentage of genes with significantly different expression levels between the treatment group and the control group in the exposed culture is X. If X < 2%, the biotoxicity is low; if 2% ≤ X < 5%, the biotoxicity is relatively low; if 5% ≤ X < 10%, the biotoxicity is moderate; if 10% ≤ X < 20%, the biotoxicity is relatively high; if X ≥ 20%, the biotoxicity is high. S4. The overall biotoxicity of the refining wastewater was determined by weighting the mortality rate and morphological malformation rate measured in S1, the ROS and SOD contents measured in S2, and the genomic perspective of the biotoxicity determined in S3. The specific calculation process is as follows: The evaluation results in S1 to S3 are summarized. Among them, low biotoxicity is scored as 0, low biotoxicity as 25, medium biotoxicity as 50, high biotoxicity as 75, and high biotoxicity as 100. The mortality rate score is a1, with a weight b1 of 0.22, and the deformity rate score is a2, with a weight b2 of 0.15. The score for ROS is a3, with a weight of b4 of 0.18; the score for SOD is a4, with a weight of b4 of 0.18. The score of the differentially expressed gene X is recorded as a5, and the weight b5 is 0.27; The final overall biotoxicity evaluation standard for the refining wastewater samples to be tested is as follows: L=a1*b1+a2*b2+a3*b3+a4*b4+a5*b5 If 0 ≤ L < 20, the biological toxicity of the refining wastewater sample is low; if 20 ≤ L < 40, the biological toxicity of the refining wastewater sample is relatively low; if 40 ≤ L < 60, the biological toxicity of the refining wastewater sample is moderate; if 60 ≤ L < 80, the biological toxicity of the refining wastewater sample is relatively high; and if 80 ≤ L < 100, the biological toxicity of the refining wastewater sample is very high.

[0025] Example 2 The difference between this embodiment and Embodiment 1 is that: In S1, 2 mL of culture medium was added to each well of a 6-well plate. The culture medium was a 1:1 volume mixture of embryo culture water and the refined wastewater sample to be tested. The pH of the embryo culture water was 6.8, the density of the exposed culture was 30 embryos / well, and several parallel groups were set up. The 6-well plate was placed in a light incubator, and the culture conditions were: temperature 27 ℃, light-dark ratio 14:10. The culture medium was changed every 24 h to obtain zebrafish embryos. The mortality rate and morphological malformation rate of the zebrafish embryos were determined.

[0026] Example 3 The difference between this embodiment and Embodiment 1 is that: In S1, 6 mL of culture medium was added to each well of a 6-well plate. The culture medium was a 1:1 volume mixture of embryo culture water and the refined wastewater sample to be tested. The pH of the embryo culture water was 7.5, the density of the exposed culture was 50 embryos / well, and several parallel groups were set up. The 6-well plate was placed in a light incubator, and the culture conditions were: temperature 29 ℃, light-dark ratio 14:10. The culture medium was changed every 24 h to obtain zebrafish embryos. The mortality rate and morphological malformation rate of the zebrafish embryos were determined.

[0027] Example 4 The difference between this embodiment and Embodiment 1 is that: In S2, the ROS content was determined as follows: using the Beyotime S0033 kit, zebrafish embryos cultured at 72 hpf were washed twice with PBS solution, each time soaked for 1 min. The washed embryos were then added to a 6-well plate containing 2 ml of the reactive oxygen species (ROS) fluorescent probe DCFH-DA working solution and incubated in a 26℃ incubator in the dark for 30 min. After that, the embryos were washed three times with PBS solution to remove free probes that had not entered the embryos. The washed embryos were then placed in a black 96-well plate, with 3 embryos and 100 μL of PBS solution added to each well. Each group was set up with 3 replicates. The fluorescence intensity was detected using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and the fluorescence intensity data were recorded. The method for determining SOD content is as follows: Using the Beyotime S0109 kit, zebrafish embryos cultured at 96 hpf were washed twice with PBS solution at 4℃, each time soaked for 1 min. The washed embryos were then added to PBS solution at 4℃ and centrifuged at 12000 rpm for 10 min. The supernatant was used as the test sample. The test sample was placed in a 96-well plate for detection. 20 μL of supernatant was taken from each well, and 20 μL of reaction start-up solution and 160 μL of working solution were added. The plate was incubated at 37℃ for 20 min, and the absorbance value at 560 nm was read on a microplate reader.

[0028] Example 5 The difference between this embodiment and Embodiment 1 is that: In S2, the ROS content was determined as follows: using the Beyotime S0033 kit, zebrafish embryos cultured at 72 hpf were washed three times with PBS solution, each time soaking for 2 min. The washed embryos were then added to a 6-well plate containing 2 ml of the reactive oxygen species (ROS) fluorescent probe DCFH-DA working solution and incubated in a 28℃ incubator in the dark for 60 min. After that, the embryos were washed three times with PBS solution to remove the free probes that had not entered the embryos. The washed embryos were then placed in a black 96-well plate, with 5 embryos and 100 μL of PBS solution added to each well. Each group was set up with 3 replicates. The fluorescence intensity was detected using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and the fluorescence intensity data were recorded. The method for determining SOD content is as follows: Using the Beyotime S0109 kit, zebrafish embryos cultured at 96 hpf were washed three times with PBS solution at 4℃, each time soaked for 2 min. The washed embryos were then added to PBS solution at 4℃ and centrifuged at 12000 rpm for 15 min. The supernatant was used as the test sample. The test sample was placed in a 96-well plate for detection. 20 μL of supernatant was taken from each well, and 20 μL of reaction starter solution and 160 μL of working solution were added. The plate was incubated at 37℃ for 30 min, and the absorbance value at 560 nm was read on a microplate reader.

[0029] Experimental Example The following experiments demonstrate the biotoxicity detection method for refining wastewater based on a dual-level zebrafish embryo method of the present invention. Taking the method in Example 1 as an example, different types of wastewater were tested. The test results of water samples from each stage of the chemical wastewater treatment unit are as follows: Figures 1-3As shown, the water samples from each section of the chemical wastewater treatment unit caused certain mortality and malformation rates in zebrafish embryos, and the impact varied from unit to unit. For most of the units, which were classified as having low to medium biotoxicity, except for the wastewater from the high-density sedimentation tank which was directly assessed as having high biotoxicity, the remaining wastewater needed to undergo further analysis of oxidative stress levels and gene transcriptomics to ultimately obtain the biotoxicity of each wastewater from the chemical wastewater treatment unit. The test results of water samples from each section of the refinery wastewater treatment unit are as follows: Figures 4-6 As shown, the water samples from each section of the refinery wastewater treatment unit caused certain mortality and malformation rates in zebrafish embryos, and the impact varied from unit to unit. For most of the units, which were classified as having low to medium biotoxicity, the wastewater entering the refinery unit was directly assessed as having high biotoxicity. The remaining wastewater required further analysis of oxidative stress levels and transcriptomics to ultimately determine the biotoxicity of each wastewater from the refinery wastewater treatment unit. The test results of water samples from each section of the recycled water wastewater treatment unit are as follows: Figures 7-9 As shown, the water samples from each section of the recycled water wastewater treatment unit caused certain mortality and malformation rates in zebrafish embryos, and the effects varied from unit to unit. For most of the units, which were in the low to medium biotoxicity range, further analysis of oxidative stress levels and gene transcriptomics is needed to obtain the biotoxicity of each wastewater in the recycled water wastewater treatment unit. The test results of water samples from each section of the refinery wastewater treatment unit are as follows: Figures 10-12 As shown, the water samples from each section of the concentrated wastewater treatment unit caused certain mortality and malformation rates in zebrafish embryos, and the impact varied among different units. For most of the units, which were classified as having low to medium biotoxicity, except for the wastewater from the neutralization reaction tank which was directly assessed as having high biotoxicity, the remaining wastewater needed to undergo further analysis of oxidative stress levels and gene transcriptomics to ultimately determine the biotoxicity of each wastewater from the concentrated wastewater treatment unit.

Claims

1. A method for detecting the biotoxicity of refining wastewater based on a dual-level approach using zebrafish embryos, characterized in that... Includes the following steps: S1. Mortality analysis: Zebrafish fertilized eggs were placed in the water sample of the refining wastewater to be tested for exposure culture. After 120 hpf, zebrafish embryos were obtained. The mortality rate and morphological malformation rate of the zebrafish embryos were determined. During the culture period, a portion of the zebrafish embryos cultured to 72 hpf and 96 hpf were retained for later use. S2. Analysis of oxidative stress level: Zebrafish embryos cultured to 72 hpf and 96 hpf retained in S1 were taken, and the ROS and SOD contents of the zebrafish embryos were measured by ROS and SOD kits, respectively, to determine the oxidative stress level of the zebrafish embryos. S3. Transcriptomic analysis: Zebrafish embryos that were not exposed to the wastewater sample to be tested were used as a control group. Total RNA was extracted from the two types of zebrafish embryos and subjected to comparative transcriptomic analysis. RNA sequences were compared and gene expression levels were quantified. Genes with significant differences were screened out. By analyzing the differential expression of affected genes and signaling pathways, the biotoxicity of the wastewater from a gene perspective was determined. S4. The overall biotoxicity of the refining wastewater is determined by weighting the mortality rate and morphological malformation rate measured in S1, the ROS and SOD content measured in S2, and the biotoxicity of the refining wastewater from a genetic perspective determined in S3.

2. The method for detecting the biotoxicity of refining wastewater based on a dual-level zebrafish embryo method according to claim 1, characterized in that, In S1, the container used for the exposure culture is a 6-well plate. 2-6 mL of culture medium is added to each well of the 6-well plate. The culture medium is a mixture of embryo culture water and the refined wastewater sample to be tested at a volume ratio of 1:

1. The pH of the embryo culture water is 6.8-7.

5. The density of the exposure culture is 30-50 embryos / well, and several parallel groups are set up. The 6-well plate is placed in a light incubator, and the culture conditions are: temperature 28±1 ℃, light-dark ratio 14:10, and the culture medium is changed every 24 h.

3. The method for detecting the biotoxicity of refining wastewater based on a dual-level zebrafish embryo method according to claim 1, characterized in that, In S1, zebrafish fertilized eggs are fertilized eggs produced by adult zebrafish within 1 hour after disinfection and in good condition.

4. The method for detecting the biotoxicity of refining wastewater based on a dual-level zebrafish embryo according to claim 1, characterized in that, In S2, the ROS content was determined as follows: Using the S0033 kit, zebrafish embryos cultured at 72 hpf were washed 2-3 times with PBS solution, each time soaking for 1-2 min. The washed embryos were then added to a 6-well plate containing 2 ml of reactive oxygen species (ROS) fluorescent probe working solution and incubated in a 26-28℃ incubator in the dark for 30-60 min. After that, the embryos were washed 3 times with PBS solution to remove free probes that had not entered the embryos. The washed embryos were then placed in a black 96-well plate, with 3-5 embryos and 100 μL of PBS solution added to each well. Each group was set up with 3 replicates. The fluorescence intensity was detected using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and the fluorescence intensity data were recorded.

5. The method for detecting the biotoxicity of refining wastewater based on a dual-level zebrafish embryo method according to claim 1, characterized in that, In S2, the SOD content was determined as follows: using the S0109 kit, zebrafish embryos cultured at 96 hpf were washed 2-3 times with PBS solution at 4℃, each time soaking for 1-2 min. The washed embryos were then added to PBS solution at 4℃ and centrifuged at 12000 rpm for 10-15 min. The supernatant was used as the test sample. The test sample was placed in a 96-well plate for detection. 20 μL of supernatant was taken from each well, and 20 μL of reaction starter solution and 160 μL of working solution were added. The plate was incubated at 37℃ for 20-30 min, and the absorbance value at 560 nm was read on a microplate reader.

6. The method for detecting the biotoxicity of refining wastewater based on a dual-level zebrafish embryo method according to claim 1, characterized in that, In S3, the method for extracting total RNA from two types of zebrafish embryos was as follows: zebrafish embryos were homogenized using a tissue homogenizer at room temperature (26-28℃). Total RNA was extracted using the Trizol method in a clean bench. The OD260 / OD280 ratio was first detected using a UV spectrophotometer, and the integrity of the 28S and 18S rRNA bands was checked by agarose gel electrophoresis. Finally, the RNA concentration was accurately measured using a Qubit fluorescence quantitative quantitation system. An mRNA-Seq library was constructed using the Library Prep Kit for Illumina. Raw short sequence fragments were generated using the Illumina HiSeq™ platform. After filtering with FASTP software, sequences containing primer / adapter contamination, low-quality sequences, or unidentified nucleotides exceeding 10% were removed to obtain high-quality sequences from the raw data. The obtained sequences were compared, and gene expression levels were quantified. Analysis was performed using R-packages to screen genes with significantly different expression levels between the exposed culture treatment group and the control group, and functional enrichment analysis was conducted to interpret the biological functions and pathways of the differentially expressed genes.

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